A High-Throughput Screen for Tuberculosis Progression
Publication Date
February 16, 2011
Journal
PLOS ONE
Authors
Ralph Carvalho, Jan De Sonneville, Oliver W. Stockhammer, Nigel D. L. Savage, et al
Volume
6
Issue
2
Pages
e16779
DOI
http://doi.org/10.1371/journal.pone.0016779
Publisher URL
http://journals.plos.org/plosone/article?id=10.1371%2Fjournal.pone.0016779
PubMed
http://www.ncbi.nlm.nih.gov/pubmed/21390204
PubMed Central
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3040195
Europe PMC
http://europepmc.org/abstract/MED/21390204
Web of Science
000287392700024
Scopus
79951889177
Mendeley
http://www.mendeley.com/research/highthroughput-screen-tuberculosis-progression
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Mendeley | Further Information

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CrossRef

Scopus | Further Information

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Figshare

  • {"files"=>["https://ndownloader.figshare.com/files/399125", "https://ndownloader.figshare.com/files/399141", "https://ndownloader.figshare.com/files/399171", "https://ndownloader.figshare.com/files/399208"], "description"=>"<div><p>One-third of the world population is infected with <em>Mycobacterium tuberculosis</em> and multi-drug resistant strains are rapidly evolving. The noticeable absence of a whole organism high-throughput screening system for studying the progression of tuberculosis is fast becoming the bottleneck in tuberculosis research. We successfully developed such a system using the zebrafish <em>Mycobacterium marinum</em> infection model, which is a well-characterized model for tuberculosis progression with biomedical significance, mimicking hallmarks of human tuberculosis pathology. Importantly, we demonstrate the suitability of our system to directly study <em>M. tuberculosis</em>, showing for the first time that the human pathogen can propagate in this vertebrate model, resulting in similar early disease symptoms to those observed upon <em>M. marinum</em> infection. Our system is capable of screening for disease progression via robotic yolk injection of early embryos and visual flow screening of late-stage larvae. We also show that this system can reliably recapitulate the standard caudal vein injection method with a throughput level of 2,000 embryos per hour. We additionally demonstrate the possibility of studying signal transduction leading to disease progression using reverse genetics at high-throughput levels. Importantly, we use reference compounds to validate our system in the testing of molecules that prevent tuberculosis progression, making it highly suited for investigating novel anti-tuberculosis compounds <em>in vivo</em>.</p> </div>", "links"=>[], "tags"=>["high-throughput", "tuberculosis", "progression"], "article_id"=>138836, "categories"=>["Microbiology", "Cancer", "Biochemistry", "Developmental Biology", "Immunology"], "users"=>["Ralph Carvalho", "Jan de Sonneville", "Oliver W. Stockhammer", "Nigel D. L. Savage", "Wouter J. Veneman", "Tom H. M. Ottenhoff", "Ron P. Dirks", "Annemarie H. Meijer", "Herman P. Spaink"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0016779.s001", "https://dx.doi.org/10.1371/journal.pone.0016779.s002", "https://dx.doi.org/10.1371/journal.pone.0016779.s003", "https://dx.doi.org/10.1371/journal.pone.0016779.s004"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/A_High_Throughput_Screen_for_Tuberculosis_Progression/138836", "title"=>"A High-Throughput Screen for Tuberculosis Progression", "pos_in_sequence"=>0, "defined_type"=>4, "published_date"=>"2011-02-16 02:27:16"}
  • {"files"=>["https://ndownloader.figshare.com/files/797810"], "description"=>"<p>(<b>A</b>) 5 dpi fli1-egfp larva with gfp-labelled vasculature showing spread of bacteria (red) throughout the body (scale bar: 250 µm). (<b>B</b> and <b>D</b>) Bright-field/fluorescence overlay and (<b>C</b> and <b>E</b>) confocal z-stack of red-fluorescent bacteria showing activation of green-fluorescent <i>gags</i> at the (<b>B</b> and <b>C</b>) edge of the yolk extension and on the (<b>D</b> and <b>E</b>) tail of a 7 day-old larva (scale bar: 25 µm). (<b>F</b> and <b>H</b>) Bright-field confocal plane and (<b>G</b> and <b>I</b>) confocal z-stack of red-fluorescent bacteria co-localizing with green-fluorescent leukocytes detected by L-plastin immunostaining (scale bar: 25 µm). The lesions caused by the granulomas can be clearly seen in <b>F</b> and <b>H</b>.</p>", "links"=>[], "tags"=>["yolk", "sac", "injection", "embryos", "16-", "512-cell"], "article_id"=>468170, "categories"=>["Microbiology", "Biochemistry", "Infectious Diseases", "Developmental Biology", "Immunology"], "users"=>["Ralph Carvalho", "Jan de Sonneville", "Oliver W. Stockhammer", "Nigel D. L. Savage", "Wouter J. Veneman", "Tom H. M. Ottenhoff", "Ron P. Dirks", "Annemarie H. Meijer", "Herman P. Spaink"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0016779.g001"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Outcome_of_M_marinum_yolk_sac_injection_of_embryos_between_the_16_and_the_512_cell_stage_/468170", "title"=>"Outcome of <i>M. marinum</i> yolk sac injection of embryos between the 16- and the 512-cell stage.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-02-16 02:16:10"}
  • {"files"=>["https://ndownloader.figshare.com/files/797986"], "description"=>"<p>(<b>A</b> and <b>B</b>) 3 day-old infected mpx-gfp transgenic embryos (<b>A</b>) with and (<b>B</b>) without Pu.1 morpholino (scale bar: 250 µm). Greater numbers of (extracellular) bacteria throughout body of morphant embryo seen in <b>A</b> contrast with lower amount of more localized (phagocytosed) bacteria seen in <b>B</b>. Very low number of mpx-gfp labelled neutrophils in <b>A</b> confirms Pu.1 morpholino effect. (<b>C</b>) Bright-field/fluorescence overlay and (<b>D</b>) confocal z-stack of <i>mag</i>49-GFP/mCherry bacteria in body of 2 dpi embryo (scale bar: 25 µm). Red-fluorescent bacteria form a cording structure adjacent to a few cells containing green-fluorescent (<i>mag</i>49-activated) bacteria. Lack of green fluorescence in cording bacteria indicates no phagocytosis by macrophages and extracellularity. (<b>E</b>) Close-up (digital zoom: 5.2) of cording structure formed by extracellular bacteria (scale bar: 10 µm; only red channel shown).</p>", "links"=>[], "tags"=>["yolk", "sac", "co-injection", "morpholino", "bacterial", "localization", "proliferation"], "article_id"=>468350, "categories"=>["Microbiology", "Biochemistry", "Infectious Diseases", "Developmental Biology", "Immunology"], "users"=>["Ralph Carvalho", "Jan de Sonneville", "Oliver W. Stockhammer", "Nigel D. L. Savage", "Wouter J. Veneman", "Tom H. M. Ottenhoff", "Ron P. Dirks", "Annemarie H. Meijer", "Herman P. Spaink"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0016779.g002"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Effect_of_yolk_sac_co_injection_of_Pu_1_morpholino_and_M_marinum_on_bacterial_localization_and_proliferation_within_embryos_/468350", "title"=>"Effect of yolk sac co-injection of Pu.1 morpholino and <i>M. marinum</i> on bacterial localization and proliferation within embryos.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-02-16 02:19:10"}
  • {"files"=>["https://ndownloader.figshare.com/files/798100"], "description"=>"<p>(<b>A</b>) After fertilization eggs are harvested, washed and distributed on injection plate. (<b>B</b>) Appropriate inoculum is injected in early stage embryos (up to 1,024-cell stage). (<b>C</b>) Injected embryos are dispensed into appropriate containers and drug screens take place between 3 and 6 dpi. (<b>D</b>) Groups of treated and untreated embryos are separately screened using COPAS during (when appropriate) and after drug exposure. Detailed optical analyses are performed on selected larvae.</p>", "links"=>[], "tags"=>["high-throughput", "zebrafish", "embryos"], "article_id"=>468464, "categories"=>["Microbiology", "Biochemistry", "Infectious Diseases", "Developmental Biology", "Immunology"], "users"=>["Ralph Carvalho", "Jan de Sonneville", "Oliver W. Stockhammer", "Nigel D. L. Savage", "Wouter J. Veneman", "Tom H. M. Ottenhoff", "Ron P. Dirks", "Annemarie H. Meijer", "Herman P. Spaink"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0016779.g003"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Pipeline_of_high_throughput_infection_of_zebrafish_embryos_and_subsequent_drug_testing_/468464", "title"=>"Pipeline of high-throughput infection of zebrafish embryos and subsequent drug testing.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-02-16 02:21:04"}
  • {"files"=>["https://ndownloader.figshare.com/files/798250"], "description"=>"<p>(<b>A</b>) the automatic injector system inside a laminar flow cabinet; (<b>B</b>) the embryo holder, showing the agarose grid within the steel support; (<b>C</b>) the embryo-filled grid, demonstrating the highly reproducible alignment of the embryos, with the cell mass resting to the side. Although size variation is observed, the embryos are always precisely in the centre of each well (the point of calibration for injection).</p>", "links"=>[], "tags"=>["depicting", "injector"], "article_id"=>468613, "categories"=>["Microbiology", "Biochemistry", "Infectious Diseases", "Developmental Biology", "Immunology"], "users"=>["Ralph Carvalho", "Jan de Sonneville", "Oliver W. Stockhammer", "Nigel D. L. Savage", "Wouter J. Veneman", "Tom H. M. Ottenhoff", "Ron P. Dirks", "Annemarie H. Meijer", "Herman P. Spaink"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0016779.g004"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Pictures_depicting_automatic_injector_system_/468613", "title"=>"Pictures depicting automatic injector system.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-02-16 02:23:33"}
  • {"files"=>["https://ndownloader.figshare.com/files/798429"], "description"=>"<p>(<b>A</b>) Effect of treatment on bacterial growth (measured by COPAS) in 5 and 6 day-old fli1-egfp larvae with gfp-labelled vasculature. Blue bars represent treated embryos, red bars represent untreated embryos. (<b>B–D</b>) Untreated versus (<b>E–G</b>) treated 5 day-old larvae, depicted whole in (<b>B</b> and <b>E</b>) bright-field and (<b>C</b> and <b>F</b>) fluorescent images, and (<b>D</b> and <b>G</b>) profiled by COPAS (scale bar: 250 µm). The localization of bacteria (red) in <b>C</b> and <b>F</b> correlates well with COPAS profile peaks in <b>D</b> and <b>G</b>, respectively (peaks in the tail region are shown enlarged in the inset; arrowheads depict two representative locations).</p>", "links"=>[], "tags"=>["yolk", "sac", "injection", "infected"], "article_id"=>468796, "categories"=>["Microbiology", "Biochemistry", "Infectious Diseases", "Developmental Biology", "Immunology"], "users"=>["Ralph Carvalho", "Jan de Sonneville", "Oliver W. Stockhammer", "Nigel D. L. Savage", "Wouter J. Veneman", "Tom H. M. Ottenhoff", "Ron P. Dirks", "Annemarie H. Meijer", "Herman P. Spaink"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0016779.g005"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Automatic_yolk_sac_injection_of_M_marinum_and_effect_of_treatment_on_infected_larvae_/468796", "title"=>"Automatic yolk sac injection of <i>M. marinum</i> and effect of treatment on infected larvae.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-02-16 02:26:36"}
  • {"files"=>["https://ndownloader.figshare.com/files/798573"], "description"=>"<p>(<b>A</b>) Confocal z-stack (8×2 stitching) of a 6 day-old whole larva (fli1-egfp with gfp-labelled vasculature) showing spread of bacteria (red) throughout the body (scale bar: 250 µm). (<b>B</b>) Confocal z-stack of red-fluorescent bacteria co-localizing with green-fluorescent leukocytes detected by L-plastin immunostaining (scale bar: 25 µm). (<b>C</b>) Close-up (digital zoom: 4.2) of bacteria-containing leukocyte depicted in <b>B</b> by straight arrow (scale bar: 10 µm). (<b>D</b>) Close-up (digital zoom: 4.3) of bacteria-containing leukocyte depicted in <b>B</b> by arrowhead (scale bar: 10 µm).</p>", "links"=>[], "tags"=>["yolk", "sac", "injection", "infected"], "article_id"=>468933, "categories"=>["Microbiology", "Biochemistry", "Infectious Diseases", "Developmental Biology", "Immunology"], "users"=>["Ralph Carvalho", "Jan de Sonneville", "Oliver W. Stockhammer", "Nigel D. L. Savage", "Wouter J. Veneman", "Tom H. M. Ottenhoff", "Ron P. Dirks", "Annemarie H. Meijer", "Herman P. Spaink"], "doi"=>["https://dx.doi.org/10.1371/journal.pone.0016779.g006"], "stats"=>{"downloads"=>0, "page_views"=>0, "likes"=>0}, "figshare_url"=>"https://figshare.com/articles/_Automatic_yolk_sac_injection_of_M_tuberculosis_and_effect_of_treatment_on_infected_larvae_/468933", "title"=>"Automatic yolk sac injection of <i>M. tuberculosis</i> and effect of treatment on infected larvae.", "pos_in_sequence"=>0, "defined_type"=>1, "published_date"=>"2011-02-16 02:28:53"}

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