RESUMO
The development of in vivo imaging and optogenetic tools makes it possible to control neural circuit activities in an all-optical, closed-loop manner, but such applications are limited by the lack of software for online analysis of neuronal imaging data. We developed an analysis software ORCA (Online Real-time activity and offline Cross-session Analysis), which performs image registration, neuron segmentation, and activity extraction at over 100 frames per second, fast enough to support real-time detection and readout of neural activity. Our active neuron detection algorithm is purely statistical, achieving a much higher speed than previous methods. We demonstrated closed-loop control of neurons that were identified on the fly, without prior recording or image processing. ORCA also includes a cross-session alignment module that efficiently tracks neurons across multiple sessions. In summary, ORCA is a powerful toolbox for fast imaging data analysis and provides a solution for all-optical closed-loop control of neuronal activity.
RESUMO
Galactokinases, which catalyze the phosphorylation of galactose and possible other monosaccharides, can provide an activated sugar donor to synthesize sugar-containing molecules. In this study, a novel galactokinase from human gut symbiont Akkermansia muciniphila ATCC BAA-835 (GalKAmu) was expressed and characterized. GalKAmu displayed broad substrate tolerance, with catalytic activity towards Gal (100 %), GalN (100 %), GalA (20.2 %), Glc (52.5 %), GlcNAc (15.5 %), Xyl (<5%), ManNAc (58 %), ManF (37.4 %) and l-Glc (80 %). Most interestingly, this was the first GalK isoform which can tolerate ManNAc. Thus, our characterization of GalKAmu broadens the substrate selection of galactokinases.
