The Experts below are selected from a list of 35772 Experts worldwide ranked by ideXlab platform
Stan J. J. Brouns - One of the best experts on this subject based on the ideXlab platform.
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direct visualization of native crispr target search in Live Bacteria reveals cascade dna surveillance mechanism
Molecular Cell, 2020Co-Authors: Jochem N. A. Vink, Marnix Vlot, Rebecca E. Mckenzie, Koen J. A. Martens, Boris Estrada Bonilla, Daan J.w. Brocken, Johannes Hohlbein, Cristobal Almendros, Stan J. J. BrounsAbstract:Summary CRISPR-Cas systems encode RNA-guided surveillance complexes to find and cleave invading DNA elements. While it is thought that invaders are neutralized minutes after cell entry, the mechanism and kinetics of target search and its impact on CRISPR protection levels have remained unknown. Here, we visualize individual Cascade complexes in a native type I CRISPR-Cas system. We uncover an exponential relation between Cascade copy number and CRISPR interference levels, pointing to a time-driven arms race between invader replication and target search, in which 20 Cascade complexes provide 50% protection. Driven by PAM-interacting subunit Cas8e, Cascade spends half its search time rapidly probing DNA (∼30 ms) in the nucleoid. We further demonstrate that target DNA transcription and CRISPR arrays affect the integrity of Cascade and affect CRISPR interference. Our work establishes the mechanism of cellular DNA surveillance by Cascade that allows the timely detection of invading DNA in a crowded, DNA-packed environment.
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Direct visualization of native CRISPR target search in Live Bacteria reveals Cascade DNA surveillance mechanism
bioRxiv, 2019Co-Authors: Jochem N. A. Vink, Marnix Vlot, Rebecca E. Mckenzie, Koen J. A. Martens, Boris Estrada Bonilla, Daan J.w. Brocken, Johannes Hohlbein, Cristobal Almendros, Stan J. J. BrounsAbstract:Abstract CRISPR-Cas systems encode RNA-guided surveillance complexes to find and cleave invading DNA elements. While it is thought that invaders are neutralized minutes after cell entry, the mechanism and kinetics of target search and its impact on CRISPR protection levels have remained unknown. Here we visualized individual Cascade complexes in a native type I CRISPR-Cas system. We uncovered an exponential relationship between Cascade copy number and CRISPR interference levels, pointing to a time-driven arms race between invader replication and target search, in which 20 Cascade complexes provide 50% protection. Driven by PAM-interacting subunit Cas8e, Cascade spends half its search time rapidly probing DNA (∼30 ms) in the nucleoid. We further demonstrate that target DNA transcription and CRISPR arrays affect the integrity of Cascade and impact CRISPR interference. Our work establishes the mechanism of cellular DNA surveillance by Cascade that allows the timely detection of invading DNA in a crowded, DNA-packed environment. One sentence summary The results from in vivo tracking of single CRISPR RNA-surveillance complexes in the native host cell explain their ability to rapidly recognize invader sequences.
Jochem N. A. Vink - One of the best experts on this subject based on the ideXlab platform.
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direct visualization of native crispr target search in Live Bacteria reveals cascade dna surveillance mechanism
Molecular Cell, 2020Co-Authors: Jochem N. A. Vink, Marnix Vlot, Rebecca E. Mckenzie, Koen J. A. Martens, Boris Estrada Bonilla, Daan J.w. Brocken, Johannes Hohlbein, Cristobal Almendros, Stan J. J. BrounsAbstract:Summary CRISPR-Cas systems encode RNA-guided surveillance complexes to find and cleave invading DNA elements. While it is thought that invaders are neutralized minutes after cell entry, the mechanism and kinetics of target search and its impact on CRISPR protection levels have remained unknown. Here, we visualize individual Cascade complexes in a native type I CRISPR-Cas system. We uncover an exponential relation between Cascade copy number and CRISPR interference levels, pointing to a time-driven arms race between invader replication and target search, in which 20 Cascade complexes provide 50% protection. Driven by PAM-interacting subunit Cas8e, Cascade spends half its search time rapidly probing DNA (∼30 ms) in the nucleoid. We further demonstrate that target DNA transcription and CRISPR arrays affect the integrity of Cascade and affect CRISPR interference. Our work establishes the mechanism of cellular DNA surveillance by Cascade that allows the timely detection of invading DNA in a crowded, DNA-packed environment.
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Direct visualization of native CRISPR target search in Live Bacteria reveals Cascade DNA surveillance mechanism
bioRxiv, 2019Co-Authors: Jochem N. A. Vink, Marnix Vlot, Rebecca E. Mckenzie, Koen J. A. Martens, Boris Estrada Bonilla, Daan J.w. Brocken, Johannes Hohlbein, Cristobal Almendros, Stan J. J. BrounsAbstract:Abstract CRISPR-Cas systems encode RNA-guided surveillance complexes to find and cleave invading DNA elements. While it is thought that invaders are neutralized minutes after cell entry, the mechanism and kinetics of target search and its impact on CRISPR protection levels have remained unknown. Here we visualized individual Cascade complexes in a native type I CRISPR-Cas system. We uncovered an exponential relationship between Cascade copy number and CRISPR interference levels, pointing to a time-driven arms race between invader replication and target search, in which 20 Cascade complexes provide 50% protection. Driven by PAM-interacting subunit Cas8e, Cascade spends half its search time rapidly probing DNA (∼30 ms) in the nucleoid. We further demonstrate that target DNA transcription and CRISPR arrays affect the integrity of Cascade and impact CRISPR interference. Our work establishes the mechanism of cellular DNA surveillance by Cascade that allows the timely detection of invading DNA in a crowded, DNA-packed environment. One sentence summary The results from in vivo tracking of single CRISPR RNA-surveillance complexes in the native host cell explain their ability to rapidly recognize invader sequences.
Yair Rivenson - One of the best experts on this subject based on the ideXlab platform.
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early detection and classification of Live Bacteria using time lapse coherent imaging and deep learning
Light-Science & Applications, 2020Co-Authors: Hongda Wang, Hatice Ceylan Koydemir, Yunzhe Qiu, Bijie Bai, Yibo Zhang, Yiyin Jin, Sabiha Tok, Enis Cagatay Yilmaz, Esin Gumustekin, Yair RivensonAbstract:Early identification of pathogenic Bacteria in food, water, and bodily fluids is very important and yet challenging, owing to sample complexities and large sample volumes that need to be rapidly screened. Existing screening methods based on plate counting or molecular analysis present various tradeoffs with regard to the detection time, accuracy/sensitivity, cost, and sample preparation complexity. Here, we present a computational Live Bacteria detection system that periodically captures coherent microscopy images of Bacterial growth inside a 60-mm-diameter agar plate and analyses these time-lapsed holograms using deep neural networks for the rapid detection of Bacterial growth and the classification of the corresponding species. The performance of our system was demonstrated by the rapid detection of Escherichia coli and total coliform Bacteria (i.e., Klebsiella aerogenes and Klebsiella pneumoniae subsp. pneumoniae) in water samples, shortening the detection time by >12 h compared to the Environmental Protection Agency (EPA)-approved methods. Using the preincubation of samples in growth media, our system achieved a limit of detection (LOD) of ~1 colony forming unit (CFU)/L in ≤9 h of total test time. This platform is highly cost-effective (~$0.6/test) and has high-throughput with a scanning speed of 24 cm2/min over the entire plate surface, making it highly suitable for integration with the existing methods currently used for Bacteria detection on agar plates. Powered by deep learning, this automated and cost-effective Live Bacteria detection platform can be transformative for a wide range of applications in microbiology by significantly reducing the detection time and automating the identification of colonies without labelling or the need for an expert. A novel automated system quickly detects and classifies Live Bacteria in water by using deep neural networks to analyze holographic images. Water-borne pathogens affect billions of people, but current gold standard methods for counting and identifying Live Bacteria in water take 24 hours or more, highlighting the need for fast, accurate, automatic methods that can handle large sample sizes. Aydogan Ozcan at the University of California Los Angeles, USA, and co-workers developed a system that analyzes lensfree holographic microscopy images of Bacteria growing on agar plates. After training and testing their algorithms with >16000 Bacterial colonies from three different species, the team was able to detect Bacterial growth and classify species in <12 hours. The system will not only improve monitoring of food and water quality, but also provides a powerful tool for microbiology research.
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early detection and classification of Live Bacteria using time lapse coherent imaging and deep learning
arXiv: Instrumentation and Detectors, 2020Co-Authors: Hongda Wang, Hatice Ceylan Koydemir, Yibo Zhang, Enis Cagatay Yilmaz, Esin Gumustekin, Yair Rivenson, Aydogan OzcanAbstract:We present a computational Live Bacteria detection system that periodically captures coherent microscopy images of Bacterial growth inside a 60 mm diameter agar-plate and analyzes these time-lapsed holograms using deep neural networks for rapid detection of Bacterial growth and classification of the corresponding species. The performance of our system was demonstrated by rapid detection of Escherichia coli and total coliform Bacteria (i.e., Klebsiella aerogenes and Klebsiella pneumoniae subsp. pneumoniae) in water samples. These results were confirmed against gold-standard culture-based results, shortening the detection time of Bacterial growth by >12 h as compared to the Environmental Protection Agency (EPA)-approved analytical methods. Our experiments further confirmed that this method successfully detects 90% of Bacterial colonies within 7-10 h (and >95% within 12 h) with a precision of 99.2-100%, and correctly identifies their species in 7.6-12 h with 80% accuracy. Using pre-incubation of samples in growth media, our system achieved a limit of detection (LOD) of ~1 colony forming unit (CFU)/L within 9 h of total test time. This computational Bacteria detection and classification platform is highly cost-effective (~$0.6 per test) and high-throughput with a scanning speed of 24 cm2/min over the entire plate surface, making it highly suitable for integration with the existing analytical methods currently used for Bacteria detection on agar plates. Powered by deep learning, this automated and cost-effective Live Bacteria detection platform can be transformative for a wide range of applications in microbiology by significantly reducing the detection time, also automating the identification of colonies, without labeling or the need for an expert.
Robert K Poole - One of the best experts on this subject based on the ideXlab platform.
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differential pulse polarography a method for the direct study of biosorption of metal ions by Live Bacteria from mixed metal solutions
Antonie Van Leeuwenhoek International Journal of General and Molecular Microbiology, 2003Co-Authors: Ioannis Savvaidis, Martin N Hughes, Robert K PooleAbstract:The technique of differential pulse polarography is shown here to be applicable to the monitoring directly the biosorption of metal ions from solution by Live Bacteria from mixed metal solutions. Biosorption of Cd(II), Zn(II) and Ni(II) by P. cepacia was followed using data obtained at the potential which is characteristic of the metal ion in the absence and presence of cells. Hepes buffer (pH 7.4, 50 mM) was used as a supporting electrolyte in the polarographic chamber and metal ion peaks in the presence of cells of lower amplitude were obtained due to metal-binding by the cells. Well defined polarographic peaks were obtained in experiments involving mixtures of metal ions of Cd(II)-Zn(II), Cu(II)-Zn(II), Cu(II)-Cd(II) and Cd(II)-Ni(II). Biosorption of Cd(II), Zn(II) increased with solution pH. The method was also tested as a rapid technique for assessing removal of metal ions by Live Bacteria and the ability of the polarographic technique in measuring biosorption of metal ions from mixed metal solutions is demonstrated. Cu(II) was preferentially bound and removal of metals was in the order Cu(II) > Ni(II) > Zn(II), Cd(II) by intact cells of P. cepacia.
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differential pulse polarography a method of directly measuring uptake of metal ions by Live Bacteria without separation of biomass and medium
Fems Microbiology Letters, 1992Co-Authors: Ioannis Savvaidis, Martin N Hughes, Robert K PooleAbstract:The technique of differential pulse polarography is shown here for the first time to be applicable to monitoring directly the uptake of metal ions from solution by Live Bacteria in the chamber of the polarograph. The potential at which the polarographic current peak is observed is characteristic of the metal, whereas peak height is proportional to metal concentration. Adding solutions of Cd(II) or Zn(II) to a suspension of Pseudomonas cepacia in 50 mM Hepes buffer (pH 7.4) in the chamber gave polarographic peaks of lower amplitude than those observed when these metal solutions were added to buffer alone, due to metal binding or uptake by cells. Langmuir plots gave binding capacities of 0.13 and 0.20 mmol metal (Dd or Zn, respectively) per g (dry weight) biomass. Ni(II) uptake was biphasic. Metal uptake increased with pH. The value of polarography for rapid assessment of metal removal by cells and the ability to measure uptake from multi-metal solutions is demonstrated.
Johannes Hohlbein - One of the best experts on this subject based on the ideXlab platform.
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direct visualization of native crispr target search in Live Bacteria reveals cascade dna surveillance mechanism
Molecular Cell, 2020Co-Authors: Jochem N. A. Vink, Marnix Vlot, Rebecca E. Mckenzie, Koen J. A. Martens, Boris Estrada Bonilla, Daan J.w. Brocken, Johannes Hohlbein, Cristobal Almendros, Stan J. J. BrounsAbstract:Summary CRISPR-Cas systems encode RNA-guided surveillance complexes to find and cleave invading DNA elements. While it is thought that invaders are neutralized minutes after cell entry, the mechanism and kinetics of target search and its impact on CRISPR protection levels have remained unknown. Here, we visualize individual Cascade complexes in a native type I CRISPR-Cas system. We uncover an exponential relation between Cascade copy number and CRISPR interference levels, pointing to a time-driven arms race between invader replication and target search, in which 20 Cascade complexes provide 50% protection. Driven by PAM-interacting subunit Cas8e, Cascade spends half its search time rapidly probing DNA (∼30 ms) in the nucleoid. We further demonstrate that target DNA transcription and CRISPR arrays affect the integrity of Cascade and affect CRISPR interference. Our work establishes the mechanism of cellular DNA surveillance by Cascade that allows the timely detection of invading DNA in a crowded, DNA-packed environment.
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Direct visualization of native CRISPR target search in Live Bacteria reveals Cascade DNA surveillance mechanism
bioRxiv, 2019Co-Authors: Jochem N. A. Vink, Marnix Vlot, Rebecca E. Mckenzie, Koen J. A. Martens, Boris Estrada Bonilla, Daan J.w. Brocken, Johannes Hohlbein, Cristobal Almendros, Stan J. J. BrounsAbstract:Abstract CRISPR-Cas systems encode RNA-guided surveillance complexes to find and cleave invading DNA elements. While it is thought that invaders are neutralized minutes after cell entry, the mechanism and kinetics of target search and its impact on CRISPR protection levels have remained unknown. Here we visualized individual Cascade complexes in a native type I CRISPR-Cas system. We uncovered an exponential relationship between Cascade copy number and CRISPR interference levels, pointing to a time-driven arms race between invader replication and target search, in which 20 Cascade complexes provide 50% protection. Driven by PAM-interacting subunit Cas8e, Cascade spends half its search time rapidly probing DNA (∼30 ms) in the nucleoid. We further demonstrate that target DNA transcription and CRISPR arrays affect the integrity of Cascade and impact CRISPR interference. Our work establishes the mechanism of cellular DNA surveillance by Cascade that allows the timely detection of invading DNA in a crowded, DNA-packed environment. One sentence summary The results from in vivo tracking of single CRISPR RNA-surveillance complexes in the native host cell explain their ability to rapidly recognize invader sequences.