Increasing frequencies of site-specific mutagenesis and gene targeting in Arabidopsis by manipulating DNA repair pathways.

Improved methods for engineering sequence-specific nucleases, including zinc finger nucleases (ZFNs) and TAL effector nucleases (TALENs), have made it possible to precisely modify plant genomes. However, the success of genome modification is largely dependent on the intrinsic activity of the engineered nucleases. In this study, we sought to enhance ZFN-mediated targeted mutagenesis and gene targeting (GT) in Arabidopsis by manipulating DNA repair pathways. Using a ZFN that creates a double-strand break (DSB) at the endogenous ADH1 locus, we analyzed repair outcomes in the absence of DNA repair proteins such as KU70 and LIG4 (both involved in classic nonhomologous end-joining, NHEJ) and SMC6B (involved in sister-chromatid-based homologous recombination, HR). We achieved a fivefold to 16-fold enhancement in HR-based GT in a ku70 mutant and a threefold to fourfold enhancement in GT in the lig4 mutant. Although the NHEJ mutagenesis frequency was not significantly changed in ku70 or lig4, DNA repair was shifted to microhomology-dependent alternative NHEJ. As a result, mutations in both ku70 and lig4 were predominantly large deletions, which facilitates easy screening for mutations by PCR. Interestingly, NHEJ mutagenesis and GT at the ADH1 locus were enhanced by sixfold to eightfold and threefold to fourfold, respectively, in a smc6b mutant. The increase in NHEJ-mediated mutagenesis by loss of SMC6B was further confirmed using ZFNs that target two other Arabidopsis genes, namely, TT4 and MPK8. Considering that components of DNA repair pathways are highly conserved across species, mutations in DNA repair genes likely provide a universal strategy for harnessing repair pathways to achieve desired targeted genome modifications.

The Gos28 SNARE protein mediates intra-Golgi transport of rhodopsin and is required for photoreceptor survival.

SNARE proteins play indispensable roles in membrane fusion events in many cellular processes, including synaptic transmission and protein trafficking. Here, we characterize the Golgi SNARE protein, Gos28, and its role in rhodopsin (Rh1) transport through Drosophila photoreceptors. Mutations in gos28 lead to defective Rh1 trafficking and retinal degeneration. We have pinpointed a role for Gos28 in the intra-Golgi transport of Rh1, downstream from α-mannosidase-II in the medial- Golgi. We have confirmed the necessity of key residues in Gos28's SNARE motif and demonstrate that its transmembrane domain is not required for vesicle fusion, consistent with Gos28 functioning as a t-SNARE for Rh1 transport. Finally, we show that human Gos28 rescues both the Rh1 trafficking defects and retinal degeneration in Drosophila gos28 mutants, demonstrating the functional conservation of these proteins. Our results identify Gos28 as an essential SNARE protein in Drosophila photoreceptors and provide mechanistic insights into the role of SNAREs in neurodegenerative disease.

Outcomes of Femtosecond Laser Assisted Cataract Surgery Performed by Resident and Attending Surgeons.

Objective The study aimed to evaluate the safety and efficacy of resident surgeons performing femtosecond laser assisted cataract surgery (FLACS). Methods A retrospective chart review was conducted at the University of Wisconsin-Madison from postgraduate year four residents performing FLACS between 2017 and 2019. Data were also collected from residents performing manual cataract surgery, and attending surgeons performing FLACS for comparison. Recorded data included patient demographics, pre- and postoperative visual acuity, pre- and postoperative spherical equivalent, nuclear sclerotic cataract grade, ocular and systemic comorbidities, intraocular lens, duration of surgery, cumulative dissipated energy (CDE), and intraoperative and postoperative complications. Results A total of 90 cases were reviewed with 30 resident manual cases, 30 resident FLACS cases, and 30 attending FLACS cases. Resident manual (25.5 ± 6.8 minutes) and resident FLACS (17.5 ± 7.1 minutes) cases took a significantly longer time to complete compared with attending FLACS cases (13.6 ± 4.4 minutes; p < 0.001). There was higher CDE in resident FLACS and resident manual cases compared with attending FLACS cases, but the difference was not statistically significant ( p = 0.06). Postoperative visual acuity was not statistically different at 1-day and 1-month after surgery among the three groups. Resident FLACS complications, which included one case requiring an intraoperative suture to close the wound, two cases with intraoperative corneal abrasions, two cases with postoperative ocular hypertension, and one case with cystoid macular edema, were not significantly greater than attending FLACS complications ( p = 0.30). Conclusion The FLACS performed by resident surgeons had comparable visual acuity outcomes to FLACS performed by attending surgeons, and to manual cataract surgery performed by resident surgeons. However, resident FLACS cases took significantly longer time to complete, and they were associated with a higher CDE and minor complication rate compared with attending FLACS cases. Introducing advanced technologies into surgical training curricula improves resident preparedness for independent practice, and this study suggests FLACS can be incorporated safely and effectively into resident education.

Quantification of Geographic Atrophy Using Spectral Domain OCT in Age-Related Macular Degeneration.

PURPOSE: To compare geographic atrophy (GA) area and enlargement rate measured with spectral domain OCT (SD-OCT) with fundus autofluorescence (FAF), color fundus photography (CFP), and infrared reflectance (IR) imaging. DESIGN: Retrospective, multicenter, natural history case series. PARTICIPANTS: A total of 70 eyes with GA from 48 participants were included. METHODS: Participants underwent SD-OCT, FAF, CFP, and IR imaging at baseline and 12 months in the study eye. Spectral domain OCT images were graded for GA area using 2 distinct criteria: (1) complete retinal pigment epithelium and outer retinal atrophy (cRORA) and (2) hypertransmission through Bruch's membrane. Areas were measured with SD-OCT using a custom-developed tool that allows for mapping areas of retinal layer loss on SD-OCT cross-sectional scans with co-registered IR images. Circularity index was calculated from area and perimeter. Spectral domain OCT images were also assessed for presence of reticular pseudodrusen, outer-retinal tubules, and hyporeflective wedge-shaped bands. Area of GA was measured in millimeters squared from FAF, CFP, and IR images. MAIN OUTCOME MEASURES: Geographic atrophy mean baseline area and enlargement rate measured with 2 SD-OCT criteria, FAF, CFP, and IR. RESULTS: At baseline, the mean GA area was 6.9 (standard deviation [SD], 4.7) mm2 using the SD-OCT cRORA criteria and 7.3 (SD, 4.7) mm2 using the SD-OCT hypertransmission criteria. The mean annual enlargement rate of GA was 1.6 (SD, 1.1) mm2 using the SD-OCT cRORA criteria and 1.5 (SD, 1.0) mm2 using the SD-OCT hypertransmission criteria. When comparing both SD-OCT grading criteria with FAF, CFP, and IR, there were no statistically significant differences in baseline area or annual enlargement rate of GA. Circularity index was identified as the risk factor for increased annual enlargement rate. CONCLUSIONS: Measuring GA using retinal layer morphology provides a novel means of obtaining area measurements. Area measurements tend to vary based on criteria used and are comparable to other imaging modalities.

Reticular Pseudodrusen Characteristics and Associations in the Carotenoids in Age-Related Eye Disease Study 2 (CAREDS2), an Ancillary Study of the Women's Health Initiative.

PURPOSE: To determine the prevalence and morphologic features of reticular pseudodrusen (RPD) and their association with participant demographics and age-related macular degeneration (AMD) status in the Carotenoids in Age-Related Eye Disease Study 2 (CAREDS2) sample, an ancillary study of the Women's Health Initiative Observational Study. DESIGN: Cross-sectional, multicenter, natural history study. PARTICIPANTS: Nine hundred and twenty-seven eyes from 466 postmenopausal women 69 to 101 years of age. METHODS: Multimodal imaging, including spectral-domain (SD) OCT and infrared reflectance (IR), were used to identify RPD characteristics, including location (within or outside the 6-mm diameter circle centered at the macula), presence of peripapillary RPD, pattern of RPD, and RPD area. Age-related macular degeneration features from SD OCT, IR, and color photographs also were assessed and AMD severity was categorized. MAIN OUTCOME MEASURES: Reticular pseudodrusen prevalence using SD OCT and IR imaging and AMD status. RESULTS: Reticular pseudodrusen were present in 130 eyes (14% of eyes, 16% of participants), with increasing prevalence with age: 7% in those younger than 78 years, 14% in those 78 to 83 years of age, and 30% in those older than 83 years. Using clinical classification of AMD with color photography, RPD were seen in 2.4% of eyes with no AMD or aging changes, 11.5% in early AMD, 25.1% in intermediate AMD, and 51.1% in late AMD. Mean RPD area was 17.4 mm2 (standard deviation, 14.7 mm2). Ribbon morphologic RPD (53%) was more common than dot morphologic RPD (36%). Reticular pseudodrusen mostly were located both within and outside the 6-mm circle with primarily superior retinal distribution. Reticular pseudodrusen were visualized with corresponding color fundus photography in only 38 eyes (4% of total eyes). Participants with and without RPD had a visual acuity±standard error of 77.9 ± 1.4 letters and 81.3 ± 0.4 letters, respectively (P = 0.02). CONCLUSIONS: The prevalence of RPD in CAREDS2 increased with age and was associated with AMD severity. Reticular pseudodrusen were detected in eyes without other features of AMD and could represent an earlier disease state. Multimodal imaging with SD OCT and IR has significantly greater sensitivity for visualizing RPD than color fundus photography.