Hydrophilic trans-Cyclooctenylated Noncanonical Amino Acids for Fast Intracellular Protein Labeling.

Introduction of bioorthogonal functionalities (e.g., trans-cyclooctene-TCO) into a protein of interest by site-specific genetic encoding of non-canonical amino acids (ncAAs) creates uniquely targetable platforms for fluorescent labeling schemes in combination with tetrazine-functionalized dyes. However, fluorescent labeling of an intracellular protein is usually compromised by high background, arising from the hydrophobicity of ncAAs; this is typically compensated for by hours-long washout to remove excess ncAAs from the cellular interior. To overcome these problems, we designed, synthesized, and tested new, hydrophilic TCO-ncAAs. One derivative, DOTCO-lysine was genetically incorporated into proteins with good yield. The increased hydrophilicity shortened the excess ncAA washout time from hours to minutes, thus permitting rapid labeling and subsequent fluorescence microscopy.

Debugging Eukaryotic Genetic Code Expansion for Site-Specific Click-PAINT Super-Resolution Microscopy.

Super-resolution microscopy (SRM) greatly benefits from the ability to install small photostable fluorescent labels into proteins. Genetic code expansion (GCE) technology addresses this demand, allowing the introduction of small labeling sites, in the form of uniquely reactive noncanonical amino acids (ncAAs), at any residue in a target protein. However, low incorporation efficiency of ncAAs and high background fluorescence limit its current SRM applications. Redirecting the subcellular localization of the pyrrolysine-based GCE system for click chemistry, combined with DNA-PAINT microscopy, enables the visualization of even low-abundance proteins inside mammalian cells. This approach links a versatile, biocompatible, and potentially unbleachable labeling method with residue-specific precision. Moreover, our reengineered GCE system eliminates untargeted background fluorescence and substantially boosts the expression yield, which is of general interest for enhanced protein engineering in eukaryotes using GCE.

Anterior cruciate ligament reconstruction using remnant preservation and a femoral tensioning technique: clinical and magnetic resonance imaging results.

PURPOSE: The purpose of this study was to investigate the clinical and magnetic resonance imaging (MRI) results of anterior cruciate ligament (ACL) reconstruction with autogenous hamstring tendon by use of remnant preservation and a femoral tensioning technique. METHODS: A total of 53 patients who had ACL reconstruction by use of remnant ACL stump preservation and a femoral tensioning technique were evaluated. Clinical evaluation at a minimum of 2 years after surgery included range of motion, Lachman test, pivot-shift test, KT-2000 arthrometer testing (MEDmetric, San Diego, CA), and clinical scores. Plain radiographs were evaluated for tunnel enlargement. MRI was obtained for evaluation of graft continuity, cyclops-like mass lesion, and positioning of the tibial tunnel. Second-look arthroscopy was performed in 33 patients. RESULTS: The clinical scores improved postoperatively. There were statistically significant differences between preoperative and postoperative Lachman tests, pivot-shift tests, and KT-2000 arthrometer measurements. Postoperative MRI was available in 48 patients, and it showed intact graft in 45 patients, 2 partial tears, and 1 complete loss of graft. There were cyclops-like mass lesions in 12 patients, but none showed an extension limitation or pain at extension. The position of the tibial tunnel on the sagittal and coronal view was similar to the position of the normal ACL tibial insertion. The measured tibial tunnel widening on the radiographs at final follow-up was 2.2 ± 1.5 mm. CONCLUSIONS: Reconstruction of the ACL by use of preservation and femoral tensioning of the remnant tissue showed good clinical results without increased concerns regarding incorrect tunnel formation. Postoperative MRI showed an increased incidence of cyclops-like mass lesions, but no clinical significance was observed. LEVEL OF EVIDENCE: Level IV, case series.

Labeling proteins on live mammalian cells using click chemistry.

We describe a protocol for the rapid labeling of cell-surface proteins in living mammalian cells using click chemistry. The labeling method is based on strain-promoted alkyne-azide cycloaddition (SPAAC) and strain-promoted inverse-electron-demand Diels-Alder cycloaddition (SPIEDAC) reactions, in which noncanonical amino acids (ncAAs) bearing ring-strained alkynes or alkenes react, respectively, with dyes containing azide or tetrazine groups. To introduce ncAAs site specifically into a protein of interest (POI), we use genetic code expansion technology. The protocol can be described as comprising two steps. In the first step, an Amber stop codon is introduced--by site-directed mutagenesis--at the desired site on the gene encoding the POI. This plasmid is then transfected into mammalian cells, along with another plasmid that encodes an aminoacyl-tRNA synthetase/tRNA (RS/tRNA) pair that is orthogonal to the host's translational machinery. In the presence of the ncAA, the orthogonal RS/tRNA pair specifically suppresses the Amber codon by incorporating the ncAA into the polypeptide chain of the POI. In the second step, the expressed POI is labeled with a suitably reactive dye derivative that is directly supplied to the growth medium. We provide a detailed protocol for using commercially available ncAAs and dyes for labeling the insulin receptor, and we discuss the optimal surface-labeling conditions and the limitations of labeling living mammalian cells. The protocol involves an initial cloning step that can take 4-7 d, followed by the described transfections and labeling reaction steps, which can take 3-4 d.

Patellar instability with and without trochlear dysplasia: new arthroscopic medial soft tissue plication with pullout technique.

The aim of this study was to analyze clinical and radiologic results of an arthroscopic medial plication with the pullout technique and to define indications and limitations of this procedure for patellar instability. Records of 45 patients treated for patellar instability with arthroscopic medial plication with the pullout technique were reviewed. The mean patient age was 22.8±8.3 years. The tibial tuberosity-trochlear groove distance, trochlear depth (TD), and Insall-Salvati ratio were measured using magnetic resonance imaging taken preoperatively. Patients were evaluated clinically by the Insall and Kujala score and radiographically by measuring the congruence angle, lateral patellofemoral angle, and lateral patellar translation pre- and postoperatively. Patients were classified into 2 groups: group 1 comprised 23 patients with TD greater than or equal to 3.0 mm and group 2 comprised 22 patients with TD less than 3.0 mm. A significant improvement (P=.007) in postoperative clinical scores compared with preoperative values was observed. The congruence angle improved to 11.0°±20.6° (P=.006), the lateral patellofemoral angle improved to -1.6°±7.7° (P≤.0001), and the lateral patellar translation improved to 8.7±5.3 mm (P≤.0001) postoperatively. There were 5 (11%) failure cases. No significant difference existed in the number of failure cases and clinical scores between the 2 groups. This arthroscopic medial soft tissue pullout technique showed good clinical and radiologic results for patellar instability even in the presence of mild to moderate trochlear dysplasia. However, the technique showed limited success in severe trochlear dysplasia cases.

Isolation of Soil Microorganisms Having Antibacterial Activity and Antimigratory Effects on Sphingosylphosphorylcholine-induced Migration of PANC-1 Cells.

To obtain soil microorganisms producing antimigratory activity which is important in controlling the metastasis of cancer cells, more than three hundreds of soil microbes were isolated from sixteen soil sources including Namsan mountain and designated as DGU1001-10338. At first, their antibiotic activities were examined by paper-disc method. More than 40 soil microbes produced compounds with antibiotic activity. Then, antimigratory activities of selected soil microorganisms were examined in a sphingosylphosphorylcholine-induced migration assay in PANC-1 cells. Six of 42 soil microorganisms having antibacterial activity also had more than 45% inhibitory activity on migration of PANC-1 cells. These results suggested that selected soil microorganisms were a useful starting point to find compounds for controlling metastasis of cancer cells.