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1.
Methods Mol Biol ; 2555: 1-12, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36306075

RESUMO

The vast majority of the Earth's biological diversity are hidden in uncultured and yet uncharacterized microbial genomes. The construction of metagenomic libraries is one cultivation-independent molecular approach to assess this unexplored genetic reservoir. High numbers of novel biocatalysts have been identified by function-based or sequence-based screening of metagenomic libraries derived from various environments. Here, we describe detailed protocols for the construction of metagenomic small-insert and large-insert libraries in plasmids and fosmids, respectively, from environmental DNA.


Assuntos
Metagenoma , Metagenômica , Biblioteca Gênica , Metagenômica/métodos , Biodiversidade , Plasmídeos/genética
2.
Methods Mol Biol ; 2555: 23-49, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36306077

RESUMO

The marine ecosystem covers more than 70% of the world's surface, and oceans represent a source of varied types of organisms due to the diversified environment. Consequently, the marine environment is an exceptional depot of novel bioactive natural products, with structural and chemical features generally not found in terrestrial habitats. Here, in particular, microbes represent a vast source of unknown and probably new physiological characteristics. They have evolved during extended evolutionary processes of physiological adaptations under various environmental conditions and selection pressures. However, to date, the biodiversity of marine microbes and the versatility of their bioactive compounds and metabolites have not been fully explored. Thus, metagenomic tools are required to exploit the untapped marine microbial diversity and their bioactive compounds. This chapter focuses on function-based marine metagenomics to screen for bioactive molecules of value for biotechnology. Functional metagenomic strategies are described, including sampling in the marine environment, constructing marine metagenomic large-insert libraries, and examples on function-based screens for quorum quenching and anti-biofilm activities.


Assuntos
Ecossistema , Metagenômica , Metagenoma , Biotecnologia , Biodiversidade
3.
Methods Mol Biol ; 2555: 73-90, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36306079

RESUMO

Microbial secondary metabolites have been an important source of bioactive compounds with diverse applications from medicine to agriculture, noticeably those encoded by polyketide synthase (PKS) clusters due to their astounding chemical diversity. While most discovered compounds originate from culturable microorganisms, yet-to-be cultured microbes represent a reservoir of previously inaccessible compounds. The advent and development of metagenomics have allowed not only the characterization of these microorganisms but also their metabolic potential, making viable the prospection of environmental PKS for natural product discovery.Study of environmental PKSs often relies on the construction of metagenomic libraries and their mining, with clones containing PKS clusters identified via amplification of conserved domains and then screened for an activity of interest. Compounds produced by clones exhibiting the desired bioactivity can be isolated and characterized. However, these approaches can be less sensitive and biased against more divergent clusters, in addition to precluding the use of bioinformatics for cluster characterization prior to expression. While direct shotgun sequencing of metagenomes has identified and profiled a great number of PKSs from different environments and yet-to-be cultured microorganisms, it does not lend itself well to heterologous expression, the cruxes of natural product discovery.Here, we describe a strategy for sequencing entire metagenomic libraries while maintaining correspondence between sequence and clone, allowing the full characterization and annotation of all clusters present in a library using bioinformatic tools and then seamlessly passing clones of interest for activity screening through heterologous expression. Once a library is sequenced, the methods herein can be adapted for the mining of any biosynthetic gene cluster of interest within a metagenomic library.


Assuntos
Produtos Biológicos , Policetídeo Sintases , Policetídeo Sintases/genética , Metagenoma , Metagenômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Família Multigênica
4.
Methods Mol Biol ; 2555: 91-101, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36306080

RESUMO

Here, we outline how to identify hydrogenase enzymes from metagenomic fosmid libraries through an activity-based screening approach. A metagenomic fosmid library is constructed in E. coli and the fosmids are transferred into a hydrogenase deletion mutant of Shewanella oneidensis MR-1 (ΔhyaB) via triparental mating. If a fosmid clone exhibits hydrogen-uptake activity, S. oneidensis' phenotype is restored and hydrogenase activity is indicated by a color change of the medium from yellow to colorless. The screen enables screening of 48 metagenomic fosmid clones in parallel.


Assuntos
Hidrogenase , Hidrogenase/genética , Hidrogênio , Escherichia coli/genética , Metagenômica , Metagenoma , Biblioteca Gênica
5.
Methods Mol Biol ; 2555: 103-114, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36306081

RESUMO

Phosphate release from inorganic and organic phosphorus compounds can be enzymatically mediated. Phosphate-releasing enzymes, comprising acid and alkaline phosphatases, are recognized as useful biocatalysts in applications such as plant and animal nutrition, bioremediation, and diagnostic analysis. Here, we describe a functional metagenomics approach enabling rapid identification of genes encoding these enzymes. The target genes are detected based on small- and large-insert metagenomic libraries derived from diverse environments. This approach has the potential to unveil entirely new phosphatase families or subfamilies and members of known enzyme classes that hydrolyze phosphomonoester bonds such as phytases. Additionally, we provide a strategy for efficient heterologous expression of phosphatase genes.


Assuntos
6-Fitase , Metagenômica , Metagenoma , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , 6-Fitase/genética , Fosfatos
6.
Methods Mol Biol ; 2555: 115-123, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36306082

RESUMO

The ability to produce high-value products using bacteria will increasingly rely on continued research to make large-scale bacterial fermentation cost-efficient. Engineering bacteria to use alternate carbon sources as feedstock provides an opportunity to reduce production costs. Using inexpensive carbon sources from various forms of waste provides an opportunity to substantially reduce feedstock costs. Functional carbon metabolism pathways can be identified by the introduction of metagenomic libraries into the organism of interest followed by screening for the desired phenotype. We present here a method to transfer metagenomic libraries from E. coli to Pseudomonas alloputida, followed by screening for use of galactose as a sole carbon source.


Assuntos
Carbono , Microbiota , Carbono/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Metagenoma , Metagenômica , Fermentação
7.
Methods Mol Biol ; 2555: 125-137, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36306083

RESUMO

Sustainable use of natural products is one of the key challenges for the future. An increasing focus is on marine organic matter, mostly algae. New biotechnological tools for processing high amounts of micro- and macroalgae are necessary for efficient industrial degradation of marine matter. Secreted glycosyl hydrolases can be enriched and tested on the specific algae cell wall polymers of all algae groups (Rhodophyta; Phaeophyceae; Chlorophyta/Charophyta). Metagenomic analyses established new possibilities to screen algae-associated microbiomes for novel degrading enzymes in combination with sequence-based function prediction.


Assuntos
Metagenoma , Rodófitas , Hidrolases/metabolismo , Rodófitas/metabolismo , Carboidratos , Parede Celular
8.
Methods Mol Biol ; 2555: 139-151, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36306084

RESUMO

Against the background of the steadily increasing amount of plastic waste in the sea and on land, it is more important than ever to find ways out of this situation. In recent years, microorganisms have been discovered that are capable of degrading artificial polymers such as polyethylene terephthalate (PET). Even if the turnover rates of the enzymes responsible for this reaction may be too low to solve the global plastic pollution problem, it is still of great societal interest to find microorganisms that are able to degrade the polymer. The corresponding enzymes, PET esterases (PETases) can be used in biotechnological processes and could contribute to a resource-saving circular economy. In this chapter, we present a sequence-based in silico screening method to find new PETases in metagenomic datasets. This method can easily be adapted to find other enzyme classes. We also list a number of assays that can be used to test the enzymes for activity on PET as well as other substrates.


Assuntos
Metagenoma , Polietilenotereftalatos , Polietilenotereftalatos/química , Polietilenotereftalatos/metabolismo , Esterases/genética , Esterases/química , Metagenômica , Plásticos , Hidrolases/genética , Hidrolases/metabolismo
9.
Methods Mol Biol ; 2555: 167-179, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36306086

RESUMO

Metagenomic screening is a widely applied biotechnological approach for screening of novel industrial enzymes. The traditional method of metagenomic screening is based on the functional analyses of heterologously expressed environmental genes in a suitable host, which is the bottleneck of this method. To avoid limitation from the clone-dependent system, an in vitro expression technology has been developed in combination with next-generation sequencing and bioinformatics. First, the sequence profile of a target enzyme, e.g., poly(ethylene terephthalate) esterase in this protocol, is constructed according to the sequences of well-characterized enzymes. Then, the sequence screening is performed with this computationally generated profile among all available metagenomic databases. Afterwards, the candidate genes are synthesized and expressed in vitro with RNA polymerase and translation machinery from special cell extract. Finally, such in vitro produced enzymes are directly applied for the functional analyses. Comparing to the traditional screening methods, this in vitro screening technology can not only save time and materials, but also be easily developed for high-throughput screening with an automatic pipetting robot.


Assuntos
Esterases , Metagenoma , Esterases/genética , Esterases/metabolismo , Metagenômica , Polietilenotereftalatos/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala
10.
Methods Mol Biol ; 2555: 195-203, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36306088

RESUMO

Bacteriophages, also called phages, are viruses of bacteria. They are the most common and diverse biological entities on this planet. For metagenomic investigation, their diversity is also their biggest obstacle. The direct metagenomic sequence of environmental phage communities often leads to short genomic fragments limiting the investigation to a few individual aspects of phage biology and diversity.The presented protocol for generating a host-associated metagenome reduces the phage diversity to a concise and accessible size. Metagenome sequencing often leads to complete genomes, and the availability of a suitable host system ensures further experimental investigation.


Assuntos
Bacteriófagos , Metagenoma , Bacteriófagos/genética , Metagenômica/métodos , Bactérias/genética , Genômica , Genoma Viral
11.
Methods Mol Biol ; 2555: 205-212, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36306089

RESUMO

Phages are viruses of bacteria and have been known for over a century. They do not have a metabolism or protein synthesis machinery and rely on host cells for replication. The model organism Bacillus subtilis has served as a host strain for decades and enabled the isolation of many unique viral strains. However, many viral species representatives remained orphans as no, or only a few, related phages were ever re-isolated.The presented protocol describes how a CRISPR-Cas9 system with an artificial CRISPR-array can be set up and used to discriminate abundant and well-known B. subtilis phage from a host-based metagenome enrichment. The obtained viral suspension can be used for metagenome sequencing and isolating new viral strains.


Assuntos
Bacillus subtilis , Bacteriófagos , Bacillus subtilis/genética , Sistemas CRISPR-Cas/genética , Metagenoma
12.
Methods Mol Biol ; 2601: 379-401, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36445596

RESUMO

The construction and screening of metagenomic expression libraries have a great potential to identify novel genes with desired functions. Here, we describe metagenomic library preparation from fecal DNA, screening of libraries for antibiotic resistance genes (ARGs), massively parallel DNA sequencing of the enriched DNA fragments, and a computational pipeline for high-throughput assembly and annotation of functionally selected DNA.


Assuntos
Metagenoma , Metagenômica , Resistência Microbiana a Medicamentos/genética , Biblioteca Gênica , Análise de Sequência de DNA
13.
Nat Commun ; 13(1): 7251, 2022 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-36456547

RESUMO

Antimicrobial resistance (AMR) is a major threat to global health. Understanding the emergence, evolution, and transmission of individual antibiotic resistance genes (ARGs) is essential to develop sustainable strategies combatting this threat. Here, we use metagenomic sequencing to analyse ARGs in 757 sewage samples from 243 cities in 101 countries, collected from 2016 to 2019. We find regional patterns in resistomes, and these differ between subsets corresponding to drug classes and are partly driven by taxonomic variation. The genetic environments of 49 common ARGs are highly diverse, with most common ARGs carried by multiple distinct genomic contexts globally and sometimes on plasmids. Analysis of flanking sequence revealed ARG-specific patterns of dispersal limitation and global transmission. Our data furthermore suggest certain geographies are more prone to transmission events and should receive additional attention.


Assuntos
Antibacterianos , Esgotos , Antibacterianos/farmacologia , Farmacorresistência Bacteriana/genética , Genômica , Metagenoma
14.
Microbiome ; 10(1): 209, 2022 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-36457010

RESUMO

BACKGROUND: The accurate and comprehensive analyses of genome-resolved metagenomics largely depend on the reconstruction of reference-quality (complete and high-quality) genomes from diverse microbiomes. Closing gaps in draft genomes have been approaching with the inclusion of Nanopore long reads; however, genome quality improvement requires extensive and time-consuming high-accuracy short-read polishing. RESULTS: Here, we introduce NanoPhase, an open-source tool to reconstruct reference-quality genomes from complex metagenomes using only Nanopore long reads. Using Kit 9 and Q20+ chemistries, we first evaluated the feasibility of NanoPhase using a ZymoBIOMICS gut microbiome standard (including 21 strains), then sequenced the complex activated sludge microbiome and reconstructed 275 MAGs with median completeness of ~ 90%. As a result, NanoPhase improved the MAG contiguity (median MAG N50: 735 Kb, 44-86X compared to conventional short-read-based methods) while maintaining high accuracy, allowing for a full and accurate investigation of target microbiomes. Additionally, leveraging these high-contiguity reference-quality genomes, we identified 165 prophages within 111 MAGs, with 5 as active prophages, indicating the prophage was a neglected source of genetic diversity within microbial populations and influencer in shaping microbial composition in the activated sludge microbiome. CONCLUSIONS: Our results demonstrated that NanoPhase enables reference-quality genome reconstruction from complex metagenomes directly using only Nanopore long reads. Furthermore, besides the 16S rRNA genes and biosynthetic gene clusters, the generated high-accuracy and high-contiguity MAGs improved the host identification of critical mobile genetic elements, e.g., prophage, serving as a genomic blueprint to investigate the microbial potential and ecology in the activated sludge ecosystem. Video Abstract.


Assuntos
Microbiota , Nanoporos , Metagenoma/genética , Metagenômica , RNA Ribossômico 16S/genética , Esgotos , Microbiota/genética , Prófagos
15.
Arch Microbiol ; 204(12): 726, 2022 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-36427112

RESUMO

To improve the sensory quality of aged flue-cured tobacco (FCT), Bacillus subtilis subsp, H11 was inoculated on aged FCT leaves named Pingdingshan DCFB. The metagenome and thecharacteristic aroma substances of aged FCT with different fermentation times (0 h, 12 h, 24 h, and 36 h) were systematically analyzed. The results showed that the content of aroma components and sensory quality of aged FCT were significantly improved when the strain was treated at 35 °C with 25% moisture for 24 h. The inoculation of H11 had a strong influence on the microbial composition and metabolism of the aged FCT leaf surface. Five microorganisms Pantoea (35.04%, 20.12-56.95%), Enterobacter (22.16, 13.60-39.82%), Pseudomonas (12.12, 3.13-26.17%), Terribacillus (8.00%, 4.65-13.01%) and Bacillus (6.54%, 0.67-16.96%) accounted for the largest proportion during the process of fermentation. The content of most neutral flavor components such as ketones and aldehydes in FCT after fermentation was higher than that priorto fermentation. After 24 h fermentation, 3-furfural, 5-methylfurfural, dihydrokiwi lactone and megalotrienone increased by 71.42%, 49.19%, 21.09%, and 10.56%, respectively. Correlation analysis between groups showed that Pseudomonas was significantly correlated with (E, E)-2, 4-heptadienal (P < 0.05), Franconibacter was correlated with damascus ketone (P < 0.05), and Terribacillus was related to the production of ß-citral (P < 0.05). GH9 may be involved in the formation of damasone (P < 0.05), and 4-cyclopentene-1, 3-dione was significantly correlated with glycoside hydrolase family 5 (GH5) (P < 0.05). The correlation between 4-oxyisophorone and GH31, GH103, GH73, and GH3 was significant (P < 0.05). Microorganisms and GHs may play important roles in FCT fermentation.


Assuntos
Metagenoma , Microbiota , Tabaco , Odorantes , Bacillus subtilis/genética , Folhas de Planta
16.
BMC Bioinformatics ; 23(1): 513, 2022 Nov 30.
Artigo em Inglês | MEDLINE | ID: mdl-36451083

RESUMO

BACKGROUND: The assembly of metagenomes decomposes members of complex microbe communities and allows the characterization of these genomes without laborious cultivation or single-cell metagenomics. Metagenome assembly is a process that is memory intensive and time consuming. Multi-terabyte sequences can become too large to be assembled on a single computer node, and there is no reliable method to predict the memory requirement due to data-specific memory consumption pattern. Currently, out-of-memory (OOM) is one of the most prevalent factors that causes metagenome assembly failures. RESULTS: In this study, we explored the possibility of using Persistent Memory (PMem) as a less expensive substitute for dynamic random access memory (DRAM) to reduce OOM and increase the scalability of metagenome assemblers. We evaluated the execution time and memory usage of three popular metagenome assemblers (MetaSPAdes, MEGAHIT, and MetaHipMer2) in datasets up to one terabase. We found that PMem can enable metagenome assemblers on terabyte-sized datasets by partially or fully substituting DRAM. Depending on the configured DRAM/PMEM ratio, running metagenome assemblies with PMem can achieve a similar speed as DRAM, while in the worst case it showed a roughly two-fold slowdown. In addition, different assemblers displayed distinct memory/speed trade-offs in the same hardware/software environment. CONCLUSIONS: We demonstrated that PMem is capable of expanding the capacity of DRAM to allow larger metagenome assembly with a potential tradeoff in speed. Because PMem can be used directly without any application-specific code modification, these findings are likely to be generalized to other memory-intensive bioinformatics applications.


Assuntos
Metagenoma , Microbiota , Metagenômica , Software , Biologia Computacional
17.
Nat Microbiol ; 7(12): 2128-2150, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36443458

RESUMO

Despite advances in sequencing, lack of standardization makes comparisons across studies challenging and hampers insights into the structure and function of microbial communities across multiple habitats on a planetary scale. Here we present a multi-omics analysis of a diverse set of 880 microbial community samples collected for the Earth Microbiome Project. We include amplicon (16S, 18S, ITS) and shotgun metagenomic sequence data, and untargeted metabolomics data (liquid chromatography-tandem mass spectrometry and gas chromatography mass spectrometry). We used standardized protocols and analytical methods to characterize microbial communities, focusing on relationships and co-occurrences of microbially related metabolites and microbial taxa across environments, thus allowing us to explore diversity at extraordinary scale. In addition to a reference database for metagenomic and metabolomic data, we provide a framework for incorporating additional studies, enabling the expansion of existing knowledge in the form of an evolving community resource. We demonstrate the utility of this database by testing the hypothesis that every microbe and metabolite is everywhere but the environment selects. Our results show that metabolite diversity exhibits turnover and nestedness related to both microbial communities and the environment, whereas the relative abundances of microbially related metabolites vary and co-occur with specific microbial consortia in a habitat-specific manner. We additionally show the power of certain chemistry, in particular terpenoids, in distinguishing Earth's environments (for example, terrestrial plant surfaces and soils, freshwater and marine animal stool), as well as that of certain microbes including Conexibacter woesei (terrestrial soils), Haloquadratum walsbyi (marine deposits) and Pantoea dispersa (terrestrial plant detritus). This Resource provides insight into the taxa and metabolites within microbial communities from diverse habitats across Earth, informing both microbial and chemical ecology, and provides a foundation and methods for multi-omics microbiome studies of hosts and the environment.


Assuntos
Microbiota , Animais , Microbiota/genética , Metagenoma , Metagenômica , Planeta Terra , Solo
18.
Genome Biol ; 23(1): 242, 2022 Nov 14.
Artigo em Inglês | MEDLINE | ID: mdl-36376928

RESUMO

Evaluating the quality of metagenomic assemblies is important for constructing reliable metagenome-assembled genomes and downstream analyses. Here, we present metaMIC ( https://github.com/ZhaoXM-Lab/metaMIC ), a machine learning-based tool for identifying and correcting misassemblies in metagenomic assemblies. Benchmarking results on both simulated and real datasets demonstrate that metaMIC outperforms existing tools when identifying misassembled contigs. Furthermore, metaMIC is able to localize the misassembly breakpoints, and the correction of misassemblies by splitting at misassembly breakpoints can improve downstream scaffolding and binning results.


Assuntos
Metagenoma , Metagenômica , Análise de Sequência de DNA/métodos , Metagenômica/métodos , Aprendizado de Máquina , Benchmarking , Software , Algoritmos
19.
BMC Genomics ; 23(1): 748, 2022 Nov 11.
Artigo em Inglês | MEDLINE | ID: mdl-36368923

RESUMO

BACKGROUND: Shotgun metagenome analysis provides a robust and verifiable method for comprehensive microbiome analysis of fungal, viral, archaeal and bacterial taxonomy, particularly with regard to visualization of read mapping location, normalization options, growth dynamics and functional gene repertoires. Current read classification tools use non-standard output formats, or do not fully show information on mapping location. As reference datasets are not perfect, portrayal of mapping information is critical for judging results effectively. RESULTS: Our alignment-based pipeline, Wochenende, incorporates flexible quality control, trimming, mapping, various filters and normalization. Results are completely transparent and filters can be adjusted by the user. We observe stringent filtering of mismatches and use of mapping quality sharply reduces the number of false positives. Further modules allow genomic visualization and the calculation of growth rates, as well as integration and subsequent plotting of pipeline results as heatmaps or heat trees. Our novel normalization approach additionally allows calculation of absolute abundance profiles by comparison with reads assigned to the human host genome. CONCLUSION: Wochenende has the ability to find and filter alignments to all kingdoms of life using both short and long reads, and requires only good quality reference genomes. Wochenende automatically combines multiple available modules ranging from quality control and normalization to taxonomic visualization. Wochenende is available at https://github.com/MHH-RCUG/nf_wochenende .


Assuntos
Metagenoma , Microbiota , Humanos , Metagenômica/métodos , Software , Microbiota/genética , Genoma Humano , Análise de Sequência de DNA/métodos , Algoritmos
20.
Sci Rep ; 12(1): 19405, 2022 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-36371463

RESUMO

This study revealed how Bacteria and Archaea communities and their metabolic functions differed between two groups of black deposits identified in gorge and cave environments. Scanning electron microscopy coupled with energy dispersive spectroscopy was used to analyse the presence of microbial biosignatures and the elemental composition of samples. Metabarcoding of the V3-V4 regions of 16S rRNA was used to investigate Bacteria and Archaea communities. Based on 16S rRNA sequencing results, PICRUSt software was used to predict metagenome functions. Micrographs showed that samples presented microbial biosignatures and microanalyses highlighted Mn concretions and layers on Al-Si surfaces. The 16S rRNA metabarcoding alpha-diversity metrics showed similar Simpson's and Shannon indices and different values of the Chao-1 index. The amplicon sequence variants (ASVs) analysis at the different taxonomic levels showed a diverse genera composition. However, the communities of all samples shared the presence of uncultured ASVs belonging to the Gemmatales family (Phylogenesis: Gemmataceae; Planctomycetes; Planctomycetota; Bacteria). The predicted metagenome functions analysis revealed diverse metabolic profiles of the Cave and Gorge groups. Genes coding for essential Mn metabolism were present in all samples. Overall, the findings on structure, microbiota, and predicted metagenome functions showed a similar microbial contribution to epigean and hypogean black deposits Mn metabolism.


Assuntos
Metagenoma , Microbiota , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/metabolismo , Biologia Computacional , Microbiota/genética , Bactérias , Filogenia , Archaea/genética
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