Sensitivity optimization of a rhodopsin-based fluorescent voltage indicator.

The ability to optically image cellular transmembrane voltages at millisecond-timescale resolutions can offer unprecedented insight into the function of living brains in behaving animals. Here, we present a point mutation that increases the sensitivity of Ace2 opsin-based voltage indicators. We use the mutation to develop Voltron2, an improved chemigeneic voltage indicator that has a 65% higher sensitivity to single APs and 3-fold higher sensitivity to subthreshold potentials than Voltron. Voltron2 retained the sub-millisecond kinetics and photostability of its predecessor, although with lower baseline fluorescence. In multiple in vitro and in vivo comparisons with its predecessor across multiple species, we found Voltron2 to be more sensitive to APs and subthreshold fluctuations. Finally, we used Voltron2 to study and evaluate the possible mechanisms of interneuron synchronization in the mouse hippocampus. Overall, we have discovered a generalizable mutation that significantly increases the sensitivity of Ace2 rhodopsin-based sensors, improving their voltage reporting capability.

Fast and sensitive GCaMP calcium indicators for imaging neural populations.

Calcium imaging with protein-based indicators1,2 is widely used to follow neural activity in intact nervous systems, but current protein sensors report neural activity at timescales much slower than electrical signalling and are limited by trade-offs between sensitivity and kinetics. Here we used large-scale screening and structure-guided mutagenesis to develop and optimize several fast and sensitive GCaMP-type indicators3-8. The resulting 'jGCaMP8' sensors, based on the calcium-binding protein calmodulin and a fragment of endothelial nitric oxide synthase, have ultra-fast kinetics (half-rise times of 2 ms) and the highest sensitivity for neural activity reported for a protein-based calcium sensor. jGCaMP8 sensors will allow tracking of large populations of neurons on timescales relevant to neural computation.

Direct detection of SARS-CoV-2 RNA using high-contrast pH-sensitive dyes.

The worldwide coronavirus disease 2019 pandemic has had devastating effects on health, healthcare infrastructure, social structure, and economics. One of the limiting factors in containing the spread of this virus has been the lack of widespread availability of fast, inexpensive, and reliable methods for testing of individuals. Frequent screening for infected and often asymptomatic people is a cornerstone of pandemic management plans. Here, we introduce 2 pH-sensitive "LAMPshade" dyes as novel readouts in an isothermal Reverse Transcriptase Loop-mediated isothermal AMPlification amplification assay for severe acute respiratory syndrome coronavirus 2 RNA. The resulting JaneliaLAMP assay is robust, simple, inexpensive, and has low technical requirements, and we describe its use and performance in direct testing of contrived and clinical samples without RNA extraction.

jYCaMP: an optimized calcium indicator for two-photon imaging at fiber laser wavelengths.

Femtosecond lasers at fixed wavelengths above 1,000 nm are powerful, stable and inexpensive, making them promising sources for two-photon microscopy. Biosensors optimized for these wavelengths are needed for both next-generation microscopes and affordable turn-key systems. Here we report jYCaMP1, a yellow variant of the calcium indicator jGCaMP7 that outperforms its parent in mice and flies at excitation wavelengths above 1,000 nm and enables improved two-color calcium imaging with red fluorescent protein-based indicators.

High-performance calcium sensors for imaging activity in neuronal populations and microcompartments.

Calcium imaging with genetically encoded calcium indicators (GECIs) is routinely used to measure neural activity in intact nervous systems. GECIs are frequently used in one of two different modes: to track activity in large populations of neuronal cell bodies, or to follow dynamics in subcellular compartments such as axons, dendrites and individual synaptic compartments. Despite major advances, calcium imaging is still limited by the biophysical properties of existing GECIs, including affinity, signal-to-noise ratio, rise and decay kinetics and dynamic range. Using structure-guided mutagenesis and neuron-based screening, we optimized the green fluorescent protein-based GECI GCaMP6 for different modes of in vivo imaging. The resulting jGCaMP7 sensors provide improved detection of individual spikes (jGCaMP7s,f), imaging in neurites and neuropil (jGCaMP7b), and may allow tracking larger populations of neurons using two-photon (jGCaMP7s,f) or wide-field (jGCaMP7c) imaging.

Discovery of novel inhibitors of multidrug-resistant Acinetobacter baumannii.

The recent emergence of multidrug-resistant Acinetobacter baumannii strains and the non-efficacy of currently available antibiotics against such infections have led to an urgent need for the development of novel antibacterials. In an effort to address this problem, we have identified three novel inhibitors, namely, D5, D12 and D6 using in silico screening with a homology model of the outer membrane protein W2 (OmpW2) from A. baumannii, as the proposed new drug target. OmpW is an eight-stranded ß-barrel protein involved in the transport of hydrophobic molecules across the outer membrane and maintenance of homeostasis under cellular stress. The antimicrobial activities of compounds D5, D12 and D6 were evaluated against a panel of clinical isolates of A. baumannii strains. These compounds inhibited the growth of the strains with minimum inhibitory concentration (MIC) ranges of 1-32µg/mL. Time-kill kinetic studies with the highly virulent and multidrug-resistant strain, A. baumannii 5075, indicated that D6 exhibited the highest bactericidal activity asa≥3log10 CFU/mL (99.9%) reduction in colony count from the initial inoculum was observed after 30min incubation. D5 and D12 reduced at least 1log10 CFU/mL (90%) of the initial inoculum after 24h. In conclusion, these three lead inhibitors have provided two distinct chemical scaffolds for further analog design and optimizations, using chemical synthesis, to develop more potent inhibitors of the pathogen.

Francisella tularensis 2-C-methyl-D-erythritol 4-phosphate cytidylyltransferase: kinetic characterization and phosphoregulation.

Deliberate and natural outbreaks of infectious disease, the prevalence of antibiotic resistant strains, and the ease by which antibiotic resistant bacteria can be intentionally engineered all underscore the necessity of effective vaccines and continued development of novel antimicrobial/antiviral therapeutics. Isoprenes, a group of molecules fundamentally involved in a variety of crucial biological functions, are derived from either the mevalonic acid (MVA) or methylerythritol phosphate (MEP) pathway. While mammals utilize the MVA pathway, many bacteria utilize the MEP pathway, highlighting the latter as an attractive target for antibiotic development. In this report we describe the cloning and characterization of Francisella tularensis MEP cytidylyltransferase, a MEP pathway enzyme and potential target for antibiotic development. Size exclusion chromatography indicates the protein exists as a dimer in solution. Enzyme assays produced an apparentK(MEP)(M) = 178 µM, K(CTP)(M) = 73 µM , k(MEP)(cat) = 1(s-1), k(CTP)(cat) = 0.8( s-1), and a k(MEP)(cat)/ K(MEP)(M) = 3.4 x 10(5) M(-1) min(-1). The enzyme exhibits a strict preference for Mg(+2) as a divalent cation and CTP as the nucleotide. Titanium dioxide chromatography-tandem mass spectrometry identified Thr141 as a site of phosphorylation. T141D and T141E site-directed mutants are catalytically inactive, suggesting a mechanism for post-translational control of metabolic flux through the F. tularensis MEP pathway. Overall, our study suggests that MEP cytidylyltransferase is an excellent target for the development of novel antibiotics against F. tularensis.