Cardiac Myosin Binding Protein-C (MyBP-C) is a thick filament-associated protein of the sarcomere and a potential therapeutic target for treating heart failure. We hypothesize that PKA-mediated phosphorylation of human MyBP-C alters the structure of its N terminus, which leads to reduced myofilament binding and enhanced contractility. Mimicking the structural state of phosphorylated MyBP-C by small-molecule drugs could lead to therapy that optimizes MyBP-C function in the failing heart. Therefore, we have developed site-directed probes in MyBP-C capable of detecting structural changes due to phosphorylation using Time-Resolved FRET (TR-FRET). Site-directed mutagenesis of MyBP-C N-terminal domains C0 through C2 (C0-C2) was used to remove endogenous cysteines and introduce a pair of cysteines at strategic positions for labeling with donor and acceptor dyes. Replacement of native residues did not perturb binding to actin or myosin, verifying that the mutant C0-C2 was functional. TR-FRET results revealed that phosphorylation-mediated structural changes were most pronounced between a positively-charged loop in the C1 domain that interacts with tropomyosin and the tri-helix bundle region in the phosphorylatable M-domain, suggesting an important role for these regions in MyBP-C function. FRET efficiency changed by ∼17% between unphosphorylated and phosphorylated samples with a standard deviation of ∼0.5%. Fit to a Gaussian model, this corresponds to an increase in the distance between the two probes by ∼1.3 Å and an increase in the width of the distribution by ∼50%. The dynamic range and precision of the phosphorylation-mediated changes demonstrate excellent assay functionality for the purposes of high-throughput screening. These findings provide new molecular insight into the regulatory role of MyBP-C in contractility and demonstrate the potential of C0-C2 biosensors for structure-based screening of compounds in search of MyBP-C-targeted therapies for heart failure.

PURPOSE:To describe a case of corneal ulceration associated with Nivolumab use.OBSERVATIONS:An 80-year-old woman treated with Nivolumab for metastatic melanoma developed an intractable corneal ulcer in her left eye, refractory to all therapies - including surgery to cover the ulcer with a conjunctival flap - until topical prednisolone acetate was tried, which was curative.CONCLUSIONS AND IMPORTANCE:Nivolumab use may be associated with a form of steroid-responsive corneal ulceration.

Cytotoxic lymphocytes have the capacity to kill microbes directly; however, the mechanisms involved are poorly understood. Using Cryptococcus neoformans, which causes a potentially fatal fungal infection in HIV-infected patients, our previous studies showed that granulysin is necessary, while perforin is dispensable, for CD8 T lymphocyte fungal killing. By contrast, the mechanisms by which NK cells exert their antimicrobial activity are not clear, and in particular, the contribution of granulysin and perforin to NK-mediated antifungal activity is unknown. Primary human NK cells and a human NK cell line YT were found to constitutively express granulysin and perforin, and possessed anticryptococcal activity, in contrast to CD8 T lymphocytes, which required stimulation. When granulysin protein and mRNA were blocked by granulysin small interfering RNA, the NK cell-mediated antifungal effect was not affected in contrast to the abrogated activity observed in CD8 T lymphocytes. However, when perforin was inhibited by concanamycin A, and silenced using hairpin small interfering RNA, the anticryptococcal activities of NK cells were abrogated. Furthermore, when granulysin and perforin were both inhibited, the anticryptococcal activities of the NK cells were not reduced further than by silencing perforin alone. These results indicate that the antifungal activity is constitutively expressed in NK cells in contrast to CD8 T lymphocytes, in which it requires prior activation, and perforin, but not granulysin, plays the dominant role in NK cell anticryptococcal activity, in contrast to CD8 T lymphocytes, in which granulysin, but not perforin, plays the dominant role in anticryptococcal activity.