ABSTRACT
L-Lactate is increasingly appreciated as a key metabolite and signaling molecule in mammals. However, investigations of the inter- and intra-cellular dynamics of L-lactate are currently hampered by the limited selection and performance of L-lactate-specific genetically encoded biosensors. Here we now report a spectrally and functionally orthogonal pair of high-performance genetically encoded biosensors: a green fluorescent extracellular L-lactate biosensor, designated eLACCO2.1, and a red fluorescent intracellular L-lactate biosensor, designated R-iLACCO1. eLACCO2.1 exhibits excellent membrane localization and robust fluorescence response. To the best of our knowledge, R-iLACCO1 and its affinity variants exhibit larger fluorescence responses than any previously reported intracellular L-lactate biosensor. We demonstrate spectrally and spatially multiplexed imaging of L-lactate dynamics by coexpression of eLACCO2.1 and R-iLACCO1 in cultured cells, and in vivo imaging of extracellular and intracellular L-lactate dynamics in mice.
Subject(s)
Biosensing Techniques , Lactic Acid , Mice , Animals , Biosensing Techniques/methods , Fluorescence Resonance Energy Transfer , Cells, Cultured , Optical Imaging , MammalsABSTRACT
L-Lactate, traditionally considered a metabolic waste product, is increasingly recognized as an important intercellular energy currency in mammals. To enable investigations of the emerging roles of intercellular shuttling of L-lactate, we now report an intensiometric green fluorescent genetically encoded biosensor for extracellular L-lactate. This biosensor, designated eLACCO1.1, enables cellular resolution imaging of extracellular L-lactate in cultured mammalian cells and brain tissue.
Subject(s)
Bacterial Proteins/metabolism , Biosensing Techniques/methods , Green Fluorescent Proteins/metabolism , Lactic Acid/analysis , Periplasmic Proteins/metabolism , Recombinant Fusion Proteins/metabolism , Bacterial Proteins/genetics , Binding Sites/genetics , Cell Line, Tumor , Crystallography, X-Ray , Fluorescence , Green Fluorescent Proteins/chemistry , Green Fluorescent Proteins/genetics , HEK293 Cells , HeLa Cells , Humans , Lactic Acid/metabolism , Microscopy, Fluorescence , Periplasmic Proteins/genetics , Protein Binding , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Reproducibility of ResultsABSTRACT
The quantitative description of the Meissner effect can be done by means of the current-density functional theory for superconductors (sc-CDFT), as pointed out by W Kohn. Here, we propose a calculation scheme of the sc-CDFT. In this scheme, the superconducting gap and attractive interaction between electrons are treated as variables, while experimental data are used for the penetration depth. The variables are determined by solving the gap equation of the sc-CDFT simultaneously with the relation between energy gains of the superconducting state in the zero and nonzero magnetic field cases. This scheme is applied to homogeneous electron gas systems immersed in a magnetic field that correspond to simple models for aluminum, tin and indium immersed in a magnetic field. The magnetic field and temperature dependences of the superconducting gap are well reproduced for each case. It is also found that the attractive interaction changes with the magnetic field and temperature, which is consistent with the change in the superconducting gap.
ABSTRACT
In this manuscript, we developed a screening system that employs the difference in density between liquid water and ice (0.9998 g/cm3 vs 0.9168 g/cm3 at 0 °C) to identify ice-nucleating agents (INAs) that are encapsulated into droplets of water suspended in silicone oil of intermediate density (0.939 g/cm3). Droplets of liquid water stably reside at the interface of the silicone oil and perfluoro oil (1.6658 g/cm3); freezing causes the aqueous droplets to float to the top of the silicone oil layer. We demonstrated the feasibility of this screening system by using droplets that contained well-defined ice-nucleator Snomax. The droplets with and without Snomax froze at different temperatures and separated into two groups in our system. We employed the screening system to test samples that have different ice-nucleating activities. Starting from known ice-nucleating active bacteria Pseudomonas syringae, we confirmed that droplets that contain an increasing amount of ice-nucleating bacteria per droplet exhibit a dose-dependent increase in ice nucleation. When droplets containing different amounts of P. syringae were separated using a freeze-float setup, we observed that the droplets that floated at higher temperature contained more ice-nucleating active bacteria. The outlined system, thus, permits simple power-free separation of droplets that contain effective INA from those that contain weak or no INA. Such a setup can be used as a starting point for the development of high-throughput approaches for the discovery of new INAs.
