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Dataset Inducible RPE-specific GPX4 knockout causes oxidative stress and retinal degeneration with features of age-related macular degenerationWojciechowski, Alaina M; Bell, Brent A; Song, Ying; Anderson, Brandon D; Conomikes, Alexa; Petruconis, Cecilia; Dunaief, Joshua LAge-related macular degeneration (AMD) is one of the leading causes of vision loss in the elderly. This disease involves oxidative stress burden in the retina leading to death of retinal pigment epithelial (RPE) cells and photoreceptors. The retina is susceptible to oxidative stress, in part due to high metabolic activity and high concentration of polyunsaturated fatty acids that undergo lipid peroxidation chain reactions. Antioxidant enzymes exist in the retina to combat this stress, including glutathione peroxidase 4 (GPX4). GPX4 specifically reduces oxidized lipids, protecting against lipid peroxidation-induced oxidative stress, which is noted in dry AMD. We hypothesize that Gpx4 knockout within the RPE will result in an environment of chronic oxidative stress yielding degeneration akin to AMD. C57BL/6J mice with a floxed Gpx4 gene were mated with Rpe65Cre/ER mice. Offspring containing Rpe65Cre ± alleles and either Gpx4 WT or Gpx4 fl/fl alleles were administered tamoxifen to induce Gpx4 knockout in Gpx4 fl/fl mice. At sequential timepoints, retinal phenotypes were assessed via in vivo imaging utilizing confocal scanning laser ophthalmoscopy and optical coherence tomography (OCT), and visual function was probed by electroretinography. Retinas were studied post-mortem by immunohistochemical analyses, electron microscopy, plastic sectioning, and quantitative polymerase chain reaction and Western analyses. The RPE-specific Gpx4 knockout model was validated via Western analysis indicating diminished GPX4 protein only within the RPE and not the neural retina. Following Gpx4 knockout, RPE cells became dysfunctional and died, with significant cell loss occurring 2 weeks post-knockout. Progressive thinning of the photoreceptor layer followed RPE degeneration and was accompanied by loss of visual function. OCT and light microscopy showed hyperreflective foci and enlarged, pigmented cells in and above the RPE layer. Electron microscopy revealed decreased mitochondrial cristae and loss of basal and apical RPE ultrastructure. Finally, there was increased carboxyethylpyrrole staining, indicating oxidation of docosahexaenoic acid, and increased levels of mRNAs encoding oxidative stress-associated genes in the RPE and photoreceptors. Overall, we show that RPE-localized GPX4 is necessary for the health of the RPE and outer retina, and that knockout recapitulates phenotypes of dry AMD.Dataset Disorder Enhances the Fracture Toughness of Two-Dimensional Mechanical Metamaterials(2025-01-20) Fulco, SageMechanical metamaterials with engineered failure properties typically rely on periodic unit cell geometries or bespoke microstructures to achieve their unique properties. We demonstrate that intelligent use of disorder in metamaterials leads to distributed damage during failure, resulting in enhanced fracture toughness with minimal losses of strength. Toughness depends on the level of disorder, not a specific geometry, and the confined lattices studied exhibit a maximum toughness enhancement at an optimal level of disorder. A mechanics model that relates disorder to toughness without knowledge of the crack path is presented. The model is verified through finite element simulations and experiments utilizing photoelasticity to visualize damage during failure. At the optimal level of disorder, the toughness is more than 2.6 times of an ordered lattice of equivalent density.Dataset Supplementary Figures for "A mechanism of CALHM1 channel gating"James Kevin Foskett; Z. MaThe Calcium Homeostasis Modulator (CALHM) proteins comprise a family of six genes, some of which have been demonstrated to function as ion channels. CALHM1, the founding member, is an extracellular Ca2+- and voltage-gated large-pore non-selective ion channel. The mechanisms by which Ca2+ and voltage regulate CALHM1 channel gating are unknown. Cryo-electron microscopic structures of CALHM1 and its paralogs have provided little insights into these features, although they have suggested that the amino-termini, including an amino-terminal helix (NTH) and the first transmembrane helix (TM1) may possess significant flexibility. Here we investigated the role of the amino terminus in gating regulation of human CALHM1 channels expressed in Xenopus oocytes. Deletion of the NTH and the proximal end of TM1 markedly reduced the voltage-dependence of channel gating, whereas extracellular Ca2+ retained ability to close the channel, indicating that the amino-terminus is not the Ca2+-regulated gate. Furthermore, inhibition of channel currents by ruthenium red was independent of the presence of the amino terminus and was mediated by effects on channel gating rather than pore block. Introduction of a cysteine residue into the proximal end of TM1 enabled complete inhibition of the channel by a crosslinking reagent under conditions in which the channel was in a closed state. Our findings indicate that while the NTH plays a role in voltage-dependent gating, it does not act as the gate itself. Instead, our results suggest that the gate in CALHM1 is formed by proximal regions of the first transmembrane domain.Dataset A mechanism of CALHM1 ion channel gating(2024-12-03) James Kevin FoskettThe Calcium Homeostasis Modulator (CALHM) proteins comprise a family of six genes, some of which have been demonstrated to function as ion channels. CALHM1, the founding member, is an extracellular Ca2+- and voltage-gated large-pore ion channel. The mechanisms by which Ca2+ and voltage regulate CALHM1 channel gating are unknown. Cryo-electron microscopic structures of CALHM1 and its paralogs have provided little insights into these features, although they have suggested that the amino-termini, including an amino-terminal helix (NTH) and the first transmembrane helix (TM1) may possess significant flexibility. Here we investigated the role of the amino terminus in gating regulation of human CALHM1 channels expressed in Xenopus oocytes. Deletion of the NTH and the proximal end of TM1 reduced the voltage-dependence of channel gating, whereas extracellular Ca2+ retained ability to close the channel, indicating that the amino-terminus is not the Ca2+-regulated gate. Furthermore, inhibition of channel currents by ruthenium red was independent of the presence of the amino terminus and was mediated by effects on channel gating rather than pore block. Introduction of a cysteine residue into the proximal end of TM1 enabled complete inhibition of the channel by a crosslinking reagent under conditions in which the channel was in a closed state. Our findings indicate that while the NTH plays a role in voltage-dependent gating, it does not act as the gate itself. Instead, our results suggest that the gate in CALHM1 is most likely formed by proximal regions of the first transmembrane domain.Dataset Evaluation of digital radiographic measurements for the diagnosis of acute laminitis(2024-05-14) Andrew van Eps; Georgia Skelton; Darko Stefanovski; Elizabeth AcuttBackground: Traditional radiographic measurements of distal phalanx (Pd) displacement are not useful for diagnosis and monitoring of acute laminitis. Objectives: To compare LLZ between healthy horses and those with acute or subacute laminitis. We hypothesized that the distance between the inner hoof wall and Pd (“lamellar lucent zone”; LLZ) measured on lateromedial digital radiographs would be increased in acute and subacute laminitis. Study design: Retrospective and in vivo experimental study. Methods: Forelimb radiographs from 32 healthy and 18 laminitic mixed-breed horses were analyzed retrospectively. Laminitis was defined by at least 2 clinical signs (acute multi-limb lameness, increased digital pulse amplitude and/or persistently warm hooves) for ≤ 3 days (acute), or >3 and ≤14 days (subacute) duration, without overt palmar rotation (≥ 3°), remodeling, or distal displacement of the Pd. Twelve measurements including LLZ at proximal, middle, and distal locations were performed on 126 forelimb radiographs that met the criteria for acute laminitis (n=39), sub-acute laminitis (n=23) or healthy control (n=64). A mixed effects linear regression model was used for comparisons and a receiver-operator characteristic (ROC) curve created for select measurements. Results: The marginal mean [95% confidence interval] LLZ (mm) was increased in acute and subacute laminitis compared to control in the proximal (acute=8.8 [8.4-9.2]; subacute=9 [8.4-9.5]; control=7.3 [6.9-7.7]), middle (acute=8.9 [8.5-9.3] ; subacute=9.1 [8.6-9.6]; control=6.9 [6.5-7.2]), and distal (acute=9.2 [8.6-9.7]; subacute=10.2 [9.5-10.9]; control=7.5 [7-8]) dorsal lamellar regions (P<0.001). At a cut-off of >7.5 mm for middle LLZ, sensitivity was 87% and specificity 91% for diagnosis of acute laminitis. Using the ratio of middle LLZ to distal phalanx cortical length, sensitivity and specificity were 95% (cutoff >0.11). Main Limitations: Lack of histological confirmation of acute laminitis. Conclusions and clinical importance: LLZ measurements are potentially useful for radiographic diagnosis of acute and subacute laminitis.