We report the site-specific
and covalent bioconjugation of fluorescent
polymer chains to proteins in live cells using the HaloTag technology.
Polymer chains bearing a Halo-ligand precisely located at their α-chain-end
were synthesized in a controlled manner owing to the RAFT polymerization
process. They were labeled in lateral position by several organic
fluorophores such as AlexaFluor 647. The resulting Halo-ligand polymer
probe was finally shown to selectively recognize and label HaloTag
proteins present at the membrane of live cells using confocal fluorescence
microscopy. Such a polymer bioconjugation approach holds great promises
for various applications ranging from cell imaging to cell surface
functionalization.
MALDI-TOF mass spectrometry analyses revealed the oxidation of thiol-containing polymer chain-ends during sample preparation using THF as solvent. In these conditions, the extent of oxidation was hardly reproducible, and led to various types of oxidized compounds. Preparing the samples at the last minute using commercial THF stabilized with an anti-oxidant led to more reproducible results, with the least oxidation. However, it is demonstrated herein that thiol oxidation can be advantageously taken into profit to further ascertain the presence of the thiol at the polymer chain-end. To force thiol oxidation we used THF without any anti-oxidant stabilizer, thus more prone to form peroxides. Thiol-containing polymer chains can thereby be indirectly evidenced by the formation of oxidation products such as chain-chain disulfide bonds and sulfonic acid chains-ends. More importantly, in these oxidizing conditions and in the negative mode, sulfonic acid-terminated polymer chains can be more sensitively detected than thiol ones (the low pKa of sulfonic acids facilitating their anionization in MALDI source). In conclusion, performing MALDI-TOF mass spectrometry analyses in oxidizing conditions, as complement to regular analyses, was found to be very useful for the chain-end identification of different thiol-containing polymer chains.
The aim of this study was to determine the mechanisms leading to the refractive shift and intraocular lens calculation error induced by Descemet membrane endothelial keratoplasty (DMEK), using ocular biometry and corneal elevation tomography data.Methods: This is a retrospective, monocentric cohort study. Eyes which underwent uncomplicated DMEK surgery with available pre-DMEK and post-DMEK Scheimpflug rotating camera data (Pentacam, Oculus, Wetzlar, Germany) were considered for inclusion with an age-matched control group of healthy corneas. Cataract surgery data were collected for triple-DMEK cases. DMEK-induced refractive shift (DIRS) and intraocular lens calculation error (DICE) were calculated. Pearson r correlation coefficient was calculated between each corneal parameter variation and both DIRS and DICE.Results: DIRS was calculable for 49 eyes from 43 patients. It was 30.61% neutral, 53.06% hyperopic (36.73% . 1D), and 16.32% myopic (6.12% . 1 D). DICE was calculable for 30 eyes of 26 patients: It was 46.67% neutral, 40.00% hyperopic (10.00% . 1D), and 13.33% myopic (3.33% . 1D). DIRS and DICE were mainly associated with variations in PRC/ARC ratio, anterior average radii of curvature (ARC), posterior average radii of curvature (PRC), and posterior Q.
Conclusions:Our results suggest that ARC variations, PRC/ARC ratio variations, PRC variations, and posterior Q variations are the most influential parameters for both DIRS and DICE. We suggest that a distinction between those different phenomenons, both currently described as "hyperopic shift" in the literature, should be made by researchers and clinicians.
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