Survey of ESCRS members' attitudes toward operating room waste.

In a survey of ESCRS member cataract surgeons, 92% felt that operating room waste is excessive and should be reduced; 99% were concerned about global warming and climate change. Most respondents cited restrictions on reuse by manufacturers and regulatory bodies as major drivers of this waste. There was a strong desire to have more reusable options for instruments, devices, and supplies. In comparable percentages with an earlier survey of North American cataract surgeons using the identical questionnaire, there was a strong willingness to reuse many surgical supplies, as well as topical and intraocular medications. This was true even though ESCRS members were much more likely to practice in hospitals (68% vs 35%). The similarities of these results to the North American survey suggest that these attitudes toward sustainability are in fact global and universal. The strong concordance between the 2 surveys suggests that global collaboration is both possible and necessary.

Rapid assay to detect possible natural substrates of proteases in living cells.

Proteolysis is a regulatory step in many physiological processes, but which proteases in what cellular sites are involved in activation or degradation of which peptides is not well known. We developed a rapid assay consisting of living cells and fluorogenic protease substrates to determine which bioactive peptides are possible natural substrates of a specific protease with the multifunctional or moonlighting protein CD26/dipeptidyl peptidase IV (DPPIV) as a model. CD26/DPPIV catalyzes cleavage of peptides from the amino terminus of peptides with proline at the penultimate position. Many biologically active peptides, such as beta-casomorphin1-5, contain proline in the penultimate position. We incubated living Jurkat cells, which are T cells that lack CD26/DPPIV, and CD26/DPPIV-transfected Jurkat cells in the presence of the fluorogenic substrate [Ala-Pro]2-cresyl violet (Magic Red) and beta-casomorphin1-5. Fluorescent cresyl violet was generated by CD26/DPPIV-transfected Jurkat cells but not by wild-type Jurkat cells with a Km of 3.7 microM. beta-Casomorphin1-5 appeared to be a possible natural substrate of CD26/DPPIV, because it inhibited production of fluorescence competitively (Ki = 60 microM). The assay using living cells and a fluorogenic protease substrate is an efficient system to determine whether specific peptides are possible natural substrates of a particular protease.

Fluorogenic substrate [Ala-Pro]2-cresyl violet but not Ala-Pro-rhodamine 110 is cleaved specifically by DPPIV activity: a study in living Jurkat cells and CD26/DPPIV-transfected Jurkat cells.

Fluorogenic substrates [Ala-Pro](2)-cresyl violet and Ala-Pro-rhodamine 110 have been tested for microscopic detection of protease activity of dipeptidyl peptidase IV (DPPIV) in living cells. DPPIV activity is one of the many functions of the multifunctional or moonlighting protein CD26/DPPIV. As a model we used Jurkat cells, which are T-cells that lack CD26/DPPIV expression, and CD26/DPPIV-transfected Jurkat cells. Ala-Pro-rhodamine 110 is not fluorescent, but after proteolytic cleavage rhodamine 110 fluoresces. [Ala-Pro](2)-cresyl violet is fluorescent by itself but proteolytic cleavage into cresyl violet induces a shift to longer wavelengths. This phenomenon enables the simultaneous determination of local (intracellular) substrate and product concentrations, which is important for analysis of kinetics of the cleavage reaction. [Ala-Pro](2)-cresyl violet, but not Ala-Pro-rhodamine 110, appeared to be specific for DPPIV. When microscopic analysis is performed on living cells during the first minutes of the enzyme reaction, DPPIV activity can be precisely localized in cells with the use of [Ala-Pro](2)-cresyl violet. Fluorescent product is rapidly internalized into submembrane granules in transfected Jurkat cells and is redistributed intracellularly via internalization pathways that have been described for CD26/DPPIV. We conclude that [Ala-Pro](2)-cresyl violet is a good fluorogenic substrate to localize DPPIV activity in living cells when the correct wavelengths are used for excitation and emission and images are captured in the early stages of the enzyme reaction.