We have used a “2-color” SERCA (sarco/endoplasmic reticulum calcium ATPase) biosensor and a unique high-throughput fluorescence lifetime plate-reader (FLT-PR) to develop a high-precision live-cell assay designed to screen for small molecules that perturb SERCA structure. A SERCA construct, in which red fluorescent protein (RFP) was fused to the N terminus and green fluorescent protein (GFP) to an interior loop, was stably expressed in an HEK cell line that grows in monolayer or suspension. Fluorescence resonance energy transfer (FRET) from GFP to RFP was measured in the FLT-PR, which increases precision 30-fold over intensity-based plate-readers without sacrificing throughput. FRET was highly sensitive to known SERCA modulators. We screened a small chemical library and identified ten compounds that significantly affected 2-color SERCA FLT. Three of these compounds reproducibly lowered FRET and inhibited SERCA in a dose-dependent manner. This assay is ready for large-scale HTS campaigns, and is adaptable to many other targets.

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Using fluorescence resonance energy transfer (FRET), we performed a high-throughput screen (HTS) in a reconstituted membrane system, seeking compounds that reverse inhibition of sarco-/endoplasmic reticulum Ca-ATPase (SERCA) by its endogenous regulator, phospholamban (PLB). Such compounds have long been sought to correct aberrant Ca2+ regulation in heart failure. Donor-SERCA was reconstituted in phospholipid membranes with or without acceptor-PLB, and FRET was measured in a steady-state fluorescence microplate reader. A 20,000-compound library was tested in duplicate. Compounds that decreased FRET by more than three standard deviations were considered hits. From 43 primary hits (0.2%), 31 (72%) were found to be false positives upon more thorough testing. The remaining 12 hits were tested in assays of Ca-ATPase activity, and six of these activated SERCA significantly, by as much as 60%, and several also enhanced cardiomyocyte contractility. These compounds directly activated SERCA from heart and other tissues. These results validate our FRET approach and set the stage for medicinal chemistry and pre-clinical testing. We were concerned about the high rate of false positives, resulting from the low precision of steady-state fluorescence. Preliminary studies with a novel fluorescence lifetime plate reader show 20-fold higher precision. This instrument can dramatically increase the quality of future HT.

Lithography‐based additive manufacturing technology is a layered manufacturing approach where liquid photopolymerizable resins are solidified with ultraviolet, visible, or infrared light. Using a system based on digital mirror devices, photopolymers can be exposed selectively in order to build parts with defined geometries. By modifying the system with a rotating building platform, suspensions with a high solid loading of ceramic powders can be processed, despite the high viscosity of these resins. Depending on the field of application, various formulations were developed for fabricating customized ceramic parts made of alumina, tricalcium phosphate, or bioactive glasses, respectively. On the one hand the influence of the ceramic filler on the mechanical properties is characterized, on the other hand the good precision and the high surface quality of the process system is discussed. For alumina filled resins a solid loading of 50 vol% was used to obtain fully dense parts (>99% of theoretical density) with high fracture strength (biaxial strength of 516 MPa).

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