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An in vitro method for examining cleavage patterns of zinc-finger nucleases (ZFNs) identifies previously unknown off-target cleavage sites. Some of the sites are present in the human genome and show evidence for ZFN-induced cleavage in cultured human cell lines.
A fluorescent protein that can be photoswitched with visible light from orange to far-red is presented. The photoconverted form has the most red-shifted excitation peak of all GFP-like fluorescent proteins to date and should be useful for many imaging applications.
A fluorescent reporter, named traffic light, reads out whether repair of a DNA break occurs by nonhomologous end-joining or by homologous recombination. It should enable the identification of factors that affect repair pathway choice and thus improved approaches for genome engineering.
Ubiquitin, an important post-translational modification that regulates a variety of biological processes is found in free and conjugated (monoubiquitin and polyubiquitin) forms in the cell. A method for precisely measuring these cellular pools using protein standard absolute quantification mass spectrometry is described; the approach should yield insights into ubiquitin signaling.
The functional role of protein phosphorylation is determined not just by whether a particular site is phosphorylated or not but also by the site's stoichiometry. A method to determine the absolute stoichiometries of protein phosphorylation on a proteomic scale is described.
Membrane protein interactions and conformational changes can be sensitively monitored with two-photon polarization microscopy, a method that takes advantage of the anisotropic absorption properties of fluorescent proteins. The authors applied the method to image G-protein activation and changes in intracellular calcium concentration.
A framework and web interface for the large-scale and automated synthesis of human neuroimaging data extracted from the literature is presented. It is used to generate a large database of mappings between neural and cognitive states and to address long-standing inferential problems in the neuroimaging literature.
In this sequencing-by-synthesis approach, the incorporation of a terminal-phosphate labeled fluorogenic nucleotide by DNA polymerase results in the generation of a fluorescent dye that is trapped in a sealed microreactor and does not require real-time detection.
Quantitative analysis of Caenorhabditis elegans chemosensory behavior is achieved in a structured arena with microfluidic delivery of stimuli with precise spatial and temporal control. Also in this issue, Swierczek et al. report software for real-time behavioral analysis in worms.
The Multi-Worm Tracker permits real-time, high-throughput, quantitative analysis of behavior in Caenorhabditis elegans. It should enable screens for genes implicated in complex worm behaviors. Also in this issue, Albrecht and Bargmann apply microfluidics to study worm chemosensory behavior with high spatial and temporal precision.
A microfluidic setup for nanoliter-volume perfused clonal culture and imaging of thousands of nonadherent cells is applied to study signaling and proliferation in hematopoietic stem cells.
Both identified and unidentified peptide mass spectra can be clustered and represented as consensus spectra in a spectral archive, offering new ways of interpreting proteomics data. A software tool for clustering billions of spectra is presented, as is a 1.18-billion spectra spectral archive.
Three-dimensional structural RNA modules, defined as ensembles of stacked arrays of ordered non-Watson-Crick base pairs, are found in many RNAs and play important functional roles. The presented computational tool, RMDetect, allows the identification of common RNA modules from sequence alone.
Judicious choice of probes and imaging conditions allows two-dimensional super-resolution imaging of live cells at speeds up to 2 Hz with ~25-nm resolution and three-dimensional super-resolution imaging at ~1 Hz with ~30 nm x-y and ~50 nm z dimension resolution using stochastic optical reconstruction microscopy (STORM).
A protein-trap mutagenesis system efficiently disrupts expression and reports endogenous expression patterns of genes in the zebrafish. The authors used it to generate an initial collection of 350 freely available fish lines.
Software for the automated and accurate registration of multiple images of Drosophila melanogaster brain is reported. It is used to build a preliminary atlas of gene expression in the fly brain.
Semantically typed data hypercubes (SDCubes) are described for the flexible management and organization of stored experimental data and are applied to high-content imaging data using the software package ImageRail.
A defined and simplified culture system for the derivation and growth of human induced pluripotent stem cells is reported. It permits increased efficiency of human reprogramming with an episomal approach. Also in this issue, Okita et al. describe methods for more efficient episomal reprogramming of human cells.
Analyzing the first and higher-order moments of the diffraction spot of a 4Pi fluorescence detection scheme facilitates two-color, three-dimensional super-resolution microscopy with ~6 nm axial and ~8–23 nm lateral resolution in a layer ~650 nm thick.
