Nature Methods (Br J Pharmacol)

Publications in this journal

• Article: The Caenorhabditis elegans Lifespan Machine.

  Nicholas Stroustrup, Bryne E Ulmschneider, Zachary M Nash, Isaac F López-Moyado, Javier Apfeld, Walter Fontana
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  ABSTRACT: The measurement of lifespan pervades aging research. Because lifespan results from complex interactions between genetic, environmental and stochastic factors, it varies widely even among isogenic individuals. The actions of molecular mechanisms on lifespan are therefore visible only through their statistical effects on populations. Indeed, survival assays in Caenorhabditis elegans have provided critical insights into evolutionarily conserved determinants of aging. To enable the rapid acquisition of survival curves at an arbitrary statistical resolution, we developed a scalable imaging and analysis platform to observe nematodes over multiple weeks across square meters of agar surface at 8-μm resolution. The automated method generates a permanent visual record of individual deaths from which survival curves are constructed and validated, producing data consistent with results from the manual method of survival curve acquisition for several mutants in both standard and stressful environments. Our approach permits rapid, detailed reverse-genetic and chemical screens for effects on survival and enables quantitative investigations into the statistical structure of aging.
  Nature Methods 05/2013;
• Article: Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM.

  Xueming Li, Paul Mooney, Shawn Zheng, Christopher R Booth, Michael B Braunfeld, Sander Gubbens, David A Agard, Yifan Cheng
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  ABSTRACT: In recent work with large high-symmetry viruses, single-particle electron cryomicroscopy (cryo-EM) has achieved the determination of near-atomic-resolution structures by allowing direct fitting of atomic models into experimental density maps. However, achieving this goal with smaller particles of lower symmetry remains challenging. Using a newly developed single electron-counting detector, we confirmed that electron beam-induced motion substantially degrades resolution, and we showed that the combination of rapid readout and nearly noiseless electron counting allow image blurring to be corrected to subpixel accuracy, restoring intrinsic image information to high resolution (Thon rings visible to ∼3 Å). Using this approach, we determined a 3.3-Å-resolution structure of an ∼700-kDa protein with D7 symmetry, the Thermoplasma acidophilum 20S proteasome, showing clear side-chain density. Our method greatly enhances image quality and data acquisition efficiency-key bottlenecks in applying near-atomic-resolution cryo-EM to a broad range of protein samples.
  Nature Methods 05/2013;
• Article: Live mammalian cell arrays.

  Kristina Woodruff, Luis M Fidalgo, Samy Gobaa, Matthias P Lutolf, Sebastian J Maerkl
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  ABSTRACT: High-content assays have the potential to drastically increase throughput in cell biology and drug discovery, but handling and culturing large libraries of cells such as primary tumor or cancer cell lines requires expensive, dedicated robotic equipment. We developed a simple yet powerful method that uses contact spotting to generate high-density nanowell arrays of live mammalian cells for the culture and interrogation of cell libraries.
  Nature Methods 05/2013;
• Article: Live Mammalian Cell Arrays

  Kristina Woodruff, Luis M Fidalgo, Samy Gobaa, Matthias P Lutolf, Sebastian J Maerkl
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  ABSTRACT: High-content assays have the potential to drastically increase throughput in cell biology and drug discovery, but handling and culturing large libraries of cells such as primary tumor or cancer cell lines requires expensive, dedicated robotic equipment. We developed a simple yet powerful method that uses contact spotting to generate high-density nanowell arrays of live mammalian cells for the culture and interrogation of cell libraries.
  Nature Methods 05/2013;
• Article: Nonhybrid, finished microbial genome assemblies from long-read SMRT sequencing data.

  Chen-Shan Chin, David H Alexander, Patrick Marks, Aaron A Klammer, James Drake, Cheryl Heiner, Alicia Clum, Alex Copeland, John Huddleston, Evan E Eichler, Stephen W Turner, Jonas Korlach
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  ABSTRACT: We present a hierarchical genome-assembly process (HGAP) for high-quality de novo microbial genome assemblies using only a single, long-insert shotgun DNA library in conjunction with Single Molecule, Real-Time (SMRT) DNA sequencing. Our method uses the longest reads as seeds to recruit all other reads for construction of highly accurate preassembled reads through a directed acyclic graph-based consensus procedure, which we follow with assembly using off-the-shelf long-read assemblers. In contrast to hybrid approaches, HGAP does not require highly accurate raw reads for error correction. We demonstrate efficient genome assembly for several microorganisms using as few as three SMRT Cell zero-mode waveguide arrays of sequencing and for BACs using just one SMRT Cell. Long repeat regions can be successfully resolved with this workflow. We also describe a consensus algorithm that incorporates SMRT sequencing primary quality values to produce de novo genome sequence exceeding 99.999% accuracy.
  Nature Methods 05/2013;
• Article: Reply to: "Reanalysis of Richter et al. (2010) on reproducibility".

  Hanno Würbel, S Helene Richter, Joseph P Garner
  Nature Methods 05/2013; 10(5):374.
• Article: Exploring long-range genome interactions using the WashU Epigenome Browser.

  Xin Zhou, Rebecca F Lowdon, Daofeng Li, Heather A Lawson, Pamela A F Madden, Joseph F Costello, Ting Wang
  Nature Methods 05/2013; 10(5):375-6.
• Article: Scaling up the systematic hunt for mammalian genetic interactions.

  Traver Hart, Jason Moffat
  Nature Methods 05/2013; 10(5):397-9.
• Article: Reanalysis of Richter et al. (2010) on reproducibility.

  Russell D Wolfinger
  Nature Methods 05/2013; 10(5):373-4.
• Article: PCR: living life amplified and standardized.

  Vivien Marx
  Nature Methods 05/2013; 10(5):391-5.
• Article: Inner-outer beauty: DNA-binding surface tags as cellular barcodes.

  Garry Nolan
  Nature Methods 05/2013; 10(5):399-401.
• Article: Does systematic variation improve the reproducibility of animal experiments?

  Rudy M Jonker, Anja Guenther, Leif Engqvist, Tim Schmoll
  Nature Methods 05/2013; 10(5):373.
• Article: Measuring image resolution in optical nanoscopy.

  Robert P J Nieuwenhuizen, Keith A Lidke, Mark Bates, Daniela Leyton Puig, David Grünwald, Sjoerd Stallinga, Bernd Rieger
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  ABSTRACT: Resolution in optical nanoscopy (or super-resolution microscopy) depends on the localization uncertainty and density of single fluorescent labels and on the sample's spatial structure. Currently there is no integral, practical resolution measure that accounts for all factors. We introduce a measure based on Fourier ring correlation (FRC) that can be computed directly from an image. We demonstrate its validity and benefits on two-dimensional (2D) and 3D localization microscopy images of tubulin and actin filaments. Our FRC resolution method makes it possible to compare achieved resolutions in images taken with different nanoscopy methods, to optimize and rank different emitter localization and labeling strategies, to define a stopping criterion for data acquisition, to describe image anisotropy and heterogeneity, and even to estimate the average number of localizations per emitter. Our findings challenge the current focus on obtaining the best localization precision, showing instead how the best image resolution can be achieved as fast as possible.
  Nature Methods 04/2013;
• Article: Comprehensive macromolecular conformations mapped by quantitative SAXS analyses.

  Greg L Hura, Helen Budworth, Kevin N Dyer, Robert P Rambo, Michal Hammel, Cynthia T McMurray, John A Tainer
  Nature Methods 04/2013;
• Article: Absolute quantification of transcription factors during cellular differentiation using multiplexed targeted proteomics.

  Jovan Simicevic, Adrien W Schmid, Paola A Gilardoni, Benjamin Zoller, Sunil K Raghav, Irina Krier, Carine Gubelmann, Frédérique Lisacek, Felix Naef, Marc Moniatte, Bart Deplancke
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  ABSTRACT: The cellular abundance of transcription factors (TFs) is an important determinant of their regulatory activities. Deriving TF copy numbers is therefore crucial to understanding how these proteins control gene expression. We describe a sensitive selected reaction monitoring-based mass spectrometry assay that allowed us to determine the copy numbers of up to ten proteins simultaneously. We applied this approach to profile the absolute levels of key TFs, including PPARγ and RXRα, during terminal differentiation of mouse 3T3-L1 pre-adipocytes. Our analyses revealed that individual TF abundance differs dramatically (from ∼250 to >300,000 copies per nucleus) and that their dynamic range during differentiation can vary up to fivefold. We also formulated a DNA binding model for PPARγ based on TF copy number, binding energetics and local chromatin state. This model explains the increase in PPARγ binding sites during the final differentiation stage that occurs despite a concurrent saturation in PPARγ copy number.
  Nature Methods 04/2013;
• Article: A simple tool to improve pluripotent stem cell differentiation.

  Sundari Chetty, Felicia Walton Pagliuca, Christian Honore, Anastasie Kweudjeu, Alireza Rezania, Douglas A Melton
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  ABSTRACT: We describe a method to help overcome restrictions on the differentiation propensities of human pluripotent stem cells. Culturing pluripotent stem cells in dimethylsulfoxide (DMSO) activates the retinoblastoma protein, increases the proportion of cells in the early G1 phase of the cell cycle and, in more than 25 embryonic and induced pluripotent stem cell lines, improves directed differentiation into multiple lineages. DMSO treatment also improves differentiation into terminal cell types in several cell lines.
  Nature Methods 04/2013;
• Article: Mapping genetic interactions in human cancer cells with RNAi and multiparametric phenotyping.

  Christina Laufer, Bernd Fischer, Maximilian Billmann, Wolfgang Huber, Michael Boutros
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  ABSTRACT: Genetic interactions influence many phenotypes and can be used as a powerful experimental tool to discover functional relationships between genes. Here we describe a robust and scalable method to systematically map genetic interactions in human cancer cells using combinatorial RNAi and high-throughput imaging. Through automated, single-cell phenotyping, we measured genetic interactions across a broad spectrum of phenotypes, including cell count, cell eccentricity and nuclear area. We mapped genetic interactions of epigenetic regulators in colon cancer cells, recovering known protein complexes. Our study also revealed the prospects and challenges of studying genetic interactions in human cells using multiparametric phenotyping.
  Nature Methods 04/2013;
• Article: Adhesion strength-based, label-free isolation of human pluripotent stem cells.

  Ankur Singh, Shalu Suri, Ted Lee, Jamie M Chilton, Marissa T Cooke, Weiqiang Chen, Jianping Fu, Steven L Stice, Hang Lu, Todd C McDevitt, Andrés J García
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  ABSTRACT: We demonstrate substantial differences in 'adhesive signature' between human pluripotent stem cells (hPSCs), partially reprogrammed cells, somatic cells and hPSC-derived differentiated progeny. We exploited these differential adhesion strengths to rapidly (over ∼10 min) and efficiently isolate fully reprogrammed induced hPSCs (hiPSCs) as intact colonies from heterogeneous reprogramming cultures and from differentiated progeny using microfluidics. hiPSCs were isolated label free, enriched to 95%-99% purity with >80% survival, and had normal transcriptional profiles, differentiation potential and karyotypes. We also applied this strategy to isolate hPSCs (hiPSCs and human embryonic stem cells) during routine culture and show that it may be extended to isolate hPSC-derived lineage-specific stem cells or differentiated cells.
  Nature Methods 04/2013;