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Isolation of Ustilago bromivora Strains from Infected Spikelets through Spore Recovery and Germination

Featured protocol,  Authors: Jason Bosch
Jason BoschAffiliation: Gregor Mendel Institute (GMI), Austrian Academy of Sciences, Vienna Biocenter (VBC), Dr. Bohr-Gasse 3, 1030 Vienna, Austria
Bio-protocol author page: a4828
 and Armin Djamei
Armin DjameiAffiliation: Gregor Mendel Institute (GMI), Austrian Academy of Sciences, Vienna Biocenter (VBC), Dr. Bohr-Gasse 3, 1030 Vienna, Austria
For correspondence: armin.djamei@gmi.oeaw.ac.at
Bio-protocol author page: a4829
date: 7/20/2017, 13 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2392.

Brief version appeared in Elife, Nov 2016
Ustilago bromivora is a biotrophic smut fungus infecting Brachypodium sp. It is closely related to the barley-infecting smut Ustilago hordei, and related to the well-studied, gall-inducing model pathogen Ustilago maydis. Upon flowering, the spikelets of U. bromivora-infected plants are filled with black fungal spores. While it is possible to directly use this spore material to infect Brachypodium seeds, in many cases it is more useful to isolate individual strains of U. bromivora for a genetically homogenous population. This protocol describes how to collect and germinate the spores of U. bromivora on plate in order to obtain strains derived from a single cell.

Using CRISPR/Cas9 for Large Fragment Deletions in Saccharomyces cerevisiae

Featured protocol,  Authors: Huanhuan Hao
Huanhuan HaoAffiliation: Key Laboratory of Microbial Diversity Research and Application of Hebei Province, Key discipline of biological engineering of Hebei province, College of Life Sciences, Hebei University, Baoding, China
Bio-protocol author page: a4919
Jing Huang
Jing HuangAffiliation: Key Laboratory of Microbial Diversity Research and Application of Hebei Province, Key discipline of biological engineering of Hebei province, College of Life Sciences, Hebei University, Baoding, China
Bio-protocol author page: a2481
Tongtong Liu
Tongtong LiuAffiliation: Key Laboratory of Microbial Diversity Research and Application of Hebei Province, Key discipline of biological engineering of Hebei province, College of Life Sciences, Hebei University, Baoding, China
Bio-protocol author page: a4923
Hui Tang
Hui TangAffiliation: Key Laboratory of Microbial Diversity Research and Application of Hebei Province, Key discipline of biological engineering of Hebei province, College of Life Sciences, Hebei University, Baoding, China
Bio-protocol author page: a4921
 and Liping Zhang
Liping ZhangAffiliation: Key Laboratory of Microbial Diversity Research and Application of Hebei Province, Key discipline of biological engineering of Hebei province, College of Life Sciences, Hebei University, Baoding, China
For correspondence: zhanglphbu@sohu.com
Bio-protocol author page: a4920
date: 7/20/2017, 7 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2415.

Brief version appeared in Anal Biochem, Sep 2016
CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9) systems have emerged as a powerful tool for genome editing in many organisms. The wide use of CRISPR/Cas9 systems may be due to the fact that these systems contain a simple guide RNA (sgRNA) that is relatively easy to design and they are very versatile with the ability to simultaneously target multiple genes within a cell (Varshney et al., 2015). We have developed a CRISPR/Cas9 system to delete large genomic fragments (exceeding 30 kb) in Saccharomyces cerevisiae. One application of this technology is to study the effects of large-scale deletions of non-essential genes which may give insight into the function of gene clusters within chromosomes at the molecular level. In this protocol, we describe the general procedures for large fragment deletion in S. cerevisiae using CRISPR/Cas9 including: how to design CRISPR arrays and how to construct Cas9-crRNA expression plasmids as well as how to detect mutations introduced by the system within S. cerevisiae cells.

Assaying Glycogen and Trehalose in Yeast

Featured protocol,  Authors: Yuping Chen
Yuping ChenAffiliation: Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, NY, USA
Bio-protocol author page: a4763
 and Bruce Futcher
Bruce FutcherAffiliation: Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, NY, USA
For correspondence: bfutcher@gmail.com
Bio-protocol author page: a4764
date: 7/5/2017, 167 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2371.

Brief version appeared in Mol Cell, May 2016
Organisms store carbohydrates in several forms. In yeast, carbohydrates are stored in glycogen (a multi-branched polysaccharide) and in trehalose (a disaccharide). As in other organisms, the amount of stored carbohydrate varies dramatically with physiological state, and accordingly, an assay of stored carbohydrate can help reveal physiological state. Here, we describe relatively easy and streamlined assays for glycogen and trehalose in yeast that can be applied either to a few samples, or in a moderately high-throughput fashion (dozens to hundreds of samples).

Isolation of Ustilago bromivora Strains from Infected Spikelets through Spore Recovery and Germination

Authors: Jason Bosch
Jason BoschAffiliation: Gregor Mendel Institute (GMI), Austrian Academy of Sciences, Vienna Biocenter (VBC), Dr. Bohr-Gasse 3, 1030 Vienna, Austria
Bio-protocol author page: a4828
 and Armin Djamei
Armin DjameiAffiliation: Gregor Mendel Institute (GMI), Austrian Academy of Sciences, Vienna Biocenter (VBC), Dr. Bohr-Gasse 3, 1030 Vienna, Austria
For correspondence: armin.djamei@gmi.oeaw.ac.at
Bio-protocol author page: a4829
date: 7/20/2017, 13 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2392.

[Abstract] Ustilago bromivora is a biotrophic smut fungus infecting Brachypodium sp. It is closely related to the barley-infecting smut Ustilago hordei, and related to the well-studied, gall-inducing model pathogen Ustilago maydis. Upon flowering, the spikelets of U. bromivora-infected plants are filled with black ...

Using CRISPR/Cas9 for Large Fragment Deletions in Saccharomyces cerevisiae

Authors: Huanhuan Hao
Huanhuan HaoAffiliation: Key Laboratory of Microbial Diversity Research and Application of Hebei Province, Key discipline of biological engineering of Hebei province, College of Life Sciences, Hebei University, Baoding, China
Bio-protocol author page: a4919
Jing Huang
Jing HuangAffiliation: Key Laboratory of Microbial Diversity Research and Application of Hebei Province, Key discipline of biological engineering of Hebei province, College of Life Sciences, Hebei University, Baoding, China
Bio-protocol author page: a2481
Tongtong Liu
Tongtong LiuAffiliation: Key Laboratory of Microbial Diversity Research and Application of Hebei Province, Key discipline of biological engineering of Hebei province, College of Life Sciences, Hebei University, Baoding, China
Bio-protocol author page: a4923
Hui Tang
Hui TangAffiliation: Key Laboratory of Microbial Diversity Research and Application of Hebei Province, Key discipline of biological engineering of Hebei province, College of Life Sciences, Hebei University, Baoding, China
Bio-protocol author page: a4921
 and Liping Zhang
Liping ZhangAffiliation: Key Laboratory of Microbial Diversity Research and Application of Hebei Province, Key discipline of biological engineering of Hebei province, College of Life Sciences, Hebei University, Baoding, China
For correspondence: zhanglphbu@sohu.com
Bio-protocol author page: a4920
date: 7/20/2017, 7 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2415.

[Abstract] CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9) systems have emerged as a powerful tool for genome editing in many organisms. The wide use of CRISPR/Cas9 systems may be due to the fact that these systems contain a simple guide RNA (sgRNA) that is relatively ...

Assaying Glycogen and Trehalose in Yeast

Authors: Yuping Chen
Yuping ChenAffiliation: Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, NY, USA
Bio-protocol author page: a4763
 and Bruce Futcher
Bruce FutcherAffiliation: Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, NY, USA
For correspondence: bfutcher@gmail.com
Bio-protocol author page: a4764
date: 7/5/2017, 167 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2371.

[Abstract] Organisms store carbohydrates in several forms. In yeast, carbohydrates are stored in glycogen (a multi-branched polysaccharide) and in trehalose (a disaccharide). As in other organisms, the amount of stored carbohydrate varies dramatically with physiological state, and accordingly, an assay of stored ...

Serial Immunoprecipitation of 3xFLAG/V5-tagged Yeast Proteins to Identify Specific Interactions with Chaperone Proteins

Authors: Xu Zheng
Xu ZhengAffiliation: Whitehead Institute for Biomedical Research, Cambridge, USA
Bio-protocol author page: a4703
 and David Pincus
David PincusAffiliation: Whitehead Institute for Biomedical Research, Cambridge, USA
For correspondence: pincus@wi.mit.edu
Bio-protocol author page: a4704
date: 6/20/2017, 261 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2348.

[Abstract] This method was generated to isolate high affinity protein complexes from yeast lysate by performing serial affinity purification of doubly tagged 3xFLAG/V5 proteins. First, the bait protein of interest is immunoprecipitated by anti-FLAG-conjugated magnetic beads and gently eluted by 3xFLAG antigen ...

Thermostability Measurement of an α-Glucosidase Using a Classical Activity-based Assay and a Novel Thermofluor Method

Authors: Karin Ernits
Karin ErnitsAffiliation: Institute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia
Bio-protocol author page: a4705
Katrin Viigand
Katrin ViigandAffiliation: Institute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia
Bio-protocol author page: a4706
Triinu Visnapuu
Triinu VisnapuuAffiliation: Institute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia
Bio-protocol author page: a4707
Kristina Põšnograjeva
Kristina PõšnograjevaAffiliation: Institute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia
Bio-protocol author page: a4708
 and Tiina Alamäe
Tiina AlamäeAffiliation: Institute of Molecular and Cell Biology, University of Tartu, Tartu, Estonia
For correspondence: talamae@ebc.ee
Bio-protocol author page: a4709
date: 6/20/2017, 247 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2349.

[Abstract] α-glucosidases (including maltases and isomaltases) are enzymes which release glucose from a set of α-glucosidic substrates. Their catalytic activity, substrate specificity and thermostability can be assayed using this trait. Thermostability of proteins can also be determined using a high-throughput ...

Induction and Quantification of Patulin Production in Penicillium Species

Authors: Yong Chen
Yong ChenAffiliation: Key Laboratory of Plant Resources, Institute of Botany, the Chinese Academy of Sciences, Xiangshan Nanxincun 20, Haidian Districy, Beijing 100093, China
Bio-protocol author page: a4471
Boqiang Li
Boqiang LiAffiliation: Key Laboratory of Plant Resources, Institute of Botany, the Chinese Academy of Sciences, Xiangshan Nanxincun 20, Haidian Districy, Beijing 100093, China
Bio-protocol author page: a1847
Zhanquan Zhang
Zhanquan ZhangAffiliation: Key Laboratory of Plant Resources, Institute of Botany, the Chinese Academy of Sciences, Xiangshan Nanxincun 20, Haidian Districy, Beijing 100093, China
Bio-protocol author page: a1845
 and Shiping Tian
Shiping TianAffiliation: Key Laboratory of Plant Resources, Institute of Botany, the Chinese Academy of Sciences, Xiangshan Nanxincun 20, Haidian Districy, Beijing 100093, China
For correspondence: tsp@ibcas.ac.cn
Bio-protocol author page: a1848
date: 6/5/2017, 309 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2324.

[Abstract] Patulin, a worldwide regulated mycotoxin, is primarily produced by Penicillium and Aspergillus species during fruit spoilage. Patulin contamination is a great concern with regard to human health because exposure of the mycotoxin can result in severe acute and chronic toxicity, including neurotoxic, ...

A Reliable Assay to Evaluate the Virulence of Aspergillus nidulans Using the Alternative Animal Model Galleria mellonella (Lepidoptera)

Authors: Caroline Mota Fernandes
Caroline Mota FernandesAffiliation: Instituto de Biofisica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
For correspondence: cmota@biof.ufrj.br
Bio-protocol author page: a4626
Fernanda Lopes Fonseca
Fernanda Lopes FonsecaAffiliation: Centro de Desenvolvimento Tecnológico em Saúde, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil
Bio-protocol author page: a4627
Gustavo Henrique Goldman
Gustavo Henrique GoldmanAffiliation: Faculdade de Ciências Farmacêuticas de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto, Brazil
Bio-protocol author page: a4628
Marcos Dias Pereira*
Marcos Dias PereiraAffiliation: Departamento de Bioquímica, Instituto de Química, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
For correspondence: marcosdp@iq.ufrj.br
Bio-protocol author page: a4629
 and Eleonora Kurtenbach*
Eleonora KurtenbachAffiliation: Instituto de Biofisica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil
Bio-protocol author page: a4630
 (*contributed equally to this work) date: 6/5/2017, 318 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2329.

[Abstract] The greater wax moth Galleria mellonella has emerged as an effective heterologous host to study fungal pathogenesis and the efficacy of promising antifungal drugs (Mylonakis et al., 2005; Li et al., 2013). Here, a methodology describing the Aspergillus nidulans infection in G. mellonella larvae, along ...

Single-molecule Analysis of DNA Replication Dynamics in Budding Yeast and Human Cells by DNA Combing

Authors: Hélène Tourrière
Hélène TourrièreAffiliation: Institute of Human Genetics, CNRS UMR 9002 and University of Montpellier, Equipe labéllisée LIGUE 2017, Montpellier, France
For correspondence: helene.tourriere@igh.cnrs.fr
Bio-protocol author page: a4580
Julie Saksouk
Julie SaksoukAffiliation: Institute of Human Genetics, CNRS UMR 9002 and University of Montpellier, Equipe labéllisée LIGUE 2017, Montpellier, France
Bio-protocol author page: a4581
Armelle Lengronne
Armelle LengronneAffiliation: Institute of Human Genetics, CNRS UMR 9002 and University of Montpellier, Equipe labéllisée LIGUE 2017, Montpellier, France
Bio-protocol author page: a4582
 and Philippe Pasero
Philippe Pasero Affiliation: Institute of Human Genetics, CNRS UMR 9002 and University of Montpellier, Equipe labéllisée LIGUE 2017, Montpellier, France
For correspondence: philippe.pasero@igh.cnrs.fr
Bio-protocol author page: a4583
date: 6/5/2017, 416 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2305.

[Abstract] The DNA combing method allows the analysis of DNA replication at the level of individual DNA molecules stretched along silane-coated glass coverslips. Before DNA extraction, ongoing DNA synthesis is labeled with halogenated analogues of thymidine. Replication tracks are visualized by immunofluorescence ...

Fluorescently Labelled Aerolysin (FLAER) Labelling of Candida albicans Cells

Authors: Sneh Lata Singh
Sneh Lata SinghAffiliation: School of Life Sciences, Jawaharlal Nehru University, New Delhi, India
Bio-protocol author page: a4573
 and Sneha Sudha Komath
Sneha Sudha KomathAffiliation: School of Life Sciences, Jawaharlal Nehru University, New Delhi, India
For correspondence: sskomath@mail.jnu.ac.in
Bio-protocol author page: a4574
date: 6/5/2017, 346 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2303.

[Abstract] In this protocol we describe a nonradiolabelled labelling of GPI anchor in Candida albicans. The method uses a fluorescent probe to bind specifically to GPI anchors so that the level of GPI-anchored proteins at the cell surface can be measured. The labelling does not need permeabilization of cells and ...

CRISPR-PCS Protocol for Chromosome Splitting and Splitting Event Detection in Saccharomyces cerevisiae

Authors: Yu Sasano
Yu SasanoAffiliation: Department of Applied Microbial Technology, Faculty of Biotechnology and Life Science, Sojo University, Kumamoto City, Japan
Bio-protocol author page: a4563
 and Satoshi Harashima
Satoshi HarashimaAffiliation: Department of Applied Microbial Technology, Faculty of Biotechnology and Life Science, Sojo University, Kumamoto City, Japan
For correspondence: harashima@bio.sojo-u.ac.jp
Bio-protocol author page: a4564
date: 5/20/2017, 451 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.2306.

[Abstract] Chromosome engineering is an important technology with applications in basic biology and biotechnology. Chromosome splitting technology called PCS (PCR-mediated Chromosome Splitting) has already been developed as a fundamental chromosome engineering technology in the budding yeast. However, the splitting ...
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In vitro Protein Kinase Assay

Author: Yuehua Wei
Yuehua WeiAffiliation: Department of Pharmacology, Cancer Institute of New Jersey, UMDNJ Robert Wood Johnson Medical School, Piscataway, USA
For correspondence: weiyh.sjtu.edu@gmail.com
Bio-protocol author page: a49
date: 6/5/2012, 22750 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.212.

[Abstract] This protocol will describe experimental procedures for an in vitro kinase assay of the yeast protein kinase Sch9. This protocol can be tailored to detect kinase activity of other yeast protein kinase....

[Bio101] Making Yeast Competent Cells and Yeast Cell Transformation

Author: Yongxian Lu
Yongxian LuAffiliation: Carnegie Institution for Science, Stanford University, Stanford, USA
For correspondence: yxlu@stanford.edu
Bio-protocol author page: a28
date: 7/20/2011, 22348 views, 2 Q&A
DOI: https://doi.org/10.21769/BioProtoc.96.

[Abstract] This is a quite simple but reliable protocol to make very high transformation efficiency yeast competent cells. By express your gene of interest, protein function can be studied in yeast cells....

[Bio101] Protocol for Whole Cell Lysis of Yeast

Author: Zongtian Tong
Zongtian TongAffiliation: Department of Cell Biology, Center for Metabolism and Obesity Research, Johns Hopkins School of Medicine, Baltimore, USA
For correspondence: tongzong@gmail.com
Bio-protocol author page: a14
date: 1/5/2011, 19891 views, 4 Q&A
DOI: https://doi.org/10.21769/BioProtoc.14.

[Abstract] This protocol describes how to perform lysis on whole yeast cell samples using NaOH. The lysed cells can then be used for downstream applications such as the extraction of total proteins. ...

Spot Assay for Yeast

Author: Zongtian Tong
Zongtian TongAffiliation: Department of Cell Biology, Center for Metabolism and Obesity Research, Johns Hopkins School of Medicine, Baltimore, USA
For correspondence: tongzong@gmail.com
Bio-protocol author page: a14
date: 1/5/2012, 19642 views, 3 Q&A
DOI: https://doi.org/10.21769/BioProtoc.16.

[Abstract] This protocol can be used to compare the cell growth rate of yeast under different growth conditions. It involves the serial dilution and spotting of yeast colonies....

[Bio101] Yeast Vacuole Staining with FM4-64

Author: Zongtian Tong
Zongtian TongAffiliation: Department of Cell Biology, Center for Metabolism and Obesity Research, Johns Hopkins School of Medicine, Baltimore, USA
For correspondence: tongzong@gmail.com
Bio-protocol author page: a14
date: 1/5/2011, 15404 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.18.

[Abstract] The lipophilic probe, FM 4-64 does not fluoresce much in water but fluoresces strongly after binding to the outer plasma membrane, providing clear and distinguishable plasma membrane staining. The binding is rapid and reversible. In this protocol vacuoles in yeast cells are stained with the FM4-64 dye, ...

[Bio101] How to Use an Avestin Emulsiflex C3 Homogenizer to Disrupt Cells

Author: Zongtian Tong
Zongtian TongAffiliation: Department of Cell Biology, Center for Metabolism and Obesity Research, Johns Hopkins School of Medicine, Baltimore, USA
For correspondence: tongzong@gmail.com
Bio-protocol author page: a14
date: 1/5/2011, 12279 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.11.

[Abstract] The EmulsiFlex-C3 homogenizer is powered by an electric motor. The pump does not require a compressor for it to run. This equipment can be used to disrupt cells at a large scale. The EmulsiFlex-C3 has a fixed flow-through capacity of 3 L/h. It has the ability to process samples as small as 10 ml. The ...

[Bio101] Large Scale Native Affinity Purifications of Solubilized Membrane Proteins from Yeast

Author: Zongtian Tong
Zongtian TongAffiliation: Department of Cell Biology, Center for Metabolism and Obesity Research, Johns Hopkins School of Medicin, Baltimore , USA
For correspondence: tongzong@gmail.com
Bio-protocol author page: a14
date: 1/5/2011, 11563 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.12.

[Abstract] This protocol can be used to purify membrane proteins from yeast samples under native conditions at a large scale. This protocol has been developed primarily for FLAG-tagged proteins. This protocol can however be slightly modified and applied to other tags, such as GST or HA....

[Bio101] Small Scale Native Affinity Purifications of Solubilized Membrane Proteins from Yeast

Author: Zongtian Tong
Zongtian TongAffiliation: Department of Cell Biology, Center for Metabolism and Obesity Research, Johns Hopkins School of Medicine, Baltimore, USA
For correspondence: tongzong@gmail.com
Bio-protocol author page: a14
date: 1/5/2011, 11088 views, 1 Q&A
DOI: https://doi.org/10.21769/BioProtoc.15.

[Abstract] In this protocol, we show how to purify membrane proteins from yeast using affinity purification under native conditions at a small scale. ...

Illumina Sequencing Library Construction from ChIP DNA

Author: Wei Zheng
Wei ZhengAffiliation: Keck Biotech Services, Yale University, New Haven, USA
For correspondence: wei.zheng.madison@gmail.com
Bio-protocol author page: a10
date: 2/20/2012, 10740 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.91.

[Abstract] The Illumina sequencing platform is very popular among next-generation sequencing platforms. However, the DNA sequencing library construction kit provided by Illumina is considerably expensive. The protocol described here can be used to construct high-quality sequencing libraries from chromatin immunoprecipitated ...

Co-immunoprecipitation in Yeast

Author: Olesya O. Panasenko
Olesya O. PanasenkoAffiliation: Department of Microbiology and Molecular Medicine, University of Geneva, Faculty of Medicine, CMU, Geneva, Switzerland
For correspondence: olesya.panasenko@unige.ch
Bio-protocol author page: a88
date: 8/20/2012, 10055 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.250.

[Abstract] This protocol describes investigation of protein-protein interactions in baker yeast by co-immunoprecipitation (CoIP). CoIP is a technique to identify physiologically relevant protein-protein interactions in the cell. The interesting protein can be isolated out of solution using antibody that specifically ...
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