In Silico Identification of a Key Residue for Substrate Recognition of the Riboflavin Membrane Transporter RFVT3.

Because of its specific physicochemical properties (fluorescence, photosensitizing, and redox reactions), vitamin B2, also called riboflavin (RF), has been generating a lot of interest in the fields of nanotechnology and bioengineering in the last decade. RF, by targeting its riboflavin transporters (RFVTs) overexpressed in some cancers, is particularly used to functionalize nanovectors for anticancer drug delivery. From a physiopathological point of view, an RF deficiency has been implicated in various pathologies, including mendelian diseases. RF deficiency is mainly due to natural variants of its RFVTs that make them inactive and therefore prevent RF transport. The lack of structural data about RFVT is a major drawback for a better understanding of the role of the mutations in the molecular mechanism of these transporters. In this context, this work was aimed at investigating the 3D structure of RFVT3 and its interactions with RF. For this purpose, we used an in silico procedure including protein threading, docking, and molecular dynamics. Our results propose that the natural variant W17R, known to be responsible for the Brown-Vialetto-Van Laere syndrome, prevents the recognition of RF by RFVT3 and thus blocks its transport. This in silico procedure could be used for elucidating the impact of pathogenic mutations of other proteins. Moreover, the identification of RF binding sites will be useful for the design of RF-functionalized nanovectors.

Identification and visualization of protein binding regions with the ArDock server.

ArDock (ardock.ibcp.fr) is a structural bioinformatics web server for the prediction and the visualization of potential interaction regions at protein surfaces. ArDock ranks the surface residues of a protein according to their tendency to form interfaces in a set of predefined docking experiments between the query protein and a set of arbitrary protein probes. The ArDock methodology is derived from large scale cross-docking studies where it was observed that randomly chosen proteins tend to dock in a non-random way at protein surfaces. The method predicts interaction site of the protein, or alternate interfaces in the case of proteins with multiple interaction modes. The server takes a protein structure as input and computes a score for each surface residue. Its output focuses on the interactive visualization of results and on interoperability with other services.

Corneal transplantation: study of the data of a regional eye bank for the year 2013 and analysis of the evolution of the adverse events reported in France since 2010.

To analyze the data of the adverse events collected in a single major eye bank (EFS Bourgogne Franche Comté, Besançon, France) for the year 2013 and to report the French data of biovigilance provided by the French National Agency for Medicines and Health Products Safety (ANSM) between 2010 and 2013. we have set up a study of adverse events in 2013, in collaboration with a single eye bank (EFS Bourgogne Franche Comté, Besançon, France). A survey was sent to the surgeon for each delivered corneal button by the eye bank in 2013. They were asked for each grafted patient performed in their center, the type of graft (penetrating keratoplasty, anterior keratoplasty or endothelial keratoplasty), the occurrence of adverse events (primary failure, infectious keratis, endophthalmitis, immune rejection, and other events) and the time interval between surgery and events (Less than 1 postoperative month, between 1 month and 1 year postoperatively, >1 year postoperatively). In 2013, 407 corneal buttons were delivered by the eye bank of Besançon in 21 medical centers which performed corneal grafts and we sent 407 surveys. We received 243 completed questionnaires (59.75%) from 11 centers (52.38%). The global reported rate of adverse events was 27.54% of the graft (n = 65/236 corneal grafts performed in 11 centers in 2013; 20% of Primary graft failure, 11% of infectious keratitis, 1% of endophthalmitis, 34% of rejection, 34% of other incidents). 30.16% of complications were noticed before the first month after surgery versus 52.38% of complications noticed between the first month and the first year after surgery and 17.46% of complications noticed after the post-operative first year The most common causes of adverse events after PK were Immune rejection (13.17%), surgical causes (5.98%) and infection (4.79%) and after EK were Primary graft failure (8.2%) and surgical causes (19.67%). In 2013, in France 0.83% of adverse events were notified in ANSM. For the 236 performed graft issued from a major eye bank (EFS Besançon) in 2013 the global reported rate of post-graft adverse events was 27.54% of the grafts (20% of Primary graft failure, 11% of infectious keratitis, 1% of endophthalmitis, 34% of rejection and 34% of other incidents). Compared to the ANSM data (0.83% of adverse events reported in 2013) this rate is high. This difference can be explained by the low rate of annual notification to the ANSM and shows that biovigilance in France must be more developed. Since biovigilance needs constant improvement for the safety of the graft system, training, information for practitioners, simplifications of procedures and international standardization of the definition are the main points that could be improved.

Choroidal thickness measurement in children using optical coherence tomography.

PURPOSE: To measure choroidal thickness (CT) in children of various ages by using spectral optical coherence tomography with enhanced depth imaging and to investigate the association between subfoveal CT and ocular axial length, age, gender, weight, and height in children. METHODS: Healthy children were prospectively included between May and August 2012. Optical coherence tomography with the enhanced depth imaging system (Spectralis, Heidelberg, Germany) was used for choroidal imaging at nine defined points of the macula of both eyes. Axial length was measured using IOLMaster (Carl Zeiss Meditec, Dublin, CA). Height, weight, and refraction were recorded. Interobserver agreement in readings was also assessed by the Bland-Altman Method. RESULTS: Three hundred and forty-eight eyes from 174 children aged 3.5 years to 14.9 years were imaged. The mean subfoveal CT in right eyes was 341.96 ± 74.7 µm. Choroidal thickness increased with age (r = 0.24, P = 0.017), height, and weight but not with gender (P > 0.05). It was also inversely correlated to the axial length (r = 0.24, P = 0.001). The nasal choroid appeared thinner than in the temporal area (analysis of variance, P < 0.0001). CONCLUSION: In children, CT increases with age and is inversely correlated to axial length. There is a significant variation of CT between children of the same age.

Reliability of cone counts using an adaptive optics retinal camera.

BACKGROUND: To assess the reproducibility and repeatability of cone imaging in healthy human eyes, using the RTx-1 Adaptive Optics Retinal Camera and its proprietary cone-counting software. DESIGN: Single-centre, prospective study. PARTICIPANTS: Ten healthy adults. METHODS: Macular cones were imaged. Intrasession repeatability was assessed by comparing 10 consecutive acquisitions obtained by the same operator from each subject. For the intersession study, each subject was imaged five consecutive days. Interoperator reproducibility was also evaluated by comparing the images obtained from 10 different subjects by two independent operators. Finally, intergrader agreement was evaluated by comparing the cone counts measured by two masked graders. MAIN OUTCOME MEASURES: Mean cone density (cells/mm(2) ), spacing between cells (µm) and percentage of cones with six neighbours calculated on Voronoi diagrams were measured. Correlation coefficients, intraclass correlation coefficients, and coefficients of variation were calculated. RESULTS: Correlation coefficient and intraclass correlation coefficient were respectively 0.81 and 0.96 between operators, and 0.97 and 0.98 between the two graders. The intrasession and intersession coefficients of variation were under 7%. The percentage of cells with six neighbours and the spacing between cones varied in the same proportion (coefficients of variation ranged from 1.66 to 10.05%). CONCLUSIONS: Overall, the test-retest variability of RTx-1 and its software was good in normal human eyes. Further studies in the normal clinical setting are mandatory.

The Det.Belt Server: A Tool to Visualize and Estimate Amphipathic Solvent Belts around Membrane Proteins.

Detergents wrap around membrane proteins to form a belt covering the hydrophobic part of the protein serving for membrane insertion and interaction with lipids. The number of detergent monomers forming this belt is usually unknown to investigators, unless dedicated detergent quantification is undertaken, which for many projects is difficult to setup. Yet, having an approximate knowledge of the amount of detergent forming the belt is extremely useful, to better grasp the protein of interest in interaction with its direct environment rather than picturing the membrane protein "naked". We created the Det.Belt server to dress up membrane proteins and represent in 3D the bulk made by detergent molecules wrapping in a belt. Many detergents are included in a database, allowing investigators to screen in silico the effect of different detergents around their membrane protein. The input number of detergents is changeable with fast recomputation of the belt for interactive usage. Metrics representing the belt are readily available together with scripts to render quality 3D images for publication. The Det.Belt server is a tool for biochemists to better grasp their sample.

Evolution of Corneal Graft Survival Over a 30-Year Period and Comparison of Surgical Techniques: A Cohort Study.

PURPOSE: To compare graft survival between 3 10-year periods and according to surgical techniques performed in the last years. DESIGN: Cohort study. METHODS: setting: Regional center (Besançon University Hospital, France). PATIENTS: All 1132 patients operated on between 1983 and 2014. Graft and patient baseline characteristics, risk factors for failure, surgical procedures, and postoperative corneal status were collected. MAIN OUTCOME MEASURES: Five-year survival rate in the whole cohort; 1-year and 3-year survival rates, respectively, among 88 patients with endothelial dystrophy (ED) or postoperative bullous keratopathy (PBK) operated on using endothelial lamellar keratoplasty (ELK) or penetrating keratoplasty (PK), and among 56 patients with keratoconus operated on using anterior lamellar keratoplasty (ALK) or PK. RESULTS: Between the 1983-1993 and the 2004-2014 periods, overall 5-year graft survival rate increased from 61.4% to 76.5% (P = .0004). The main prognostic factors were preoperative diagnosis, graft endothelial density, and postoperative lens status. After adjusting for these factors, difference in survival rates was no longer significant (hazard ratio 0.90 for the second and 1.17 for the third period, compared to the first, P = .4191). Only 1 graft failure, after PK, occurred among the 56 patients with keratoconus. Among the 88 patients with ED or PBK, the 1-year graft survival was higher with PK (90.6%) than with ELK (60.8%) (P = .0025) but no significance remained after adjustment (hazard ratio 3.22, P = .1304). CONCLUSIONS: Despite numerous changes in graft procedures and surgical techniques, no noticeable improvement in graft survival was found during the last 30 years while taking into account other prognostic factors.

Assessment of parafoveal cone density in patients taking hydroxychloroquine in the absence of clinically documented retinal toxicity.

PURPOSE: To measure cone density in patients taking hydroxychloroquine (HCQ), with no clinical evidence of maculopathy. METHODS: Patients visiting for HCQ macular toxicity screening in the Besançon University Hospital Ophthalmology Department (France) were studied. They underwent routine examination including spectral-domain optical coherence tomography, fundus autofluorescence and multifocal electroretinogram to detect HCQ-induced retinal toxicity. Cone metrics (density, spacing and percentage of cones with six neighbours) were obtained using an adaptive optics camera (RTX1, Imagine Eyes, Orsay, France). The region of interest corresponded to a 0.3° × 0.3° square placed nasally and temporally at 2° of eccentricity from the fovea. RESULTS: Forty eyes of 23 patients were studied. The majority of the patients (21/23) were female. They were aged from 25 to 60 years (mean age ± SD: 40.1 years ± 10). The cumulative dose for HCQ ranged from 24 to 2160 g (777 ± 558 g). None of them displayed HCQ toxicity on screening tests. Bivariate analysis showed moderate cone loss with escalating doses of HCQ (linear regression, r² = 0.23, p = 0.018). Cone spacing also increased with increasing cumulative dose (r² = 0.17, p = 0.008). Cone packing remained unchanged (p > 0.05). Multivariate analysis showed that age and cumulative dose were additive and independent factors of cone dropout. CONCLUSIONS: In this pilot study, we observed moderate cone loss as HCQ cumulative doses increased. The early detection of parafoveal cone metric changes may represent the earliest sign of HCQ macular toxicity during screening.