A mutation in the myostatin gene increases muscle mass and enhances racing performance in heterozygote dogs

Double muscling is a trait previously described in several mammalian species including cattle and sheep and is caused by mutations in the myostatin (MSTN) gene (previously referred to as GDF8). Here we describe a new mutation in MSTN found in the whippet dog breed that results in a double-muscled phenotype known as the “bully” whippet. Individuals with this phenotype carry two copies of a two-base-pair deletion in the third exon of MSTN leading to a premature stop codon at amino acid 313. Individuals carrying only one copy of the mutation are, on average, more muscular than wild-type individuals (p = 7.43 × 10⁻⁶; Kruskal-Wallis Test) and are significantly faster than individuals carrying the wild-type genotype in competitive racing events (Kendall's nonparametric measure, τ = 0.3619; p ≈ 0.00028). These results highlight the utility of performance-enhancing polymorphisms, marking the first time a mutation in MSTN has been quantitatively linked to increased athletic performance.     

Assessing enzyme activities using stable isotope labeling and mass spectrometry

Activity-based protein profiling has emerged as a valuable technology for labeling, enriching, and assessing protein activities from complex mixtures. This is primarily accomplished via a two-step identification and quantification process. Here we show a highly quantitative and streamlined method, termed catch-and-release activity profiling of enzymes (CAPE), which reduces this procedure to a single step. Furthermore the CAPE approach has the ability to detect small quantitative changes that may have been missed by alternative mass spectrometry-based techniques.     

Experimental Test of Connector Rotation during DNA Packaging into Bacteriophage ϕ29 Capsids

The bacteriophage ϕ29 generates large forces to compact its double-stranded DNA genome into a protein capsid by means of a portal motor complex. Several mechanical models for the generation of these high forces by the motor complex predict coupling of DNA translocation to rotation of the head-tail connector dodecamer. Putative connector rotation is investigated here by combining the methods of single-molecule force spectroscopy with polarization-sensitive single-molecule fluorescence. In our experiment, we observe motor function in several packaging complexes in parallel using video microscopy of bead position in a magnetic trap. At the same time, we follow the orientation of single fluorophores attached to the portal motor connector. From our data, we can exclude connector rotation with greater than 99% probability and therefore answer a long-standing mechanistic question.     

Fluorescence imaging of amyloid formation in living cells by a functional, tetracysteine-tagged alpha-synuclein

Alpha-synuclein is a major component of intraneuronal protein aggregates constituting a distinctive feature of Parkinson disease. To date, fluorescence imaging of dynamic processes leading to such amyloid deposits in living cells has not been feasible. To address this need, we generated a recombinant alpha-synuclein (alpha-synuclein-C4) bearing a tetracysteine target for fluorogenic biarsenical compounds. The biophysical, biochemical and aggregation properties of alpha-synuclein-C4 matched those of the wild-type protein in vitro and in living cells. We observed aggregation of alpha-synuclein-C4 transfected or microinjected into cells, particularly under oxidative stress conditions. Fluorescence resonance energy transfer (FRET) between FlAsH and ReAsH confirmed the close association of fibrillized alpha-synuclein-C4 molecules. Alpha-synuclein-C4 offers the means for directly probing amyloid formation and interactions of alpha-synuclein with other proteins in living cells, the response to cellular stress and screening drugs for Parkinson disease.     

In vivo gene regulation in Salmonella spp. by a salicylate-dependent control circuit

Systems allowing tightly regulated expression of prokaryotic genes in vivo are important for performing functional studies of bacterial genes in host-pathogen interactions and establishing bacteria-based therapies. We integrated a regulatory control circuit activated by acetyl salicylic acid (ASA) in attenuated Salmonella enterica that carries an expression module with a gene of interest under control of the XylS2-dependent Pm promoter. This resulted in 20-150-fold induction ex vivo. The regulatory circuit was also efficiently induced by ASA when the bacteria resided in eukaryotic cells, both in vitro and in vivo. To validate the circuit, we administered Salmonella spp., carrying an expression module encoding the 5-fluorocytosine-converting enzyme cytosine deaminase in the bacterial chromosome or in a plasmid, to mice with tumors. Induction with ASA before 5-fluorocytosine administration resulted in a significant reduction of tumor growth. These results demonstrate the usefulness of the regulatory control circuit to selectively switch on gene expression during bacterial infection.