Emergence of enhancers at late DNA replicating regions.

Enhancers are fast-evolving genomic sequences that control spatiotemporal gene expression patterns. By examining enhancer turnover across mammalian species and in multiple tissue types, we uncover a relationship between the emergence of enhancers and genome organization as a function of germline DNA replication time. While enhancers are most abundant in euchromatic regions, enhancers emerge almost twice as often in late compared to early germline replicating regions, independent of transposable elements. Using a deep learning sequence model, we demonstrate that new enhancers are enriched for mutations that alter transcription factor (TF) binding. Recently evolved enhancers appear to be mostly neutrally evolving and enriched in eQTLs. They also show more tissue specificity than conserved enhancers, and the TFs that bind to these elements, as inferred by binding sequences, also show increased tissue-specific gene expression. We find a similar relationship with DNA replication time in cancer, suggesting that these observations may be time-invariant principles of genome evolution. Our work underscores that genome organization has a profound impact in shaping mammalian gene regulation.

DNA-Diffusion: Leveraging Generative Models for Controlling Chromatin Accessibility and Gene Expression via Synthetic Regulatory Elements.

The challenge of systematically modifying and optimizing regulatory elements for precise gene expression control is central to modern genomics and synthetic biology. Advancements in generative AI have paved the way for designing synthetic sequences with the aim of safely and accurately modulating gene expression. We leverage diffusion models to design context-specific DNA regulatory sequences, which hold significant potential toward enabling novel therapeutic applications requiring precise modulation of gene expression. Our framework uses a cell type-specific diffusion model to generate synthetic 200 bp regulatory elements based on chromatin accessibility across different cell types. We evaluate the generated sequences based on key metrics to ensure they retain properties of endogenous sequences: transcription factor binding site composition, potential for cell type-specific chromatin accessibility, and capacity for sequences generated by DNA diffusion to activate gene expression in different cell contexts using state-of-the-art prediction models. Our results demonstrate the ability to robustly generate DNA sequences with cell type-specific regulatory potential. DNA-Diffusion paves the way for revolutionizing a regulatory modulation approach to mammalian synthetic biology and precision gene therapy.

The potential of epigenetic therapy to target the 3D epigenome in endocrine-resistant breast cancer.

Three-dimensional (3D) epigenome remodeling is an important mechanism of gene deregulation in cancer. However, its potential as a target to counteract therapy resistance remains largely unaddressed. Here, we show that epigenetic therapy with decitabine (5-Aza-mC) suppresses tumor growth in xenograft models of pre-clinical metastatic estrogen receptor positive (ER+) breast tumor. Decitabine-induced genome-wide DNA hypomethylation results in large-scale 3D epigenome deregulation, including de-compaction of higher-order chromatin structure and loss of boundary insulation of topologically associated domains. Significant DNA hypomethylation associates with ectopic activation of ER-enhancers, gain in ER binding, creation of new 3D enhancer-promoter interactions and concordant up-regulation of ER-mediated transcription pathways. Importantly, long-term withdrawal of epigenetic therapy partially restores methylation at ER-enhancer elements, resulting in a loss of ectopic 3D enhancer-promoter interactions and associated gene repression. Our study illustrates the potential of epigenetic therapy to target ER+ endocrine-resistant breast cancer by DNA methylation-dependent rewiring of 3D chromatin interactions, which are associated with the suppression of tumor growth.

Genome-wide transcription factor-binding maps reveal cell-specific changes in the regulatory architecture of human HSPCs.

Hematopoietic stem and progenitor cells (HSPCs) rely on a complex interplay among transcription factors (TFs) to regulate differentiation into mature blood cells. A heptad of TFs (FLI1, ERG, GATA2, RUNX1, TAL1, LYL1, LMO2) bind regulatory elements in bulk CD34+ HSPCs. However, whether specific heptad-TF combinations have distinct roles in regulating hematopoietic differentiation remains unknown. We mapped genome-wide chromatin contacts (HiC, H3K27ac, HiChIP), chromatin modifications (H3K4me3, H3K27ac, H3K27me3) and 10 TF binding profiles (heptad, PU.1, CTCF, STAG2) in HSPC subsets (stem/multipotent progenitors plus common myeloid, granulocyte macrophage, and megakaryocyte erythrocyte progenitors) and found TF occupancy and enhancer-promoter interactions varied significantly across cell types and were associated with cell-type-specific gene expression. Distinct regulatory elements were enriched with specific heptad-TF combinations, including stem-cell-specific elements with ERG, and myeloid- and erythroid-specific elements with combinations of FLI1, RUNX1, GATA2, TAL1, LYL1, and LMO2. Furthermore, heptad-occupied regions in HSPCs were subsequently bound by lineage-defining TFs, including PU.1 and GATA1, suggesting that heptad factors may prime regulatory elements for use in mature cell types. We also found that enhancers with cell-type-specific heptad occupancy shared a common grammar with respect to TF binding motifs, suggesting that combinatorial binding of TF complexes was at least partially regulated by features encoded in DNA sequence motifs. Taken together, this study comprehensively characterizes the gene regulatory landscape in rare subpopulations of human HSPCs. The accompanying data sets should serve as a valuable resource for understanding adult hematopoiesis and a framework for analyzing aberrant regulatory networks in leukemic cells.

Sox7-positive endothelial progenitors establish coronary arteries and govern ventricular compaction.

The cardiac endothelium influences ventricular chamber development by coordinating trabeculation and compaction. However, the endothelial-specific molecular mechanisms mediating this coordination are not fully understood. Here, we identify the Sox7 transcription factor as a critical cue instructing cardiac endothelium identity during ventricular chamber development. Endothelial-specific loss of Sox7 function in mice results in cardiac ventricular defects similar to non-compaction cardiomyopathy, with a change in the proportions of trabecular and compact cardiomyocytes in the mutant hearts. This phenotype is paralleled by abnormal coronary artery formation. Loss of Sox7 function disrupts the transcriptional regulation of the Notch pathway and connexins 37 and 40, which govern coronary arterial specification. Upon Sox7 endothelial-specific deletion, single-nuclei transcriptomics analysis identifies the depletion of a subset of Sox9/Gpc3-positive endocardial progenitor cells and an increase in erythro-myeloid cell lineages. Fate mapping analysis reveals that a subset of Sox7-null endothelial cells transdifferentiate into hematopoietic but not cardiomyocyte lineages. Our findings determine that Sox7 maintains cardiac endothelial cell identity, which is crucial to the cellular cross-talk that drives ventricular compaction and coronary artery development.

Quantitative trait and transcriptome analysis of genetic complexity underpinning cardiac interatrial septation in mice using an advanced intercross line.

Unlike single-gene mutations leading to Mendelian conditions, common human diseases are likely to be emergent phenomena arising from multilayer, multiscale, and highly interconnected interactions. Atrial and ventricular septal defects are the most common forms of cardiac congenital anomalies in humans. Atrial septal defects (ASD) show an open communication between the left and right atria postnatally, potentially resulting in serious hemodynamic consequences if untreated. A milder form of atrial septal defect, patent foramen ovale (PFO), exists in about one-quarter of the human population, strongly associated with ischaemic stroke and migraine. The anatomic liabilities and genetic and molecular basis of atrial septal defects remain unclear. Here, we advance our previous analysis of atrial septal variation through quantitative trait locus (QTL) mapping of an advanced intercross line (AIL) established between the inbred QSi5 and 129T2/SvEms mouse strains, that show extremes of septal phenotypes. Analysis resolved 37 unique septal QTL with high overlap between QTL for distinct septal traits and PFO as a binary trait. Whole genome sequencing of parental strains and filtering identified predicted functional variants, including in known human congenital heart disease genes. Transcriptome analysis of developing septa revealed downregulation of networks involving ribosome, nucleosome, mitochondrial, and extracellular matrix biosynthesis in the 129T2/SvEms strain, potentially reflecting an essential role for growth and cellular maturation in septal development. Analysis of variant architecture across different gene features, including enhancers and promoters, provided evidence for the involvement of non-coding as well as protein-coding variants. Our study provides the first high-resolution picture of genetic complexity and network liability underlying common congenital heart disease, with relevance to human ASD and PFO.

Decoding enhancer complexity with machine learning and high-throughput discovery.

Enhancers are genomic DNA elements controlling spatiotemporal gene expression. Their flexible organization and functional redundancies make deciphering their sequence-function relationships challenging. This article provides an overview of the current understanding of enhancer organization and evolution, with an emphasis on factors that influence these relationships. Technological advancements, particularly in machine learning and synthetic biology, are discussed in light of how they provide new ways to understand this complexity. Exciting opportunities lie ahead as we continue to unravel the intricacies of enhancer function.

Effect of Low-Concentration Atropine Eyedrops vs Placebo on Myopia Incidence in Children: The LAMP2 Randomized Clinical Trial.

Importance: Early onset of myopia is associated with high myopia later in life, and myopia is irreversible once developed. Objective: To evaluate the efficacy of low-concentration atropine eyedrops at 0.05% and 0.01% concentration for delaying the onset of myopia. Design, Setting, and Participants: This randomized, placebo-controlled, double-masked trial conducted at the Chinese University of Hong Kong Eye Centre enrolled 474 nonmyopic children aged 4 through 9 years with cycloplegic spherical equivalent between +1.00 D to 0.00 D and astigmatism less than -1.00 D. The first recruited participant started treatment on July 11, 2017, and the last participant was enrolled on June 4, 2020; the date of the final follow-up session was June 4, 2022. Interventions: Participants were assigned at random to the 0.05% atropine (n = 160), 0.01% atropine (n = 159), and placebo (n = 155) groups and had eyedrops applied once nightly in both eyes over 2 years. Main Outcomes and Measures: The primary outcomes were the 2-year cumulative incidence rate of myopia (cycloplegic spherical equivalent of at least -0.50 D in either eye) and the percentage of participants with fast myopic shift (spherical equivalent myopic shift of at least 1.00 D). Results: Of the 474 randomized patients (mean age, 6.8 years; 50% female), 353 (74.5%) completed the trial. The 2-year cumulative incidence of myopia in the 0.05% atropine, 0.01% atropine, and placebo groups were 28.4% (33/116), 45.9% (56/122), and 53.0% (61/115), respectively, and the percentages of participants with fast myopic shift at 2 years were 25.0%, 45.1%, and 53.9%. Compared with the placebo group, the 0.05% atropine group had significantly lower 2-year cumulative myopia incidence (difference, 24.6% [95% CI, 12.0%-36.4%]) and percentage of patients with fast myopic shift (difference, 28.9% [95% CI, 16.5%-40.5%]). Compared with the 0.01% atropine group, the 0.05% atropine group had significantly lower 2-year cumulative myopia incidence (difference, 17.5% [95% CI, 5.2%-29.2%]) and percentage of patients with fast myopic shift (difference, 20.1% [95% CI, 8.0%-31.6%]). The 0.01% atropine and placebo groups were not significantly different in 2-year cumulative myopia incidence or percentage of patients with fast myopic shift. Photophobia was the most common adverse event and was reported by 12.9% of participants in the 0.05% atropine group, 18.9% in the 0.01% atropine group, and 12.2% in the placebo group in the second year. Conclusions and Relevance: Among children aged 4 to 9 years without myopia, nightly use of 0.05% atropine eyedrops compared with placebo resulted in a significantly lower incidence of myopia and lower percentage of participants with fast myopic shift at 2 years. There was no significant difference between 0.01% atropine and placebo. Further research is needed to replicate the findings, to understand whether this represents a delay or prevention of myopia, and to assess longer-term safety. Trial Registration: Chinese Clinical Trial Registry: ChiCTR-IPR-15006883.

Distal regulation, silencers, and a shared combinatorial syntax are hallmarks of animal embryogenesis.

The chromatin environment plays a central role in regulating developmental gene expression in metazoans. Yet, the ancestral regulatory landscape of metazoan embryogenesis is unknown. Here, we generate chromatin accessibility profiles for six embryonic, plus larval and adult stages in the sponge Amphimedon queenslandica These profiles are reproducible within stages, reflect histone modifications, and identify transcription factor (TF) binding sequence motifs predictive of cis-regulatory elements operating during embryogenesis in other metazoans, but not the unicellular relative Capsaspora Motif analysis of chromatin accessibility profiles across Amphimedon embryogenesis identifies three major developmental periods. As in bilaterian embryogenesis, early development in Amphimedon involves activating and repressive chromatin in regions both proximal and distal to transcription start sites. Transcriptionally repressive elements ("silencers") are prominent during late embryogenesis. They coincide with an increase in cis-regulatory regions harboring metazoan TF binding motifs, as well as an increase in the expression of metazoan-specific genes. Changes in chromatin state and gene expression in Amphimedon suggest the conservation of distal enhancers, dynamically silenced chromatin, and TF-DNA binding specificity in animal embryogenesis.

Global retinoblastoma survival and globe preservation: a systematic review and meta-analysis of associations with socioeconomic and health-care factors.

BACKGROUND: Despite advancements in globe-preserving treatments, improvements in retinoblastoma outcomes are inconsistent across income levels and geographical locations. We aimed to investigate trends in global retinoblastoma survival and globe preservation during the past 40 years. We also examined associated socioeconomic and health-care factors and global survival disparity. METHODS: We did a systematic review and meta-analysis by screening articles in any language in nine databases (PubMed, Embase, ScienceDirect, Web of Science, OpenGrey, Global Burden of Disease, Global Health Data Exchange, Global Index Medicus, and International Agency for the Prevention of Blindness) published between Jan 1, 1981, and Oct 8, 2021. We screened for articles that described retinoblastoma overall survival or globe salvage, or both. All reported studies were subsequently stratified into four periods: 1980-89, 1990-99, 2000-09, and 2010-20. Indicators on socioeconomic and health-care factors were extracted from the World Bank and WHO. Ophthalmology-related indicators were further parsed from the International Agency for the Prevention of Blindness. Between-study heterogeneities by income level were assessed by mixed-effect meta-analysis. Associations of retinoblastoma outcome with socioeconomic and health-care factors and factors for survival prediction were investigated by multivariable linear regressions. This study is registered with PROSPERO, number CRD42020221556. FINDINGS: Our search identified 14 621 articles, of which 314 studies were included for analysis after screening, including 38 130 patients from 80 regions globally presenting during 1980-2020. 255 articles were entered for time-trend meta-analysis, covering 29 106 patients from 73 countries. Both overall survival (from 79% [95% CI 74-84] to 88% [83-93]; p=0·017) and globe salvage rate (from 22% [14-32] to 44% [36-52]; p=0·0003) improved significantly over the four decades. Wide disparities were observed between higher-income and lower-income countries. Overall survival, globe salvage, and globe salvage for advanced intraocular disease correlated positively with income level. Higher overall survival was associated with lower Gini index (p=0·0001) and with populations that had smaller percentages living in rural areas (p=0·0005). Higher globe salvage was associated with better health-care financing and accessibility (p=0·030). Overall survival (p=0·0024) and globe salvage (p=0·022) were both associated positively with education level. Survival gaps were observed in sub-Saharan Africa and southeast and southwest Asia. INTERPRETATION: Retinoblastoma treatment outcomes have improved globally over the past four decades but large disparities persist between higher-income and lower-income countries, with some areas having major survival gaps. Targeted health-care policy making with increased health-care financing and accessibility are needed in low-income and lower-middle-income countries to improve retinoblastoma outcomes worldwide. FUNDING: Health and Medical Research Fund (Hong Kong) and Children Cancer's Foundation (Hong Kong).

Delayed Diagnosis of Amblyopia in Children of Lower Socioeconomic Families: The Hong Kong Children Eye Study.

AIMS: To determine the prevalence of amblyopia and the factors associated with delayed diagnosis among school-aged children in Hong Kong. METHODS: Completed comprehensive ophthalmoscopic examinations were conducted among 4.273 children aged 6-8 years in the population-based Hong Kong Children Eye Study. New and previous diagnoses of amblyopia were identified. Socioeconomic status was surveyed through questionnaires. RESULTS: According to criteria from the Multi-Ethnic Pediatric Eye Disease Study (MEPEDS), amblyopia was present in 43 out of 4,273 children (1.01%; 95% CI, 0.71-1.31). Among them, 33 (0.77%) were newly detected cases that were more likely from families living in Public Rental Housing or subdivided flats (OR, 4.22; P = .012), with lower paternal (OR 6.68, P = .008) and maternal education levels (OR, 4.38; P = .039). Multivariate analysis revealed associations of amblyopia with hyperopia (≥+2.00D; OR, 6.23; 95% CI, 2.86-13.56, P < .001), astigmatism (≥+2.00D; OR, 24.88; 95% CI, 10.69-57.91, P < .001) and anisometropia (≥2.00D; OR, 14.49; 95% CI, 5.93-37.63, P < .001). CONCLUSION: These children with delayed diagnoses of amblyopia were more likely to come from lower socioeconomic backgrounds. Public education aimed at increasing awareness and utilization of child vision screening among such families is needed.

Non-genetic determinants of malignant clonal fitness at single-cell resolution.

All cancers emerge after a period of clonal selection and subsequent clonal expansion. Although the evolutionary principles imparted by genetic intratumour heterogeneity are becoming increasingly clear1, little is known about the non-genetic mechanisms that contribute to intratumour heterogeneity and malignant clonal fitness2. Here, using single-cell profiling and lineage tracing (SPLINTR)-an expressed barcoding strategy-we trace isogenic clones in three clinically relevant mouse models of acute myeloid leukaemia. We find that malignant clonal dominance is a cell-intrinsic and heritable property that is facilitated by the repression of antigen presentation and increased expression of the secretory leukocyte peptidase inhibitor gene (Slpi), which we genetically validate as a regulator of acute myeloid leukaemia. Increased transcriptional heterogeneity is a feature that enables clonal fitness in diverse tissues and immune microenvironments and in the context of clonal competition between genetically distinct clones. Similar to haematopoietic stem cells3, leukaemia stem cells (LSCs) display heritable clone-intrinsic properties of high, and low clonal output that contribute to the overall tumour mass. We demonstrate that LSC clonal output dictates sensitivity to chemotherapy and, although high- and low-output clones adapt differently to therapeutic pressure, they coordinately emerge from minimal residual disease with increased expression of the LSC program. Together, these data provide fundamental insights into the non-genetic transcriptional processes that underpin malignant clonal fitness and may inform future therapeutic strategies.

Prevalence of statin utilization and adherence among privately insured subjects in the Commonwealth of Puerto Rico.

BACKGROUND: Puerto Ricans are the Hispanic subgroup with the highest adjusted prevalence of statin-eligible patients. However, no study has described statin utilization and adherence among subjects living on the island of Puerto Rico. OBJECTIVES: To (a) estimate the prevalence of beneficiaries with diabetes aged between 40 and 75 years; (b) estimate the prevalence of statin utilization among beneficiaries with diabetes; and (c) estimate secondary adherence to statins among beneficiaries with diabetes. METHODS: With pharmacy claims data from a commercial pharmacy benefit manager (PBM) in the Commonwealth of Puerto Rico, this study used a retrospective longitudinal design to analyze all pharmacy claims generated by 115,674 beneficiaries aged between 40 and 75 years with continuous enrollment during 2018. Beneficiaries with diabetes were defined by having ≥ 2 pharmacy claims for antidiabetic agents during 2018. Statin utilization was defined by having ≥ 1 pharmacy claim for statins among beneficiaries with diabetes. The proportion of days covered (PDC) was used to measure secondary adherence to statins. Parametric and nonparametric statistics were used to describe statin utilization and adherence. RESULTS: The prevalence of beneficiaries with diabetes was 7.8%. Of the 8,975 beneficiaries with diabetes, 5,129 (57.1%) received ≥ 1 prescription for a statin. Older males with diabetes were more likely to receive prescriptions for statins. The median PDC for the 4,553 beneficiaries with ≥ 2 prescriptions for statins was 63.4%; 3,306 (72.6%) beneficiaries filled their statin prescriptions for a 30-day supply only; and 1,252 (27.5%) beneficiaries had a PDC ≥ 80%. The highest PDC (92.3%) was observed for beneficiaries who received statins for a 90-day supply only. CONCLUSIONS: This is the first study that has measured statin utilization and adherence among patients with diabetes living in Puerto Rico. The utilization and adherence to statins among privately insured beneficiaries with diabetes in Puerto Rico are suboptimal. Future studies should focus on understanding the reasons for the suboptimal use of statins and on potential interventions at the beneficiary and provider level to increase statin utilization. DISCLOSURES: No outside funding supported this study. The authors have no conflicts of interest or financial disclosures to disclose related to this study.

Cis-acting variation is common across regulatory layers but is often buffered during embryonic development.

Precise patterns of gene expression are driven by interactions between transcription factors, regulatory DNA sequences, and chromatin. How DNA mutations affecting any one of these regulatory "layers" are buffered or propagated to gene expression remains unclear. To address this, we quantified allele-specific changes in chromatin accessibility, histone modifications, and gene expression in F1 embryos generated from eight Drosophila crosses at three embryonic stages, yielding a comprehensive data set of 240 samples spanning multiple regulatory layers. Genetic variation (allelic imbalance) impacts gene expression more frequently than chromatin features, with metabolic and environmental response genes being most often affected. Allelic imbalance in cis-regulatory elements (enhancers) is common and highly heritable, yet its functional impact does not generally propagate to gene expression. When it does, genetic variation impacts RNA levels through two alternative mechanisms involving either H3K4me3 or chromatin accessibility and H3K27ac. Changes in RNA are more predictive of variation in H3K4me3 than vice versa, suggesting a role for H3K4me3 downstream from transcription. The impact of a substantial proportion of genetic variation is consistent across embryonic stages, with 50% of allelic imbalanced features at one stage being also imbalanced at subsequent developmental stages. Crucially, buffering, as well as the magnitude and evolutionary impact of genetic variants, is influenced by regulatory complexity (i.e., number of enhancers regulating a gene), with transcription factors being most robust to cis-acting, but most influenced by trans-acting, variation.

Deep conservation of the enhancer regulatory code in animals.

Interactions of transcription factors (TFs) with DNA regulatory sequences, known as enhancers, specify cell identity during animal development. Unlike TFs, the origin and evolution of enhancers has been difficult to trace. We drove zebrafish and mouse developmental transcription using enhancers from an evolutionarily distant marine sponge. Some of these sponge enhancers are located in highly conserved microsyntenic regions, including an Islet enhancer in the Islet-Scaper region. We found that Islet enhancers in humans and mice share a suite of TF binding motifs with sponges, and that they drive gene expression patterns similar to those of sponge and endogenous Islet enhancers in zebrafish. Our results suggest the existence of an ancient and conserved, yet flexible, genomic regulatory syntax that has been repeatedly co-opted into cell type-specific gene regulatory networks across the animal kingdom.

Association of Optical Coherence Tomography Angiography Metrics With Detection of Impaired Macular Microvasculature and Decreased Vision in Amblyopic Eyes: The Hong Kong Children Eye Study.

Importance: Microvascular abnormalities in amblyopia are becoming evident with high-resolution imaging, such as optical coherence tomography angiography (OCT-A); however, to our knowledge, the clinical significance and use of these findings are unknown. Objective: To assess changes in quantitative OCT-A metrics in amblyopic eyes and explore their association with visual acuity in children. Design, Setting, and Participants: This population-based nested case-control study included children aged 6 to 8 years who were consecutively recruited between January 2016 and July 2017 from the population-based Hong Kong Children Eye Study (HKCES) at the Chinese University of Hong Kong Eye Centre. All participants underwent OCT-A with a swept-source OCT and detailed ophthalmic investigations. Macular microvasculature of the superficial capillary plexus was quantified by a customized automated image analysis program. A multivariable linear regression was conducted to evaluate the differences in OCT-A metrics between amblyopic and nonamblyopic eyes after adjustment for all known confounders. Data analysis was conducted from September to November 2018. Main Outcomes and Measures: Differences in OCT-A metric (foveal avascular zone [FAZ]) area, FAZ circularity, vessel density, vessel diameter index, and fractal dimension between amblyopic and nonamblyopic eyes. Results: There were 30 participants with amblyopia (mean [SD] age, 7.57 [1.2] years; 16 girls [53.3%]) and 1045 controls (mean [SD] age, 7.65 [1.0] years; 580 girls [55.5%]) in this cohort. Compared with control eyes, amblyopic eyes had decreased FAZ circularity (-0.058; 95% CI, -0.096 to -0.021, P = .002), decreased fractal dimension (-0.014; 95% CI, -0.024 to -0.003; P = .01), and increased vessel diameter index (0.002; 95% CI, 0.002 to 0.003; P < .001). A difference was not identified between FAZ area and vessel density. LogMAR visual acuity was associated with FAZ circularity (sß, -0.133; P < .001) and vessel diameter index (sß, 0.097; P = .001) but not with vessel density nor FAZ area. Conclusions and Relevance: The results of this population-based study in children supports the presence of macular microvascular abnormalities in amblyopic eyes. Such changes as measured by OCT-A metrics are associated with visual acuity, inferring retinal involvement in the development of amblyopia and suggesting a potential role of quantitative OCT-A metrics in the diagnosis and recognition of amblyopia.

Single-Cell Transcriptomic Analysis of Cardiac Differentiation from Human PSCs Reveals HOPX-Dependent Cardiomyocyte Maturation.

Cardiac differentiation of human pluripotent stem cells (hPSCs) requires orchestration of dynamic gene regulatory networks during stepwise fate transitions but often generates immature cell types that do not fully recapitulate properties of their adult counterparts, suggesting incomplete activation of key transcriptional networks. We performed extensive single-cell transcriptomic analyses to map fate choices and gene expression programs during cardiac differentiation of hPSCs and identified strategies to improve in vitro cardiomyocyte differentiation. Utilizing genetic gain- and loss-of-function approaches, we found that hypertrophic signaling is not effectively activated during monolayer-based cardiac differentiation, thereby preventing expression of HOPX and its activation of downstream genes that govern late stages of cardiomyocyte maturation. This study therefore provides a key transcriptional roadmap of in vitro cardiac differentiation at single-cell resolution, revealing fundamental mechanisms underlying heart development and differentiation of hPSC-derived cardiomyocytes.

L-arginine and Arginase Products Potentiate Dexmedetomidine-induced Contractions in the Rat Aorta.

BACKGROUND: The α2-adrenergic sedative/anesthetic agent dexmedetomidine exerts biphasic effects on isolated arteries, causing endothelium-dependent relaxations at concentrations at or below 30 nM, followed by contractions at higher concentrations. L-arginine is a common substrate of endothelial nitric oxide synthase and arginases. This study was designed to investigate the role of L-arginine in modulating the overall vascular response to dexmedetomidine. METHODS: Isometric tension was measured in isolated aortic rings of Sprague Dawley rats. Cumulative concentrations of dexmedetomidine (10 nM to 10 µM) were added to quiescent rings (with and without endothelium) after previous incubation with vehicle, N-nitro-L-arginine methyl ester hydrochloride (L-NAME; nitric oxide synthase inhibitor), prazosin (α1-adrenergic antagonist), rauwolscine (α2-adrenergic antagonist), L-arginine, (S)-(2-boronethyl)-L-cysteine hydrochloride (arginase inhibitor), N-hydroxy-L-arginine (arginase inhibitor), urea and/or ornithine. In some preparations, immunofluorescent staining, immunoblotting, or measurement of urea content were performed. RESULTS: Dexmedetomidine did not contract control rings with endothelium but evoked concentration-dependent increases in tension in such rings treated with L-NAME (Emax 50 ± 4%) or after endothelium-removal (Emax 74 ± 5%; N = 7 to 12). Exogenous L-arginine augmented the dexmedetomidine-induced contractions in the presence of L-NAME (Emax 75 ± 3%). This potentiation was abolished by (S)-(2-boronethyl)-L-cysteine hydrochloride (Emax 16 ± 4%) and N-hydroxy-L-arginine (Emax 18 ± 4%). Either urea or ornithine, the downstream arginase products, had a similar potentiating effect as L-arginine. Immunoassay measurements demonstrated an upregulation of arginase I by L-arginine treatment in the presence of L-NAME (N = 4). CONCLUSIONS: These results suggest that when vascular nitric oxide homeostasis is impaired, the potentiation of the vasoconstrictor effect of dexmedetomidine by L-arginine depends on arginase activity and the production of urea and ornithine.

Corneal backward scattering and higher-order aberrations in children with vernal keratoconjunctivitis and normal topography.

PURPOSE: To investigate the corneal backward scattering and higher-order aberrations (HOAs) in children with vernal keratoconjunctivitis (VKC) and normal topography. METHODS: Thirty-six eyes of 22 patients with VKC and 54 eyes of 34 normal subjects were included. All participants had clear cornea, absence of dry eyes and a normal corneal tomography. Scheimpflug imaging was used to measure corneal backward scattering in zones centred on the corneal apex (central 2-mm zone and paracentral 2- to 6-mm zone), and HOAs were compared between VKC and normal control. RESULTS: The mean age of participants was 12.0 ± 4.1 years in VKC group and 11.2 ± 4.1 years in control group (p = 0.339). There was no significant intergroup difference in mean keratometry, astigmatism and apex pachymetry (p ≥ 0.076). Total corneal backscatter was higher in the VKC group compared to the control group (p ≤ 0.012). Anterior and posterior cornea displayed a higher level of backward scattering in the VKC group (p < 0.001 for anterior; p ≤ 0.048 for posterior). Patients with VKC exhibited higher total HOAs and coma (p ≤ 0.036). There were significant correlations between total anterior HOAs and backward scattering measured at the central (r = 0.500; p = 0.032) and paracentral zones (r = 0.470; p = 0.024) for VKC. CONCLUSION: The current study showed optical quality changes in patients with clear corneas and quiescent VKC. An increase in corneal backward scattering and HOAs was noted in patients with VKC as compared to normal patients.

Primary vs Secondary Endoscopic Dacryocystorhinostomy for Acute Dacryocystitis With Lacrimal Sac Abscess Formation: A Randomized Clinical Trial.

Importance: Endoscopic dacryocystorhinostomy (EN-DCR) is emerging as the preferred procedure in the management of nasolacrimal duct obstructions. However, its safety and long-term efficacy in the setting of acute dacryocystitis with lacrimal sac abscess have not been well studied. Objective: To compare outcomes of EN-DCR as primary treatment with EN-DCR as a secondary treatment after percutaneous drainage of lacrimal sac abscess in acute dacryocystitis. Design, Setting, and Participants: This randomized clinical trial was conducted from October 1, 2012, to October 31, 2015, at a tertiary ophthalmic center. The assessors of success at postoperative year 1 were masked to the procedures received by the participants. All surgical procedures were performed by 2 oculoplastic surgeons with different levels of EN-DCR experience. Eligible participants had acute dacryocystitis and lacrimal sac abscess presenting within 2 weeks of onset, who were 18 to 90 years of age. Analysis was of the intention-to-treat population. Interventions: Patients were allocated by block randomization to receive either percutaneous drainage of lacrimal sac abscess followed by EN-DCR after the acute episode subsided (control group) or primary EN-DCR within 2 weeks of presentation (intervention group). Both groups received a course of empirical systemic antibiotics (amoxicillin and clavulanic acid, 375 mg, to be taken 3 times a day for 1 week). Main Outcomes and Measures: Primary outcomes were time from presentation to documentation of symptom resolution and recurrence within 3 months. Results: Thirty-two patients were randomized equally into 2 treatment arms (control and intervention). The mean (SD) age of patients was 61 (13) years, and there was a predominance of women (27 [84%]). The mean (SD) time to symptom resolution was 13.8 (5.8) days in the intervention group compared with 31.7 (27.1) days in the control group (mean difference, 17.9; 95% CI, 3.71-32.01; P = .02). The mean (SD) time to surgery in the intervention group was shorter at 11.9 (6.3) days compared with 45.6 (30.1) days in the control group (mean difference, 33.6; 95% CI, 17.92-49.33; P < .001). Recurrences occurred once in the control group and did not occur in the intervention group. No differences in operation time and complications between the 2 groups were identified. The anatomical and functional success was 87.5% (14 of 16 cases) in both groups at postoperative year 1. Conclusions and Relevance: Primary EN-DCR in acute dacryocystitis with lacrimal sac abscess results in faster resolution compared with secondary treatment. No differences in recurrence, safety, or outcomes at postoperative year 1 were noted between the 2 treatment groups.