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A nonleaky and efficient DiCre-based conditional knockout system enables the study of essential genes in Toxoplasma gondii, revealing that the actin-myosin motor is not required for host invasion by the parasite.
Tagging of single transcripts with two fluorescent markers can be used to study many aspects of gene expression, including intrinsic noise in transcription or polymerase dynamics at a single gene.
Binding studies with systematically mutagenized RNA and RNA-binding proteins allow insight into the relationship between an RNA sequence, its structure and its function.
Using the red shifted opsin C1V1T and simple raster-scanning illumination, this work shows two-photon optogenetic stimulation of single cells, dendrites and spines. The method is also applied to map synaptic circuits in mouse brain slices and, using holographic photostimulation, for the simultaneous activation of two neurons located in different planes. Also online, Prakash et al. present a collection of opsins for two-photon excitation, inhibition and bistable control of neuronal activity in vitro and in vivo.
A method for phase contrast imaging of unstained thick tissue samples is presented. It is based on oblique back-illumination and can image at depths of several tens of microns.
The resolution achievable with single-molecule–based super-resolution fluorescence imaging is increased via a fluorophore caging procedure that uses a reducing agent to convert dyes to a long-lived dark state from which they can be activated with UV light and emit high numbers of photons.
Silica-coated silver nanocube suspensions provide an easy, rapid and label-free way to quantify protein binding to supported lipid bilayers by localized plasmon resonance measurement with a standard laboratory UV spectrophotometer.
A method for staining and embedding the entire mouse brain for electron microscopy is reported. The method results in uniform myelin staining and will allow reconstructions of myelinated long-range axons using serial block-face electron microscopy.
Modifications to an Orbitrap-based mass spectrometer enable analysis of large protein complexes in native-like states by mass spectrometry with very high sensitivity and mass resolution.
A unique multiple cloning site (MCS) and defined genetic components without MCS restriction sites allow for the rapid construction and iterative tuning of synthetic genetic circuits.
Removing phosphopantetheine-tagged labels from acyl carrier proteins (ACPs) and ACP fusion proteins contributes to a versatile labeling method in which tags can be iteratively swapped.
A high-resolution 4C-seq protocol involving two restriction digests and a revised analysis pipeline allows robust detection of physical interactions between regulatory DNA elements.
An integrated system composed of a microfluidic device, computer-vision tools and statistical methods for automatically handling, imaging, classifying and sorting C. elegans organisms is presented. The system performs automated screens of subcellular phenotypes and is used here to identify genes involved in synaptogenesis.
Gene synthesis is currently limited by the need to use error-prone oligonucleotide building blocks. Dial-out PCR overcomes the sequence verification bottleneck by using unique sequence tags and massively parallel sequencing to identify and selectively retrieve error-free DNA molecules of interest from complex mixtures.
A combination of protein correlation profiling–stable isotope labeling by amino acids in cell culture and size-exclusion chromatography allows stoichiometric analysis of changes in the human interactome in response to a growth factor.
Precise mass measurement on single mammalian cells is combined with imaging to monitor changes in cellular mass during the cell cycle over many generations.