CRX ChIP-seq reveals the cis-regulatory architecture of mouse photoreceptors.

Approximately 98% of mammalian DNA is noncoding, yet we understand relatively little about the function of this enigmatic portion of the genome. The cis-regulatory elements that control gene expression reside in noncoding regions and can be identified by mapping the binding sites of tissue-specific transcription factors. Cone-rod homeobox (CRX) is a key transcription factor in photoreceptor differentiation and survival, but its in vivo targets are largely unknown. Here, we used chromatin immunoprecipitation with massively parallel sequencing (ChIP-seq) on CRX to identify thousands of cis-regulatory regions around photoreceptor genes in adult mouse retina. CRX directly regulates downstream photoreceptor transcription factors and their target genes via a network of spatially distributed regulatory elements around each locus. CRX-bound regions act in a synergistic fashion to activate transcription and contain multiple CRX binding sites which interact in a spacing- and orientation-dependent manner to fine-tune transcript levels. CRX ChIP-seq was also performed on Nrl(-/-) retinas, which represent an enriched source of cone photoreceptors. Comparison with the wild-type ChIP-seq data set identified numerous rod- and cone-specific CRX-bound regions as well as many shared elements. Thus, CRX combinatorially orchestrates the transcriptional networks of both rods and cones by coordinating the expression of photoreceptor genes including most retinal disease genes. In addition, this study pinpoints thousands of noncoding regions of relevance to both Mendelian and complex retinal disease.

Novel Guarded Needle Trans-Scleral Biopsy for Uveal Melanoma: A Pilot Study.

INTRODUCTION: Trans-scleral biopsy of uveal melanoma (UM) poses an inherent risk of tumor and possibly retinal perforation. We describe a novel technique for trans-scleral biopsy of UM and evaluate its safety and efficacy in an initial cohort of patients. METHODS: A retrospective, consecutive observational case series was conducted from October 14, 2019, to April 15, 2020, at Kaiser Permanente, San Francisco, CA among patients with UM of the ciliary body or anterior choroid undergoing trans-scleral fine-needle aspiration biopsy using a novel guarded needle technique. RESULTS: A total of 6 patients were included in the study, with a mean age of 64.3 (range 35-77) years (5 women 83%). Mean (±SD) tumor thickness and maximal basal diameter were 6.4 (±2.66) and 11.9 (±2.13) mm, respectively. Five out of 6 patients achieved a successful biopsy with reliable gene expression profiling (GEP) results. The only failure to obtain specimen occurred in the first attempted patient and, after a minor technique modification, all subsequent biopsies were successful. No intraoperative or short-term postoperative complications were observed in any patient. CONCLUSION: This novel trans-scleral biopsy technique appears to be safe and effective when obtaining UM tissue for GEP. This method may provide a more controlled biopsy depth thereby minimizing the risk of tumor perforation and its associated complications while still obtaining adequate biopsy yield.

Measuring Forward Light Scatter by the Boston Keratoprosthesis in Various Configurations.

PURPOSE: Light scatter results in degradation of visual function. An optical bench model was used to identify the origins of scatter in the setting of a Boston keratoprosthesis (KPro). The effect of various modifications in the device design and light-blocking configurations was explored. METHODS: A KPro was mounted on a contact lens holder on a bench, and forward light scatter was recorded with a camera attached to a rotating goniometer arm. Scattered light was recorded at different angles for different KPro modifications, and the point-spread function (PSF) curves were recorded. The area under the curve (AUC) was calculated for each PSF curve. RESULTS: The isolated KPro optical cylinder in a totally blackened holding lens had a tight PSF (AUC = 3.3). Additional blackening of the walls of the KPro stem did not further diminish forward scatter significantly. If the holding lens is made translucent by sandblasting (to simulate an in vivo carrier cornea) and the KPro is inserted without a backplate, forward scatter is substantial (AUC = 11.3). If a standard backplate (with holes) is added, light scatter is considerably reduced regardless of whether the backplate is made of polymethyl methacrylate or titanium (AUC = 5.3 and 4.4, respectively). Addition of an acrylic intraocular lens behind the KPro (the pseudophakic KPro setup) did not increase scatter. CONCLUSIONS: Most of the scattered light in eyes implanted with a KPro originates from the surrounding hazy corneal graft. The standard addition of a backplate reduces light scatter. There was no difference in forward light scatter between the aphakic and the pseudophakic KPro.