Melanopsin, an atypical vertebrate visual pigment, mediates non-image forming light responses including circadian photoentrainment and pupillary light reflexes, and contrast detection for image formation. Melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs), are characterized by sluggish activation and deactivation of their light responses. The molecular determinants of mouse melanopsin's deactivation have been characterized (i.e. C-terminal phosphorylation and βarrestin binding), but a detailed analysis of melanopsin's activation is lacking. We propose that an extended 3 rd cytoplasmic loop is adjacent to the proximal C-terminal region of mouse melanopsin in the inactive conformation which is stabilized by ionic interaction of these two regions. This model is supported by site-directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy of melanopsin, the results of which suggests a high degree of steric freedom at the 3 rd cytoplasmic loop, which is increased upon C-terminus truncation, supporting the idea that these two regions are close in 3dimensional space in wild-type melanopsin. To test for a functionally critical C-terminal conformation, calcium imaging of melanopsin mutants including a proximal C-terminus truncation (at residue 365) and proline mutation of this proximal region (H377P, L380P, Y382P) delayed melanopsin's activation rate.Mutation of all potential phosphorylation sites, including a highly conserved tyrosine residue (Y382), into alanines also delayed the activation rate. A comparison of mouse melanopsin with armadillo melanopsin-which has substitutions of various potential phosphorylation sites and a substitution of the conserved tyrosine-indicates that substitution of these potential phosphorylation sites and the tyrosine residue result in dramatically slower activation kinetics, a finding that also supports the role of phosphorylation in signaling activation. We therefore propose that melanopsin's C-terminus is proximal to intracellular loop 3 and C-terminal phosphorylation permits the ionic interaction between these two regions, thus forming a stable structural conformation that is critical for initiating G-protein signaling.

Background The effects of vitamin C on clinical outcomes in critically ill patients remain controversial due to inconclusive studies. This retrospective observational cohort study evaluated the effects of vitamin C therapy on acute kidney injury (AKI) and mortality among septic patients. Methods Electronic medical records of 1390 patients from an academic hospital who were categorized as Treatment (received at least one dose of 1.5 g IV vitamin C, n = 212) or Comparison (received no, or less than 1.5 g IV vitamin C, n = 1178) were reviewed. Propensity score matching was conducted to balance a number of covariates between groups. Multivariate logistic regressions were conducted predicting AKI and in-hospital mortality among the full sample and a sub-sample of patients seen in the ICU. Results Data revealed that vitamin C therapy was associated with increases in AKI (OR = 2.07 95% CI [1.46–2.93]) and in-hospital mortality (OR = 1.67 95% CI [1.003–2.78]) after adjusting for demographic and clinical covariates. When stratified to examine ICU patients, vitamin C therapy remained a significant risk factor of AKI (OR = 1.61 95% CI [1.09–2.39]) and provided no protective benefit against mortality (OR = 0.79 95% CI [0.48–1.31]). Conclusion Ongoing use of high dose vitamin C in sepsis should be appraised due to observed associations with AKI and death.

27Melanopsin, an atypical vertebrate visual pigment, mediates non-image forming light responses 28 including circadian photoentrainment and pupillary light reflexes, and contrast detection for image 29formation. Melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs), are 30 characterized by sluggish activation and deactivation of their light responses. The molecular determinants 31 of mouse melanopsin's deactivation have been characterized (i.e. C-terminal phosphorylation and β-32 arrestin binding), but a detailed analysis of melanopsin's activation is lacking. We propose that an 33 extended 3 rd cytoplasmic loop is adjacent to the proximal C-terminal region of mouse melanopsin in the 34 inactive conformation which is stabilized by ionic interaction of these two regions. This model is 35supported by site-directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy of 36 melanopsin, the results of which suggests a high degree of steric freedom at the 3 rd cytoplasmic loop, 37which is increased upon C-terminus truncation, supporting the idea that these two regions are close in 3-38 dimensional space in wild-type melanopsin. To test for a functionally critical C-terminal conformation, 39 calcium imaging of melanopsin mutants including a proximal C-terminus truncation (at residue 365) and 40 proline mutation of this proximal region (H377P, L380P, Y382P) delayed melanopsin's activation rate. 41Mutation of all potential phosphorylation sites, including a highly conserved tyrosine residue (Y382), into 42 alanines also delayed the activation rate. A comparison of mouse melanopsin with armadillo 43 melanopsin-which has substitutions of various potential phosphorylation sites and a substitution of the 44 conserved tyrosine-indicates that substitution of these potential phosphorylation sites and the tyrosine 45 residue result in dramatically slower activation kinetics, a finding that also supports the role of 46 phosphorylation in signaling activation. We therefore propose that melanopsin's C-terminus is proximal 47 to intracellular loop 3 and C-terminal phosphorylation permits the ionic interaction between these two 48 regions, thus forming a stable structural conformation that is critical for initiating G-protein signaling. 49 50 STATEMENT OF SIGNIFICANCE 51 Melanopsin Structure and Activation 3 Melanopsin is an important visual pigment in the mammalian retina that mediates non-image 52forming responses such as circadian photoentrainment and pupil constriction, and supports contrast 53 detection for image formation. In this study, we detail two critical structural features of mouse 54 melanopsin-its 3 rd cytoplasmic loop and C-terminus-that are important in the activation of 55 melanopsin's light responses. Furthermore, we propose that these two regions directly participate in 56 coupling mouse melanopsin to its G-protein. These findings contribute to further understanding of GPCR-57 G-protein coupling, and given recent findings suggesting flexibility of melanopsin signal trans...

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