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Milan Esner, Felix Meyenhofer, Marc Bickle
Live-Cell High Content Screening in Drug Development.
Methods Mol Biol, 1683 149-164 (2018)
PubMed Source   

In the past decade, automated microscopy has become an important tool for the drug discovery and development process. The establishment of imaging modalities as screening tools depended on technological breakthroughs in the domain of automated microscopy and automated image analysis. These types of assays are often referred to as high content screening or high content analysis (HCS/HCA). The driving force to adopt imaging for drug development is the quantity and quality of cellular information that can be collected and the enhanced physiological relevance of cellular screening compared to biochemical screening. Most imaging in drug development is performed on fixed cells as this allows uncoupling the preparation of the cells from the acquisition of the images. Live-cell imaging is technically challenging, but is very useful for many aspects of the drug development pipeline such as kinetic studies of compound mode of action or to analyze the motion of cellular components. Most vendors of HCS microscopy systems offer the option of environmental chambers and onboard pipetting on their platforms. This reflects the wish and desire of many customers to have the ability to perform live-cell assays on their HCS automated microscopes. This book chapter summarizes the challenges and advantages of live-cell imaging in drug discovery. Examples of applications are presented and the motivation to perform these assays in kinetic mode is discussed.
@article{Esner6982,
author={Milan Esner, Felix Meyenhofer, Marc Bickle},
title={Live-Cell High Content Screening in Drug Development.},
journal={Methods in molecular biology (Clifton, N.J.)},
volume={1683},
pages={149--164},
year=2018
}

L Carine Stapel, Coleman Broaddus, Nadine Vastenhouw
Detection and Automated Analysis of Single Transcripts at Subcellular Resolution in Zebrafish Embryos.
Methods Mol Biol, 1649 143-162 (2018)
PubMed Source   

Single molecule fluorescence in situ hybridization (smFISH) is a method to visualize single mRNA molecules. When combined with cellular and nuclear segmentation, transcripts can be assigned to different cellular compartments resulting in quantitative information on transcript levels at subcellular resolution. The use of smFISH in zebrafish has been limited by the lack of protocols and an automated image analysis pipeline for samples of multicellular organisms. Here we present a protocol for smFISH on zebrafish cryosections. The protocol includes a method to obtain high-quality sections of zebrafish embryos, an smFISH protocol optimized for zebrafish cryosections, and a user-friendly, automated analysis pipeline for cell segmentation and transcript detection. The software is freely available and can be used to analyze sections of any multicellular organism.
@article{Stapel6984,
author={L Carine Stapel, Coleman Broaddus, Nadine Vastenhouw},
title={Detection and Automated Analysis of Single Transcripts at Subcellular Resolution in Zebrafish Embryos.},
journal={Methods in molecular biology (Clifton, N.J.)},
volume={1649},
pages={143--162},
year=2018
}

Martina Ugrinic, Adrian Zambrano, Simon Berger, Stephen Mann, T-Y Dora Tang, Andrew deMello
Microfluidic formation of proteinosomes.
Chem. Commun. (Camb.), Art. No. doi: 10.1039/c7cc08466h (2017)
PubMed Source   

Herein we describe a novel microfluidic method for the generation of proteinosome micro-droplets, based on bovine serum albumin and glucose oxidase conjugated to PNIPAAm chains. The size of such water-in-oil droplets is regulated via control of the input reagent flow rate, with generated proteinosome populations exhibiting narrower size distributions than those observed when using standard bulk methodologies. Importantly, proteinosomes transferred from an oil to an aqueous-environment remain intact, become fully hydrated and exhibit an increase in average size. Moreover, functional proteinosomes prepared via microfluidics exhibit lower Km values and higher enzymatic activities than proteinosomes produced by bulk methodologies.
@article{Ugrinic7002,
author={Martina Ugrinic, Adrian Zambrano, Simon Berger, Stephen Mann, T-Y Dora Tang, Andrew deMello},
title={Microfluidic formation of proteinosomes.},
journal={Chemical communications (Cambridge, England)},
volume={},
pages={1--1},
year=2017
}

Julia Brenmoehl, Christina Walz, Marion Spitschak, Elisa Wirthgen, Michael Walz, Martina Langhammer, Armin Tuchscherer, Ronald Naumann, Andreas Hoeflich
Partial phenotype conversion and differential trait response to conditions of husbandry in mice.
J. Comp. Physiol. B, Biochem. Syst. Environ. Physiol., 1-1 (2017)
PubMed Source   

Functional genome analysis usually is performed on the level of genotype-phenotype interaction. However, phenotypes also depend on the relations between genomes and environment. In our experimental system, we observed differential response to environmental factors defined by different conditions of husbandry in a semi-barrier unit or in a SPF (specific pathogen free) barrier unit, which resulted in partial reversal of phenotypes previously observed under semi-barrier conditions. To provide an update of basic phenotypes in unselected and randomly mated controls (DUC) and long-term selected DUhTP (Dummerstorf high treadmill performance) mice in the SPF facility, we compared growth parameters, reproductive performance, the accretion of muscle and fat mass, physical activity, and running performance as well as food intake in all experimental groups. For selected parameters, the comparative analysis spans more than 30 generations. In DUC mice, under SPF conditions a more than threefold (P < 0.0001) higher subcutaneous fat mass, higher muscle mass by about 25% (P < 0.0001), but lower epididymal fat mass in DUhTP mice by about 20% (P < 0.0001) were observed. In SPF husbandry, body weight increased to a stronger extent in adult DUC mice (≈ 20%; P < 0.0001) than in DUhTP mice (≈ 8%; P = 0.001). The concentrations of IGF-1 and IGFBPs in the serum as well as the liver weights were similar in all experimental groups, indicating growth effects independent of the somatotropic axis. Under SPF conditions the litter size at birth increased in DUC mice (P < 0.001) but not in DUhTP mice. The differential effect of husbandry on body weights at day 21 and concentrations of triglycerides in the serum of our model were due to the different diets used in the semi-barrier and in the SPF facility. Our results demonstrate differential trait response to environmental factors resulting in partial phenotype conversion in our experimental system. The existence of conditional phenotypes as a result of genotype-environment interactions points to the importance of environmental factors in functional genome analysis.
@article{Brenmoehl7003,
author={Julia Brenmoehl, Christina Walz, Marion Spitschak, Elisa Wirthgen, Michael Walz, Martina Langhammer, Armin Tuchscherer, Ronald Naumann, Andreas Hoeflich},
title={Partial phenotype conversion and differential trait response to conditions of husbandry in mice.},
journal={Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology},
volume={},
pages={1--1},
year=2017
}

Kai Schuhmann, Kristina Srzentić, Konstantin O Nagornov, Henrik Thomas, Theresia Gutmann, Ünal Coskun, Yury O Tsybin, Andrej Shevchenko
Monitoring Membrane Lipidome Turnover by Metabolic 15N Labeling and Shotgun Ultra-High-Resolution Orbitrap Fourier Transform Mass Spectrometry.
Anal Chem, 89(23) 12857-12865 (2017)
PubMed Source   

Lipidomes undergo permanent extensive remodeling, but how the turnover rate differs between lipid classes and molecular species is poorly understood. We employed metabolic 15N labeling and shotgun ultra-high-resolution mass spectrometry (sUHR) to quantify the absolute (molar) abundance and determine the turnover rate of glycerophospholipids and sphingolipids by direct analysis of total lipid extracts. sUHR performed on a commercial Orbitrap Elite instrument at the mass resolution of 1.35 × 106 (m/z 200) baseline resolved peaks of 13C isotopes of unlabeled and monoisotopic peaks of 15N labeled lipids (Δm = 0.0063 Da). Therefore, the rate of metabolic 15N labeling of individual lipid species could be determined without compromising the scope, accuracy, and dynamic range of full-lipidome quantitative shotgun profiling. As a proof of concept, we employed sUHR to determine the lipidome composition and fluxes of 62 nitrogen-containing membrane lipids in human hepatoma HepG2 cells.
@article{Schuhmann6996,
author={Kai Schuhmann, Kristina Srzentić, Konstantin O Nagornov, Henrik Thomas, Theresia Gutmann, Ünal Coskun, Yury O Tsybin, Andrej Shevchenko},
title={Monitoring Membrane Lipidome Turnover by Metabolic 15N Labeling and Shotgun Ultra-High-Resolution Orbitrap Fourier Transform Mass Spectrometry.},
journal={Analytical chemistry},
volume={89},
issue ={23},
pages={12857--12865},
year=2017
}

Oleksandr Ostrenko, Pietro Incardona, Rajesh Ramaswamy, Lutz Brusch, Ivo F. Sbalzarini
pSSAlib: The partial-propensity stochastic chemical network simulator.
PLoS Comput Biol, 13(12) Art. No. e1005865 (2017)
PubMed Source   

Chemical reaction networks are ubiquitous in biology, and their dynamics is fundamentally stochastic. Here, we present the software library pSSAlib, which provides a complete and concise implementation of the most efficient partial-propensity methods for simulating exact stochastic chemical kinetics. pSSAlib can import models encoded in Systems Biology Markup Language, supports time delays in chemical reactions, and stochastic spatiotemporal reaction-diffusion systems. It also provides tools for statistical analysis of simulation results and supports multiple output formats. It has previously been used for studies of biochemical reaction pathways and to benchmark other stochastic simulation methods. Here, we describe pSSAlib in detail and apply it to a new model of the endocytic pathway in eukaryotic cells, leading to the discovery of a stochastic counterpart of the cut-out switch motif underlying early-to-late endosome conversion. pSSAlib is provided as a stand-alone command-line tool and as a developer API. We also provide a plug-in for the SBMLToolbox. The open-source code and pre-packaged installers are freely available from http://mosaic.mpi-cbg.de.
@article{Ostrenko7001,
author={Oleksandr Ostrenko, Pietro Incardona, Rajesh Ramaswamy, Lutz Brusch, Ivo F. Sbalzarini},
title={pSSAlib: The partial-propensity stochastic chemical network simulator.},
journal={PLoS computational biology},
volume={13},
issue ={12},
pages={null--null},
year=2017
}

Marcus Braun, Zdenek Lansky, Agata Szuba, Friedrich W Schwarz, Aniruddha Mitra, Mengfei Gao, Annemarie Lüdecke, Pieter Rein Ten Wolde, Stefan Diez
Changes in microtubule overlap length regulate kinesin-14-driven microtubule sliding.
Nat Chem Biol, 13(12) 1245-1252 (2017)
PubMed Source   

Microtubule-crosslinking motor proteins, which slide antiparallel microtubules, are required for the remodeling of microtubule networks. Hitherto, all microtubule-crosslinking motors have been shown to slide microtubules at a constant velocity until no overlap remains between them, leading to the breakdown of the initial microtubule geometry. Here, we show in vitro that the sliding velocity of microtubules, driven by human kinesin-14 HSET, decreases when microtubules start to slide apart, resulting in the maintenance of finite-length microtubule overlaps. We quantitatively explain this feedback using the local interaction kinetics of HSET with overlapping microtubules that cause retention of HSET in shortening overlaps. Consequently, the increased HSET density in the overlaps leads to a density-dependent decrease in sliding velocity and the generation of an entropic force that antagonizes the force exerted by the motors. Our results demonstrate that a spatial arrangement of microtubules can regulate the collective action of molecular motors through the local alteration of their individual interaction kinetics.
@article{Braun6989,
author={Marcus Braun, Zdenek Lansky, Agata Szuba, Friedrich W Schwarz, Aniruddha Mitra, Mengfei Gao, Annemarie Lüdecke, Pieter Rein Ten Wolde, Stefan Diez},
title={Changes in microtubule overlap length regulate kinesin-14-driven microtubule sliding.},
journal={Nature chemical biology},
volume={13},
issue ={12},
pages={1245--1252},
year=2017
}

Andreas Müller, Martin Neukam, Anna Ivanova, Anke Sönmez, Carla Münster, Susanne Kretschmar, Yannis Kalaidzidis, Thomas Kurth, Jean-Marc Verbavatz, Michele Solimena
A Global Approach for Quantitative Super Resolution and Electron Microscopy on Cryo and Epoxy Sections Using Self-labeling Protein Tags.
Sci Rep, 7 Art. No. 23 (2017)
  PubMed Source   

Correlative light and electron microscopy (CLEM) is a powerful approach to investigate the molecular ultrastructure of labeled cell compartments. However, quantitative CLEM studies are rare, mainly due to small sample sizes and the sensitivity of fluorescent proteins to strong fixatives and contrasting reagents for EM. Here, we show that fusion of a self-labeling protein to insulin allows for the quantification of age-distinct insulin granule pools in pancreatic beta cells by a combination of super resolution and transmission electron microscopy on Tokuyasu cryosections. In contrast to fluorescent proteins like GFP organic dyes covalently bound to self-labeling proteins retain their fluorescence also in epoxy resin following high pressure freezing and freeze substitution, or remarkably even after strong chemical fixation. This enables for the assessment of age-defined granule morphology and degradation. Finally, we demonstrate that this CLEM protocol is highly versatile, being suitable for single and dual fluorescent labeling and detection of different proteins with optimal ultrastructure preservation and contrast.
@article{Müller6796,
author={Andreas Müller, Martin Neukam, Anna Ivanova, Anke Sönmez, Carla Münster, Susanne Kretschmar, Yannis Kalaidzidis, Thomas Kurth, Jean-Marc Verbavatz, Michele Solimena},
title={A Global Approach for Quantitative Super Resolution and Electron Microscopy on Cryo and Epoxy Sections Using Self-labeling Protein Tags.},
journal={Scientific reports},
volume={7},
pages={null--null},
year=2017
}

Sandra Richter, Ulrike Schulze, Pavel Tomançak, Andrew C. Oates
Small molecule screen in embryonic zebrafish using modular variations to target segmentation.
Nat Commun, 8(1) 1901-1901 (2017)
PubMed Source   

Small molecule in vivo phenotypic screening is used to identify drugs or biological activities by directly assessing effects in intact organisms. However, current screening designs may not exploit the full potential of chemical libraries due to false negatives. Here, we demonstrate a modular small molecule screen in embryonic zebrafish that varies concentration, genotype and timing to target segmentation disorders, birth defects that affect the spinal column. By testing each small molecule in multiple interrelated ways, this screen recovers compounds that a standard screening design would have missed, increasing the hit frequency from the chemical library three-fold. We identify molecular pathways and segmentation phenotypes, which we share in an open-access annotated database. These hits provide insight into human vertebral segmentation disorders and myopathies. This modular screening strategy is applicable to other developmental questions and disease models, highlighting the power of relatively small chemical libraries to accelerate gene discovery and disease study.
@article{Richter6998,
author={Sandra Richter, Ulrike Schulze, Pavel Tomançak, Andrew C. Oates},
title={Small molecule screen in embryonic zebrafish using modular variations to target segmentation.},
journal={Nature communications},
volume={8},
issue ={1},
pages={1901--1901},
year=2017
}

Vladimir Ulman, Martin Maška, Klas E. G. Magnusson, Olaf Ronneberger, Carsten Haubold, Nathalie Harder, Pavel Matula, Pavel Matula, David Svoboda, Miroslav Radojevic, Ihor Smal, Karl Rohr, Joakim Jaldén, Helen M. Blau, Oleh Dzyubachyk, Boudewijn Lelieveldt, Pengdong Xiao, Yuexiang Li, Siu-Yeung Cho, Alexandre C Dufour, Jean-Christophe Olivo-Marin, Constantino C Reyes-Aldasoro, Jose A Solis-Lemus, Robert Bensch, Thomas Brox, Johannes Stegmaier, Ralf Mikut, Steffen Wolf, Fred A Hamprecht, Tiago Esteves, Pedro Quelhas, Ömer Demirel, Lars Malmström, Florian Jug, Pavel Tomancak, Erik Meijering, Arrate Muñoz-Barrutia, Michal Kozubek, Carlos Ortiz-de-Solorzano
An objective comparison of cell-tracking algorithms.
Nat Methods, 14(12) 1141-1152 (2017)
  PubMed Source   

We present a combined report on the results of three editions of the Cell Tracking Challenge, an ongoing initiative aimed at promoting the development and objective evaluation of cell segmentation and tracking algorithms. With 21 participating algorithms and a data repository consisting of 13 data sets from various microscopy modalities, the challenge displays today's state-of-the-art methodology in the field. We analyzed the challenge results using performance measures for segmentation and tracking that rank all participating methods. We also analyzed the performance of all of the algorithms in terms of biological measures and practical usability. Although some methods scored high in all technical aspects, none obtained fully correct solutions. We found that methods that either take prior information into account using learning strategies or analyze cells in a global spatiotemporal video context performed better than other methods under the segmentation and tracking scenarios included in the challenge.
@article{Ulman6956,
author={Vladimir Ulman, Martin Maška, Klas E. G. Magnusson, Olaf Ronneberger, Carsten Haubold, Nathalie Harder, Pavel Matula, Pavel Matula, David Svoboda, Miroslav Radojevic, Ihor Smal, Karl Rohr, Joakim Jaldén, Helen M. Blau, Oleh Dzyubachyk, Boudewijn Lelieveldt, Pengdong Xiao, Yuexiang Li, Siu-Yeung Cho, Alexandre C Dufour, Jean-Christophe Olivo-Marin, Constantino C Reyes-Aldasoro, Jose A Solis-Lemus, Robert Bensch, Thomas Brox, Johannes Stegmaier, Ralf Mikut, Steffen Wolf, Fred A Hamprecht, Tiago Esteves, Pedro Quelhas, Ömer Demirel, Lars Malmström, Florian Jug, Pavel Tomancak, Erik Meijering, Arrate Muñoz-Barrutia, Michal Kozubek, Carlos Ortiz-de-Solorzano},
title={An objective comparison of cell-tracking algorithms.},
journal={Nature methods},
volume={14},
issue ={12},
pages={1141--1152},
year=2017
}