Brief Communications in 2008

A combination of in vitro protein synthesis and microfluidics is used to measure protein-protein interactions between 43 proteins in Streptococcus pneumoniae. The method does not require expression within cells and is amenable to large-scale experiments.

An infrared laser is used to activate gene expression from a heat shock promoter in single cells in Caenorhabditis elegans, and is shown to be more effective and less detrimental to cells than a visible laser used for this purpose.

A web-based protein-protein interaction (PPI) analysis platform called PINA integrates PPI data from six public databases and provides tools to aid in the construction and analysis of PPI networks, including local recuration and annotation of existing records and manual addition of new records.

This variant ascertainment algorithm, or VAAL, uses short sequence reads of haploid bacterial genomes to first locally assemble the reads and then compare these assemblies to the reference genome. This allows VAAL to detect all types of variants ranging from single-nucleotide polymorphisms to large insertions or deletions.

To study microRNA function in vivo, the authors optimize lentiviral-driven expression of microRNA target sequences in mice and show dose-dependent inhibition of microRNA-mediated regulation of reporter constructs as well as of natural microRNA targets. With the inhibition of a miR-223, they can phenocopy the knockout of this microRNA.

A simple modification to the optical configuration used for fluorescence photoactivation localization microscopy (FPALM) allows the fluorescence anisotropies of each individual molecule in a nanoscale image to be measured. The method was used to obtain position and orientation information for fluorescently labeled actin or hemagglutinin molecules in fixed fibroblasts.

The combination of a glass window placed on top of a mouse mammary gland with photoswitchable fluorescent protein labeling of implanted tumor cells allows tumor-cell tracking over multiple imaging sessions in orthotopic tumors. Results show the existence of two distinct microenvironments with different tumor-cell invasion and intravasation characteristics.

The algorithm Sylamer finds over- or underrepresented nucleotide motifs, such as microRNA seeds, in a gene list ranked according to expression levels and thus establishes whether a microRNA is directly affecting gene expression.

Many different red fluorescent proteins display cytotoxicity substantially higher than EGFP when used for whole-cell labeling of bacterial and mammalian cells with standard high-level expression systems. An improved tetrameric red fluorescent protein called DsRed-Express2 allows high-level labeling with minimal cytotoxicity comparable to that of EGFP.

To determine long-range linkage between single-nucleotide polymorphisms (SNPs) and the repeat-containing region of a disease-related gene, Liu et al. develop SNP linkage by circularization (SLiC) and lay the groundwork for using allele-specific RNA interference to target insertion or deletion mutations in disease-associated genes.

Directed evolution experiments usually rely on high-throughput screening of very large libraries of mutants, but most of the mutants do not even yield stable, functional proteins. The concept of neutral drift can be used to generate small but highly polymorphic and stable mutant libraries as a starting point for further evolution.

A miniature epifluorescence microscope that can be carried by a freely-moving adult mouse allows cellular-level imaging of neuronal spiking or measurement of microcirculation during normal behavioral activities.

A strategy using 48 or more singly labeled fluorescent oligonucleotide probes targeted to individual mRNA molecules allows the simultaneous localization and quantification of three mRNA species in fixed cells. mRNA visualization in whole animals and other organisms is also demonstrated.

Spectral searching, based on matching experimental peptide spectra to reference spectral libraries, is gaining interest as an alternative to traditional sequence-database searching in mass spectrometry–based proteomics. A software tool, SpectraST, now allows users to build their own high-quality spectral libraries from raw data.

A simple yet powerful super-resolution imaging approach based on switching off ordinary fluorophores and localizing those remaining or regaining fluorescence is illustrated using continuous widefield illumination and imaging of fixed and living cells labeled with rhodamine-derived dyes or fluorescent proteins. Biteen et al., also in this issue, describe related work using the ordinary fluorophore of EYFP for super-resolution imaging.

Previous work showed that the commonly used fluorescent protein EYFP can be bleached and reactivated. Exploiting this property allows super-resolution in vivo imaging of EYFP-labeled structures in living bacteria. Fölling et al., also in this issue, describe a related approach for super-resolution imaging using other ordinary fluorophores.

An automated sorting method using the COPAS Biosort machine allows the isolation of mutant C. elegans displaying differences in GFP expression in small numbers of cells. Compared to manual methods this increases the efficiency of the phenotypic selection step in cell-fate screens.

A simplified strategy to enzymatically preadenylate bar-coded oligonucleotides to be used for capturing microRNAs in biological samples is described. This efficient method should greatly facilitate multiplex analysis and profiling of microRNAs.

Identifying the molecular lesions in mutants isolated in forward genetic screens can be a laborious process. A proof-of-principle study in Caenorhabditis elegans now shows that this can be achieved rapidly by whole-genome deep sequencing.

Designing fluorescent protein-based sensors that display large changes in fluorescence resonance energy transfer (FRET) is challenging. Redesign of a FRET-based voltage sensor using new fluorescent proteins increased the sensor response to changes in membrane voltage and measurements at warmer temperatures displayed faster kinetics comparable to action potentials.