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While the bioinformatics resource-tool iSyTE (integrated Systems Tool for Eye gene discovery) effectively identifies human cataract-associated genes, it is currently based on just transcriptome data, and thus it is necessary to include protein-level information to gain greater confidence in gene prioritization. Here we expand iSyTE through development of a novel proteome-based resource on the lens and demonstrate its utility in cataract gene discovery. We applied highthroughput tandem mass spectrometry (MS/MS) to generate a global protein expression profile of mouse lens at embryonic day (E)14.5, which identified 2371 lens-expressed proteins. A major challenge of high-throughput expression profiling is identification of high-priority candidates among the thousands of expressed proteins. To address this problem, we generated new MS/MS proteome data on mouse whole embryonic body (WB). WB proteome was then used as a reference dataset for performing "in silico WB-subtraction" comparative analysis with the lens proteome, which effectively identified 422 proteins with lens-enriched expression at ≥2.5 average spectral counts, ≥2.0 fold-enrichment (FDR <0.01) cut-off. These top 20% candidates represent a rich pool of high-priority proteins in the lens including known human cataract-linked genes and many new potential regulators of lens development and homeostasis. This rich information is made publicly accessible through iSyTE (https://research.bioinformatics.udel.edu/iSyTE/), which enables user-*

Purpose Age-related cataracts affect the majority of older adults and are a leading cause of blindness worldwide. Treatments that delay cataract onset or severity have the potential to delay cataract surgery, but require relevant animal models that recapitulate the major types of cataracts for their development. Unfortunately, few such models are available. Here, we report the lens phenotypes of aged mice lacking the critical antioxidant transcription factor Nfe2l2 (designated as Nrf2 −/−). Methods Three independent cohorts of Nrf2 −/− and wild-type C57BL/6J mice were evaluated for cataracts using combinations of slit lamp imaging, photography of freshly dissected lenses, and histology. Mice were fed high glycemic diets, low glycemic diets, regular chow ad libitum, or regular chow with 30% caloric restriction. Results Nrf2 −/− mice developed significant opacities between 11 and 15 months and developed advanced cortical, posterior subcapsular, anterior subcapsular, and nuclear cataracts. Cataracts occurred similarly in male mice fed high or low glycemic diets, and were also observed in 21-month male and female Nrf2 −/− mice fed ad libitum or 30% caloric restriction. Histological observation of 18-month cataractous lenses revealed significant disruption to fiber cell architecture and the retention of nuclei throughout the cortical region of the lens. However, fiber cell denucleation and initiation of lens differentiation was normal at birth, with the first abnormalities observed at 3 months. Conclusions Nrf2 −/− mice offer a tool to understand how defective antioxidant signaling causes multiple forms of cataract and may be useful for screening drugs to prevent or delay cataractogenesis in susceptible adults.

The transparent ocular lens in the anterior chamber of the eye is responsible for fine focusing of light onto the retina. The lens is entirely cellular with bulk of the tissue composed of fiber cells, and the anterior hemisphere of the lens is covered by a monolayer of epithelial cells. Lens epithelial cells are important for maintaining fiber cell homeostasis and for continual growth of the lens tissue throughout life. Cataracts, defined as any opacity in the lens, remain the leading cause of blindness in the world. Following cataract surgery, lens epithelial cells can undergo a process of epithelial-to-mesenchymal transition (EMT), leading to secondary cataracts due to posterior capsular opacification (PCO). Since the epithelial cells make up only a small fraction of the lens, specialized techniques are required to study lens epithelial cell biology and pathology. Studies using native lens epithelial cells often require pooling of samples to obtain enough cells to make sufficient samples for traditional molecular biology techniques. Here, we provide detailed protocols that enable the study of native mouse lens epithelial cells, including immunostaining of the native lens epithelium in flat mounts, extraction of RNA and proteins from pairs of lens epithelial monolayers, and isolation of lens epithelial cells for primary culture. These protocols will enable researchers to gain better insight on representative molecular expression and cellular structure of lens epithelial cells. We also provide comparative data between native, primary culture, and immortalized lens epithelial cells and discuss the advantages and disadvantages of each technique presented.