Kynurenine pathway metabolites and ascorbate degradation products are present in human lenses. In this study, we showed that erythrulose, a major ascorbate degradation product, reacts spontaneously with 3-hydroxykynurenine to form a fluorescent product. Structural characterization of the product revealed it to be 2-amino-4-(2-hydroxy-3-(2-hydroxyethyl)-2H-benzo[b][1, 4]oxazin-5-yl)-4-oxobutanoic acid, which we named kynoxazine. Unlike 3-hydroxykynurenine, 3-hydroxykynurenine glucoside and kynurenine were unable to form a kynoxazine-like compound, which suggested that the aminophenol moiety in 3-hydroxykynurenine is essential for the formation of kynoxazine. This reasoning was confirmed using a model compound, 1-(2-amino-3-hydroxyphenyl)ethan-1-one, which is an aminophenol lacking the amino acid moiety of 3-hydroxykynurenine. Ultra-performance liquid chromatography-tandem mass spectrometry analyses showed that kynoxazine is present in the human lens at levels ranging from 0 to 64 pmol/mg lens. Kynoxazine as well as erythrulose degraded under physiological conditions to generate 3-deoxythreosone, which modified and cross-linked proteins through the formation of an arginine adduct, 3-deoxythreosone-derived hydroimidazolone, and a lysine-arginine cross-linking adduct, 3-deoxythreosone-derived hydroimidazolimine cross-link. Ultra-performance liquid chromatography-tandem mass spectrometry quantification showed that 32-169 pmol/mg protein of 3-deoxythreosone-derived hydroimidazolone and 1.1-11.2 pmol/mg protein of 3-deoxythreosone-derived hydroimidazolimine cross-link occurred in aging lenses. Taken together, these results demonstrate a novel biochemical mechanism by which ascorbate oxidation and the kynurenine pathway intertwine, which could promote protein modification and cross-linking in aging human lenses.

With aging, human lenses lose the ability to focus on nearby objects due to decreases in accommodative ability, a condition known as presbyopia. An increase in stiffness or decrease in lens elasticity due to protein aggregation and insolubilization are the primary reasons for presbyopia. In this study, we tested aggrelyte-1 (S, N-diacetyl glutathione diethyl ester) for its ability to promote protein solubility and decrease the stiffness of lenses through its dual property of lysine acetylation and disulfide reduction. Treatment of water-insoluble proteins from aged human lenses (58–75 years) with aggrelyte-1 significantly increased the solubility of those proteins. A control compound that did not contain the S-acetyl group (aggrelyte-1C) was substantially less efficient in solubilizing water-insoluble proteins. Aggrelyte-1-treated solubilized protein had significant amounts of acetyllysine, as measured by Western blotting and LC-MS/MS. Aggrelytes increased the protein-free thiol content in the solubilized protein. Aged mouse (7 months) and human (44-66 years) lenses treated with aggrelyte-1 showed reduced stiffness accompanied by higher free thiol and acetyllysine levels compared with those treated with aggrelyte-1C or untreated controls. Our results suggested that aggrelyte-1 reduced lens stiffness through acetylation followed by disulfide reduction. This proof-of-concept study paves the way for developing aggrelyte-1 and related compounds to reverse presbyopia.

Objective To allow medical undergraduate students an exposure to ophthalmology in the preclinical years as well as introduce concepts of basic and clinical science in ophthalmology for medical students. Methods The 10-session elective was offered to 2nd year medical students in the fall of 2016 and to 1st and 2nd year medical students in the fall of 2017 at the University of Colorado School of Medicine. The curriculum included a dissection laboratory, lectures, and journal reviews of key topics in ophthalmology with a basic scientist and clinician. At the conclusion of the sessions, the students evaluated this course by survey. Results Six medical students participated in the fall of 2016 and 11 students in the fall of 2017. The response rate was 83.33 and 100%, respectively. On a five- point Likert's scale, the students in both fall 2016 and 2017 rated the course as 4.7, indicating a positive reaction. Quality of learning objectives was rated as 4.4 and 4.5 in 2016 and 2017, respectively. Course management had a score of 4.4 and 4.6 in 2016 and 2017, respectively. Comments included: “I learned a lot about the eye I would not have known if I had not taken the course,” “I enjoyed the interplay between the clinical and basic science experts,” and “I liked the model of learning about a subject then looking at the research [sic].” Conclusions Based on the students' responses and level of satisfaction, we concluded that the elective course was successful at increasing medical students' exposure to ophthalmology at the University of Colorado School of Medicine while incorporating both basic and clinical science. Based on review of the students' feedback, modifications to the course included, expanding the course to 1st year medical students, limiting presentation times, simplifying presentation topics, and adding worksheets to guide article review sessions.

The original version of this article unfortunately contained a mistake in Fig. 5a. The author inadvertently used the same lower panels for Figs. 4 and 5a.The online version of the original article can be found at http://dx.doi.