Light-activateable apoptosis via genetic code expansion as an in-vivo single-cell ablation tool in Caenorhabditis elegans

Natural proteins are biopolymers built from a limited variety of canonical amino acids that are encoded by corresponding triplet codons. Genetic code expansion via amber suppression enables me to install (incorporate) in a target protein a “designer” amino acid beyond canonical amino acids, at the site of a pre-introduced amber stop codon. This is through expression in the host cell of an orthogonal pair, consisting of an aminoacyl-tRNA synthetase and an amber-suppressing tRNA (tRNACUA) evolved for the non-canonical amino acids (NCAA). Site-specific incorporation of NCAA endows target proteins with new properties, enabling protein measurement and/or manipulation in ways that are otherwise impossible. Photo-caged cysteine (PCCys) as a useful NCAA has not been used in any animal before. By using a PCCRS/tRNAPylCUA pair evolved from a PylRS/tRNAPylCUA pair, I introduced (PCCys) into protein synthesis of multicellular model species Caenorhabditis elegans (C. elegans). I demonstrated this incorporation of PCCys by expressing a fluorescent reporter either throughout the nematode or in two different neuronal classes. I used site-specific PCCys incorporation to develop a light-activatable caspase for precisely ablating cells (especially neurons) in living worms. Cell ablation has been widely adopted in studies on C. elegans cell lineage and cell functions. Common ablation methods include high-powered laser ablation, genetic ablation and optogenetic ablation. However, they are unable to ablate single cells in fully developed worms. Caspase is a core executor of apoptosis of both C. elegans and human cells. I designed and engineered a photo-caged caspase from human Caspase-3 by replacing its catalytic cysteine with PCCys. 365-nm UVA illumination removes the caging group of PCCys in the caged caspase, thereby activating the caspase to induce apoptosis of the cell(s) targeted. I succeeded in using global UV illumination to activate respective apoptosis events of oxygen-sensing neurons, touch receptor neurons and muscular cells in adult worms. Also, I demonstrate that individual adult neurons can be selectively targeted and efficiently killed with the use of a microscope-mounted 365-nm laser. With this better spatiotemporal control than other ablation methods, our approach is likely to facilitate future C. elegans studies with unprecedented specificity and precision

Precise optical control of gene expression in C elegans using improved genetic code expansion and Cre recombinase.

Synthetic strategies for optically controlling gene expression may enable the precise spatiotemporal control of genes in any combination of cells that cannot be targeted with specific promoters. We develop an improved genetic code expansion system in Caenorhabditis elegans and use it to create a photoactivatable Cre recombinase. We laser-activate Cre in single neurons within a bilaterally symmetric pair to selectively switch on expression of a loxP-controlled optogenetic channel in the targeted neuron. We use the system to dissect, in freely moving animals, the individual contributions of the mechanosensory neurons PLML/PLMR to the C. elegans touch response circuit, revealing distinct and synergistic roles for these neurons. We thus demonstrate how genetic code expansion and optical targeting can be combined to break the symmetry of neuron pairs and dissect behavioural outputs of individual neurons that cannot be genetically targeted

Evolution of Sequence Type 4821 Clonal Complex Hyperinvasive and Quinolone-Resistant Meningococci

Expansion of quinolone-resistant Neisseria meningitidis clone ChinaCC4821-R1-C/B from sequence type (ST) 4821 clonal complex (CC4821) caused a serogroup shift from serogroup A to serogroup C invasive meningococcal disease (IMD) in China. To determine the relationship among globally distributed CC4821 meningococci, we analyzed whole-genome sequence data from 173 CC4821 meningococci isolated from 4 continents during 1972-2019. These meningococci clustered into 4 sublineages (1-4); sublineage 1 primarily comprised of IMD isolates (41/50, 82%). Most isolates from outside China (40/49, 81.6%) formed a distinct sublineage, the Europe-USA cluster, with the typical strain designation B:P1.17-6,23:F3-36:ST-3200(CC4821), harboring mutations in penicillin-binding protein 2. These data show that the quinolone-resistant clone ChinaCC4821-R1-C/B has expanded to other countries. The increasing distribution worldwide of serogroup B CC4821 raises the concern that CC4821 has the potential to cause a pandemic that would be challenging to control, despite indirect evidence that the Trumenba vaccine might afford some protection

Potential of Core-Collapse Supernova Neutrino Detection at JUNO

JUNO is an underground neutrino observatory under construction in Jiangmen, China. It uses 20kton liquid scintillator as target, which enables it to detect supernova burst neutrinos of a large statistics for the next galactic core-collapse supernova (CCSN) and also pre-supernova neutrinos from the nearby CCSN progenitors. All flavors of supernova burst neutrinos can be detected by JUNO via several interaction channels, including inverse beta decay, elastic scattering on electron and proton, interactions on C12 nuclei, etc. This retains the possibility for JUNO to reconstruct the energy spectra of supernova burst neutrinos of all flavors. The real time monitoring systems based on FPGA and DAQ are under development in JUNO, which allow prompt alert and trigger-less data acquisition of CCSN events. The alert performances of both monitoring systems have been thoroughly studied using simulations. Moreover, once a CCSN is tagged, the system can give fast characterizations, such as directionality and light curve

Detection of the Diffuse Supernova Neutrino Background with JUNO

As an underground multi-purpose neutrino detector with 20 kton liquid scintillator, Jiangmen Underground Neutrino Observatory (JUNO) is competitive with and complementary to the water-Cherenkov detectors on the search for the diffuse supernova neutrino background (DSNB). Typical supernova models predict 2-4 events per year within the optimal observation window in the JUNO detector. The dominant background is from the neutral-current (NC) interaction of atmospheric neutrinos with 12C nuclei, which surpasses the DSNB by more than one order of magnitude. We evaluated the systematic uncertainty of NC background from the spread of a variety of data-driven models and further developed a method to determine NC background within 15\% with {\it{in}} {\it{situ}} measurements after ten years of running. Besides, the NC-like backgrounds can be effectively suppressed by the intrinsic pulse-shape discrimination (PSD) capabilities of liquid scintillators. In this talk, I will present in detail the improvements on NC background uncertainty evaluation, PSD discriminator development, and finally, the potential of DSNB sensitivity in JUNO

Real-time Monitoring for the Next Core-Collapse Supernova in JUNO

Core-collapse supernova (CCSN) is one of the most energetic astrophysical events in the Universe. The early and prompt detection of neutrinos before (pre-SN) and during the SN burst is a unique opportunity to realize the multi-messenger observation of the CCSN events. In this work, we describe the monitoring concept and present the sensitivity of the system to the pre-SN and SN neutrinos at the Jiangmen Underground Neutrino Observatory (JUNO), which is a 20 kton liquid scintillator detector under construction in South China. The real-time monitoring system is designed with both the prompt monitors on the electronic board and online monitors at the data acquisition stage, in order to ensure both the alert speed and alert coverage of progenitor stars. By assuming a false alert rate of 1 per year, this monitoring system can be sensitive to the pre-SN neutrinos up to the distance of about 1.6 (0.9) kpc and SN neutrinos up to about 370 (360) kpc for a progenitor mass of 30$M_{\odot}$ for the case of normal (inverted) mass ordering. The pointing ability of the CCSN is evaluated by using the accumulated event anisotropy of the inverse beta decay interactions from pre-SN or SN neutrinos, which, along with the early alert, can play important roles for the followup multi-messenger observations of the next Galactic or nearby extragalactic CCSN.Comment: 24 pages, 9 figure

Identification of Novel 4′-<i>O</i>-Demethyl-epipodophyllotoxin Derivatives as Antitumor Agents Targeting Topoisomerase II

C4 variation of 4′-O-demethyl-epipodophyllotoxin (DMEP) is an effective approach to optimize the antitumor spectra of this compound class. Accordingly, two series of novel DMEP derivatives were synthesized, and as expected, the antitumor spectra of these derivatives varied with different C4 substituents. Notably, most compounds showed significant inhibition against the etoposide (2)-resistant KBvin cells. Four of the compounds (11, 18, 27 and 28) induced protein-linked DNA break (PLDB) levels higher than those of GL-331 (6) and 2, and are assumed to be topoisomerase II (topo II) poisons more potent than 6 and 2. Compound 28, a potent topo II poison highly effective against KBvin cells, was further evaluated with a panel of tumor cells and was most active against HepG2. This compound also exhibited apparent in vivo antitumor efficacy in hepatoma 22 (H22) mouse model. The results indicated that C4 derivation of DMEP is a feasible approach to identify potent topo II inhibitors with optimized antitumor profiles

Purification, Identification and Molecular Docking of Immunomodulatory Peptides from the Heads of <i>Litopenaeus vannamei</i>

In order to realize the high-value utilization of Litopenaeus vannamei (L. vannamei) heads, immunomodulatory peptides were prepared from the enzymatic hydrolysate of L. vannamei heads, and the action mechanism of immunomodulatory peptides was determined by molecular docking. The results showed that six proteases were used to hydrolyze L. vannamei head proteins, with the animal protease hydrolysate exhibiting the highest macrophage relative proliferation rate (MRPR). The enzymatic products were then sequentially purified by ultrafiltration, Sephadex G-15 gel chromatography, identified by liquid chromatography-mass spectrometry (LC-MS/MS), and finally selected for six immunomodulatory peptides (PSPFPYFT, SAGFPEGF, GPQGPPGH, QGF, PGMR, and WQR). These peptides maintained good immune activity under heat treatment, pH treatment, and in vitro gastrointestinal digestion. Molecular docking analysis indicated that these peptides showed great binding to both toll-like receptor 2 and 4 (TLR2 and TLR4/MD-2), leading to immunomodulation. The discarded L. vannamei heads in this article are considered to be promising food-borne immunomodulators that contribute to enhancing the immune function of the body

Three-Dimensional CFD Simulations of Start-Up Processes of a Pump-Turbine Considering Governor Regulation

The pumped-storage power station is an efficient stability regulator of the power grid. However, due to the instability of the pump-turbine in the S-shaped characteristic region, rotational speed fluctuation is easy to occur in the speed no-load condition, making synchronization with and connection to the grid difficult. To investigate the key factors of these difficult grid connections, the start-up processes of a practical pump-turbine under the lowest head condition were simulated by using the three-dimensional CFD method, in which the governor regulating equations with different regulating parameters were integrated successfully. The results show that the working points oscillate with the fluctuations of rotational speed, discharge, and torque, and different regulating parameters have a significant influence on the dynamic histories. In addition, the internal flow patterns, especially the backflows at the runner inlet, keep apparent values at the middle span (0.5 span) but have regular transitions near the shroud side (0.7&ndash;0.8 span). The faster the guide vanes adjust, the faster the backflows change, and the larger the macro parameters fluctuate. Overall, the instability of the start-up is the result of the periodical evolutions of backflows at the runner inlet, because the trend and period of the radial velocities at different inlet span locations are consistent with those of the discharge