Direct multiplex imaging and optogenetics of Rho GTPases enabled by near-infrared FRET

Direct visualization and light control of several cellular processes is a challenge, owing to the spectral overlap of available genetically encoded probes. Here we report the most red-shifted monomeric near-infrared (NIR) fluorescent protein, miRFP720, and the fully NIR Forster resonance energy transfer (FRET) pair miRFP670-miRFP720, which together enabled design of biosensors compatible with CFP-YFP imaging and blue-green optogenetic tools. We developed a NIR biosensor for Rac1 GTPase and demonstrated its use in multiplexed imaging and light control of Rho GTPase signaling pathways. Specifically, we combined the Rac1 biosensor with CFP-YFP FRET biosensors for RhoA and for Rac1-GDI binding, and concurrently used the LOV-TRAP tool for upstream Rac1 activation. We directly observed and quantified antagonism between RhoA and Rac1 dependent on the RhoA-downstream effector ROCK; showed that Rac1 activity and GDI binding closely depend on the spatiotemporal coordination between these two molecules; and simultaneously observed Rac1 activity during optogenetic manipulation of Rac1.Peer reviewe

Two-phase flow metering using a large coriolis mass flow meter applied to ship fuel bunkering

With rising oil prices, there is growing interest in more accurate measurement during transfers of marine fuel oil, whether for the purposes of environmental monitoring or for validating custody transfer. Air entrainment is a common problem in marine fuel transfer (bunkering) operations, which can induce measurement errors in many metering technologies. The latest generation of Coriolis mass flow meters is able to operate in both single- and two-phase (oil-air) flow conditions. Witnessed trials have taken place at the National Engineering Laboratory in the UK using a large (200mm diameter) Coriolis flowmeter. The accuracies of the mass flow and density measurements were assessed for steady single-phase and two-phase flow on high and low viscosity oils. Meter accuracy was further assessed over a series of simulated bunkering operations, in which the meter started and finished empty of oil, with periods of both single and two-phase operation. Further trials of a metering skid are currently being carried on a bunkering vessel in Singapore

Two-Phase Flow Metering Using A Large Coriolis Mass Flow Meter Applied To Ship Fuel Bunkering

With rising oil prices, there is growing interest in more accurate measurement during transfers of marine fuel oil, whether for the purposes of environmental monitoring or for validating custody transfer. Air entrainment is a common problem in marine fuel transfer (bunkering) operations, which can induce measurement errors in many metering technologies. The latest generation of Coriolis mass flow meters is able to operate in both single- and two-phase (oil-air) flow conditions. Witnessed trials have taken place at the National standards laboratories at TUV NEL Ltd in the UK using a large (200mm diameter) Coriolis flowmeter. These independent facilities have a unique combination of multiphase and heavy oil flow research capabilities. The accuracies of the mass flow and density measurements were assessed for steady single-phase and two-phase flow on high and low viscosity oils. Meter accuracy was further assessed over a series of simulated bunkering operations, in which the meter started and finished empty of oil, with periods of both single and two-phase operation. Further trials of a metering skid are currently being carried on a bunkering vessel in Singapore