Descemet Membrane Endothelial Keratoplasty-Induced Refractive Shift and Descemet Membrane Endothelial Keratoplasty-Induced Intraocular Lens Calculation Error.

PURPOSE: 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.

Fluorescent Polymer-AS1411-Aptamer Probe for dSTORM Super-Resolution Imaging of Endogenous Nucleolin.

Nucleolin is a multifunctional protein involved in essential biological processes. To precisely localize it and unravel its different roles in cells, fluorescence imaging is a powerful tool, especially super-resolution techniques. Here, we developed polymer-aptamer probes, both small and bright, adapted to direct stochastic optical reconstruction microscopy (dSTORM). Well-defined fluorescent polymer chains bearing fluorophores (AlexaFluor647) and a reactive end group were prepared via RAFT polymerization. The reactive end-group was then used for the oriented conjugation with AS1411, a DNA aptamer that recognizes nucleolin with high affinity. Conjugation via strain-promoted alkyne/azide click chemistry (SPAAC) between dibenzylcyclooctyne-ended fluorescent polymer chains and 3'-azido-functionalized nucleic acids proved to be the most efficient approach. In vitro and in cellulo evaluations demonstrated that selective recognition for nucleolin was retained. Their brightness and small size make these polymer-aptamer probes an appealing alternative to immunofluorescence, especially for super-resolution (10-20 nm) nanoscopy. dSTORM imaging demonstrated the ability of our fluorescent polymer-aptamer probe to provide selective and super-resolved detection of cell surface nucleolin.

Changes of Contamination Rate and Microorganism Evaluation in Organ-Cultured Human Corneas: A 14-Year Review From a French Regional Eye Bank.

PURPOSE: This study aimed to assess how the contamination rate of organ-cultured corneas has evolved and to analyze the evolution of microorganisms involved. METHODS: Data from the Besançon eye bank were reviewed over 14 years (2005-2018). The changes in the contamination rate and the contaminant species found during the organ culture storage were analyzed. Microbiological tests were performed twice on the storage media-at day 5 and before the deswelling phase. RESULTS: Among the 17,979 donor corneas collected, 1240 corneas were microbiological-test positive. The average annual contamination rate was 6.8% (range: 5.2%-8.9%). Seventy-five percent of contaminations were bacterial. The most frequently found bacterium was Staphylococcus spp. (31.3%), followed by non-Enterobacteriaceae Gram-negative Bacilli (GNB) (27.3%), with most Sphingomonas spp. and Pseudomonas spp. Fungal contamination (21.9%) was dominated by Candida (82.7%). Seventy-seven types of microorganisms were identified. The Staphylococcus rate tended to decrease, whereas non-Enterobacteriaceae GNB rate has increased in the past few years to reach 46% of bacteria. Most of the contaminations were detected in the early phase of organ culture at day 5 (89.2%). The second microbiological test found 44.8% of fungal contaminations (predominantly Candida spp.). CONCLUSIONS: The annual contamination rate was stable and remains low, but the types of contaminating microorganisms varied from year to year. Staphylococcus spp. and non-Enterobacteriaceae GNB accounted for a significant proportion of the contaminations. We found a significant proportion of contamination, especially fungal, at the late phase of storage. Reassessing the antibiotics and antifungals in the storage medium may be useful to limit corneal disposal.

Taking Advantage of Oxidation to Characterize Thiol-Containing Polymer Chains by MALDI-TOF Mass Spectrometry.

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 antioxidant 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 antioxidant 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.

Advanced Fluorescent Polymer Probes for the Site-Specific Labeling of Proteins in Live Cells Using the HaloTag Technology.

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.