Regulation of basal autophagy by calmodulin availability.

Macroautophagy (hereafter autophagy) is a process that degrades cellular components to maintain homeostasis. The Ca2+ sensor calmodulin (CaM) regulates numerous cell functions but is a limiting factor due to its insufficient availability for all target proteins. However, evidence that CaM availability regulates basal autophagy is lacking. Here, we have tested this hypothesis. CaM antagonists W-7, trifluoperazine and CGS9343b cause autophagosome accumulation and inhibit basal autophagic flux in the same manner as does chloroquine. These reagents promote the activity of AMP-activated protein kinase (AMPK) but not that of the mechanistic target of rapamycin (mTOR). Competitive binding assays using CaM sensors with different Ca2+ dependencies showed that chloroquine directly binds CaM in a Ca2+ -dependent fashion. The CaM antagonists have disparate effects on cytoplasmic Ca2+ , triggering from none to robust signals, indicating that their consistent inhibition of autophagy is due to inhibition of CaM and not Ca2+ . Chelating intracellular Ca2+ reduces the effect of the CaM antagonists to accumulate LC3-II, indicating that they do so by inhibiting CaM-dependent activities at basal Ca2+ level. The CaM antagonists cause lysosomal alkalinisation. Consistently, buffering CaM with a high-affinity CaM-binding protein that binds CaM at resting Ca2+ level increases lysosomal pH. Enhanced CaM buffering using a chimeric protein that contains two high-affinity CaM-binding sites that can collectively bind CaM at a large range of Ca2+ further increases lysosomal pH and increases LC3-II accumulation and AMPK activity, but not that of mTOR. These data demonstrate that CaM availability is required for basal autophagy.

First Reported Case of Donor Related Candida Endophthalmitis after Descemet Membrane Endothelial Keratoplasty.

PURPOSE: To report the first case of Candida donor to host transmission following descemet membrane endothelial keratoplasty (DMEK). METHODS: A retrospective case report. RESULTS: A patient underwent uneventful DMEK. Following surgery the donor rim was culture positive for Candida. The patient developed fungal endophthalmitis that was treated medically with multiple injections of voriconazole and amphotericin. Medical treatment was unable to clear the infection and removal of the donor material was required. Following removal the infection subsided. CONCLUSION: Candida interface keratitis and endophthalmitis can occur following DMEK and may be difficult to treat medically. Early removal of the donor material should be considered.