The cellular proportion of free and protein-bound NADH complexes is increasingly recognized as a metabolic indicator and biomarker. Because free and bound forms exhibit different fluorescence spectra, we consider whether autofluorescence shape sufficiently correlates with mitochondrial metabolism to be useful for monitoring in cellular suspensions. Several computational approaches for rapidly quantifying spectrum shape are used to detect Saccharomyces cereviseae response to oxygenation, and to the addition of mitochondrial functional modifiers and metabolic substrates. Observed changes appear consistent with previous studies probing free/protein-bound proportions, making this a potentially useful approach for the real-time monitoring of metabolism. (© 2015 WILEY-VCH Verlag GmbH &Co. KGaA, Weinheim).

Cellular NADH conformation is increasingly recognized as an endogenous optical biomarker and metabolic indicator. Recently, we reported a real-time approach for tracking metabolism on the basis of the quantification of UV-excited autofluorescence spectrum shape. Here, we use nanosecond-gated spectral acquisition, combined with spectrum-shape quantification, to monitor the long excited-state lifetime autofluorescence (usually associated with protein-bound NADH conformations) separately from the autofluorescence signal as a whole. We observe that the autofluorescence response induced by two NADH-oxidation inhibitors—cyanide and ethanol—are similar in Saccharomyces cerevisiae when monitored using time-integrated detection but easily distinguished using time-gated detection. Results are consistent with the observation of multiple NADH conformations as assessed using spectral phasor analysis. Further, because well-known oxidation inhibitors are used, changes in spectrum shape can be associated with NADH conformations involved in the different metabolic pathways, giving bioanalytic utility to the spectral responses.

We construct a micro-perfusion system using piston screw pump generators for use during real-time, high-pressure physiological studies. Perfusion is achieved using two generators, with one generator being compressed while the other is retracted, thus maintaining pressurization while producing fluid flow. We demonstrate control over perfusion rates in the 10-μl/s range and the ability to change between fluid reservoirs at up to 50 MPa. We validate the screw-pump approach by monitoring the cyanide-induced response of UV-excited autofluorescence from Saccharomyces cerevisiae under pressurization.

with high affinity and exhibit a robust open probability~0.4. Substituting dual alanines for the 'IQ' residues in the IQ element significantly weakens apocalmodulin binding, and markedly diminishes peak open probability (~0.1). Overexpressing apocalmodulin fully rescues open probability, demonstrating that the effects on opening reflect apocalmodulin binding per se. This extensive structural and functional similarity substantiates a striking conservation of calmodulin regulation across Nav and Cav channels, joint investigation of which now presents as a genuinely synergistic endeavor.

the ToxR/TcpP/toxT protein-DNA complex important in early pathogenesis. In addition to elucidating the regulatory pathway of V. cholerae, the impact of this work will be to further provide a general model for outermembrane-bound transcription control in bacteria and nuclear-membranebound transcription in eukaryotic cells.