The genetic incorporation of unnatural amino acids (UAAs) with high efficiency and fidelity is a powerful tool for the study of protein structure and dynamics with site-specificity in a relatively nonintrusive manner. Here, we illustrate the ability of L-4-nitrophenylalanine to serve as a sensitive IR probe of local protein environments in the 247 residue superfolder green fluorescent protein (sfGFP). Specifically, the nitro symmetric stretching frequency of L-4-nitrophenylalanine was shown to be sensitive to both solvents that mimic different protein environments and (15)N isotopic labeling of the three-atom nitro group of this UAA. (14)NO(2) and (15)NO(2) variants of this UAA were incorporated utilizing an engineered orthogonal aminoacyl-tRNA synthetase/tRNA pair into a solvent exposed and a partially buried position in sfGFP with high efficiency and fidelity. The combination of isotopic labeling and difference FTIR spectroscopy permitted the nitro symmetric stretching frequency of L-4-nitrophenylalanine to be experimentally measured at either site in sfGFP. The (14)NO(2) symmetric stretching frequency red-shifted 7.7 cm(-1) between the solvent exposed and partially buried position, thus illustrating the ability of this UAA to serve as an effective IR probe of local protein environments.

CALI and the labelling specificity that fluorescent proteins provide is very useful to avoid uncontrolled photodamage. Indeed, fluorescent proteins have been successfully used in CALI, although of the inactivation mechanisms by ROS are dependent on the fluorescent protein used and are not fully understood [2,3]. Here, we present a quantitative study of the ability of TagRFP to produce ROS, in particular singlet oxygen. TagRFP is able to photosensitize singlet oxygen with an estimated quantum yield of 0.004 [4]. This is the first estimation of a quantum yield of singlet oxygen production value for a GFP-like protein. We also find that TagRFP has a short triplet lifetime, which reflects relatively high oxygen accessibility to the chromophore compared to EGFP. Our results provide photophysical insight that allows the understanding of the mechanism behind CALI. Moreover, it has implications in improving photobleaching in fluorescent proteins.[1] K.

OBJECTIVE To evaluate radiation exposure of dogs and cats undergoing procedures requiring intraoperative fluoroscopy and for operators performing those procedures. SAMPLE 360 fluoroscopic procedures performed at 2 academic institutions between 2012 and 2015. PROCEDURES Fluoroscopic procedures were classified as vascular, urinary, respiratory, cardiac, gastrointestinal, and orthopedic. Fluoroscopy operators were classified as interventional radiology-trained clinicians, orthopedic surgeons, soft tissue surgeons, internists, and cardiologists. Total radiation exposure in milligrays and total fluoroscopy time in minutes were obtained from dose reports for 4 C-arm units. Kruskal-Wallis equality of populations rank tests and Dunn pairwise comparisons were used to compare differences in time and exposure among procedures and operators. RESULTS Fluoroscopy time (median, 35.80 minutes; range, 0.60 to 84.70 minutes) was significantly greater and radiation exposure (median, 137.00 mGy; range, 3.00 to 617.51 mGy) was significantly higher for vascular procedures than for other procedures. Median total radiation exposure was significantly higher for procedures performed by interventional radiology-trained clinicians (16.10 mGy; range, 0.44 to 617.50 mGy), cardiologists (25.82 mGy; range, 0.33 to 287.45 mGy), and internists (25.24 mGy; range, 3.58 to 185.79 mGy). CONCLUSIONS AND CLINICAL RELEVANCE Vascular fluoroscopic procedures were associated with significantly longer fluoroscopy time and higher radiation exposure than were other evaluated fluoroscopic procedures. Future studies should focus on quantitative radiation monitoring for patients and operators, importance of operator training, intraoperative safety measures, and protocols for postoperative monitoring of patients.

People with calcium oxalate (CaOx) urolithiasis and idiopathic hypercalciuria (IH) often have evidence of increased bone resorption, but bone turnover has not previously been investigated in dogs with these conditions. The aim of this study was to determine whether a marker of bone resorption, β-crosslaps, differs between dogs with CaOx urolithiasis and IH compared to controls. This retrospective, cross-sectional study used a canine specific ELISA to measure β-crosslaps concentrations in stored frozen serum samples from 20 dogs with CaOx urolithiasis and IH and 20 breed-, sex-, and age-matched stone-free controls (18 Miniature Schnauzers, 14 Bichons Frise, and 8 Shih Tzus). Dogs with CaOx urolithiasis and IH had lower β-crosslaps concentrations relative to controls (P = 0.0043), and β-crosslaps had a moderate negative correlation with urinary calciumto-creatinine ratios (r = −0.44, P = 0.0044). Miniature Schnauzers had lower β-crosslaps concentrations than the other two breeds (P = 0.0035). The ELISA had acceptable intra-assay precision, but concentrations decreased when samples were repeatedly assayed over time. Assay recovery rates were also below acceptance criteria. In conclusion, Miniature Schnauzers, Bichons Frise, and Shih Tzus with CaOx urolithiasis and IH have evidence of decreased bone resorption compared to stone-free controls. This suggests that other causes of IH, such as intestinal hyperabsorption of calcium, underlie risk for CaOx urolithiasis in these breeds. Results should be confirmed in larger populations and with other β-crosslaps assays and additional biomarkers of bone turnover. The stability of canine serum β-crosslaps after freeze-thaw cycles and storage at various temperatures requires investigation.

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