Pyrrolysine is the 22nd amino acid. An unresolved question has been how this atypical genetically encoded residue is inserted into proteins, because all previously described naturally occurring aminoacyl-tRNA synthetases are specific for one of the 20 universally distributed amino acids. Here we establish that synthetic L-pyrrolysine is attached as a free molecule to tRNA(CUA) by PylS, an archaeal class II aminoacyl-tRNA synthetase. PylS activates pyrrolysine with ATP and ligates pyrrolysine to tRNA(CUA) in vitro in reactions specific for pyrrolysine. The addition of pyrrolysine to Escherichia coli cells expressing pylT (encoding tRNA(CUA)) and pylS results in the translation of UAG in vivo as a sense codon. This is the first example from nature of direct aminoacylation of a tRNA with a non-canonical amino acid and shows that the genetic code of E. coli can be expanded to include UAG-directed pyrrolysine incorporation into proteins.

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Background: The modified Jobe technique of ulnar collateral ligament (UCL) reconstruction has previously been biomechanically compared with primary repair augmented with internal bracing. However, the docking technique has not been compared with repair with internal bracing. Hypothesis: Load to failure, gapping, and valgus opening angle are similar under valgus loading at 90° of flexion between repair with internal bracing and the docking technique for the UCL. Study Design: Controlled laboratory study. Methods: Nine matched pairs of fresh-frozen cadaveric elbows were potted with the forearm in neutral rotation. The palmaris longus tendon graft was harvested, and the bone was sectioned 14 cm proximal and distal to the elbow joint. First, native UCL testing was performed at 90° of flexion with 0.5 N·m preload, followed by a 5 N·m valgus moment to the elbow in cycles of 1, 10, 100, and 1000 at 1 Hz. The specimens were then loaded to failure at a rate of 0.2 mm/s. Next, the elbows were randomly divided into matched pairs to undergo either UCL reconstruction with docking technique or UCL repair augmented with internal bracing. Last, these specimens underwent testing as aforementioned. Results: Load to failure, gapping, and valgus opening angle did not differ significantly between native ligaments that underwent reconstruction or repair with internal bracing, paired native ligaments and reconstructions, paired native ligaments and repairs augmented with internal bracing, or reconstructions and repairs augmented with internal bracing. Conclusion: UCL reconstruction with docking technique and repair augmented with internal bracing provides valgus stability to the medial elbow comparable to the native ligament at 90°. No significant differences were noted between docking reconstruction and repair techniques for load to failure, gapping, or valgus opening angle during cyclic loading at time zero. Clinical Relevance: Our results suggest that UCL repair with internal bracing has a similar biomechanical profile at the time of initial fixation compared with the docking technique of UCL reconstruction.

A structure-activity relationship for symmetrical cyanine inhibitors of human tau aggregation was elaborated using a filter trap assay. Antagonist activity depended on cyanine heterocycle, polymethine bridge length, and the nature of meso-and N-substituents. One potent member of the series, 3,3'-diethyl-9-methylthiacarbocyanine iodide (compound 11), retained submicromolar potency and had calculated physical properties consistent with blood-brain barrier and cell membrane penetration. Exposure of organotypic slices prepared from JNPL3 transgenic mice (which express human tau harboring the aggregation prone P301L tauopathy mutation) to compound 11 for one week revealed a biphasic dose response relationship. Low nanomolar concentrations decreased insoluble tau aggregates to half those observed in slices treated with vehicle alone. In contrast, high concentrations (≥300 nM) augmented tau aggregation and produced abnormalities in tissue tubulin levels. These data suggest that certain symmetrical carbocyanine dyes can modulate tau aggregation in the slice biological model at concentrations well below those associated with toxicity.