Imaging and Quantifying Ribosomal Frameshifting Dynamics with Single-RNA Precision in Live Cells.

Recent advances in fluorescence microscopy have now made it possible to measure the translation dynamics of individual RNA in living cells and in multiple colors. Here we describe a protocol that exploits these recent advances to simultaneously image the translation of two open reading frames encoded on a single reporter RNA yet frameshifted with respect to each other. This enables precise measurements of frameshifting dynamics and efficiency from specific frameshift stimulatory sequences, all with single-RNA precision.

Live-Cell Single RNA Imaging Reveals Bursts of Translational Frameshifting.

Ribosomal frameshifting during the translation of RNA is implicated in human disease and viral infection. While previous work has uncovered many details about single RNA frameshifting kinetics in vitro, little is known about how single RNA frameshift in living systems. To confront this problem, we have developed technology to quantify live-cell single RNA translation dynamics in frameshifted open reading frames.

Multicolour single-molecule tracking of mRNA interactions with RNP granules.

Ribonucleoprotein (RNP) granules are non-membrane-bound organelles that have critical roles in the stress response, maternal messenger RNA storage, synaptic plasticity, tumour progression and neurodegeneration. However, the dynamics of their mRNA components within and near the granule surface remain poorly characterized, particularly in the context and timing of mRNAs exiting translation. Herein, we used multicolour single-molecule tracking to quantify the precise timing and kinetics of single mRNAs as they exit translation and enter RNP granules during stress.

Single-stranded telomere-binding protein employs a dual rheostat for binding affinity and specificity that drives function.

ssDNA, which is involved in numerous aspects of chromosome biology, is managed by a suite of proteins with tailored activities. The majority of these proteins bind ssDNA indiscriminately, exhibiting little apparent sequence preference. However, there are several notable exceptions, including the Cdc13 protein, which is vital for yeast telomere maintenance.

Minimotif Miner 4: a million peptide minimotifs and counting.

Minimotif Miner (MnM) is a database and web system for analyzing short functional peptide motifs, termed minimotifs. We present an update to MnM growing the database from ∼300 000 to >1 000 000 minimotif consensus sequences and instances. This growth comes largely from updating data from existing databases and annotation of articles with high-throughput approaches analyzing different types of post-translational modifications.

Imaging Translational and Post-Translational Gene Regulatory Dynamics in Living Cells with Antibody-Based Probes.

Antibody derivatives, such as antibody fragments (Fabs) and single-chain variable fragments (scFvs), are now being used to image traditionally hard-to-see protein subpopulations, including nascent polypeptides being translated and post-translationally modified proteins. This has allowed researchers to directly image and quantify, for the first time, translation initiation and elongation kinetics with single-transcript resolution and the temporal ordering and kinetics of post-translational histone and RNA polymerase II modifications. Here, we review these developments and discuss the strengths and weaknesses of live-cell imaging with antibody-based probes.

Real-time quantification of single RNA translation dynamics in living cells.

Although messenger RNA (mRNA) translation is a fundamental biological process, it has never been imaged in real time in vivo with single-molecule precision. To achieve this, we developed nascent chain tracking (NCT), a technique that uses multi-epitope tags and antibody-based fluorescent probes to quantify protein synthesis dynamics at the single-mRNA level. NCT reveals an elongation rate of ~10 amino acids per second, with initiation occurring stochastically every ~30 seconds.

The Functional Human C-Terminome.

All translated proteins end with a carboxylic acid commonly called the C-terminus. Many short functional sequences (minimotifs) are located on or immediately proximal to the C-terminus. However, information about the function of protein C-termini has not been consolidated into a single source.

Natural variability of minimotifs in 1092 people indicates that minimotifs are targets of evolution.

Since the function of a short contiguous peptide minimotif can be introduced or eliminated by a single point mutation, these functional elements may be a source of human variation and a target of selection. We analyzed the variability of ∼300 000 minimotifs in 1092 human genomes from the 1000 Genomes Project. Most minimotifs have been purified by selection, with a 94% invariance, which supports important functional roles for minimotifs.

Evaluation of calciotropic hormones in cats with odontoclastic resorptive lesions.

Objective: To assess associations between epidemiologic and laboratory variables and calciotropic hormones in cats with odontoclastic resorptive lesions (ORLs).

Animals: 182 client-owned cats older than 1 year of age with oral disease.

Procedure: Information on medical history, behavior, living environment, and feeding management was assessed by use of a questionnaire.

Gingivostomatitis.

Gingivostomatitis (GS) with various patterns of disease may require antiviral therapy, steroids, laser fulguration, immunomodulation drugs, or nonsteroidal anti-inflammatory drugs. The use of cyclosporine as an immunomodulation drug has long-term benefits in reduction of the immunologic events that contribute to GS. Whole-mouth extraction or partial extraction (premolars and molars), with radiographic conformation that all root remnants have been removed, may be the most viable option in nonresponsive and or intractably painful stomatitis in noncompliant cats or dogs.

Prediction of the Viscosities of "Soda-Lime" Silica Glasses.

Published data are used to develop factors for predicting the viscosity-temperature relationship from the compositions of "soda-lime" type silicate glasses at specific temperatures in the range of 600 to 1300 °C. The effects of NaO, KO, CaO, MgO, AlO and their interactions are evaluated. The influence of minor amounts of BaO, BO, LiO, and F, in the temperature range of 700 to 1300 °C, is estimated.