BEST1 encodes Bestrophin-1 (Best1), a homo-oligomeric, integral membrane protein localized to the basolateral plasma membrane of the retinal pigment epithelium. Mutations in BEST1 cause five distinct retinal degenerative diseases, including adult vitelliform macular dystrophy (AVMD), autosomal recessive bestrophinopathy (ARB), autosomal dominant vitreoretinochoroidopathy (ADVIRC), and retinitis pigmentosa (RP). The mechanisms underlying these diseases and why mutations cause one disease over another are, for the most part, unknown. To gain insights into these four diseases, we expressed 28 Best1 mutants fused to YFP in polarized MDCK monolayers and, via confocal microscopy and immunofluorescence, live-cell FRET, and reciprocal co-immunoprecipitation experiments, screened these mutants for defects in localization and oligomerization. All 28 mutants exhibited comparable FRET efficiencies to and co-immunoprecipitated with WT Best1, indicating unimpaired oligomerization. RP- and ADVIRC-associated mutants were properly localized to the basolateral plasma membrane of cells, while two AVMD and most ARB mutants were mislocalized. When co-expressed, all mislocalized mutants caused mislocalization of WT Best1to intracellular compartments. Our current and past results indicate that mislocalization of Best1 is not an absolute feature of any individual bestrophinopathy, occurring in AVMD, BVMD, and ARB. Furthermore, some ARB mutants that do not also cause dominant disease cause mislocalization of Best1, indicating that mislocalization is not a cause of disease, and that absence of Best1 activity from the plasma membrane is tolerated. Lastly, we find that the ARB truncation mutants L174Qfs*57 and R200X can form oligomers with WT Best1, indicating that the first ~174 amino acids of Best1 are sufficient for oligomerization to occur.

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Background Light's criteria are ratios of pleural fluid to serum total protein (TP), pleural fluid to serum lactate dehydrogenase (LDH) and pleural fluid LDH to the upper reference limit for serum LDH. They are used to classify pleural effusions into an exudate or transudate when pleural fluid protein is 25-35 g/L. We evaluated the impact of between analytical platforms on the classification of pleural effusions using Light's criteria. Methods Light's criteria were used to classify pleural effusions with fluid TP between 25 and 35 g/L into exudate and transudate. LDH and TP were analysed using an Abbott ARCHITECT c16000 analyser using a lactate to pyruvate method for LDH and two Roche Cobas 800 c702 analysers, one using a lactate to pyruvate method (laboratory B) and one a lactate to pyruvate method (laboratory C). Results Eighty-three paired serum and pleural fluid samples were analysed. Of these, 44 samples had a pleural fluid TP between 25 and 35 g/L and were classified according to Light's criteria. Classification of pleural fluid into transudate or exudate using different analytical platforms was 82% concordant. The LDH ratio and TP ratio were similar in laboratory B and laboratory C, but these were respectively lower ( p<0.001) and higher ( p<0.001) than those at laboratory A. Conclusions Although Light's criteria are ratios, which should minimise interassay variability, we report 18% discordance between different analytical platforms. The discordance was largely due to the performance of LDH and to a lesser extent protein assays in pleural fluid. Laboratories should be aware that assays may perform differently in serum and pleural fluid.

Background In acutely ill patients with new onset hyperglycaemia, plasma glucose cannot reliably distinguish between stress hyperglycaemia and undiagnosed diabetes mellitus. We, therefore, investigated the diagnostic reliability of glycated haemoglobin (HbA1c) in acute illness by prospectively evaluating the effect of the systemic inflammatory response, as provoked by elective orthopaedic surgery, on HbA. Methods HbA and serum C-reactive protein concentrations were compared before and two days after elective knee or hip surgery in 30 patients without diabetes. C-reactive protein was used to assess the systemic inflammatory response. Results The mean (standard deviation) serum C-reactive protein increased following surgery (4.8 [7.5] vs. 179.7 [61.9] mg/L; P<0.0001). HbA was similar before and after surgery (39.2 [5.4] vs. 38.1 [5.1] mmol/moL, respectively; P = 0.4363). Conclusions HbA is unaffected within two days of a systemic inflammatory response as provoked by elective orthopaedic surgery. This suggests that HbA may be able to differentiate newly presenting type 2 diabetes mellitus from stress hyperglycaemia in acutely ill patients with new onset hyperglycaemia.

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