Sensitivity optimization of a rhodopsin-based fluorescent voltage indicator

The ability to optically image cellular transmembrane voltage at millisecond-timescale resolution can offer unprecedented insight into the function of living brains in behaving animals. The chemigenetic voltage indicator Voltron is bright and photostable, making it a favorable choice for long in vivo imaging of neuronal populations at cellular resolution. Improving the voltage sensitivity of Voltron would allow better detection of spiking and subthreshold voltage signals. We performed site saturation mutagenesis at 40 positions in Voltron and screened for increased ΔF/F0 in response to action potentials (APs) in neurons. Using a fully automated patch-clamp system, we discovered a Voltron variant (Voltron.A122D) that increased the sensitivity to a single AP by 65% compared to Voltron. This variant (named Voltron2) also exhibited approximately 3-fold higher sensitivity in response to sub-threshold membrane potential changes. Voltron2 retained the sub-millisecond kinetics and photostability of its predecessor, with lower baseline fluorescence. Introducing the same A122D substitution to other Ace2 opsin-based voltage sensors similarly increased their sensitivity. We show that Voltron2 enables improved sensitivity voltage imaging in mice, zebrafish and fruit flies. Overall, we have discovered a generalizable mutation that significantly increases the sensitivity of Ace2 rhodopsin-based sensors, improving their voltage reporting capability.

Central Carbon Metabolism, Sodium-Motive Electron Transfer, and Ammonium Formation by the Vaginal Pathogen Prevotella bivia

Replacement of the Lactobacillus dominated vaginal microbiome by a mixed bacterial population including Prevotella bivia is associated with bacterial vaginosis (BV). To understand the impact of P. bivia on this microbiome, its growth requirements and mode of energy production were studied. Anoxic growth with glucose depended on CO2 and resulted in succinate formation, indicating phosphoenolpyruvate carboxylation and fumarate reduction as critical steps. The reductive branch of fermentation relied on two highly active, membrane-bound enzymes, namely the quinol:fumarate reductase (QFR) and Na+-translocating NADH:quinone oxidoreductase (NQR). Both enzymes were characterized by activity measurements, in-gel fluorography, and VIS difference spectroscopy, and the Na+-dependent build-up of a transmembrane voltage was demonstrated. NQR is a potential drug target for BV treatment since it is neither found in humans nor in Lactobacillus. In P. bivia, the highly active enzymes L-asparaginase and aspartate ammonia lyase catalyze the conversion of asparagine to the electron acceptor fumarate. However, the by-product ammonium is highly toxic. It has been proposed that P. bivia depends on ammonium-utilizing Gardnerella vaginalis, another typical pathogen associated with BV, and provides key nutrients to it. The product pattern of P. bivia growing on glucose in the presence of mixed amino acids substantiates this notion.

A FEM Study of Molecular Transport Through a Single Nanopore in a Spherical Cell

Electroporation has a specific application in the delivery of drugs into the cells. In addition, the challenge is to be able to deliver the drugs effectively. The key to the electroporation-based delivery method is regulated induced transmembrane voltage (ITMV). Recently, with the advent of COVID-19, there has been an increase in clinical trials on the delivery of DNA plasmids by electroporation. As a result, the substantial number of laboratory experiments are not feasible, thereby increasing the dependency on simulation-based research. Simulations of delivery of extracellular material into the cell depend upon molecular transport modeling in an electroporated cell. In this paper, molecular transport through a single nanopore is being studied theoretically. The closed-form expression of molecular transport is used in COMSOL Multiphysics simulation to obtain extracellular concentration variation as a function of time. Sinusoidal pulses with the varying magnitude of electric field (8kV/cm and 10 kV/cm) and time duration were used to understand pulse parameters' effect on molecular transport. The simulation results match the empirical result from the literature hence validate the simulation study.

P–035 Mathematical model of the signaling cascade during mouse sperm capacitation

Study question Modeling the feedback loop controlling sperm capacitation to study the reversibility/irreversibility of the process Summary answer We demonstrate the existence of a feedback loop between pH increase and hyperpolarization, inducing bistability and possibly the reversibility of the capacitation process. What is known already The process of mammalian capacitation does need an influx of calcium ions through the transmembrane Catsper Channel Complex. This plasmic membrane channel, which is activated by an increase in either the intracellular pH or the membrane potential, is a sperm-specific protein that is localized in the sperm flagella. Two other sperm specific transporters, sNHE and SLO3, found in the flagella, have been shown to control protons influx and transmembrane voltage respectively, and could work together in a feedback loop keeping the sperm in a high pH state with hyperpolarized membrane. Study design, size, duration Mathematical model Participants/materials, setting, methods Not applicable Main results and the role of chance The results given by this minimal theoretical model are robust as the predicted qualitative behavior can accommodate to a wide span of physiological parameters variations. Limitations, reasons for caution The model and its parameters are mainly based on in vitro results of the literature, so that the in vivo implications should be inferred carefully. Wider implications of the findings: The possible reversibility of the capacitation process could have major clinical implications, relevant for the optimization of sperm preparation in assisted reproductive techniques and cryopreservation procedures. Trial registration number Not applicable

A Mathematical Model to Expedite Electroporation Based Vaccine Development for COVID-19

Electroporation has an application in the selective delivery of drugs explicitly into cells. However, the challenge is to achieve efficiency in delivering the drugs. The key parameter responsible for successful electroporation-mediated drug delivery is induced transmembrane voltage (ITMV). The Food & Drug Administration (FDA) has recently approved the clinical trials of DNA plasmid delivery of the COVID-19 vaccine through electroporation. The requirement is to develop a COVID-19 vaccine within a limited time. Hence, the extensive amount of laboratory experiments are not feasible. It has increased dependency on simulation-based analysis. The simulations of electroporation depend on ITMV expression for the specified cell and the environment. In this paper, we have derived the closed-form expression of ITMV (∆Vm). The closed-form expression is used in COMSOL Multiphysics simulation to obtain extracellular concentration variation as a function of time. The simulation results match the empirical results from the literature and hence validate the closed-form expression. The closed-form expression will reduce the development time of electroporation-assisted COVID-19 vaccine delivery.

Water Pores in Planar Lipid Bilayers at Fast and Slow Rise of Transmembrane Voltage

Basic understanding of the barrier properties of biological membranes can be obtained by studying model systems, such as planar lipid bilayers. Here, we study water pores in planar lipid bilayers in the presence of transmembrane voltage. Planar lipid bilayers were exposed to fast and slow linearly increasing voltage and current signals. We measured the capacitance, breakdown voltage, and rupture time of planar lipid bilayers composed of 1-pamitoyl 2-oleoyl phosphatidylcholine (POPC), 1-pamitoyl 2-oleoyl phosphatidylserine (POPS), and a mixture of both lipids in a 1:1 ratio. Based on the measurements, we evaluated the change in the capacitance of the planar lipid bilayer corresponding to water pores, the radius of water pores at membrane rupture, and the fraction of the area of the planar lipid bilayer occupied by water pores.planar lipid bilayer capacitance, which corresponds to water pores, water pore radius at the membrane rupture, and a fraction of the planar lipid bilayer area occupied by water pores. The estimated pore radii determining the rupture of the planar lipid bilayer upon fast build-up of transmembrane voltage are 0.101 nm, 0.110 nm, and 0.106 nm for membranes composed of POPC, POPS, and POPC:POPS, respectively. The fraction of the surface occupied by water pores at the moment of rupture of the planar lipid bilayer The fraction of an area that is occupied by water pores at the moment of planar lipid bilayer rupture is in the range of 0.1–1.8%.

Lateral voltage as a new input for artificial lipid bilayer systems

In this work, we propose lateral voltage as a new input for use in artificial lipid bilayer systems in addition to the commonly used transmembrane voltage. To apply a lateral...