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Mutations in ABCA4 are the most common cause of Mendelian retinal disease. Clinical evaluation of this gene is challenging because of its extreme allelic diversity, the large fraction of non-exomic mutations, and the wide range of associated disease. We used patient-derived retinal organoids as well as DNA samples and clinical data from a large cohort of patients with ABCA4-associated retinal disease to investigate the pathogenicity of a variant in ABCA4 (IVS30 + 1321 A>G) that occurs heterozygously in 2% of Europeans. We found that this variant causes mis-splicing of the gene in photoreceptor cells such that the resulting protein contains 36 incorrect amino acids followed by a premature stop. We also investigated the phenotype of 10 patients with compound genotypes that included this mutation. Their median age of first vision loss was 39 years, which is in the mildest quintile of a large cohort of patients with ABCA4 disease. We conclude that the IVS30 + 1321 A>G variant can cause disease when paired with a sufficiently deleterious opposing allele in a sufficiently permissive genetic background., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

This study uses Texas's 2017 integration of the state disability and mental health agencies as a case study, combining interviews with Texas agency and advocacy organization leaders to examine perceptions of agency integration and augmented synthetic control analyses of 2014-2020 Medical Expenditure Panel Survey to examine impacts on mental health service use among individuals with co-occurring cognitive disabilities (including intellectual and developmental disabilities) and mental health conditions. Interviewees described the intensive process of agency integration and identified primarily positive (e.g., decreased administrative burden) impacts of integration. Quantitative analyses indicated no effects of integration on receipt of mental health-related services among people with co-occurring conditions. While leaders identified some potentially beneficial impacts of state agency integration, the limited impact of integration beyond the agency suggests that interventions at multiple levels of the service system, including those targeting providers, are needed to better meet the mental health service needs for this population., (© 2024. The Author(s).)

Objective: The authors aimed to identify barriers to and strategies for supporting coordination between state agencies for intellectual and developmental disability (IDD) or mental health to meet the mental health needs of people with co-occurring IDD and mental health conditions., Methods: Forty-nine employees of state agencies as well as advocacy and service delivery organizations across 11 U.S. states with separate IDD and mental health agencies were interviewed between April 2022 and April 2023. Data were analyzed with a thematic analysis approach., Results: Interviewees reported that relationships between the IDD and mental health agencies have elements of both competition and coordination and that coordination primarily takes place in response to crisis events. Barriers to interagency coordination included a narrow focus on the populations targeted by each agency, within-state variation in agency structures, and a lack of knowledge about co-occurring IDD and mental health conditions. Interviewees also described both administrative (e.g., memorandums of understanding) and agency culture (e.g., focusing on whole-person care) strategies that are or could be used to improve coordination to provide mental health services for people with both IDD and a mental health condition., Conclusions: Strategies that support state agencies in moving away from crisis response toward a focus on whole-person care should be prioritized to support coordination of mental health services for individuals with co-occurring IDD and mental health conditions., Competing Interests: The authors report no financial relationships with commercial interests.

Purpose: To investigate the distribution of genotypes and natural history of ABCA4-associated retinal disease in a large cohort of patients seen at a single institution., Design: Retrospective, single-institution cohort review., Participants: Patients seen at the University of Iowa between November 1986 and August 2022 clinically suspected to have disease caused by sequence variations in ABCA4., Methods: DNA samples from participants were subjected to a tiered testing strategy progressing from allele-specific screening to whole genome sequencing. Charts were reviewed, and clinical data were tabulated. The pathogenic severity of the most common alleles was estimated by studying groups of patients who shared 1 allele. Groups of patients with shared genotypes were reviewed for evidence of modifying factor effects., Main Outcome Measures: Age at first uncorrectable vision loss, best-corrected visual acuity, and the area of the I2e isopter of the Goldmann visual field., Results: A total of 460 patients from 390 families demonstrated convincing clinical features of ABCA4-associated retinal disease. Complete genotypes were identified in 399 patients, and partial genotypes were identified in 61. The median age at first vision loss was 16 years (range, 4-76 years). Two hundred sixty-five families (68%) harbored a unique genotype, and no more than 10 patients shared any single genotype. Review of the patients with shared genotypes revealed evidence of modifying factors that in several cases resulted in a > 15-year difference in age at first vision loss. Two hundred forty-one different alleles were identified among the members of this cohort, and 161 of these (67%) were found in only a single individual., Conclusions: ABCA4-associated retinal disease ranges from a very severe photoreceptor disease with an onset before 5 years of age to a late-onset retinal pigment epithelium-based condition resembling pattern dystrophy. Modifying factors frequently impact the ABCA4 disease phenotype to a degree that is similar in magnitude to the detectable ABCA4 alleles themselves. It is likely that most patients in any cohort will harbor a unique genotype. The latter observations taken together suggest that patients' clinical findings in most cases will be more useful for predicting their clinical course than their genotype., Financial Disclosure(s): Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article., (Copyright © 2024 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.)

Background: There are many surgical techniques to treat chronic subdural hematomas (CSHs). However, they all have high recurrence rates. Recently, embolization of the middle meningeal artery (MMA) following surgical evacuation of CSH has reduced the recurrence rate. We investigated the feasibility of combining the surgical obliteration of the MMA at the same time as the placement of a burr hole for evacuation of the CSH., Case Description: We report on nine patients who underwent 11 of these combined procedure by the same surgeon in two hospitals, including clinical data and images during the perioperative and postoperative periods. Cardoso had previously reported details of the surgical technique. Two patients underwent bilateral procedures. Two patients had two burr holes because the hematomas did not extend caudally to the pterion, where the MMA enters the calvarium. Intraoperative fluoroscopy was used to locate the point of entry of the MMA into the calvarium in most cases, except in two instances when navigation was utilized., Conclusion: This small series of nine cases suggests the feasibility of using this combined procedure as an additional option to the treatment of CSHs, especially where endovascular treatment might not be readily available. Furthermore, it has the potential advantages of safety, efficacy, avoidance of a second endovascular procedure, faster disappearance of the subdural collection, lesser exposure to radiation, and cost containment. Larger prospective controlled series are needed to identify its potential usefulness., Competing Interests: There are no conflicts of interest., (Copyright: © 2024 Surgical Neurology International.)

Purpose: Autoimmune retinopathy (AIR) is a poorly characterized disease with a wide phenotypic spectrum, complicating investigations of its underlying pathophysiology. We sought to analyze optical coherence tomography (OCT) retinal thickness changes in AIR patients., Methods: A retrospective chart review from 2007 to 2017 was performed evaluating AIR patients at a single academic, tertiary referral center. OCT retinal sublayer analysis was performed, and paradoxical thickening phenotypes were reviewed., Results: Twenty-nine AIR patients with positive anti-retinal antibodies and OCT imaging were identified. Overall, AIR patients had thinner retinal sublayers compared to controls; however, 12 patients (41.4%) had paradoxical thickening of the outer plexiform layer (OPL). This revealed two distinct OCT phenotypes. No association was found between retinal sublayer thickness and specific antiretinal antibodies., Conclusions: While the pathogenicity of antiretinal antibodies remains unclear, the OCT phenotypes observed underscore the potential for identifying clues in the underlying disease processes and clinical diagnosis.

Changes in chronic noncancer pain treatment have led to decreases in prescribing of opioids and increases in the availability of medical cannabis, despite its federal prohibition. Patients may face barriers to establishing new care with a physician based on use of these treatments. We compared physician willingness to accept patients based on prescription opioid, cannabis, or other pain treatment use. This study of 36 states and Washington, DC, with active medical cannabis programs surveyed physicians who treat patients with chronic noncancer pain between July 13 and August 4, 2023. Of 1000 physician respondents (34.5% female, 63.2% White, 78.1% primary care), 852 reported accepting new patients with chronic pain. Among those accepting new patients with chronic pain, more physicians reported that they would not accept new patients taking prescription opioids (20.0%) or cannabis (12.7%) than those taking nonopioid prescription analgesics (0.1%). In contrast, 68.1% reported willingness to accept new patients using prescribed opioids on a daily basis. For cannabis, physicians were more likely to accept new patients accessing cannabis through medical programs (81.6%) than from other sources (60.2%). Access to care for persons with chronic noncancer pain appears to be the most restricted among those taking prescription opioids, although patients taking cannabis may also encounter reduced access., Competing Interests: Conflicts of interest Please see ICMJE form(s) for author conflicts of interest. These have been provided as supplementary materials., (© The Author(s) 2024. Published by Oxford University Press on behalf of Project HOPE - The People-To-People Health Foundation, Inc.)

Background and Objective: Color fundus photography is an important imaging modality that is currently limited by a narrow dynamic range. We describe a post-image processing technique to generate high dynamic range (HDR) retinal images with enhanced detail., Patients and Methods: This was a retrospective, observational case series evaluating fundus photographs of patients with macular pathology. Photographs were acquired with three or more exposure values using a commercially available camera (Topcon 50-DX). Images were aligned and imported into HDR processing software (Photomatix Pro). Fundus detail was compared between HDR and raw photographs., Results: Sixteen eyes from 10 patients (5 male, 5 female; mean age 59.4 years) were analyzed. Clinician graders preferred the HDR image 91.7% of the time (44/48 image comparisons), with good grader agreement (81.3%, 13/16 eyes)., Conclusions: HDR fundus imaging is feasible using images from existing fundus cameras and may be useful for enhanced visualization of retinal detail in a variety of pathologic states. [ Ophthalmic Surg Lasers Imaging Retina 2024;55:263-269.] .

While dysfunction and death of light-detecting photoreceptor cells underlie most inherited retinal dystrophies, knowledge of the species-specific details of human rod and cone photoreceptor cell development remains limited. Here, we generated retinal organoids carrying retinal disease-causing variants in NR2E3, as well as isogenic and unrelated controls. Organoids were sampled using single-cell RNA sequencing (scRNA-Seq) across the developmental window encompassing photoreceptor specification, emergence, and maturation. Using scRNA-Seq data, we reconstruct the rod photoreceptor developmental lineage and identify a branch point unique to the disease state. We show that the rod-specific transcription factor NR2E3 is required for the proper expression of genes involved in phototransduction, including rhodopsin, which is absent in divergent rods. NR2E3-null rods additionally misexpress several cone-specific phototransduction genes. Using joint multimodal single-cell sequencing, we further identify putative regulatory sites where rod-specific factors act to steer photoreceptor cell development. Finally, we show that rod-committed photoreceptor cells form and persist throughout life in a patient with NR2E3-associated disease. Importantly, these findings are strikingly different from those observed in Nr2e3 rodent models. Together, these data provide a road map of human photoreceptor development and leverage patient induced pluripotent stem cells to define the specific roles of rod transcription factors in photoreceptor cell emergence and maturation in health and disease.

Background: People with cognitive disabilities such as intellectual and developmental disabilities face significant barriers to accessing high-quality health care services. Barriers may be exacerbated for those with co-occurring mental health conditions., Objective: This study compares patient experiences of health care services between adults with and without cognitive disabilities and, among people with a cognitive disability, those with and without co-occurring mental health conditions., Methods: Cross-sectional analyses were conducted using 2021 Medical Expenditure Panel Survey data, a national U.S. survey, to examine differences in Consumer Assessment of Healthcare Providers and Systems measures., Results: Adults with cognitive disabilities reported lower satisfaction with health care services compared to the general population (7.62 (95% confidence interval (CI): 7.41-7.83) vs. 8.33 (95% CI: 8.29-8.38) on scale from 0 to 10). Adults with cognitive disabilities were less likely to report that providers listened carefully to them (odds ratio (OR): 0.55, 95% CI: 0.42-0.71), explained things in a way that was easy to understand (OR: 0.48, 95% CI: 0.35-0.66), showed respect for what they had to say (OR: 0.38, 95% CI: 0.29-0.51), spent enough time with them (OR: 0.52, 95% CI: 0.40-0.69), or gave advice that was easy to understand (OR: 0.40, 95% CI: 0.28-0.58) compared to the general population. Among adults with cognitive disabilities, there were no differences based on co-occurring mental health conditions., Conclusions: Adults with cognitive disabilities report lower satisfaction with health care services driven by worse experiences with the health care system. Policies to increase provider capacity to support this population should be prioritized., Competing Interests: Declaration of competing interest Authors have no conflicts of interest to report., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Quantum biological tunnelling for electron transfer is involved in controlling essential functions for life such as cellular respiration and homoeostasis. Understanding and controlling the quantum effects in biology has the potential to modulate biological functions. Here we merge wireless nano-electrochemical tools with cancer cells for control over electron transfer to trigger cancer cell death. Gold bipolar nanoelectrodes functionalized with redox-active cytochrome c and a redox mediator zinc porphyrin are developed as electric-field-stimulating bio-actuators, termed bio-nanoantennae. We show that a remote electrical input regulates electron transport between these redox molecules, which results in quantum biological tunnelling for electron transfer to trigger apoptosis in patient-derived cancer cells in a selective manner. Transcriptomics data show that the electric-field-induced bio-nanoantenna targets the cancer cells in a unique manner, representing electrically induced control of molecular signalling. The work shows the potential of quantum-based medical diagnostics and treatments., (© 2023. The Author(s).)

Rhodopsin ( RHO ) mutations such as Pro23His are the leading cause of dominantly inherited retinitis pigmentosa in North America. As with other dominant retinal dystrophies, these mutations lead to production of a toxic protein product, and treatment will require knockdown of the mutant allele. The purpose of this study was to develop a CRISPR-Cas9-mediated transcriptional repression strategy using catalytically inactive Staphylococcus aureus Cas9 (dCas9) fused to the Krüppel-associated box (KRAB) transcriptional repressor domain. Using a reporter construct carrying green fluorescent protein (GFP) cloned downstream of the RHO promoter fragment (nucleotides -1403 to +73), we demonstrate a ∼74-84% reduction in RHO promoter activity in RHOp CRISPRi-treated versus plasmid-only controls. After subretinal transduction of human retinal explants and transgenic Pro23His mutant pigs, significant knockdown of rhodopsin protein was achieved. Suppression of mutant transgene in vivo was associated with a reduction in endoplasmic reticulum (ER) stress and apoptosis markers and preservation of photoreceptor cell layer thickness.

Human induced pluripotent stem cells (hiPSCs) have demonstrated great promise for a variety of applications that include cell therapy and regenerative medicine. Production of clinical grade hiPSCs requires reproducible manufacturing methods with stringent quality-controls such as those provided by image-controlled robotic processing systems. In this paper we present an automated image analysis method for identifying and picking hiPSC colonies for clonal expansion using the CellX TM robotic cell processing system. This method couples a light weight deep learning segmentation approach based on the U-Net architecture to automatically segment the hiPSC colonies in full field of view (FOV) high resolution phase contrast images with a standardized approach for suggesting pick locations. The utility of this method is demonstrated using images and data obtained from the CellX TM system where clinical grade hiPSCs were reprogrammed, clonally expanded, and differentiated into retinal organoids for use in treatment of patients with inherited retinal degenerative blindness., Competing Interests: Declaration of Competing Interest KAP is a paid consultant and shareholder in Cell X Technologies Inc. GM is the Chief Technology Officer and shareholder in Cell X Technologies Inc. VL is an employee and shareholder in Cell X Technologies Inc., (Copyright © 2023. Published by Elsevier Inc.)

Introduction: Most Americans live in a state that has legalized cannabis as a medical treatment for pain, but it is unclear how chronic pain intensity relates to cannabis use. Our objective was to examine the association between patient-reported pain measures and cannabis among adults with chronic pain., Methods: This cross-sectional study of a representative sample of adults reporting chronic non-cancer pain in 36 states and DC with active medical cannabis programs from March to April 2022 assessed cannabis use for chronic pain, categorized as active (within 30 days), past (>31 days), or never use (referent). Measures were pain intensity (primary) and interference, Widespread Pain Index, and number of chronic pain diagnoses., Results: Among 1628 participants (57% female, 69% white), 352 (22%) actively used cannabis to treat chronic pain, 137 (8%) reported past cannabis use, and 1139 (70%) never used cannabis. In adjusted models, active cannabis use was associated with higher scores for pain intensity (score difference 1.03, 95% CI 0.05 to 2.02) and pain interference (score difference 1.82, 95% CI 0.99 to 2.65) compared with never use. Persons who actively used cannabis had higher Widespread Pain Index scores (score difference 0.56, 95% CI 0.26 to 0.86) and more chronic pain diagnoses (difference 0.45, 95% CI 0.06 to 0.83)., Conclusion: People with chronic non-cancer pain who used cannabis for pain reported non-clinically meaningful worse pain measures and greater burden of chronic pain conditions than their counterparts who never used cannabis. Alternatively, those with worse pain and greater burden of pain appear more likely to use cannabis., Competing Interests: Competing interests: MCB reports current grants from PCORI, CDC, NIH, Michigan Department of Health and Human Services and past grants from the Arnold Foundation. The other authors report no conflicts of interest., (© American Society of Regional Anesthesia & Pain Medicine 2023. No commercial re-use. See rights and permissions. Published by BMJ.)

Some human retinal diseases are characterized by pathology that is restricted to specific cell types and to specific regions of the eye. Several disease entities either selectively affect or spare the macula, the retina region at the center of the posterior pole. Photoreceptor cells in the macula are involved in high-acuity vision and require metabolic support from non-neuronal cell types. Some macular diseases involve the retinal pigment epithelium (RPE), an epithelial cell layer with several metabolic-support functions essential for the overlying photoreceptors. In the current study, the ways in which RPE confers region-specific disease susceptibility were determined by examining heterogeneity within RPE tissue from human donors. RPE nuclei from the macular and peripheral retina were profiled using joint single-nucleus RNA and ATAC sequencing. The expression of several genes differed between macular and peripheral RPE. Region-specific ATAC peaks were found, suggesting regulatory elements used exclusively by macular or peripheral RPE. Across anatomic regions, subpopulations of RPE were identified that appeared to have differential levels of expression of visual cycle genes. Finally, loci associated with age-related macular degeneration were examined for a better understanding of RPE-specific disease phenotypes. These findings showed variations in the regulation of gene expression in the human RPE by region and subpopulation, and provide a source for a better understanding of the molecular basis of macular disease., (Copyright © 2023 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Mutations in the thrombospondin 1 ( THBS1 ) gene have been previously reported in primary congenital glaucoma (PCG) pedigrees that exhibit autosomal dominant inheritance with low penetrance. We sought to determine the role of THBS1 mutations in a cohort of 20 patients with PCG and 362 normal controls from Iowa using a combination of Sanger sequencing and whole exome sequencing. We detected 16 different THBS1 variants, including 4 rare, nonsynonymous variants (p.Thr611Met, p.Asn708Lys, p.Gln1089His, and p.Glu1166Lys). However, none of these variants were judged to be disease-causing mutations based on: 1) prevalence in cases and controls from Iowa, 2) prevalence in the public database gnomAD, 3) mutation analysis algorithms, and 4) THBS1 DNA sequence conservation. These results indicate THBS1 mutations are not a common cause of PCG in patients from Iowa and may be a rare cause of PCG overall., Competing Interests: Disclosure: The authors declare no conflict of interest., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

Background: Policy evaluation studies that assess how state-level policies affect health-related outcomes are foundational to health and social policy research. The relative ability of newer analytic methods to address confounding, a key source of bias in observational studies, has not been closely examined., Methods: We conducted a simulation study to examine how differing magnitudes of confounding affected the performance of 4 methods used for policy evaluations: (1) the two-way fixed effects difference-in-differences model; (2) a 1-period lagged autoregressive model; (3) augmented synthetic control method; and (4) the doubly robust difference-in-differences approach with multiple time periods from Callaway-Sant'Anna. We simulated our data to have staggered policy adoption and multiple confounding scenarios (i.e., varying the magnitude and nature of confounding relationships)., Results: Bias increased for each method: (1) as confounding magnitude increases; (2) when confounding is generated with respect to prior outcome trends (rather than levels), and (3) when confounding associations are nonlinear (rather than linear). The autoregressive model and augmented synthetic control method had notably lower root mean squared error than the two-way fixed effects and Callaway-Sant'Anna approaches for all scenarios; the exception is nonlinear confounding by prior trends, where Callaway-Sant'Anna excels. Coverage rates were unreasonably high for the augmented synthetic control method (e.g., 100%), reflecting large model-based standard errors and wide confidence intervals in practice., Conclusions: In our simulation study, no single method consistently outperformed the others, but a researcher's toolkit should include all methodologic options. Our simulations and associated R package can help researchers choose the most appropriate approach for their data., Competing Interests: The authors report no conflicts of interest., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

Purpose: Macular neovascularization is a relatively common and potentially visually devastating complication of age-related macular degeneration. In macular neovascularization, pathologic angiogenesis can originate from either the choroid or the retina, but we have limited understanding of how different cell types become dysregulated in this dynamic process., Methods: To study how gene expression is altered in focal areas of pathology, we performed spatial RNA sequencing on a human donor eye with macular neovascularization as well as a healthy control donor. We performed differential expression to identify genes enriched within the area of macular neovascularization and used deconvolution algorithms to predict the originating cell type of these dysregulated genes., Results: Within the area of neovascularization, endothelial cells demonstrated increased expression of genes related to Rho family GTPase signaling and integrin signaling. Likewise, VEGF and TGFB1 were identified as potential upstream regulators that could drive the observed gene expression changes produced by endothelial and retinal pigment epithelium cells in the macular neovascularization donor. These spatial gene expression profiles were compared to previous single-cell gene expression experiments in human age-related macular degeneration as well as a model of laser-induced neovascularization in mice. As a secondary aim, we investigated regional gene expression patterns within the macular neural retina and between the macular and peripheral choroid., Conclusions: Overall, this study spatially analyzes gene expression across the retina, retinal pigment epithelium, and choroid in health and describes a set of candidate molecules that become dysregulated in macular neovascularization.

Purpose: Choroideremia is an X-linked choroidopathy caused by pathogenic variants in the CHM gene. It is characterized by the early appearance of multiple scotomas in the peripheral visual field that spread and coalesce, usually sparing central vision until late in the disease. These features make quantitative monitoring of visual decline particularly challenging. Here, we describe a novel computational approach to convert Goldmann visual field (GVF) data into quantitative volumetric measurements. With this approach, we analyzed visual field loss in a longitudinal, retrospective cohort of patients with choroideremia., Design: Single-center, retrospective, cohort study., Participants: We analyzed data from 238 clinic visits of 56 molecularly-confirmed male patients with choroideremia from 41 families (range, 1-27 visits per patient). Patients had a median follow up of 4 years (range, 0-56 years) with an age range of 5 to 76 years at the time of their visits., Methods: Clinical data from molecularly-confirmed patients with choroideremia, including GVF data, were included for analysis. Goldmann visual field records were traced using a tablet-based application, and the 3-dimensional hill of vision was interpolated for each trace. This procedure allowed quantification of visual field loss from data collected over decades with differing protocols, including different or incomplete isopters. Visual acuity (VA) data were collected and converted to logarithm of the minimum angle of resolution values. A delayed exponential mixed-effects model was used to evaluate the loss of visual field volume over time., Main Outcome Measures: Visual acuity and GVF volume., Results: The estimated mean age at disease onset was 12.6 years (standard deviation, 9.1 years; 95% quantile interval, 6.5-36.4 years). The mean field volume loss was 6.8% per year (standard deviation, 4.5%; 95% quantile interval, 1.9%-18.8%) based on exponential modeling. Field volume was more strongly correlated between eyes ( r 2  = 0.935) than best-corrected VA ( r 2  = 0.285)., Conclusions: Volumetric analysis of GVF data enabled quantification of peripheral visual function in patients with choroideremia and evaluation of disease progression. The methods presented here may facilitate the analysis of historical GVF data from patients with inherited retinal disease and other diseases associated with visual field loss. This work informs the creation of appropriate outcome measures in choroideremia therapeutic trials, particularly in trial designs., Financial Disclosures: Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article., (© 2023 by the American Academy of Ophthalmology.)

Importance: The p.Asp67Tyr genetic variant in the GJA3 gene is responsible for congenital cataracts in a family with a high incidence of glaucoma following cataract surgery., Objective: To describe the clinical features of a family with a strong association between congenital cataracts and glaucoma following cataract surgery secondary to a genetic variant in the GJA3 gene (NM_021954.4:c.199G>T, p.Asp67Tyr)., Design, Setting, and Participants: This was a retrospective, observational, case series, genetic association study from the University of Iowa spanning 61 years. Examined were the ophthalmic records from 1961 through 2022 of the family members of a 4-generation pedigree with autosomal dominant congenital cataracts., Main Outcomes and Measures: Frequency of glaucoma following cataract surgery and postoperative complications among family members with congenital cataract due to the p.Asp67Tyr GJA3 genetic variant., Results: Medical records were available from 11 of 12 family members (7 male [63.6%]) with congenital cataract with a mean (SD) follow-up of 30 (21.7) years (range, 0.2-61 years). Eight of 9 patients with congenital cataracts developed glaucoma, and 8 of 8 patients who had cataract surgery at age 2 years or younger developed glaucoma following cataract surgery. The only family member with congenital cataracts who did not develop glaucoma had delayed cataract surgery until 12 and 21 years of age. Five of 11 family members (45.5%) had retinal detachments after cataract extraction and vitrectomy. No patients developed retinal detachments after prophylactic 360-degree endolaser., Conclusions and Relevance: The GJA3 genetic variant, p.Asp67Tyr, was identified in a 4-generation congenital cataract pedigree from Iowa. This report suggests that patients with congenital cataract due to some GJA3 genetic variants may be at especially high risk for glaucoma following cataract surgery. Retinal detachments after cataract extraction in the first 2 years of life were also common in this family, and prophylactic retinal endolaser may be indicated at the time of surgery.

Variants within the high copy number mitochondrial genome (mtDNA) can disrupt organelle function and lead to severe multisystem disease. The wide range of manifestations observed in patients with mitochondrial disease results from varying fractions of abnormal mtDNA molecules in different cells and tissues, a phenomenon termed heteroplasmy. However, the landscape of heteroplasmy across cell types within tissues and its influence on phenotype expression in affected patients remains largely unexplored. Here, we identify nonrandom distribution of a pathogenic mtDNA variant across a complex tissue using single-cell RNA-Seq, mitochondrial single-cell ATAC sequencing, and multimodal single-cell sequencing. We profiled the transcriptome, chromatin accessibility state, and heteroplasmy in cells from the eyes of a patient with mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) and from healthy control donors. Utilizing the retina as a model for complex multilineage tissues, we found that the proportion of the pathogenic m.3243A>G allele was neither evenly nor randomly distributed across diverse cell types. All neuroectoderm-derived neural cells exhibited a high percentage of the mutant variant. However, a subset of mesoderm-derived lineage, namely the vasculature of the choroid, was near homoplasmic for the WT allele. Gene expression and chromatin accessibility profiles of cell types with high and low proportions of m.3243A>G implicate mTOR signaling in the cellular response to heteroplasmy. We further found by multimodal single-cell sequencing of retinal pigment epithelial cells that a high proportion of the pathogenic mtDNA variant was associated with transcriptionally and morphologically abnormal cells. Together, these findings show the nonrandom nature of mitochondrial variant partitioning in human mitochondrial disease and underscore its implications for mitochondrial disease pathogenesis and treatment.

Purpose: To describe the phenotypic variability and rates of progression of atrophy in patients with PROM1 -associated macular dystrophy., Methods: Patients in this retrospective, longitudinal case series from a tertiary center had clinical examination and multimodal imaging performed. Areas of retinal pigment epithelium and ellipsoid zone loss over time by optical coherence tomography were calculated by two independent graders., Results: Fifteen patients from five kindreds with an Arg373Cys mutation in PROM1 were studied. The average age was 39 years, and 80% were women. The visual acuity was 20/40 at presentation and 20/57 at last follow-up (average 4.8 years). Three distinct macular phenotypes were observed: 1) central geographic atrophy (13%), 2) multifocal geographic atrophy (20%), and 3) bull's eye maculopathy (67%). The overall rate of atrophy progression was 0.36 mm 2 /year, but the average rate of atrophy progression varied by macular phenotype: 1.08 mm 2 /year for central geographic atrophy, 0.53 mm 2 /year for multifocal geographic atrophy, and 0.23 mm 2 /year for bull's eye maculopathy., Conclusion: Patients with PROM1 -associated macular dystrophy demonstrate distinct phenotypes, with bull's eye maculopathy being the most common. The average rate of atrophy progression may be similar to reported rates for ABCA4 -related Stargardt disease and less than age-related macular degeneration. These results provide important measures for following treatment response in future gene and stem cell-based therapies., (Copyright © 2023 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the Opthalmic Communications Society, Inc.)

Prior to use, newly generated induced pluripotent stem cells (iPSC) should be thoroughly validated. While excellent validation and release testing assays designed to evaluate potency, genetic integrity, and sterility exist, they do not have the ability to predict cell type-specific differentiation capacity. Selection of iPSC lines that have limited capacity to produce high-quality transplantable cells, places significant strain on valuable clinical manufacturing resources. The purpose of this study was to determine the degree and root cause of variability in retinal differentiation capacity between cGMP-derived patient iPSC lines. In turn, our goal was to develop a release testing assay that could be used to augment the widely used ScoreCard panel. IPSCs were generated from 15 patients (14-76 years old), differentiated into retinal organoids, and scored based on their retinal differentiation capacity. Despite significant differences in retinal differentiation propensity, RNA-sequencing revealed remarkable similarity between patient-derived iPSC lines prior to differentiation. At 7 days of differentiation, significant differences in gene expression could be detected. Ingenuity pathway analysis revealed perturbations in pathways associated with pluripotency and early cell fate commitment. For example, good and poor producers had noticeably different expressions of OCT4 and SOX2 effector genes. QPCR assays targeting genes identified via RNA sequencing were developed and validated in a masked fashion using iPSCs from 8 independent patients. A subset of 14 genes, which include the retinal cell fate markers RAX, LHX2, VSX2, and SIX6 (all elevated in the good producers), were found to be predictive of retinal differentiation propensity., (© The Author(s) 2023. Published by Oxford University Press.)

Introduction: X-linked retinoschisis (XLRS) is an inherited retinal disease (IRD) caused by pathogenic mutations in the retinoschisin gene, RS1 . Affected individuals develop retinal layer separation, leading to loss of visual acuity (VA). Several XLRS gene therapy trials have been attempted but none have met their primary endpoints. An improved understanding of XLRS natural history and clinical outcomes may better inform future trials. Here, we report the long-term functional and structural outcomes of XLRS and the relevance of RS1 genotypes to the visual prognosis of affected individuals., Methods: A retrospective chart review of patients with molecularly confirmed X-linked retinoschisis was performed. Functional and structural outcomes, and RS1 genotype data, were included for analysis., Results: Fifty-two patients with XLRS from 33 families were included in the study. Median age at symptom onset was 5 years (range 0-49) and median follow-up was 5.7 years (range 0.1-56.8). Macular retinoschisis occurred in 103 of 104 eyes (99.0%), while peripheral retinoschisis occurred in 48 of 104 eyes (46.2%), most often in the inferotemporal quadrant (40.4%). Initial and final VA were similar (logMAR 0.498 vs. 0.521; p = 0.203). Fifty of 54 eyes (92.6%) developed detectable outer retinal loss by age 20, and 29 of 66 eyes (43.9%) had focal or diffuse outer retinal atrophy (ORA) by age 40. ORA but not central subfield thickness (CST) was associated with reduced VA. Inter-eye correlation was modest for VA ( r -squared = 0.03; p = 0.08) and CST ( r -squared = 0.15; p = 0.001). Carbonic anhydrase inhibitors (CAIs) improved CST ( p = 0.026), but not VA ( p = 0.380). Eight of 104 eyes (7.7%) had XLRS-related retinal detachment (RD), which was associated with poorer outcomes compared to eyes without RD (median final VA 0.875 vs. 0.487; p <0.0001). RS1 null genotypes had greater odds of at least moderate visual impairment at final follow-up (OR 7.81; 95% CI 2.17, 28.10; p = 0.002) which was independent of age at onset, initial CST, initial ORA, or previous RD., Discussion: Overall, long-term follow-up of XLRS patients demonstrated relatively stable VA, with presenting CST, development of ORA, and null RS1 mutations associated with poorer long-term visual outcomes, indicating a clinically relevant genotype-phenotype correlation in XLRS., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Fenner, Russell, Drack, Dumitrescu, Sohn, Russell, Boldt, Affatigato, Hoffmann, Andorf, Stone and Han.)

Adeno-associated virus (AAV)-mediated gene therapy has great potential for treating a wide range of retinal degenerative diseases. However, some initial enthusiasm for gene therapy has been tempered by emerging evidence of AAV-associated inflammation, which in several instances has contributed to clinical trial discontinuation. Currently, there is a paucity of data describing the variable immune responses to different AAV serotypes, and similarly, little is known regarding how these responses differ depending on route of ocular delivery, including in animal models of disease. In this study, we characterize the severity and retinal distribution of AAV-associated inflammation in rats triggered by delivery of five different AAV vectors (AAV1, AAV2, AAV6, AAV8, and AAV9), each of which contained enhanced green fluorescent protein (eGFP) driven under control of the constitutively active cytomegalovirus promoter. We further compare the inflammation across three different potential routes (intravitreal, subretinal, and suprachoroidal) of ocular delivery. Compared to buffer-injected controls for each route of delivery, AAV2 and AAV6 induced the most inflammation across all routes of delivery of vectors tested, with AAV6 inducing the highest levels of inflammation when delivered suprachoroidally. AAV1-induced inflammation was highest when delivered suprachoroidally, whereas minimal inflammation was seen with intravitreal delivery. In addition, AAV1, AAV2, and AAV6 each induce infiltration of adaptive immune cells like T cells and B cells into the neural retina, suggesting an innate adaptive response to a single dose of virus. AAV8 and AAV9 induced minimal inflammation across all routes of delivery. Importantly, the degree of inflammation was not correlated with vector-mediated transduction and expression of eGFP. These data emphasize the importance of considering ocular inflammation when selecting AAV serotypes and ocular delivery routes for the development of gene therapy strategies.

Objective: Choroidal neovascularization (CNV) is usually considered to be a late-stage complication in Best vitelliform macular dystrophy (BVMD) and can be difficult to diagnose with fluorescein angiography. This study used swept-source (SS) OCT angiography (OCTA) to evaluate the prevalence of CNV in BVMD, identify structural features associated with CNV, and provide insight into the role of CNV in vitelliform lesion evolution., Design: Institutional review board-approved, retrospective, cross-sectional, and longitudinal study., Participants: Patients with molecularly confirmed BVMD., Methods: Charts from consecutive patients with BVMD imaged with SS-OCTA (PLEX Elite 9000, Carl-Zeiss Meditec Inc) at the University of Iowa from September 2017 to October 2021 were reviewed. Clinical data, including age, gender, best-corrected visual acuity (BCVA), and treatment with intravitreal anti-VEGF injections were recorded. The presence of CNV on SS-OCTA was determined by expert graders and correlated with structural features, such as interstitial fluid, subretinal fluid, nodular subretinal pillar, focal choroidal excavation (FCE), and subfoveal choroidal thickness, with a P value of < 0.05 considered statistically significant., Main Outcome Measures: Presence of CNV on SS-OCTA and correlation with structural features on SS-OCT., Results: A total of 53 eyes from 27 patients (13 women; 48.1%) were included. The mean age was 45 years (range, 8-79 years), and the mean logarithm of the minimum angle of resolution BCVA was 0.38 (range, 0-1). Choroidal neovascularization was identified on SS-OCTA in 27 eyes (50.9%), of which 63.0% had a vitelliform (Gass stage 2) lesion. In 40.7% (11 of 27) of eyes, there was no prior clinical diagnosis of CNV. Other structural features associated with CNV included FCEs (15.1%, 8 of 53 eyes) and nodular pillars (15.1%, 8 of 53 eyes) (P < 0.01). Seven patients had available longitudinal imaging, and most of these patients had CNV visible on SS-OCTA (71.4%; 10 of 14 eyes)., Conclusion: Choroidal neovascularization is common in BVMD, including in the early stages of the disease. The presence of FCEs or nodular pillars should heighten the clinical suspicion of CNV, which may accelerate vitelliform lesion evolution., Financial Disclosure(s): Proprietary or commercial disclosure may be found after the references., (Copyright © 2022 American Academy of Ophthalmology. Published by Elsevier Inc. All rights reserved.)

Many retinal diseases involve the loss of light-sensing photoreceptor cells (rods and cones) over time. The severity and distribution of photoreceptor loss varies widely across diseases and affected individuals, so characterizing the degree and pattern of photoreceptor loss can clarify pathophysiology and prognosis. Currently, in vivo visualization of individual photoreceptors requires technology such as adaptive optics, which has numerous limitations and is not widely used. By contrast, optical coherence tomography (OCT) is nearly ubiquitous in daily clinical practice given its ease of image acquisition and detailed visualization of retinal structure. However, OCT cannot resolve individual photoreceptors, and no OCT-based method exists to distinguish between the loss of rods versus cones. Here, we present a computational model that quantitatively estimates rod versus cone photoreceptor loss from OCT. Using histologic data of human photoreceptor topography, we constructed an OCT-based reference model to simulate outer nuclear layer thinning caused by differential loss of rods and cones. The model was able to estimate rod and cone loss using in vivo OCT data from patients with Stargardt disease and healthy controls. Our model provides a powerful new tool to quantify photoreceptor loss using OCT data alone, with potentially broad applications for research and clinical care., (© 2023. The Author(s).)

In humans, mutations in the beta subunit of cGMP-phosphodiesterase type 6 (PDE6B) cause autosomal recessive retinitis pigmentosa (RP), which typically has an aggressive clinical course of early-onset severe vision loss due to rapid photoreceptor degeneration. In this study, we describe the generation of a novel Pde6b-deficient rat model using CRISPR-Cas9 genome editing. We characterize the model at multiple time points using clinical imaging modalities as well as histology with immunohistochemistry to show rapid photoreceptor degeneration compared to wild-type and heterozygous animals. We describe the manufacture of two different adeno-associated viral (AAV) vectors (AAV2/1, AAV2/5) under current Good Manufacturing Practices (cGMP) and demonstrate their ability to drive human PDE6B expression in vivo. We further demonstrate the ability of AAV-mediated subretinal gene therapy to delay photoreceptor loss in Pde6b-deficient rats compared to untreated controls. However, severe progressive photoreceptor loss was noted even in treated eyes, likely due to the aggressive nature of the disease. These data provide useful preclinical data to guide the development of potential human gene therapy for PDE6B-associated RP. In addition, the rapid photoreceptor degeneration of the Pde6b-deficient rat with intact inner retina may provide a useful model for the study of cell replacement strategies., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

Background: Pervasive social and structural barriers-including national policies-inhibit HIV testing uptake among priority populations, including adolescents. We assessed the relationship between age-of-consent policies for HIV testing and adolescent HIV testing coverage in 51 low- and middle-income countries., Methods: We pooled data from household surveys (2010-2020) and calculated the weighted country-level prevalence of lifetime HIV testing separately for adolescent girls and boys (ages 15-19). We then abstracted age-of-consent requirements for HIV testing across countries. Using multivariable linear regression, we estimated the average difference in national HIV testing coverage estimates for adolescent girls and boys by age-of-consent restrictions for HIV testing., Results: National HIV testing coverage estimates ranged from 0.7% to 72.5% among girls (median: 18.0%) and 0% to 73.2% among boys (median: 7.5%) in Pakistan and Lesotho, respectively. In adjusted models, HIV testing coverage in countries requiring parental consent for individuals <18 years was, on average, 9.4 percentage-points (pp) lower (95% confidence interval [95%CI] -17.9pp to -0.9pp) among girls and 9.3pp lower (95%CI: -17.3pp to -1.2pp) among boys, relative to countries with less restrictive policies (age-of-consent: ≤16 years). Compared to countries with less restrictive (age-of-consent: ≤14 years) policies, HIV testing prevalence was significantly lower among girls ( β -10.5pp, 95%CI: -19.7pp to -1.3pp) and boys ( β -10.5pp, 95%CI -19.2pp to -1.8pp) in countries with more restrictive (age-of-consent: 18 years) parental consent requirements., Conclusions: Age-of-consent policies are persistent obstacles to adolescent HIV testing. Repealing parental consent requirements for HIV testing is needed to expand coverage and accelerate progress towards global HIV treatment and prevention targets.

Background: Many states have adopted laws that limit the amount or duration of opioid prescriptions. These limits often focus on prescriptions for acute pain, but there may be unintended consequences for those diagnosed with chronic pain, including reduced opioid prescribing without substitution of appropriate non-opioid treatments., Objective: To evaluate the effects of state opioid prescribing cap laws on opioid and non-opioid treatment among those diagnosed with chronic pain., Design: We used a difference-in-differences approach that accounts for staggered policy adoption. Treated states included 32 states that implemented a prescribing cap law between 2017 and 2019., Population: A total of 480,856 adults in the USA who were continuously enrolled in medical and pharmacy coverage from 2013 to 2019 and diagnosed with a chronic pain condition between 2013 and 2016., Main Measures: Among individuals with chronic pain in each state: proportion with at least one opioid prescription and with prescriptions of a specific duration or dose, average number of opioid prescriptions, average opioid prescription duration and dose, proportion with at least one non-opioid chronic pain prescription, average number of such prescriptions, proportion with at least one chronic pain procedure, and average number of such procedures., Key Results: State laws limiting opioid prescriptions were not associated with changes in opioid prescribing, non-opioid medication prescribing, or non-opioid chronic pain procedures among patients with chronic pain diagnoses., Conclusions: These findings do not support an association between state opioid prescribing cap laws and changes in the treatment of chronic non-cancer pain., (© 2022. The Author(s), under exclusive licence to Society of General Internal Medicine.)

Background: Inherited retinal degeneration is a leading cause of incurable vision loss in the developed world. While autologous iPSC mediated photoreceptor cell replacement is theoretically possible, the lack of commercially available technologies designed to enable high throughput parallel production of patient specific therapeutics has hindered clinical translation., Methods: In this study, we describe the use of the Cell X precision robotic cell culture platform to enable parallel production of clinical grade patient specific iPSCs. The Cell X is housed within an ISO Class 5 cGMP compliant closed aseptic isolator (Biospherix XVivo X2), where all procedures from fibroblast culture to iPSC generation, clonal expansion and retinal differentiation were performed., Results: Patient iPSCs generated using the Cell X platform were determined to be pluripotent via score card analysis and genetically stable via karyotyping. As determined via immunostaining and confocal microscopy, iPSCs generated using the Cell X platform gave rise to retinal organoids that were indistinguishable from organoids derived from manually generated iPSCs. In addition, at 120 days post-differentiation, single-cell RNA sequencing analysis revealed that cells generated using the Cell X platform were comparable to those generated under manual conditions in a separate laboratory., Conclusion: We have successfully developed a robotic iPSC generation platform and standard operating procedures for production of high-quality photoreceptor precursor cells that are compatible with current good manufacturing practices. This system will enable clinical grade production of iPSCs for autologous retinal cell replacement., (© 2023. The Author(s).)

Inequitable experiences of community gun violence and victimization by police use of force led to nationwide calls to "reimagine public safety" in 2020. In January 2021, we examined public support among U.S. adults for 7 policy approaches to reforming policing and investing in community gun violence prevention. Using a nationally representative sample (N = 2778), with oversampling for Black Americans, Hispanic Americans, and gun owners, we assessed support overall and by racial, ethnic, and gun owner subgroups. Overall, we found majority support for funding and implementing police and mental health co-responder models (66% and 76%, respectively), diversion from incarceration for people with symptoms of mental illness (72%), stronger laws to assure police accountability (72%), and funding for community-based and hospital-based gun violence prevention programs (69% and 60%, respectively). Support for redirecting funding from the police to social services was more variable (44% overall; White: 35%, Black: 60%, Hispanic: 43%). For all survey items, support was strongest among Black Americans. Gun owners overall reported lower support for public safety reforms and investments than respondents who did not own guns, but this distinction was found to be driven by White gun owners. The views of Black gun owners were indistinguishable from Black non-owners and were similar to White non-owners on most issues. These findings suggest that broad public support exists for innovative violence reduction strategies and public safety reforms., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Gun-related deaths and gun purchases were at record highs in 2020. In light of the COVID-19 pandemic, public protests against police violence, and a tense political environment, which may influence policy preferences, we aimed to understand the current state of support for gun policies in the U.S. We fielded a national public opinion survey in January 2019 and January 2021 using an online panel to measure support for 34 gun policies among U.S. adults. We compared support over time, by gun ownership status, and by political party affiliation. Most respondents supported 33 of the 34 gun regulations studied. Support for seven restrictive policies declined from 2019 to 2021, driven by reduced support among non-gun owners. Support declined for three permissive policies: allowing legal gun carriers to bring guns onto college campuses or K-12 schools and stand your ground laws. Public support for gun-related policies decreased from 2019 to 2021, driven by decreased support among Republicans and non-gun owners., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Gun-related deaths and gun purchases were at record highs in 2020. In light of the COVID-19 pandemic, public protests against police violence, and a tense political environment, which may influence policy preferences, we aimed to understand the current state of support for gun policies in the U.S. We fielded a national public opinion survey in January 2019 and January 2021 using an online panel to measure support for 34 gun policies among U.S. adults. We compared support over time, by gun ownership status, and by political party affiliation. Most respondents supported 33 of the 34 gun regulations studied. Support for seven restrictive policies declined from 2019 to 2021, driven by reduced support among non-gun owners. Support declined for three permissive policies: allowing legal gun carriers to bring guns onto college campuses or K-12 schools and stand your ground laws. Public support for gun-related policies decreased from 2019 to 2021, driven by decreased support among Republicans and non-gun owners., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Introduction: In response to the role of opioid prescribing in the U.S. opioid crisis, states have enacted laws intended to curb high risk opioid prescribing practices. This study assessed the effects of state prescribing cap laws that limit the dose and/or duration of dispensed opioid prescriptions on opioid prescribing patterns and opioid overdose., Methods: We identified 1,414,908 adults from a large U.S. administrative insurance claims database. Treatment states included 32 states that implemented a prescribing cap law between 2017 and 2019. Comparison states included 16 states and DC without a prescribing cap law by 2019. A difference-in-differences approach with staggered policy adoption was used to assess effects of these laws on opioid analgesic prescribing and opioid overdose., Results: State opioid prescribing cap laws were not associated with changes in the proportion of people receiving opioid analgesic prescriptions, the dose or duration of opioid prescriptions, or opioid overdose. States with laws that imposed days' supply limits only versus days' supply and dosage limits, as well as with specific law provisions also showed no association with opioid prescribing or opioid overdose outcomes., Conclusions: State opioid prescribing cap laws did not appear to impact outcomes related to opioid analgesic prescribing or opioid overdose. These findings are potentially due to the limited scope of these laws, which often apply only to a subset of opioid prescriptions and include professional judgment exemptions., Competing Interests: Conflict of Interest Drs. Tormohlen, McCourt, Schmid, Stone, Stuart, Davis, and McGinty have no financial disclosures. Dr. Bicket reports past service as a consultant for Axial Healthcare and Alosa Health, both unrelated to the submitted work., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Objective: To evaluate the effects of state opioid prescribing cap laws on opioid prescribing after surgery., Data Sources: OptumLabs Data Warehouse administrative claims data covering all 50 states from July 2012 through June 2019., Study Design: We included individuals from 20 states that had implemented prescribing cap laws without exemptions for postsurgical pain by June 2019 and individuals from 16 control states plus the District of Columbia. We used a difference-in-differences approach accounting for differential timing in law implementation across states to estimate the effects of state prescribing cap laws on postsurgical prescribing of opioids. Outcome measures included filling an opioid prescription within 30 days after surgery; filling opioid prescriptions of specific doses or durations; and the number, days' supply, daily dose, and pill quantity of opioid prescriptions. To assess the validity of the parallel counterfactual trends assumption, we examined differences in outcome trends between law-implementing and control states in the years preceding law implementation using an equivalence testing framework., Data Collection/extraction Methods: We included the first surgery in the study period for opioid-naïve individuals undergoing one of eight common surgical procedures., Principal Findings: State prescribing cap laws were associated with 0.109 lower days' supply of postsurgical opioids on the log scale (95% Confidence Interval [CI]: -0.139, -0.080) but were not associated with the number (Average treatment effect on the treated [ATT]: -0.011; 95% CI: -0.043, 0.021) or daily dose of postsurgical opioid prescriptions (ATT: -0.013; 95% CI: -0.030, 0.005). The negative association observed between prescribing cap laws and the probability of filling a postsurgical opioid prescription (ATT: -0.041; 95% CI: -0.054, -0.028) was likely spurious, given differences between law-implementing and control states in the pre-law period., Conclusions: Prescribing cap laws appear to have minimal effects on postsurgical opioid prescribing., (© 2022 Health Research and Educational Trust.)

The choriocapillaris, a dense capillary network located at the posterior pole of the eye, is essential for supporting normal vision, supplying nutrients, and removing waste products from photoreceptor cells and the retinal pigment epithelium. The anatomical location, heterogeneity, and homeostatic interactions with surrounding cell types make the choroid complex to study both in vivo and in vitro. Recent advances in single-cell RNA sequencing, in vivo imaging, and in vitro cell modeling are vastly improving our knowledge of the choroid and its role in normal health and in age-related macular degeneration (AMD). Histologically, loss of endothelial cells (ECs) of the choriocapillaris occurs early in AMD concomitant with elevated formation of the membrane attack complex of complement. Advanced imaging has allowed us to visualize early choroidal blood flow changes in AMD in living patients, supporting histological findings of loss of choroidal ECs. Single-cell RNA sequencing is being used to characterize choroidal cell types transcriptionally and discover their altered patterns of gene expression in aging and disease. Advances in induced pluripotent stem cell protocols and 3D cultures will allow us to closely mimic the in vivo microenvironment of the choroid in vitro to better understand the mechanism leading to choriocapillaris loss in AMD.

Importance: High-dose and long-duration opioid prescriptions remain relatively common among children and adolescents, but there is insufficient research on the association of state laws limiting the dose and/or duration of opioid prescriptions (referred to as opioid prescribing cap laws) with opioid prescribing for this group., Objective: To examine the association between state opioid prescribing cap laws and the receipt of opioid prescriptions among children and adolescents., Design Setting and Participants: This repeated cross-sectional study used a difference-in-differences approach accounting for staggered policy adoption to assess the association of state opioid prescribing cap laws in the US from January 1, 2013, to December 31, 2019, with receipt of opioid prescriptions among children and adolescents. Analyses were conducted between March 22 and December 15, 2021. Data were obtained from the OptumLabs Data Warehouse, a national commercial insurance claims database. The analysis included 482 118 commercially insured children and adolescents aged 0 to 17 years with full calendar-year continuous insurance enrollment between 2013 and 2019. Individuals were included for every year in which they were continuously enrolled; they did not need to be enrolled for the entire 7-year study period. Those with any cancer diagnosis were excluded from analysis., Exposure: Implementation of a state opioid prescribing cap law between January 1, 2017, and July 1, 2019. This date range allowed analysis of the same number years for both pre-cap and post-cap data., Main Outcomes and Measures: Outcomes of interest included receipt of any opioid prescription and, among those with at least 1 opioid prescription, the mean number of opioid prescriptions, mean morphine milligram equivalents (MMEs) per day, and mean days' supply., Results: Among 482 118 children and adolescents (754 368 person-years of data aggregated to the state-year level), 245 178 (50.9%) were male, with a mean (SD) age of 9.8 (4.8) years at the first year included in the sample (data on race and ethnicity were not collected as part of this data set, which was obtained from insurance billing claims). Overall, 10 659 children and adolescents (2.2%) received at least 1 opioid prescription during the study period. Among those with at least 1 prescription, the mean (SD) number of filled opioid prescriptions was 1.2 (0.8) per person per year. No statistically significant association was found between state opioid prescribing cap laws and any outcome. After opioid prescribing cap laws were implemented, a -0.001 (95% CI, -0.005 to 0.002) percentage point decrease in the proportion of youths receiving any opioid prescription was observed. In addition, percentage point decreases of -0.01 (95% CI, -0.10 to 0.09) in high-dose opioid prescriptions (>50 MMEs per day) and -0.02 (95% CI, -0.12 to 0.08) in long-duration opioid prescriptions (>7 days' supply) were found after cap laws were implemented., Conclusions and Relevance: In this cross-sectional study, no association was observed between state opioid prescribing cap laws and the receipt of opioid prescriptions among children and adolescents. Alternative strategies, such as opioid prescribing guidelines tailored to youths, are needed., Competing Interests: Conflict of Interest Disclosures: Dr Bicket reported receiving grants from the Centers for Disease Control and Prevention, the Michigan Department of Health and Human Services, and the National Institutes of Health and personal fees from Alosa Health and axialHealthcare outside the submitted work. No other disclosures were reported., (Copyright 2022 Stone EM et al. JAMA Health Forum.)

The human choroid is a heterogeneous, highly vascular connective tissue that dysfunctions in age-related macular degeneration (AMD). In this study, we performed single-cell RNA sequencing on 21 human choroids, 11 of which were derived from donors with early atrophic or neovascular AMD. Using this large donor cohort, we identified new gene expression signatures and immunohistochemically characterized discrete populations of resident macrophages, monocytes/inflammatory macrophages and dendritic cells. These three immune populations demonstrated unique expression patterns for AMD genetic risk factors, with dendritic cells possessing the highest expression of the neovascular AMD-associated MMP9 gene. Additionally, we performed trajectory analysis to model transcriptomic changes across the choroidal vasculature, and we identified expression signatures for endothelial cells from choroidal arterioles and venules. Finally, we performed differential expression analysis between control, early atrophic AMD, and neovascular AMD samples, and we observed that early atrophic AMD samples had high expression of SPARCL1, a gene that has been shown to increase in response to endothelial damage. Likewise, neovascular endothelial cells harbored gene expression changes consistent with endothelial cell damage and demonstrated increased expression of the sialomucins CD34 and ENCM, which were also observed at the protein level within neovascular membranes. Overall, this study characterizes the molecular features of new populations of choroidal endothelial cells and mononuclear phagocytes in a large cohort of AMD and control human donors., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Dynamic motions of enzymes occurring on a broad range of timescales play a pivotal role in all steps of the reaction pathway, including substrate binding, catalysis, and product release. However, it is unknown whether structural information related to conformational flexibility can be exploited for the directed evolution of enzymes with higher catalytic activity. Here, we show that mutagenesis of residues exclusively located at flexible regions distal to the active site of Homo sapiens kynureninase (HsKYNase) resulted in the isolation of a variant (BF-HsKYNase) in which the rate of the chemical step toward kynurenine was increased by 45-fold. Mechanistic pre–steady-state kinetic analysis of the wild type and the evolved enzyme shed light on the underlying effects of distal mutations (>10 Å from the active site) on the rate-limiting step of the catalytic cycle. Hydrogen-deuterium exchange coupled to mass spectrometry and molecular dynamics simulations revealed that the amino acid substitutions in BF-HsKYNase allosterically affect the flexibility of the pyridoxal-5′-phosphate (PLP) binding pocket, thereby impacting the rate of chemistry, presumably by altering the conformational ensemble and sampling states more favorable to the catalyzed reaction.

Objectives. To characterize the experience and impact of pandemic-related workplace violence in the form of harassment and threats against public health officials. Methods. We used a mixed methods approach, combining media content and a national survey of local health departments (LHDs) in the United States, to identify harassment against public health officials from March 2020 to January 2021. We compared media-portrayed experiences, survey-reported experiences, and publicly reported position departures. Results. At least 1499 harassment experiences were identified by LHD survey respondents, representing 57% of responding departments. We also identified 222 position departures by public health officials nationally, 36% alongside reports of harassment. Public health officials described experiencing structural and political undermining of their professional duties, marginalization of their expertise, social villainization, and disillusionment. Many affected leaders remain in their positions. Conclusions. Interventions to reduce undermining, ostracizing, and intimidating acts against health officials are needed for a sustainable public health system. We recommend training leaders to respond to political conflict, improving colleague support networks, providing trauma-informed worker support, investing in long-term public health staffing and infrastructure, and establishing workplace violence reporting systems and legal protections. ( Am J Public Health . 2022;112(5):736-746. https://doi.org/10.2105/AJPH.2021.306649).

Activation of the alternative complement pathway is an initiating event in the pathology of age-related macular degeneration (AMD). Unchecked complement activation leads to the formation of a pro-lytic pore, the membrane attack complex (MAC). MAC deposition is observed on the choriocapillaris of AMD patients and likely causes lysis of choroidal endothelial cells (CECs). Complement factor H (FH, encoded by the gene CFH) is an inhibitor of complement. Both loss of function of FH and reduced choroidal levels of FH have been reported in AMD. It is plausible that reduced local FH availability promotes MAC deposition on CECs. FH is produced primarily in the liver; however, cells including the retinal pigment epithelium can produce FH locally. We hypothesized that CECs produce FH locally to protect against MAC deposition. We aimed to investigate the effect of reduced FH levels in the choroid to determine whether increasing local FH could protect CECs from MAC deposition. We demonstrated that siRNA knockdown of FH (CFH) in human immortalized CECs results in increased MAC deposition. We generated AMD iPSC-derived CECs and found that overexpression of FH protects against MAC deposition. These results suggest that local CEC-produced FH protects against MAC deposition, and that increasing local FH protein may be beneficial in limiting MAC deposition in AMD. © 2022 The Pathological Society of Great Britain and Ireland., (© 2022 The Pathological Society of Great Britain and Ireland.)

By combining next generation whole exome sequencing and induced pluripotent stem cell (iPSC) technology we found that an Alu repeat inserted in exon 9 of the MAK gene results in a loss of normal MAK transcript and development of human autosomal recessive retinitis pigmentosa (RP). Although a relatively rare cause of disease in the general population, the MAK variant is enriched in individuals of Jewish ancestry. In this population, 1 in 55 individuals are carriers and one third of all cases of recessive RP is caused by this gene. The purpose of this study was to determine if a viral gene augmentation strategy could be used to safely restore functional MAK protein as a step toward a treatment for early stage MAK-associated RP. Patient iPSC-derived photoreceptor precursor cells were generated and transduced with viral vectors containing the MAK transcript. One week after transduction, transcript and protein could be detected via rt-PCR and western blotting respectively. Using patient-derived fibroblast cells and mak knockdown zebra fish we demonstrate that over-expression of the retinal MAK transgene restored the cells ability to regulate primary cilia length. In addition, the visual defect in mak knockdown zebrafish was mitigated via treatment with the retinal MAK transgene. There was no evidence of local or systemic toxicity at 1-month or 3-months following subretinal delivery of clinical grade vector into wild type rats. The findings reported here will help pave the way for initiation of a phase 1 clinical trial for the treatment of patients with MAK-associated RP., (© 2021. The Author(s).)