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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.
The 3′ region extraction and deep sequencing (3′READS) method accurately identifies cleavage and polyadenylation sites, avoiding common artifacts and detecting sites in A-rich contexts. It was used to greatly expand the number of characterized sites in the mouse genome, including those in long noncoding RNAs.
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 collection of simulation tools and workflow for single-molecule Förster resonance energy transfer (smFRET) allows highly quantitative structural modeling. This hybrid approach yields a model of reverse-transcriptase binding to DNA at sub-angstrom accuracy when benchmarked against a crystal structure and can resolve a flexible single-stranded template overhang.
The structure of the membrane anchor domain of the bacterial autotransporter YadA is solved by a solid-state NMR spectroscopy approach using a uniformly 13C- and 15N-labeled microcrystalline sample.
A collection of opsins for two-photon modulation of neuronal activity in vitro and in vivo is presented in this resource. The opsins have kinetic, expression and spectral properties ideally suited to typical raster-scanning two-photon microscopy. Also online, Packer et al. use the red-shifted opsin C1V1T and simple raster-scanning illumination to stimulate individual spines and dendrites and map synaptic circuits.
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.
Cells are dosed with magnetic nanoparticles and patterned onto micromagnetic substrates, enabling the application of controlled and variable mechanical force to tens of thousands of cells.
The probe selection for imputation (PSI) approach accurately imputes global gene expression profiles from a small subset of probes that it chooses based on a training set of full profiles, allowing many more combinatorial experiments to be performed given the same resources.
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.