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Wearable haptics serve as an enhanced media to connect humans and VR/robots. The inevitable sweating issue in all wearables creates a bottleneck for wearable haptics, as the sweat/moisture accumulated in the skin/device interface can substantially affect feedback accuracy, comfortability, and create hygienic problems. Nowadays, wearable haptics typically gain performance at the cost of sacrificing the breathability, comfort, and biocompatibility. Here, we developed a fully integrated breathable haptic textile (FIBHT) to solve these trade-off issues, where the FIBHT exhibits high-level integration of 128 pixels over the palm, great stretchability of 400%, and superior permeability of over 657 g/m2/day (moisture) and 40 mm/s (air). It is a stand-alone haptic system totally composed of stretchable, breathable, and bioadhesive materials, which empowers it with precise, sweating/movement-insensitive and dynamic feedback, and makes FIBHT powerful for virtual touching in broad scenarios.

PURPOSE: To determine the reliability of a nine-point summary scale for grading intermediate age-related macular degeneration (AMD) image morphologic features based on the Early Treatment Diabetic Retinopathy Study (ETDRS) grid. METHODS: Two trained graders independently divided spectral domain-optical coherence tomography (SD-OCT) scans into nine subfields and then graded each subfield for the presence of intraretinal hyperreflective foci (HRF), reticular pseudodrusen (RPD), and incomplete or complete retinal pigment epithelium and outer retinal atrophy (iRORA or cRORA). Grading results were assessed by summing the subfield grades into a nine-point summary score and also by using an eye-level binary grade for presence of the finding in any subfield. Gwets first-order agreement coefficient (AC1) was calculated to assess intergrader agreement. RESULTS: Images of 79 eyes from 52 patients were evaluated. Intergrader agreement was higher when the OCT grades were summarized with a nine-point summary score (Gwets AC1 0.92, 0.89, 0.99, and 0.99 for HRF, RPD, iRORA, and cRORA, respectively) compared with the eye-level binary grade (Gwets AC1 0.75, 0.76, 0.97, and 0.96 for HRF, RPD, iRORA, and cRORA, respectively), with significant differences detected for HRF and RPD. CONCLUSIONS: The use of a nine-point summary score showed higher reliability in grading when compared to the binary subfield- and eye-level data, and thus may offer more precise estimation of AMD disease staging. TRANSLATIONAL RELEVANCE: These findings suggest that a nine-point summary score could be a useful means of disease staging by using findings on OCT in clinical studies of AMD.

While neurodegeneration underlies the pathological basis for permanent disability in multiple sclerosis (MS), predictive biomarkers for progression are lacking. Using an animal model of chronic MS, we find that synaptic injury precedes neuronal loss and identify thinning of the inner plexiform layer (IPL) as an early feature of inflammatory demyelination-prior to symptom onset. As neuronal domains are anatomically segregated in the retina and can be monitored longitudinally, we hypothesize that thinning of the IPL could represent a biomarker for progression in MS. Leveraging our dataset with over 800 participants enrolled for more than 12 years, we find that IPL atrophy directly precedes progression and propose that synaptic loss is predictive of functional decline. Using a blood proteome-wide analysis, we demonstrate a strong correlation between demyelination, glial activation, and synapse loss independent of neuroaxonal injury. In summary, monitoring synaptic injury is a biologically relevant approach that reflects a potential driver of progression.

PurposeChoroidal vascular changes occur with normal aging and age-related macular degeneration (AMD). Here, we evaluate choroidal thickness and vascularity in aged rhesus macaques to better understand the choroid's role in this nonhuman primate model of AMD.MethodsWe analyzed optical coherence tomography (OCT) images of 244 eyes from 122 rhesus macaques (aged 4-32 years) to measure choroidal thickness (CT) and choroidal vascularity index (CVI). Drusen number, size, and volume were measured by semiautomated annotation and segmentation of OCT images. We performed regression analyses to determine any association of CT or CVI with age, sex, and axial length and to determine if the presence and volume of soft drusen impacted these choroidal parameters.ResultsIn rhesus macaques, subfoveal CT decreased with age at 3.2 µm/y (R2 = 0.481, P < 0.001), while CVI decreased at 0.66% per year (R2 = 0.257, P < 0.001). Eyes with soft drusen exhibited thicker choroid (179.9 ± 17.5 µm vs. 162.0 ± 27.9 µm, P < 0.001) and higher CVI (0.612 ± 0.051 vs. 0.577 ± 0.093, P = 0.005) than age-matched control animals. Neither CT or CVI appeared to be associated with drusen number, size, or volume in this cohort. However, some drusen in macaques were associated with underlying choroidal vessel enlargement resembling pachydrusen in human patients with AMD.ConclusionsChanges in the choroidal vasculature in rhesus macaques resemble choroidal changes in human aging, but eyes with drusen exhibit choroidal thickening, increased vascularity, and phenotypic characteristics of pachydrusen observed in some patients with AMD.

PURPOSE: The purpose of this study was to determine the impact of prophylactic ranibizumab (PR) injections given every 3 months in eyes with intermediate nonexudative age-related macular degeneration (AMD) on drusen volume, macular layer thicknesses, and progression of geographic atrophy (GA) area over 24 months in the PREVENT trial. METHODS: This post hoc analysis of the prospective PREVENT trial compared eyes with intermediate AMD randomized to PR versus sham injections to determine rates of conversion to neovascular AMD over 24 months. Drusen area and volume, macular thickness and volume, and retinal layer thicknesses were measured on spectral-domain optical coherence tomography images and analyzed. Masked grading of GA area and subretinal drusenoid deposits (SDDs) using fundus autofluorescence images was performed. RESULTS: There were no statistical differences in drusen area and volumes between groups, and similar reductions in central subfield thickness, mean cube thickness, cube volume, and retinal sublayer thickness from baseline to 24 months (P = 0.018 to < 0.001), with no statistical differences between groups in any of these anatomic parameters. These findings were not impacted by the presence or absence of SDD. Among the 9 eyes with GA in this study, mean GA growth rate from baseline to 24 months was 1.34 +/- 0.79 mm2/year after PR and 1.95 +/- 1.73 mm2/year in sham-treated eyes (P = 0.49), and similarly showed no statistical difference with square root transformation (P = 0.61). CONCLUSIONS: Prophylactic ranibizumab given every 3 months did not appear to affect drusen volume, macular thinning, or GA progression in eyes with intermediate AMD. TRANSLATIONAL RELEVANCE: This work investigates the impact of PR on progressive retinal degeneration in a clinical trial.

Lutein, zeaxanthin, and meso-zeaxanthin are three xanthophyll carotenoid pigments that selectively concentrate in the center of the retina. Humans cannot synthesize lutein and zeaxanthin, so these compounds must be obtained from the diet or supplements, with meso-zeaxanthin being converted from lutein in the macula. Xanthophylls are major components of macular pigments that protect the retina through the provision of oxidant defense and filtering of blue light. The accumulation of these three xanthophylls in the central macula can be quantified with non-invasive methods, such as macular pigment optical density (MPOD). MPOD serves as a useful tool for assessing risk for, and progression of, age-related macular degeneration, the third leading cause of blindness worldwide. Dietary surveys suggest that the dietary intakes of lutein and zeaxanthin are decreasing. In addition to low dietary intake, pregnancy and lactation may compromise the lutein and zeaxanthin status of both the mother and infant. Lutein is found in modest amounts in some orange- and yellow-colored vegetables, yellow corn products, and in egg yolks, but rich sources of zeaxanthin are not commonly consumed. Goji berries contain the highest known levels of zeaxanthin of any food, and regular intake of these bright red berries may help protect against the development of age-related macular degeneration through an increase in MPOD. The purpose of this review is to summarize the protective function of macular xanthophylls in the eye, speculate on the compounds' role in maternal and infant health, suggest the establishment of recommended dietary values for lutein and zeaxanthin, and introduce goji berries as a rich food source of zeaxanthin.

ObjectivesTo investigate factors associated with severe COVID-19 in people with psoriasis (PsO), psoriatic arthritis (PsA) and axial spondyloarthritis (axSpA).MethodsDemographic data, clinical characteristics and COVID-19 outcome severity of adults with PsO, PsA and axSpA were obtained from two international physician-reported registries. A three-point ordinal COVID-19 severity scale was defined: no hospitalisation, hospitalisation (and no death) and death. ORs were estimated using multivariable ordinal logistic regression.ResultsOf 5045 cases, 18.3% had PsO, 45.5% PsA and 36.3% axSpA. Most (83.6%) were not hospitalised, 14.6% were hospitalised and 1.8% died. Older age was non-linearly associated with COVID-19 severity. Male sex (OR 1.54, 95% CI 1.30 to 1.83), cardiovascular, respiratory, renal, metabolic and cancer comorbidities (ORs 1.25-2.89), moderate/high disease activity and/or glucocorticoid use (ORs 1.39-2.23, vs remission/low disease activity and no glucocorticoids) were associated with increased odds of severe COVID-19. Later pandemic time periods (ORs 0.42-0.52, vs until 15 June 2020), PsO (OR 0.49, 95% CI 0.37 to 0.65, vs PsA) and baseline exposure to TNFi, IL17i and IL-23i/IL-12+23i (OR 0.57, 95% CI 0.44 to 0.73; OR 0.62, 95% CI 0.45 to 0.87; OR 0.67, 95% CI 0.45 to 0.98; respectively; vs no disease-modifying antirheumatic drug) were associated with reduced odds of severe COVID-19.ConclusionOlder age, male sex, comorbidity burden, higher disease activity and glucocorticoid intake were associated with more severe COVID-19. Later pandemic time periods, PsO and exposure to TNFi, IL17i and IL-23i/IL-12+23i were associated with less severe COVID-19. These findings will enable risk stratification and inform management decisions for patients with PsO, PsA and axSpA during COVID-19 waves or similar future respiratory pandemics.

CD3δ SCID is a devastating inborn error of immunity caused by mutations in CD3D, encoding the invariant CD3δ chain of the CD3/TCR complex necessary for normal thymopoiesis. We demonstrate an adenine base editing (ABE) strategy to restore CD3δ in autologous hematopoietic stem and progenitor cells (HSPCs). Delivery of mRNA encoding a laboratory-evolved ABE and guide RNA into a CD3δ SCID patient's HSPCs resulted in a 71.2% ± 7.85% (n = 3) correction of the pathogenic mutation. Edited HSPCs differentiated in artificial thymic organoids produced mature T cells exhibiting diverse TCR repertoires and TCR-dependent functions. Edited human HSPCs transplanted into immunodeficient mice showed 88% reversion of the CD3D defect in human CD34+ cells isolated from mouse bone marrow after 16 weeks, indicating correction of long-term repopulating HSCs. These findings demonstrate the preclinical efficacy of ABE in HSPCs for the treatment of CD3δ SCID, providing a foundation for the development of a one-time treatment for CD3δ SCID patients.

Introduction: Lower insurance reimbursements have limited the financial sustainability of remote eye screening programs. Greater utilization and insurance coverage for teleophthalmology screening during the coronavirus disease 2019 (COVID-19) pandemic in 2020 may enhance awareness and expand remote retinal imaging services. This retrospective cross-sectional study evaluates utilization and insurance coverage for remote retinal imaging in the United States in 2020. Methods: We analyzed teleretinal imaging utilization and insurance payments from January 1 to December 31, 2020, using the Optum Labs Data Warehouse, a comprehensive national database of deidentified administrative claims for commercial and Medicare Advantage enrollees in the United States. We evaluated frequency of claims and insurance payment for services using the Current Procedural Terminology codes 92227 and 92228 for remote eye imaging by any provider, and 92250 for fundus photography by non-eye care providers. Results: The use of remote retinal imaging in the United States declined rapidly during the initial COVID-19 lockdown from 3,627 claims in February 2020 to 1,414 claims in April 2020, but returned to 3,133 claims by December 2020, similar to mean prepandemic levels in 2019 (2,841 ± 174.8 claims). The proportion of insurance payments for remote imaging increased temporarily from 47.4% in February to 56.7% in April, and then returned to 45.9% in December of 2020. Discussion: Utilization of remote retinal imaging declined steeply, while the insurance coverage increased during the initial COVID-19 lockdown in 2020, but returned to prepandemic levels by end of the year. Changes in utilization and relaxed restrictions on insurance reimbursements for teleophthalmology during the COVID-19 pandemic were not sustained.

Many biomarkers in clinical neuroscience lack pathological certification. This issue is potentially a significant contributor to the limited success of neuroprotective and neurorestorative therapies for human neurological disease-and is evident even in areas with therapeutic promise such as myelin repair. Despite the identification of promising remyelinating candidates, biologically validated methods to demonstrate therapeutic efficacy or provide robust preclinical evidence of remyelination in the CNS are lacking. Therapies with potential to remyelinate the CNS constitute one of the most promising and highly anticipated therapeutic developments in the pipeline to treat multiple sclerosis and other demyelinating diseases. The optic nerve has been proposed as an informative pathway to monitor remyelination in animals and human subjects. Recent clinical trials using visual evoked potential have had promising results, but without unequivocal evidence about the cellular and molecular basis for signal changes on visual evoked potential, the interpretation of these trials is constrained. The visual evoked potential was originally developed and used in the clinic as a diagnostic tool but its use as a quantitative method for assessing therapeutic response requires certification of its biological specificity. Here, using the tools of experimental pathology we demonstrate that quantitative measurements of myelination using both histopathological measures of nodal structure and ultrastructural assessments correspond to visual evoked potential latency in both inflammatory and chemical models of demyelination. Visual evoked potential latency improves after treatment with a tool remyelinating compound (clemastine), mirroring both quantitative and qualitative myelin assessment. Furthermore, clemastine does not improve visual evoked potential latency following demyelinating injury when administered to a transgenic animal incapable of forming new myelin. Therefore, using the capacity for therapeutic enhancement and biological loss of function we demonstrate conclusively that visual evoked potential measures myelin status and is thereby a validated tool for preclinical verification of remyelination.

PurposeTo define the normal range of central corneal thickness (CCT) and corneal endothelial cell density (ECD) in rhesus macaques (Macaca mulatta) and the effects of age, body weight, sex, and intraocular pressure (IOP) on these parameters.MethodsOphthalmic examinations were performed on 144 rhesus macaques without anterior segment pathology. The CCT was measured via ultrasound pachymetry (USP) and specular microscopy, and the ECD was semiautomatically and manually counted using specular microscopy. Rebound tonometry was used to measure IOP. Linear regression and mixed-effects linear regression models were used to evaluate the effects of age, body weight, sex, and IOP on CCT and ECD.ResultsWe included 98 females and 46 males with an age range of 0.2 to 29.4 years. The mean CCT by USP and specular microscopy were 483 ± 39 and 463 ± 33 µm, respectively, and were statistically different (P < 0.001). The ECDs were 2717 ± 423 and 2747 ± 438 cells/mm2 by semiautomated and manual analysis, respectively. Corneal endothelial degeneration was identified in one aged rhesus macaque.ConclusionsThe mean USP and specular microscopy CCT values differed significantly, whereas the semiautomatic and manual ECD did not. The CCT was associated with the IOP and sex, whereas the ECD was associated with body weight and age (P < 0.05). As in humans, corneal disease in rhesus macaques is uncommon.Translational relevanceEstablishing reference values is fundamental to use rhesus macaques as a model for corneal disease or to identify toxicity in studies of ocular drugs or devices.

Astrocytes are vital support cells that ensure proper brain function. In brain disease, astrocytes reprogram into a reactive state that alters many of their cellular roles. A long-standing question in the field is whether downregulation of reactive astrocyte (RA) markers during resolution of inflammation is because these astrocytes revert back to a non-reactive state or die and are replaced. This has proven difficult to answer mainly because existing genetic tools cannot distinguish between healthy versus RAs. Here we describe the generation of an inducible genetic tool that can be used to specifically target and label a subset of RAs. Longitudinal analysis of an acute inflammation model using this tool revealed that the previously observed downregulation of RA markers after inflammation is likely due to changes in gene expression and not because of cell death. Our findings suggest that cellular changes associated with astrogliosis after acute inflammation are largely reversible.

According to a national claims database, remote retinal imaging utilization in the U.S. increased rapidly from 2011 to 2020, but insurance coverage declined, with disproportionate impact on vulnerable populations including older, Black, and lower-income patients.

ObjectiveDiseases such as age-related macular degeneration (AMD) are classified based on human rubrics that are prone to bias. Supervised neural networks trained using human-generated labels require labor-intensive annotations and are restricted to specific trained tasks. Here, we trained a self-supervised deep learning network using unlabeled fundus images, enabling data-driven feature classification of AMD severity and discovery of ocular phenotypes.DesignDevelopment of a self-supervised training pipeline to evaluate fundus photographs from the Age-Related Eye Disease Study (AREDS).ParticipantsOne hundred thousand eight hundred forty-eight human-graded fundus images from 4757 AREDS participants between 55 and 80 years of age.MethodsWe trained a deep neural network with self-supervised Non-Parametric Instance Discrimination (NPID) using AREDS fundus images without labels then evaluated its performance in grading AMD severity using 2-step, 4-step, and 9-step classification schemes using a supervised classifier. We compared balanced and unbalanced accuracies of NPID against supervised-trained networks and ophthalmologists, explored network behavior using hierarchical learning of image subsets and spherical k-means clustering of feature vectors, then searched for ocular features that can be identified without labels.Main outcome measuresAccuracy and kappa statistics.ResultsNPID demonstrated versatility across different AMD classification schemes without re-training and achieved balanced accuracies comparable with those of supervised-trained networks or human ophthalmologists in classifying advanced AMD (82% vs. 81-92% or 89%), referable AMD (87% vs. 90-92% or 96%), or on the 4-step AMD severity scale (65% vs. 63-75% or 67%), despite never directly using these labels during self-supervised feature learning. Drusen area drove network predictions on the 4-step scale, while depigmentation and geographic atrophy (GA) areas correlated with advanced AMD classes. Self-supervised learning revealed grader-mislabeled images and susceptibility of some classes within more granular AMD scales to misclassification by both ophthalmologists and neural networks. Importantly, self-supervised learning enabled data-driven discovery of AMD features such as GA and other ocular phenotypes of the choroid (e.g., tessellated or blonde fundi), vitreous (e.g., asteroid hyalosis), and lens (e.g., nuclear cataracts) that were not predefined by human labels.ConclusionsSelf-supervised learning enables AMD severity grading comparable with that of ophthalmologists and supervised networks, reveals biases of human-defined AMD classification systems, and allows unbiased, data-driven discovery of AMD and non-AMD ocular phenotypes.

Gene therapies aim to deliver a therapeutic payload to specified tissues with underlying protein deficiency. Since the 1990s, gene therapies have been explored as potential treatments for chronic conditions requiring lifetime care and medical management. Ocular gene therapies target a range of ocular disorders, but retinal diseases are of particular importance due to the prevalence of retinal disease and the current treatment burden of such diseases on affected patients, as well as the challenge of properly delivering these therapies to the target tissue. The purpose of this review is to provide an update on the most current data available for five different retinal gene therapies currently undergoing clinical trials for use against age-related macular degeneration (AMD) and the development of novel delivery routes for the administration of such therapies. Research has been performed and compiled from PubMed and the select authors of this manuscript on the treatment and effectiveness of five current retinal gene therapies: Luxturna, ADVM-022, RGX-314, GT-005, and HMR59. We present the available data of current clinical trials for the treatment of neovascular and dry age-related macular degeneration with different AAV-based gene therapies. We also present current research on the progress of developing novel routes of administration for ocular gene therapies. Retinal gene therapies offer the potential for life-changing treatment for chronic conditions like age-related macular degeneration with a single administration. In doing so, gene therapies change the landscape of treatment options for these chronic conditions for both patient and provider.

PurposeTo identify clinical and anatomic factor-associated vision loss in eyes with treatment-naïve diabetic macular edema and good initial visual acuity.MethodsRetrospective cohort study after long-term history of eyes with untreated center-involving diabetic macular edema and baseline visual acuity ≥ 20/25 seen at the University of California, Davis Eye Center between March 2007 and March 2018. We collected characteristics including diabetes type, hemoglobin A1c, presence of visual symptoms, visual acuity, and diabetic retinopathy severity; and spectral-domain optical coherence tomography biomarkers including central subfield thickness, intraretinal cyst size, intraretinal hyperreflective foci, disorganization of retinal inner layers, and outer layer disruptions to determine factors associated with vision loss as defined by DRCR Protocol V as threshold for initiating aflibercept therapy.ResultsFifty-six eyes (48 patients) with untreated diabetic macular edema and mean baseline visual acuity of logMAR 0.05 ± 0.05 (Snellen 20/22) were followed for an average of 5.1 ± 3.3 years, with a median time to vision loss of 465 days (15 months). Older age (hazard ratio [HR] 1.04/year, P = 0.0195) and eyes with severe NPDR (HR 3.0, P = 0.0353) or proliferative diabetic retinopathy (HR 7.7, P = 0.0008) had a higher risk of a vision loss event. None of the spectral-domain optical coherence tomography biomarkers were associated with vision loss except central subfield thickness (HR 0.98, P = 0.0470) and cyst diameter (HR 1.0, P = 0.0094).ConclusionIn eyes with diabetic macular edema and good initial vision, those with older age and worse diabetic retinopathy severity should be monitored closely for prompt treatment initiation when vision loss occurs.

Acetylcholinesterase (EC 3.1.1.7), a key acetylcholine-hydrolyzing enzyme in cholinergic neurotransmission, is present in a variety of states in situ, including monomers, C-terminally disulfide-linked homodimers, homotetramers, and up to three tetramers covalently attached to structural subunits. Could oligomerization that ensures high local concentrations of catalytic sites necessary for efficient neurotransmission be affected by environmental factors? Using small-angle X-ray scattering (SAXS) and cryo-EM, we demonstrate that homodimerization of recombinant monomeric human acetylcholinesterase (hAChE) in solution occurs through a C-terminal four-helix bundle at micromolar concentrations. We show that diethylphosphorylation of the active serine in the catalytic gorge or isopropylmethylphosphonylation by the RP enantiomer of sarin promotes a 10-fold increase in homodimer dissociation. We also demonstrate the dissociation of organophosphate (OP)-conjugated dimers is reversed by structurally diverse oximes 2PAM, HI6, or RS194B, as demonstrated by SAXS of diethylphosphoryl-hAChE. However, binding of oximes to the native ligand-free hAChE, binding of high-affinity reversible ligands, or formation of an SP-sarin-hAChE conjugate had no effect on homodimerization. Dissociation monitored by time-resolved SAXS occurs in milliseconds, consistent with rates of hAChE covalent inhibition. OP-induced dissociation was not observed in the SAXS profiles of the double-mutant Y337A/F338A, where the active center gorge volume is larger than in wildtype hAChE. These observations suggest a key role of the tightly packed acyl pocket in allosterically triggered OP-induced dimer dissociation, with the potential for local reduction of acetylcholine-hydrolytic power in situ. Computational models predict allosteric correlated motions extending from the acyl pocket toward the four-helix bundle dimerization interface 25 Å away.

PurposeThe purpose of this study was to characterize clinician-scientists in ophthalmology and identify factors associated with successful research funding, income, and career satisfaction.DesignCross-sectional study.MethodsA survey was conducted of clinician-scientists in ophthalmology at US academic institutions between April 17, 2019, and May 19, 2019. Collected information including 1) demographic data; 2) amount, type, and source of startup funding; first extramural grant; and first R01-equivalent independent grant; 3) starting and current salaries; and 4) Likert-scale measurements of career satisfaction were analyzed using multivariate regression.ResultsNinety-eight clinician-scientists in ophthalmology were surveyed across different ages (mean: 48 ± 11 years), research categories, institutional types, geographic regions, and academic ranks. Median startup funding ranged from $50-99k, and median starting salaries ranged from $150-199k. A majority of investigators (67%) received their first extramural award from the National Eye Institute, mainly through K-award mechanisms (82%). The median time to receiving their first independent grant was 8 years, mainly through an R01 award (70%). Greater institutional startup support (P = .027) and earlier extramural grant success (P = .022) were associated with earlier independent funding. Male investigators (P = .001) and MD degreed participants (P = .008) were associated with higher current salaries but not starting salaries. Overall career satisfaction increased with career duration (P = .011) but not with earlier independent funding (P = .746) or higher income (P = .300).ConclusionsSuccess in research funding by clinician-scientists in ophthalmology may be linked to institutional support and earlier acquisition of extramural grants but does not impact academic salaries. Nevertheless, career satisfaction among clinician-scientists improves with time, which is not necessarily influenced by research or financial success.

Dopaminergic projections exert widespread influence over multiple brain regions and modulate various behaviors including movement, reward learning, and motivation. It is increasingly appreciated that dopamine neurons are heterogeneous in their gene expression, circuitry, physiology, and function. Current approaches to target dopamine neurons are largely based on single gene drivers, which either label all dopamine neurons or mark a subset but concurrently label non-dopaminergic neurons. Here, we establish a mouse line with Flpo recombinase expressed from the endogenous Slc6a3 (dopamine active transporter [DAT]) locus. DAT-P2A-Flpo mice can be used together with Cre-expressing mouse lines to efficiently and selectively label dopaminergic subpopulations using Cre/Flp-dependent intersectional strategies. We demonstrate the utility of this approach by generating DAT-P2A-Flpo;NEX-Cre mice that specifically label Neurod6-expressing dopamine neurons, which project to the nucleus accumbens medial shell. DAT-P2A-Flpo mice add to a growing toolbox of genetic resources that will help parse the diverse functions mediated by dopaminergic circuits.

The ongoing pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently affecting millions of lives worldwide. Large retrospective studies indicate that an elevated level of inflammatory cytokines and pro-inflammatory factors are associated with both increased disease severity and mortality. Here, using multidimensional epigenetic, transcriptional, in vitro, and in vivo analyses, we report that topoisomerase 1 (TOP1) inhibition suppresses lethal inflammation induced by SARS-CoV-2. Therapeutic treatment with two doses of topotecan (TPT), an FDA-approved TOP1 inhibitor, suppresses infection-induced inflammation in hamsters. TPT treatment as late as 4 days post-infection reduces morbidity and rescues mortality in a transgenic mouse model. These results support the potential of TOP1 inhibition as an effective host-directed therapy against severe SARS-CoV-2 infection. TPT and its derivatives are inexpensive clinical-grade inhibitors available in most countries. Clinical trials are needed to evaluate the efficacy of repurposing TOP1 inhibitors for severe coronavirus disease 2019 (COVID-19) in humans.

PurposeTo use visible light optical coherence tomography (OCT) to investigate subcellular reflectivity contributions to the outermost (4th) of the retinal hyperreflective bands visualized by current clinical near-infrared (NIR) OCT.MethodsVisible light OCT, with 1.0 µm axial resolution, was performed in 28 eyes of 19 human subjects (21-57 years old) without history of ocular pathology. Two foveal and three extrafoveal hyperreflective zones were consistently depicted within band 4 in all eyes. The two outermost hyperreflective bands, occasionally visualized by NIR OCT, were presumed to be the retinal pigment epithelium (RPE) and Bruch's membrane (BM). RPE thickness, BM thickness, and RPE interior reflectivity were quantified topographically across the macula.ResultsA method for correcting RPE multiple scattering tails was found to both improve the Gaussian goodness-of-fit for the BM intensity profile and reduce the coefficient of variation of BM thickness in vivo. No major topographical differences in macular BM thickness were noted. RPE thickness decreased with increasing eccentricity. Visible light OCT signal intensity in the RPE was weighted to the apical side and attenuated more across the RPE in the fovea than peripherally.ConclusionsMorphometry of the presumed RPE and BM bands is consistent with known anatomy. Weighting of RPE reflectivity toward the apical side suggests that melanosomes are the predominant contributors to RPE backscattering and signal attenuation in young eyes.Translational relevanceBy enabling morphometric analysis of the RPE and BM, visible light OCT deciphers the main reflectivity contributions to outer retinal band 4, commonly visualized by commercial OCT systems.

PurposeTo investigate whether intraoperative retinal changes during epiretinal membrane (ERM) peeling affect anatomic or functional outcomes after surgery.MethodsWe measured retinal thickness using an intraoperative optical coherence tomography (iOCT) device in patients undergoing pars plana vitrectomy with membrane peeling for idiopathic ERM. Changes in intraoperative central macular thickness (iCMT) were compared with postoperative improvements in CMT and best-corrected visual acuity (VA).ResultsTwenty-seven eyes from 27 patients (mean age 68 years) underwent iOCT-assisted ERM peeling surgery. Before surgery, mean VA was logMAR 0.50 ± 0.36 (Snellen 20/63), and mean baseline CMT was 489 ± 82 µm. Mean iCMT before peeling was 477 ± 87 µm, which correlated well with preoperative CMT (P < 0.001). Mean change in iCMT was -39.6 ± 37 µm (range -116 to +77 µm). After surgery, VA improved to logMAR 0.40 ± 0.38 (Snellen 20/50) at month 1 and logMAR 0.27 ± 0.23 (Snellen 20/37) at month 3, whereas CMT decreased to 397 ± 44 µm and 396 ± 51 µm at months 1 and 3. Eyes that underwent greater amount of iCMT change (absolute value of iCMT change) were associated with greater CMT reduction at month 1 (P < 0.001) and month 3 (P = 0.010), whereas those with greater intraoperative thinning (actual iCMT change) showed a trend toward better VA outcomes at months 1 (P = 0.054) and 3 (P = 0.036).ConclusionsIntraoperative changes in retinal thickness may predict anatomic and visual outcomes after idiopathic ERM peeling surgery.Translational relevanceOur study suggests that intraoperative retinal tissue response to ERM peeling surgery measured by iOCT may be a prognostic indicator for restoration of retinal architecture and for visual acuity outcomes.

Age-related macular degeneration (AMD) is the third leading cause of blindness worldwide. Macular pigment optical density (MPOD), a biomarker for AMD, is a non-invasive measure to assess risk. The macula xanthophyll pigments lutein (L) and zeaxanthin (Z) protect against blue light and provide oxidant defense, which can be indexed by MPOD. This study examined the effects of Z-rich goji berry intake on MPOD and skin carotenoids in healthy individuals. A randomized, unmasked, parallel-arm study was conducted with 27 participants, aged 45-65, who consumed either 28 g of goji berries or a supplement containing 6 mg L and 4 mg Z (LZ), five times weekly for 90 days. After 90 days, MPOD was significantly increased in the goji berry group at 0.25 and 1.75 retinal eccentricities (p = 0.029 and p = 0.044, respectively), while no changes were noted in the LZ group. Skin carotenoids were significantly increased in the goji berry group at day 45 (p = 0.025) and day 90 (p = 0.006), but not in the LZ group. Regular intake of goji berries in a healthy middle-aged population increases MPOD may help prevent or delay the development of AMD.

Here we provide a counter-example to the conventional wisdom in biomedical optics that longer wavelengths aid deeper imaging in tissue. Specifically, we investigate visible light optical coherence tomography of Bruch's membrane (BM) in the non-pathologic eyes of humans and two mouse strains. Surprisingly, we find that shorter visible wavelengths improve the visualization of BM in pigmented eyes, where it is located behind a highly scattering layer of melanosomes in the retinal pigment epithelium (RPE). Monte Carlo simulations of radiative transport suggest that, while absorption and scattering are higher at shorter wavelengths, detected multiply scattered light from the RPE is preferentially attenuated relative to detected backscattered light from the BM.

A 73-year-old man was displaying symptoms of massive gastrointestinal (GI) bleed. Surgical actions were performed to control the bleed caused by an erosive duodenal ulcer with duodenal perforation. When investigating the culprit of this case, the pain medications prescribed two weeks prior by a traditional Chinese medicine doctor raised attention. The patient's admission serum sample and the pain medications from unknown sources were analyzed using a clinically validated liquid chromatography-high-resolution mass spectrometry (LC-HRMS) method. The NSAIDs diclofenac, piroxicam, and indomethacin were identified, as well as some other synthetic drugs and natural products. The patient's concurrent exposure to multiple NSAIDs significantly increased the risk of upper GI complications. It is reasonable to argue that the high-dose use of the NSAIDs was a major cause of the duodenal ulcer and GI bleed. In addition, the identified natural products such as atropine and ephedrine have well-documented toxicities. It is important to increase the visibility of unregulated medications, and the capability to perform untargeted mass spectrometry analysis provides a unique diagnostic advantage in cases where exposure to toxic substances is possible.

It is well understood that the adaptive immune response to infectious agents includes a modulating suppressive component as well as an activating component. We now show that the very early innate response also has an immunosuppressive component. Infected cells upregulate the CD47 "don't eat me" signal, which slows the phagocytic uptake of dying and viable cells as well as downstream antigen-presenting cell (APC) functions. A CD47 mimic that acts as an essential virulence factor is encoded by all poxviruses, but CD47 expression on infected cells was found to be upregulated even by pathogens, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), that encode no mimic. CD47 upregulation was revealed to be a host response induced by the stimulation of both endosomal and cytosolic pathogen recognition receptors (PRRs). Furthermore, proinflammatory cytokines, including those found in the plasma of hepatitis C patients, upregulated CD47 on uninfected dendritic cells, thereby linking innate modulation with downstream adaptive immune responses. Indeed, results from antibody-mediated CD47 blockade experiments as well as CD47 knockout mice revealed an immunosuppressive role for CD47 during infections with lymphocytic choriomeningitis virus and Mycobacterium tuberculosis Since CD47 blockade operates at the level of pattern recognition receptors rather than at a pathogen or antigen-specific level, these findings identify CD47 as a novel potential immunotherapeutic target for the enhancement of immune responses to a broad range of infectious agents.IMPORTANCE Immune responses to infectious agents are initiated when a pathogen or its components bind to pattern recognition receptors (PRRs). PRR binding sets off a cascade of events that activates immune responses. We now show that, in addition to activating immune responses, PRR signaling also initiates an immunosuppressive response, probably to limit inflammation. The importance of the current findings is that blockade of immunomodulatory signaling, which is mediated by the upregulation of the CD47 molecule, can lead to enhanced immune responses to any pathogen that triggers PRR signaling. Since most or all pathogens trigger PRRs, CD47 blockade could be used to speed up and strengthen both innate and adaptive immune responses when medically indicated. Such immunotherapy could be done without a requirement for knowing the HLA type of the individual, the specific antigens of the pathogen, or, in the case of bacterial infections, the antimicrobial resistance profile.

PurposeTo evaluate the management and long-term outcomes of patients with diabetic macular oedema (DMO) and good initial visual acuity in real-world settings.MethodsWe reviewed 122 eyes of 100 patients with treatment-naive DMO and initial best-corrected visual acuity (BCVA) of 20/25 or better. We assessed clinical characteristics, logMAR BCVA, central subfield thickness (CST), cumulative intravitreal injections and laser treatments at yearly intervals, and characteristics at time of initial treatment. Linear mixed effects models were used to identify predictors of visual outcomes.ResultsAt presentation, mean BCVA was 0.057 ± 0.048 logMAR (Snellen 20/23) and mean CST was 288 ± 57 μm. After a median follow-up of 3 years, 51% of eyes underwent treatment. More eyes underwent intravitreal injection as initial treatment (54%), but lasers were initiated at an earlier time and at better BCVA. Final BCVA was associated with better BCVA (P

PurposeAlthough the choroid contributes to the pathogenesis of age-related macular degeneration (AMD), the role of retinal perfusion is unclear. We sought to compare retinal vascular measurements between eyes with nonexudative and exudative AMD using optical coherence tomography angiography (OCT-A).DesignRetrospective, cross-sectional study.MethodsOCT-A images were analyzed from 310 eyes of 182 patients (mean age ± standard deviation [SD], 78.8 ± 8.8 years) with nonexudative (54.2%) and exudative (45.8%) AMD to measure retinal vessel density (VD) from the superficial capillary plexus in the foveal, parafoveal, and full macular regions and foveal avascular zone (FAZ) area, perimeter, and circularity. Multivariate regressions were used to compare nonexudative and exudative AMD eyes and the impact of anti-vascular endothelial growth factor (anti-VEGF) treatments or geographic atrophy (GA).ResultsIn eyes with AMD, VD decreases with age in the foveal (β = -0.211, P < .001), parafoveal (β = -0.305, P < .001), and full macular regions (β = -0.295, P < .001). Eyes with exudative AMD demonstrated lower VD, especially in the parafoveal (29.8% ± 6.3% vs 33.0% ± 5.7%, P < .001) and full regions (27.9% ± 6.2% vs 31.2% ± 5.5%, P < .001) compared with nonexudative AMD. There were no differences in FAZ area, perimeter, or circularity between the 2 groups (P = .503-.907). In eyes with exudative AMD, previous anti-VEGF treatments did not impact retinal vascular measurements (P = .324-.986). Nonexudative AMD severity and presence of central GA also impacted retinal VD and FAZ morphology.ConclusionsRetinal VD is decreased in eyes with exudative AMD compared with nonexudative AMD but is unaffected by anti-VEGF treatments, suggesting a retinal vascular contribution to the pathogenesis of AMD.

The discovery of drivers of cancer has traditionally focused on protein-coding genes1-4. Here we present analyses of driver point mutations and structural variants in non-coding regions across 2,658 genomes from the Pan-Cancer Analysis of Whole Genomes (PCAWG) Consortium5 of the International Cancer Genome Consortium (ICGC) and The Cancer Genome Atlas (TCGA). For point mutations, we developed a statistically rigorous strategy for combining significance levels from multiple methods of driver discovery that overcomes the limitations of individual methods. For structural variants, we present two methods of driver discovery, and identify regions that are significantly affected by recurrent breakpoints and recurrent somatic juxtapositions. Our analyses confirm previously reported drivers6,7, raise doubts about others and identify novel candidates, including point mutations in the 5' region of TP53, in the 3' untranslated regions of NFKBIZ and TOB1, focal deletions in BRD4 and rearrangements in the loci of AKR1C genes. We show that although point mutations and structural variants that drive cancer are less frequent in non-coding genes and regulatory sequences than in protein-coding genes, additional examples of these drivers will be found as more cancer genomes become available.

Brown adipose tissue (BAT) is highly metabolically active tissue that dissipates energy via UCP1 as heat, and BAT mass is correlated negatively with obesity. The presence of BAT/BAT-like tissue in humans renders BAT as an attractive target against obesity and insulin resistance. Here, we identify Aifm2, a NADH oxidoreductase domain containing flavoprotein, as a lipid droplet (LD)-associated protein highly enriched in BAT. Aifm2 is induced by cold as well as by diet. Upon cold or β-adrenergic stimulation, Aifm2 associates with the outer side of the mitochondrial inner membrane. As a unique BAT-specific first mammalian NDE (external NADH dehydrogenase)-like enzyme, Aifm2 oxidizes NADH to maintain high cytosolic NAD levels in supporting robust glycolysis and to transfer electrons to the electron transport chain (ETC) for fueling thermogenesis. Aifm2 in BAT and subcutaneous white adipose tissue (WAT) promotes oxygen consumption, uncoupled respiration, and heat production during cold- and diet-induced thermogenesis. Aifm2, thus, can ameliorate diet-induced obesity and insulin resistance.

PurposeTo evaluate spectral-domain (SD)-OCT features associated with baseline vision and visual outcomes in the prospective, multicenter Study Evaluating Dosing Regimens for Treatment with Intravitreal Ranibizumab Injections in Subjects with Macular Edema following Retinal Vein Occlusion (SHORE).DesignPost hoc analysis of prospective clinical trial data.ParticipantsTwo hundred two participants in the 15-month, phase 4 SHORE study comparing monthly versus pro re nata ranibizumab after 7 monthly doses in eyes with retinal vein occlusion (RVO) with macular edema.MethodsBaseline SD-OCT images were assessed for (1) central subfield thickness (CST); (2) presence of vitreomacular adhesion, vitreomacular traction, or epiretinal membrane; (3) presence, location, and amount of intraretinal fluid or subretinal fluid (SRF); (4) presence, location, and amount of hyperreflective foci (HF); (5) disorganization of retinal inner layers (DRIL); and (6) disruption of external limiting membrane (ELM), ellipsoid zone (EZ), and interdigitation zone (IZ). Univariate and multivariate regression analyses were performed to evaluate the association of these features with baseline best-corrected visual acuity (BCVA) and change in BCVA after 7 monthly ranibizumab injections.Main outcome measuresAssociation of SD-OCT features with baseline BCVA and change in BCVA after 7 monthly ranibizumab injections.ResultsBefore therapy, worse baseline BCVA was associated with ERM presence (P = 0.0045), thicker SRF (P = 0.0006), larger intraretinal cysts (P = 0.0015), and higher percentage of DRIL (P < 0.0001), percentage of ELM disruption (P < 0.0001), percentage of EZ disruption (P = 0.0003), and percentage of IZ disruption (P = 0.0018). In multivariate models, only percentage of ELM disruption independently impacted baseline BCVA (P < 0.0001). After 7 monthly ranibizumab injections, mean BCVA improved by 18.3±12.6 Early Treatment Diabetic Retinopathy Study letters in treated eyes. The only factors independently associated with BCVA gain after 7 monthly ranibizumab treatments were younger age (P < 0.0001) and worse baseline BCVA (P < 0.0001).ConclusionsAlthough SD-OCT features may be associated with presenting vision in eyes with macular edema and RVO, most eyes treated with ranibizumab achieve substantial vision gains, and only older age and better baseline BCVA limited visual improvements.

No single neuroimaging technique or sequence is capable of reflecting the functional deficits manifest in MS. Given the interest in imaging biomarkers for short- to medium-term studies, we aimed to assess which imaging metrics might best represent functional impairment for monitoring in clinical trials. Given the complexity of functional impairment in MS, however, it is useful to isolate a particular functionally relevant pathway to understand the relationship between imaging and neurological function. We therefore analyzed existing data, combining multiparametric MRI and OCT to describe MS associated visual impairment. We assessed baseline data from fifty MS patients enrolled in ReBUILD, a prospective trial assessing the effect of a remyelinating drug (clemastine). Subjects underwent 3T MRI imaging, including Neurite Orientation Dispersion and Density Imaging (NODDI), myelin content quantification, and retinal imaging, using OCT. Visual function was assessed, using low-contrast letter acuity. MRI and OCT data were studied to model visual function in MS, using a partial, least-squares, regression analysis. Measures of neurodegeneration along the entire visual pathway, described most of the observed variance in visual disability, measured by low contrast letter acuity. In those patients with an identified history of ON, however, putative myelin measures also showed correlation with visual performance. In the absence of clinically identifiable inflammatory episodes, residual disability correlates with neurodegeneration, whereas after an identifiable exacerbation, putative measures of myelin content are additionally informative.

OBJECTIVE:Consumption of excess added sugar in the form of sugar-sweetened beverages (SSBs) contributes to a wide range of health concerns in adolescents. Identification of modifiable determinants of SSB consumption based on behavioral theory may inform development of interventions aimed at reducing SSB consumption. The aim of the current study was to test the efficacy of an extended version of the Health Action Process Approach to predict adolescent SSB consumption. METHODS:Using a prospective design, adolescent students (N = 450) self-reported their outcome expectancies, perceived behavioral control, intentions, habit, action, maintenance, and recovery self-efficacy, action and coping planning, perceived affordability, and past behavior with respect to SSB consumption. One month later, participants self-reported their SSB consumption. RESULTS:A structural equation model revealed significant direct effects of action self-efficacy, outcome expectancies, and perceived behavioral control on intentions to reduce SSB consumption. Significant direct effects of action self-efficacy on maintenance self-efficacy, and maintenance self-efficacy on recovery self-efficacy, were also identified. There were significant direct effects of intentions and maintenance self-efficacy on action and coping planning. Only intentions and perceived affordability had significant direct effects on SSB consumption. There were also indirect effects of outcome expectancy and perceived behavioral control on SSB consumption mediated by intentions. Inclusion of past behavior attenuated model effects, with past behavior the only remaining predictor of SSB consumption. CONCLUSIONS:Findings indicate that adolescent SSB consumption is predicted by intentions and perceived affordability, but effects were extinguished by the inclusion of past behavior. The pervasive effects of past behavior point to the importance of identifying potential mediators of past behavior in future research, and that interventions targeting non-conscious rather than intentional processes may be most effective in reducing SSB consumption.

Structure-guided design of novel pharmacologically active molecules relies at least in part on functionally relevant accuracy of macromolecular structures for template based drug design. Currently, about 95% of all macromolecular X-ray structures available in the PDB (Protein Data Bank) were obtained from diffraction experiments at low, cryogenic temperatures. However, it is known that functionally relevant conformations of both macromolecules and pharmacological ligands can differ at higher, physiological temperatures. We describe in this article development and properties of new human acetylcholinesterase (AChE) crystals of space group P31 and a new unit cell, amenable for room-temperature X-ray diffraction studies. We co-crystallized hAChE in P31 unit cell with the reversible inhibitor 9-aminoacridine that binds at the base of the active center gorge in addition to inhibitors that span the full length of the gorge, donepezil (Aricept, E2020) and AChE specific inhibitor BW284c51. Their new low temperature P31 space group structures appear similar to those previously obtained in the different P3121 unit cell. Successful solution of the new room temperature 3.2 Å resolution structure of BW284c51*hAChE complex from large P31 crystals enables us to proceed with studying room temperature structures of lower affinity complexes, such as oxime reactivators bound to hAChE, where temperature-related conformational diversity could be expected in both oxime and hAChE, which could lead to better informed structure-based design under conditions approaching physiological temperature.

This report describes the first documented case of choriovitreal neovascularization (CVNV) occurring after infectious chorioretinitis, with OCT-angiography (OCT-A) demonstrating flow through the vitreal portion of the lesion, and response to anti-VEGF therapy.

Background/objectivesTo evaluate the long-term progression of idiopathic epiretinal membranes (iERMs) with good baseline visual acuity, and to identify predictors of visual decline.DesignRetrospective case series SUBJECTS METHODS: We reviewed records of 145 eyes with iERM and best-corrected visual acuity (BCVA) of 20/40 or greater at presentation, including BCVA, lens status, and central macular thickness (CMT) at yearly visits; as well as anatomic biomarkers including vitreomacular adhesion, pseudohole, lamellar hole, intraretinal cysts, disorganization of the inner retinal layers (DRIL), and disruption of outer retinal layers. Linear mixed effects and mixed-effects Cox proportional hazards models were used to identify clinical and anatomic predictors of vision change and time to surgery.ResultsAt presentation, mean BCVA was 0.17 ± 0.10 logMAR units (Snellen 20/30) and mean CMT was 353.3 ± 75.4 μm. After a median follow-up of 3.7 years (range 1-7 years), BCVA declined slowly at 0.012 ± 0.003 logMAR units/year, with phakic eyes declining more rapidly than pseudophakic eyes (0.019 ± 0.003 vs. 0.010 ± 0.004 logMAR units/year). Metamorphopsia, phakic lens status, lamellar hole, and inner nuclear layer cysts were associated with faster visual decline. Cumulative rates of progression to surgery were 2.9, 5.6, 12.2, and 21.1% at years 1-4. Visual symptoms, metamorphopsia, greater CMT, and disruption of outer retinal layers were associated with greater hazard for surgery.ConclusionEyes with iERM and visual acuity ≥ 20/40 experience slow visual decline, with 21% of eyes requiring surgery after 4 years. Clinical and anatomic predictors of vision loss may be distinct from factors associated with earlier surgical intervention.

ObjectiveTo evaluate the outcomes of patients undergoing pneumatic retinopexy (PR) performed by vitreoretinal fellows at 6 academic centers in the United States.DesignRetrospective, multicenter, consecutive case series.Participants483 patients with primary retinal detachments who underwent PR by 49 vitreoretinal fellows from 6 U.S. training sites between 2002 and 2016.MethodsWe reviewed medical records of patients and recorded baseline clinical characteristics (age, sex, baseline visual acuity, lens status, presence of lattice degeneration, presence of vitreous hemorrhage, location of retinal breaks, macular status, and size of detachment), visual and anatomic outcomes at 3-months after PR, as well as training level and PR experience of the fellow at the time of the procedure.Main outcome measuresSingle-procedure anatomic success and visual acuity at 3-months follow-up, and association with clinical and training-related factors.ResultsVitreoretinal fellows performed a variable number of PR, with a median of 7 cases per fellow (range 1-24). Single-procedure anatomic success was 66.8%, and mean LogMAR visual outcome was 0.43 (Snellen equivalent 20/54) at 3 months. Factors that were independently associated with single-procedure success include phakic lens status (P = 0.01), smaller size of retinal detachment (P = 0.02), and the fellow's procedure experience (P = 0.01). The only factor associated with worse visual outcome was baseline visual acuity (P < 0.001).ConclusionVitreoretinal fellows perform variably few pneumatic retinopexies but have outcomes comparable to reported rates by experienced specialists. Procedure experience of individual fellows may impact anatomic outcomes.

Inherited retinal degenerations are a common cause of untreatable blindness worldwide, with retinitis pigmentosa and cone dystrophy affecting approximately 1 in 3500 and 1 in 10,000 individuals, respectively. A major limitation to the development of effective therapies is the lack of availability of animal models that fully replicate the human condition. Particularly for cone disorders, rodent, canine, and feline models with no true macula have substantive limitations. By contrast, the cone-rich macula of a nonhuman primate (NHP) closely mirrors that of the human retina. Consequently, well-defined NHP models of heritable retinal diseases, particularly cone disorders that are predictive of human conditions, are necessary to more efficiently advance new therapies for patients. We have identified 4 related NHPs at the California National Primate Research Center with visual impairment and findings from clinical ophthalmic examination, advanced retinal imaging, and electrophysiology consistent with achromatopsia. Genetic sequencing confirmed a homozygous R565Q missense mutation in the catalytic domain of PDE6C, a cone-specific phototransduction enzyme associated with achromatopsia in humans. Biochemical studies demonstrate that the mutant mRNA is translated into a stable protein that displays normal cellular localization but is unable to hydrolyze cyclic GMP (cGMP). This NHP model of a cone disorder will not only serve as a therapeutic testing ground for achromatopsia gene replacement, but also for optimization of gene editing in the macula and of cone cell replacement in general.

In recent years, advances in ocular imaging, drug delivery, and ophthalmic surgery have allowed for better visualization and access to the suprachoroidal space. Although previously considered as only a potential space, the suprachoroidal space may serve as a route for drug delivery to the posterior pole, an egress for glaucoma drainage devices, a location for temporary buckling, and a site for prosthesis implantation. Drugs delivered to the suprachoroidal space may achieve higher concentrations in the retina while minimizing exposure to anterior segment tissues, potentially reducing risks of glaucoma or cataracts. Finally, advanced multimodal imaging now allows not only a better understanding of the physiology of the suprachoroid, but also in vivo monitoring of pathologies and drug delivery to the suprachoroidal space. Here, we discuss the newest developments in the medical and surgical applications of this space with potential.

Age-related macular degeneration (AMD) - the leading cause of vision loss in the elderly - share many risks factors as atherosclerosis, which exhibits loss of vascular compliance resulting from aging and oxidative stress. Here, we attempt to explore choroidal and retinal vascular compliance in patients with AMD by evaluating dynamic vascular changes using live ocular imaging following treatment with oral sildenafil citrate, a phosphodiesterase type 5 (PDE5) inhibitor and potent vasodilator. Enhanced-depth imaging optical coherence tomography (EDI-OCT) and OCT angiography (OCT-A) were performed on 46 eyes of 23 subjects, including 15 patients with non-exudative AMD in one eye and exudative AMD in the fellow eye, and 8 age-matched control subjects. Choroidal thickness, choroidal vascularity, and retinal vessel density were measured across the central macula at 1 and 3 hours after a 100 mg oral dose of sildenafil citrate. Baseline choroidal thickness was 172.1 ± 60.0 μm in non-exudative AMD eyes, 196.4 ± 89.8 μm in exudative AMD eyes, and 207.4 ± 77.7 μm in control eyes, with no difference between the 3 groups (P = 0.116). After sildenafil, choroidal thickness increased by 6.0% to 9.0% at 1 and 3 hours in all groups (P = 0.001-0.014). Eyes from older subjects were associated with choroidal thinning at baseline (P = 0.005) and showed less choroidal expansion at 1 hour and 3 hours after sildenafil (P = 0.001) regardless of AMD status (P = 0.666). The choroidal thickening appeared to be primarily attributed to expansion of the stroma rather than luminal component. Retinal vascular density remained unchanged after sildenafil in all 3 groups (P = 0.281-0.587). Together, our studies suggest that vascular response of the choroid to sildenafil decreases with age, but is not affected by the presence of non-exudative or exudative AMD, providing insight into changes in vessel compliance in aging and AMD.

BACKGROUND: The protection conferred by influenza vaccination is generally thought to last less than a year, necessitating annual revaccination. However, the speed with which influenza vaccine effectiveness might decline during a year is unknown, which is of particular importance for locations with year-round influenza activity. We aimed to assess how influenza vaccine effectiveness changes by time intervals between vaccination and admission to hospital, taking advantage of almost year-round circulation of influenza in Hong Kong. METHODS: In this test-negative case-control study, we analysed vaccine effectiveness in children (aged 6 months to 17 years) who were admitted to hospital in Hong Kong over 5 consecutive years (2012-17). We included those who were admitted to general wards in four public hospitals in Hong Kong with a fever (≥38°C) and any respiratory symptom, such as runny nose, cough, or sore throat. We used direct immunofluorescence assay and reverse transcription PCR to detect influenza virus infection, and recorded childrens influenza immunisation history. We compared characteristics of positive cases and negative controls and examined how vaccine effectiveness changed by time between vaccination and admission to hospital with regression analyses. FINDINGS: Between Sept 1, 2012, and Aug 31, 2017, we enrolled 15 695 children hospitalised for respiratory infections, including 2500 (15·9%) who tested positive for influenza A or B and 13 195 (84·1%) who tested negative. 159 (6·4%) influenza-positive cases and 1445 (11·0%) influenza-negative cases had been vaccinated. Most vaccinations were done by December of each influenza vaccination season. Influenza-related admissions to hospital occurred year-round, with peaks in January through March in most years and a large summer peak in 2016; pooled vaccine effectiveness for children of all ages was 79% (95% CI 42-92) for September to December, 67% (57-74) for January to April, and 43% (25-57) for May to August. Vaccine effectiveness against influenza A or B was estimated as 79% (95% CI 64-88) within 0·5-2 months of vaccination, 60% (46-71) within >2-4 months, 57% (39-70) within >4-6 months, and 45% (22-61) within >6-9 months. In separate analyses by type and subtype, we estimated that vaccine effectiveness declined by 2-5 percentage points per month. INTERPRETATION: Influenza vaccine effectiveness decreased during the 9 months after vaccination in children in Hong Kong. Our findings confirm the importance of annual vaccination in children. Influenza vaccines that provide broader and longer-lasting protection are needed to provide year-round protection in regions with irregular influenza seasonality or lengthy periods of influenza activity. FUNDING: Health and Medical Research Fund, Hong Kong and the Research Grants Council, Hong Kong.

Hippo signaling has been recognized as a key tumor suppressor pathway. Here, we perform a comprehensive molecular characterization of 19 Hippo core genes in 9,125 tumor samples across 33 cancer types using multidimensional "omic" data from The Cancer Genome Atlas. We identify somatic drivers among Hippo genes and the related microRNA (miRNA) regulators, and using functional genomic approaches, we experimentally characterize YAP and TAZ mutation effects and miR-590 and miR-200a regulation for TAZ. Hippo pathway activity is best characterized by a YAP/TAZ transcriptional target signature of 22 genes, which shows robust prognostic power across cancer types. Our elastic-net integrated modeling further reveals cancer-type-specific pathway regulators and associated cancer drivers. Our results highlight the importance of Hippo signaling in squamous cell cancers, characterized by frequent amplification of YAP/TAZ, high expression heterogeneity, and significant prognostic patterns. This study represents a systems-biology approach to characterizing key cancer signaling pathways in the post-genomic era.

PurposeTo determine if different types of retinal fluid in the central macula affect the reproducibility of choroidal thickness (CT) measurements on enhanced depth imaging optical coherence tomography (EDI-OCT).DesignRetrospective reliability analysis.MethodsEDI-OCT images were obtained and the choroidal-scleral junction was analyzed through semiautomated segmentation. CT was measured at the fovea and averaged across the central 3-mm horizontal segment. Demographic data, central macular thickness, and type of fluid present were recorded. Intragrader and intergrader repeatability were assessed using the intraclass correlation coefficient (ICC) and coefficient of repeatability (CR).ResultsOf 124 eyes analyzed, 60 (48.4%) had diabetic macular edema, 32 (25.8%) had neovascular age-related macular degeneration, and 32 (25.8%) had other causes of fluid. Intergrader ICC (CR) was 0.95 (74.1 μm) and 0.96 (63.9 μm) for subfoveal and average CT, respectively. CR was similar across various causes of retinal fluid, but was worst for subretinal fluid compared to intraretinal or sub-retinal pigment epithelial fluid. CR also worsened with increasing choroidal thickness, but was not affected by retinal thickness. Intragrader repeatability was generally greater than intergrader values, and followed the same trend.ConclusionsThe presence of macular fluid reduces CT measurement reproducibility, particularly in eyes with subretinal fluid and greater choroidal thickness. A difference of 74.1 μm in subfoveal CT or 63.9 μm in average CT may be necessary to detect true clinical change in eyes with macular fluid.

We present a new method, OMSV, for accurately and comprehensively identifying structural variations (SVs) from optical maps. OMSV detects both homozygous and heterozygous SVs, SVs of various types and sizes, and SVs with or without creating or destroying restriction sites. We show that OMSV has high sensitivity and specificity, with clear performance gains over the latest method. Applying OMSV to a human cell line, we identified hundreds of SVs >2 kbp, with 68 % of them missed by sequencing-based callers. Independent experimental validation confirmed the high accuracy of these SVs. The OMSV software is available at http://yiplab.cse.cuhk.edu.hk/omsv/ .

PurposeTo evaluate the accuracy and precision of different syringe designs for intravitreal injection of anti-VEGF agents.MethodsVolume output was measured from three syringe designs-1) 1.0 mL tuberculin syringe, 2) 1.0 mL syringe with low dead space plunger, and 3) 0.5 mL low-volume syringe-to deliver 50 µL of bevacizumab, ranibizumab, or aflibercept, each repeated four times by three different physicians for 108 total simulated injections. Volume output was calculated from difference in syringe weight before and after expelling the drug. Accuracy was determined by mean absolute percentage error (MAPE), and precision was measured by coefficient of variation (CV).ResultsVolume output from all three syringes was significantly different from 50 µL, with mean volumes of 58.0 ± 5.7 µL for the tuberculin syringe, 58.0 ± 4.0 µL for the low dead space syringe, and 55.5 ± 5.1 µL for the low-volume syringe (p < 0.00001 for all). The low-volume syringe was the most accurate (MAPE = 12.8 ± 7.8% vs. 17.3 ± 9.3% or 15.9 ± 8.1%), and the low dead space syringe was the most reproducible (CV = 0.068 vs. 0.091 or 0.097). There was no significant difference in volume output between different anti-VEGF agents.ConclusionsIntravitreal injections of anti-VEGF agents using a 1.0 mL tuberculin syringe demonstrate poor accuracy and precision. A lower capacity syringe may improve accuracy, while a low dead space plunger may improve precision.

The last decade has seen a substantial increase in research focused on the identification of blood-based biomarkers that have utility in Alzheimer's disease (AD). Blood-based biomarkers have significant advantages of being time- and cost-efficient as well as reduced invasiveness and increased patient acceptance. Despite these advantages and increased research efforts, the field has been hampered by lack of reproducibility and an unclear path for moving basic discovery toward clinical utilization. Here we reviewed the recent literature on blood-based biomarkers in AD to provide a current state of the art. In addition, a collaborative model is proposed that leverages academic and industry strengths to facilitate the field in moving past discovery only work and toward clinical use. Key resources are provided. This new public-private partnership model is intended to circumvent the traditional handoff model and provide a clear and useful paradigm for the advancement of biomarker science in AD and other neurodegenerative diseases.

PurposeTo compare cross-sectional choroidal morphology in rhesus macaque and human eyes using enhanced-depth imaging optical coherence tomography (EDI-OCT) and histologic analysis.MethodsEnhanced-depth imaging-OCT images from 25 rhesus macaque and 30 human eyes were evaluated for choriocapillaris and choroidal-scleral junction (CSJ) visibility in the central macula based on OCT reflectivity profiles, and compared with age-matched histologic sections. Semiautomated segmentation of the choriocapillaris and CSJ was used to measure choriocapillary and choroidal thickness, respectively. Multivariate regression was performed to determine the association of age, refractive error, and race with choriocapillaris and CSJ visibility.ResultsRhesus macaques exhibit a distinct hyporeflective choriocapillaris layer on EDI-OCT, while the CSJ cannot be visualized. In contrast, humans show variable reflectivities of the choriocapillaris, with a distinct CSJ seen in many subjects. Histologic sections demonstrate large, darkly pigmented melanocytes that are densely distributed in the macaque choroid, while melanocytes in humans are smaller, less pigmented, and variably distributed. Optical coherence tomography reflectivity patterns of the choroid appear to correspond to the density, size, and pigmentation of choroidal melanocytes. Mean choriocapillary thickness was similar between the two species (19.3 ± 3.4 vs. 19.8 ± 3.4 μm, P = 0.615), but choroidal thickness may be lower in macaques than in humans (191.2 ± 43.0 vs. 266.8 ± 78.0 μm, P < 0.001). Racial differences in uveal pigmentation also appear to affect the visibility of the choriocapillaris and CSJ on EDI-OCT.ConclusionsPigmented uveal melanocytes affect choroidal morphology on EDI-OCT in rhesus macaque and human eyes. Racial differences in pigmentation may affect choriocapillaris and CSJ visibility, and may influence the accuracy of choroidal thickness measurements.

PurposeTo employ type II clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 endonuclease to suppress ocular angiogenesis by genomic disruption of VEGF-A in human RPE cells.MethodsCRISPR sequences targeting exon 1 of human VEGF-A were computationally identified based on predicted Cas9 on- and off-target probabilities. Single guide RNA (gRNA) cassettes with these target sequences were cloned into lentiviral vectors encoding the Streptococcuspyogenes Cas9 endonuclease (SpCas9) gene. The lentiviral vectors were used to infect ARPE-19 cells, a human RPE cell line. Frequency of insertion or deletion (indel) mutations was assessed by T7 endonuclease 1 mismatch detection assay; mRNA levels were assessed with quantitative real-time PCR; and VEGF-A protein levels were determined by ELISA. In vitro angiogenesis was measured using an endothelial cell tube formation assay.ResultsFive gRNAs targeting VEGF-A were selected based on the highest predicted on-target probabilities, lowest off-target probabilities, or combined average of both scores. Lentiviral delivery of the top-scoring gRNAs with SpCas9 resulted in indel formation in the VEGF-A gene at frequencies up to 37.0% ± 4.0% with corresponding decreases in secreted VEGF-A protein up to 41.2% ± 7.4% (P < 0.001), and reduction of endothelial tube formation up to 39.4% ± 9.8% (P = 0.02). No significant indel formation in the top three putative off-target sites tested was detected.ConclusionsThe CRISPR-Cas9 endonuclease system may reduce VEGF-A secretion from human RPE cells and suppress angiogenesis, supporting the possibility of employing gene editing for antiangiogenesis therapy in ocular diseases.

PurposeTo assess the reliability of manual choroidal thickness measurements by comparing different posterior boundary definitions of the choroidal-scleral junction on enhanced depth imaging optical coherence tomography (EDI-OCT).DesignReliability analysis.MethodsTwo graders marked the choroidal-scleral junction with segmentation software using different posterior boundaries: (1) the outer border of the choroidal vessel lumen, (2) the outer border of the choroid stroma, and (3) the inner border of the sclera, to measure the vascular choroidal thickness (VCT), stromal choroidal thickness (SCT), and total choroidal thickness (TCT), respectively. Measurements were taken at 0.5-mm intervals from 1.5 mm nasal to 1.5 mm temporal to the fovea, and averaged continuously across the central 3 mm of the macula. Intraclass correlation coefficient (ICC) and coefficient of reliability (CR) were compared to assess intergrader and intragrader reliability.ResultsChoroidal thickness measurements varied significantly with different posterior boundaries (P < .001 for all). Intergrader ICCs were greater for SCT (0.959-0.980) than for TCT (0.928-0.963) and VCT (0.750-0.869), even in eyes where choroidal-scleral junction visibility was

Study objectiveThe emergency department (ED) is an inherently high-risk setting. Our objective is to identify the factors associated with the combined poor outcome of either death or an ICU admission shortly after ED discharge in older adults.MethodsWe conducted chart review of 600 ED visit records among adults older than 65 years that resulted in discharge from any of 13 hospitals within an integrated health system in 2009 to 2010. We randomly chose 300 patients who experienced the combined outcome within 7 days of discharge and matched case patients to controls who did not experience the outcome. Two emergency physicians blinded to the outcome reviewed the records and identified whether a number of characteristics were present. Predictors of the outcome were identified with conditional logistic regression.ResultsOf 1,442,594 ED visits to Kaiser Permanente Southern California in 2009 to 2010, 300 unique cases and 300 unique control records were randomly abstracted. Characteristics associated with the combined poor outcome included cognitive impairment (adjusted odds ratio [AOR] 2.10; 95% confidence interval [CI] 1.19 to 3.56), disposition plan change (AOR 2.71; 95% CI 1.50 to 4.89), systolic blood pressure less than 120 mm Hg (AOR 1.48; 95% CI 1.00 to 2.20), and pulse rate greater than 90 beats/min (AOR 1.66; 95% CI 1.02 to 2.71).ConclusionWe found that older patients discharged from the ED with a change in disposition from "admit" to "discharge," cognitive impairment, systolic blood pressure less than 120 mm Hg, and pulse rate greater than 90 beats/min were at increased risk of death or ICU admission shortly after discharge. Increased awareness of these high-risk characteristics may improve ED disposition decisionmaking.