Articles in 2010

A microfluidic embryo-trap array for large-scale end-on imaging of Drosophila melanogaster embryos permits quantitative analysis of dorsoventral gradients during development.

The Network-Free Stochastic Simulator (NFsim) allows the representation of complex biological systems as rule-based models and facilitates coarse-graining of the reaction mechanisms.

Fast, two-photon intravital imaging of a mechanically stabilized and physiologically intact preparation of the mouse lung is reported. It is used to monitor immune cells in the lung under normal and injured conditions.

Derivatizing glycosphingolipids, extracted from bovine brain or human erythrocytes, with a fluorescent tag allows their immobilization on an array which can be probed with glycan binding proteins.

Identification of residues critical for dimerization of the Fok1 nuclease domain of zinc-finger nucleases permits rational design of enzymes with improved cleavage activity and retained obligate heterodimerization. Also in this issue, Sander et al. report context-dependent assembly (CoDA), a simple method for designing zinc-finger nucleases.

Methods are reported for the combination of fluorescence nanoscopy using either stimulated emission depletion microscopy (STED) or photoactivated localization microscopy (PALM) with electron microscopy, to achieve correlative imaging in which the super-resolved fluorescence signal is placed in the context of cellular ultrastructure.

Defined surfaces displaying heparin-binding peptides support long-term culture of multiple human embryonic and induced pluripotent stem cell lines in defined media.

A statistical approach to quantitatively derive single and double mutant fitness from colony-based growth assays is described. The resultant SGA score permits assessment of yeast fitness and genetic interactions on the genome scale.

The mixture of isoforms model (MISO) assesses the confidence in estimates of the abundance of spliced exons or isoforms from paired-end RNA-seq data and detects their differential expression.

High-throughput sequencing of RNA fragments generated from a single-strand RNA-specific nuclease followed by novel computational analysis yields structural insights into noncoding RNA at the transcriptome level.

Readily synthesized maltose–neopentyl glycol (MNG) amphiphiles are useful reagents for stabilizing, extracting and crystallizing a variety of integral membrane proteins and have favorable properties relative to conventional detergents.

Retroviral integration is used to mark clones in human embryonic stem cell cultures and clonal distribution is assessed after functionally testing the cells with different methods. Distinct subsets of clones are detected after in vitro differentiation versus teratoma formation in vivo.

Proteins can be transferred between cells in contact, such as via trogocytosis in lymphocytes, or acquired via bacteria-host interactions during infection. A quantitative proteomics approach to identify such non-cell-autonomous proteins is described.

A targeting method for lentiviral vectors relying on the use of single-chain antibodies recognizing cell-surface antigens is applied to generate lentiviral vectors specific for endothelial cells, hematopoietic progenitors and neurons.

Generalized phase contrast and temporal focusing are combined to shape two-photon excitation patterns that elicit large photocurrents in ChR2-expressing neurons in culture and slices. This method allows precise aiming of the stimulating light at single neuronal processes, neurons or groups of neurons and can elicit simultaneous excitation of multiple cells using optogenetics.

Alternative expression analysis by sequencing (ALEXA-seq) aligns RNA-seq reads from different cell types to a database of alternative expression sequence features and quantifies isoforms that are differentially expressed between samples.

MicroRNA targets predicted by a variety of computational tools can be validated using a quantitative targeted proteomics approach, using stable isotope labeling and selected reaction monitoring mass spectrometry. The authors used this method to confirm predicted let-7 and miR-58 targets in Caenorhabditis elegans.

Single-molecule fluorescence resonance energy transfer (FRET) is a useful technique for monitoring biomolecular dynamics. A new method, termed switchable FRET, facilitates monitoring of multiple distances in single molecules, using a single donor and multiple spectrally identical acceptors that are switched on and off between a fluorescent state and a dark state.

A computational approach to both measure and infer microtubule dynamics from time-lapse images of end-labeled microtubules is described. It permits intracellular spatial patterns in microtubule behavior to be monitored.

The combination of protein display, moderate selection for protein activity and high-throughput DNA sequencing can be applied to hundreds of thousands of protein variants in parallel, enabling the derivation of sequence-function relationships.