Sugar shock: Probing Streptococcus pyogenes metabolism through bioluminescence imaging

Streptococcus pyogenes (S. pyogenes) can thrive in its host during an infection, and, as a result, it must be able to respond to external stimuli and available carbon sources. The pre-clinical use of engineered pathogens capable of constitutive light production may provide real-time information on microbial-specific metabolic processes. Here we mapped the central metabolism of a luxABCDE-modified S. pyogenes Xen20 (Strep. Xen20) to its de novo synthesis of luciferase substrates as assessed by the rate of light production in response to different environmental triggers. Previous characterization predicted that the lux operon was under the myo-inositol iolE promotor. Here we show that supplementation with myo-inositol generated increased Xen20 luminescence. Surprisingly, when supplemented with infection-relevant carbon sources, such as glucose or glycine, light production was diminished. This was presumably due to the scavenging of pyruvate by L-lactate dehydrogenase (LDH). Inhibition of LDH by its inhibitor, oxamate, partially restored luminescent signal in the presence of glucose, presumably by allowing the resulting pyruvate to proceed to acetyl-coenzyme A (CoA). This phenomenon appeared specific to the lactic acid bacterial metabolism as glucose or glycine did not reduce signal in an analogous luxABCDE-modified Gram-positive pathogen, Staph. Xen29. The Strep. Xen20 cells produced light in a concentration-dependent manner, inversely related to the amount of glucose present. Taken together, our measures of microbial response could provide new information regarding the responsiveness of S. pyogenes metabolism to acute changes in its local environments and cellular health.

Selective control of synaptically-connected circuit elements by all-optical synapses

Understanding percepts, engrams and actions requires methods for selectively modulating synaptic communication between specific subsets of interconnected cells. Here, we develop an approach to control synaptically connected elements using bioluminescent light: Luciferase-generated light, originating from a presynaptic axon terminal, modulates an opsin in its postsynaptic target. Vesicular-localized luciferase is released into the synaptic cleft in response to presynaptic activity, creating a real-time Optical Synapse. Light production is under experimenter-control by introduction of the small molecule luciferin. Signal transmission across this optical synapse is temporally defined by the presence of both the luciferin and presynaptic activity. We validate synaptic Interluminescence by multi-electrode recording in cultured neurons and in mice in vivo. Interluminescence represents a powerful approach to achieve synapse-specific and activity-dependent circuit control in vivo.

Scintillation light detection in the 6-m drift length ProtoDUNE Dual Phase liquid argon TPC

The Deep Underground Neutrino Experiment (DUNE) is a leading-edge experiment for long-baseline neutrino oscillation studies, neutrino astrophysics and nucleon decay searches. ProtoDUNE-Dual Phase (DP) is a 6 × 6 × 6 m3 liquid argon time-projection-chamber (LArTPC) operated at the CERN Neutrino Platform in 2019–2020 as a prototype of the DUNE far detector. In ProtoDUNE-DP, the scintillation and electroluminescence light produced by cosmic muons in the LArTPC is collected by photomultiplier tubes placed up to 7 m away from the ionizing track. In this paper, we present the performance of the ProtoDUNE-DP photon detection system, comparing different wavelength-shifting techniques and the use of xenon-doped LAr as a promising option for future large LArTPCs. The scintillation light production and propagation processes are analyzed and compared to simulations, improving understanding of the liquid argon properties.

Generation of bright autobioluminescent bacteria by chromosomal integration of the improved lux operon ilux2

The bacterial bioluminescence system enables light production in living cells without an external luciferin. Due to its relatively low levels of light emission, many applications of bioluminescence imaging would benefit from an increase in brightness of this system. In this report a new approach of mutagenesis and screening of the involved proteins is described that is based on the identification of mutants with improved properties under rate-limiting reaction conditions. Multiple rounds of screening in Escherichia coli resulted in the operon ilux2 that contains 26 new mutations in the fatty acid reductase complex which provides the aldehyde substrate for the bioluminescence reaction. Chromosomal integration of ilux2 yielded an autonomously bioluminescent E. coli strain with 7-fold increased brightness compared to the previously described ilux operon. The ilux2 strain produces sufficient signal for the robust detection of individual cells and enables highly sensitive long-term imaging of bacterial propagation without a selection marker.

Selective Control of Synaptically-Connected Circuit Elements by All-Optical Synapses

Understanding percepts, engrams and actions requires methods for selectively modulating synaptic communication between specific subsets of interconnected cells. Here, we develop an approach to control synaptically connected elements using bioluminescent light: Luciferase-generated light, originating from a presynaptic axon terminal, modulates an opsin in its postsynaptic target. Vesicular-localized luciferase is released into the synaptic cleft in response to presynaptic activity, creating a real-time Optical Synapse. Light production is under experimenter-control by introduction of the small molecule luciferin. Signal transmission across this optical synapse is temporally defined by the presence of both the luciferin and presynaptic activity. We validate synaptic Interluminescence by multi-electrode recording in cultured neurons and in mice in vivo. Interluminescence represents a powerful approach to achieve synapse-specific and activity-dependent circuit control during behavior in vivo.

A Novel Manufacturing Process for Glass THGEMs and First Characterisation in an Optical Gaseous Argon TPC

This paper details a novel, patent pending, abrasive machining manufacturing process for the formation of sub-millimetre holes in THGEMs, with the intended application in gaseous and dual-phase TPCs. Abrasive machining favours a non-ductile substrate such as glasses or ceramics. This innovative manufacturing process allows for unprecedented versatility in THGEM substrates, electrodes, and hole geometry and pattern. Consequently, THGEMs produced via abrasive machining can be tailored for specific properties: for example, high stiffness, low total thickness variation, radiopurity, moisture absorption/outgassing and/or carbonisation resistance. This paper specifically focuses on three glass substrate THGEMs (G-THGEMs) made from Schott Borofloat 33 and fused silica. Circular and hexagonal hole shapes are also investigated. The G-THGEM electrodes are made from indium tin oxide (ITO), with a resistivity of 150 Ω/Sq. All G-THGEMs were characterised in an optical (EMCCD) readout GArTPC and compared to a traditionally manufactured FR4 THGEM, with their charging and secondary scintillation (S2) light production behaviour analysed.

Luciferins Under Construction: A Review of Known Biosynthetic Pathways

Bioluminescence, or the ability of a living organism to generate visible light, occurs as a result of biochemical reaction where enzyme, known as a luciferase, catalyzes the oxidation of a small-molecule substrate, known as luciferin. This advantageous trait has independently evolved dozens of times, with current estimates ranging from the most conservative 40, based on the biochemical diversity found across bioluminescence systems (Haddock et al., 2010) to 100, taking into account the physiological mechanisms involved in the behavioral control of light production across a wide range of taxa (Davis et al., 2016; Verdes and Gruber, 2017; Bessho-Uehara et al., 2020a; Lau and Oakley, 2021). Chemical structures of ten biochemically unrelated luciferins and several luciferase gene families have been described; however, a full biochemical pathway leading to light emission has been elucidated only for two: bacterial and fungal bioluminescence systems. Although the recent years have been marked by extraordinary discoveries and promising breakthroughs in understanding the molecular basis of multiple bioluminescence systems, the mechanisms of luciferin biosynthesis for many organisms remain almost entirely unknown. This article seeks to provide a succinct overview of currently known luciferins’ biosynthetic pathways.

Two-Mode Squeezed and Entangled Light Production in Parametric Oscillations

We investigate the statistical and quadrature squeezings, as well as the entanglement properties, of a two-mode light generated by non-degenerate parametric oscillations coupled to a two-mode squeezed vacuum reservoir, by employing the solutions of the quantum Langevin equations. It is found that the two-mode light shows the two-mode squeezing and entanglement for all values of the time. Moreover, it is observed that the squeezed vacuum reservoir and the growing amplitude of the pump mode enhance the degrees of two-mode squeezing and entanglement. We have also shown that the amounts of squeezing and entanglement are significant in a region, where the mean photon number is higher, and the photon number correlation is lower.

Boosting background suppression in the NEXT experiment through Richardson-Lucy deconvolution

Next-generation neutrinoless double beta decay experiments aim for half-life sensitivities of ∼ 1027 yr, requiring suppressing backgrounds to < 1 count/tonne/yr. For this, any extra background rejection handle, beyond excellent energy resolution and the use of extremely radiopure materials, is of utmost importance. The NEXT experiment exploits differences in the spatial ionization patterns of double beta decay and single-electron events to discriminate signal from background. While the former display two Bragg peak dense ionization regions at the opposite ends of the track, the latter typically have only one such feature. Thus, comparing the energies at the track extremes provides an additional rejection tool. The unique combination of the topology-based background discrimination and excellent energy resolution (1% FWHM at the Q-value of the decay) is the distinguishing feature of NEXT. Previous studies demonstrated a topological background rejection factor of ∼ 5 when reconstructing electron-positron pairs in the 208Tl 1.6 MeV double escape peak (with Compton events as background), recorded in the NEXT-White demonstrator at the Laboratorio Subterráneo de Canfranc, with 72% signal efficiency. This was recently improved through the use of a deep convolutional neural network to yield a background rejection factor of ∼ 10 with 65% signal efficiency. Here, we present a new reconstruction method, based on the Richardson-Lucy deconvolution algorithm, which allows reversing the blurring induced by electron diffusion and electroluminescence light production in the NEXT TPC. The new method yields highly refined 3D images of reconstructed events, and, as a result, significantly improves the topological background discrimination. When applied to real-data 1.6 MeV e−e+ pairs, it leads to a background rejection factor of 27 at 57% signal efficiency.