Facile Production of the Pseudomonas aeruginosa Virulence Factor LasB in Escherichia coli for Structure-Based Drug Design

The human pathogen Pseudomonas aeruginosa has a number of virulence factors at its disposal that play crucial roles in the progression of infection. LasB is one of the major virulence factors and exerts its effects through elastolytic and proteolytic activities aimed at dissolving connective tissue and inactivating host defense proteins. LasB is of great interest for the development of novel pathoblockers to temper the virulence, but access has thus far largely been limited to protein isolated from Pseudomonas cultures. Here, we describe a new protocol for high-level production of native LasB in Escherichia coli. We demonstrate that this facile approach is suitable for the production of mutant, thus far inaccessible LasB variants, and characterize the proteins biochemically and structurally. We expect that easy access to LasB will accelerate the development of inhibitors for this important virulence factor

The open XXZ-chain: Bosonisation, Bethe ansatz and logarithmic corrections

We calculate the bulk and boundary parts of the free energy for an open spin-1/2 XXZ-chain in the critical regime by bosonisation. We identify the cutoff independent contributions and determine their amplitudes by comparing with Bethe ansatz calculations at zero temperature T. For the bulk part of the free energy we find agreement with Lukyanov's result [Nucl.Phys.B 522, 533 (1998)]. In the boundary part we obtain a cutoff independent term which is linear in T and determines the temperature dependence of the boundary susceptibility in the attractive regime for $T\ll 1$. We further show that at particular anisotropies where contributions from irrelevant operators with different scaling dimensions cross, logarithmic corrections appear. We give explicit formulas for these terms at those anisotropies where they are most important. We verify our results by comparing with extensive numerical calculations based on a numerical solution of the T=0 Bethe ansatz equations, the finite temperature Bethe ansatz equations in the quantum-transfer matrix formalism, and the density-matrix renormalisation group applied to transfer matrices.Comment: 35 pages, 8 figure

Plasma detachment study of high density helium plasmas in the Pilot-PSI device

We have investigated plasma detachment phenomena of high-density helium plasmas in the linear plasma device Pilot-PSI, which can realize a relevant ITER SOL/Divertor plasma condition. The experiment clearly indicated plasma detachment features such as drops in the plasma pressure and particle flux along the magnetic field lines that were observed under the condition of high neutral pressure; a feature of flux drop was parameterized using the degree of detachment (DOD) index. Fundamental plasma parameters such as electron temperature (T e) and electron density in the detached recombining plasmas were measured by different methods: reciprocating electrostatic probes, Thomson scattering (TS), and optical emission spectroscopy (OES). The T e measured using single and double probes corresponded to the TS measurement. No anomalies in the single probe I – V characteristics, observed in other linear plasma devices [16, 17, 36], appeared under the present condition in the Pilot-PSI device. A possible reason for this difference is discussed by comparing the different linear devices. The OES results are also compared with the simulation results of a collisional radiative (CR) model. Further, we demonstrated more than 90% of parallel particle and heat fluxes were dissipated in a short length of 0.5 m under the high neutral pressure condition in Pilot-PSI.</p

Substrate-Inspired Fragment Merging and Growing Affords Efficacious LasB Inhibitors

Extracellular virulence factors have emerged as attractive targets in the current antimicrobial resistance crisis. The Gram-negative pathogen Pseudomonas aeruginosa secretes the virulence factor elastase B (LasB), which plays an important role in the infection process. Here, we report a submicromolar, non-peptidic, fragment-like inhibitor of LasB discovered by careful visual inspection of structural data. Inspired by the natural LasB substrate, the original fragment was successfully merged and grown. The optimized inhibitor is accessible via simple chemistry and retained selectivity with a substantial improvement in activity, which can be rationalized by the crystal structure of LasB in complex with the inhibitor. We also demonstrate an improved in vivo efficacy of the optimized hit in Galleria mellonella larvae, highlighting the significance of this class of compounds as promising drug candidates

Soledge2D‐Eirene simulations of the Pilot‐PSI linear plasma device compared to experimental data

Predictions for the operation of tokamak divertors are reliant on edge plasma simulations typically utilizing a fluid plasma code in combination with a Monte Carlo code for neutral species. Pilot‐PSI is a linear device operating with a cascaded arc plasma source that produces plasmas comparable to those expected in the ITER divertor (Te ∼ 1 eV, ne ∼ 1021&nbsp;m−3). In this study, plasma discharges in Pilot‐PSI are modelled using the Soledge2D fluid plasma code coupled to the Eirene neutral Monte Carlo code. The plasma is generated using an external source of plasma density and power. These input parameters are tuned in order to match Thomson scattering (TS) measurements close to the cascaded arc source nozzle. The sensitivity of the simulations to different atomic physics models is explored. It is found that elastic collisions between ions and hydrogen molecules have a strong influence on calculated profiles. Without their inclusion, supersonic flow regimes are obtained with M ∼ 2 close to the target plate. Simulation results are compared with experimental findings using TS close to the target and, in the case of Pilot‐PSI, a Langmuir probe embedded in the target. Comparison between experimental trends observed in a background pressure scan and the simulations show that the inclusion of the elastic collision is mandatory for the trends to be reproduced.</p

Enhancing glycan stability via site-selective fluorination: modulating substrate orientation by molecular design

Single site OH → F substitution at the termini of maltotetraose leads to significantly improved hydrolytic stability towards α-amylase and α-glucosidase relative to the natural compound. To explore the effect of molecular editing, selectively modified oligosaccharides were prepared via a convergent α-selective strategy. Incubation experiments in purified α-amylase and α-glucosidase, and in human and murine blood serum, provide insight into the influence of fluorine on the hydrolytic stability of these clinically important scaffolds. Enhancements of ca. 1 order of magnitude result from these subtle single point mutations. Modification at the monosaccharide furthest from the probable enzymatic cleavage termini leads to the greatest improvement in stability. In the case of α-amylase, docking studies revealed that retentive C2-fluorination at the reducing end inverts the orientation in which the substrate is bound. A co-crystal structure of human α-amylase revealed maltose units bound at the active-site. In view of the evolving popularity of C(sp3)–F bioisosteres in medicinal chemistry, and the importance of maltodextrins in bacterial imaging, this discovery begins to reconcile the information-rich nature of carbohydrates with their intrinsic hydrolytic vulnerabilities

Collective Thomson scattering system for determination of ion properties in a high flux plasma beam

A collective Thomson scattering system has been developed for measuring ion temperature, plasma velocity and impurity concentration in the high density magnetized Magnum-PSI plasma beam, allowing for measurements at low temperature (4 x 10 20m3,while avoiding laser plasma heating caused by inverse Bremsstrahlung. The collective Thomson scattering system is based on the fundamental mode of a seeded Nd:YAG laser and equipped with an LIVAR M506 camera (EBABS technology). The first collective Thomson scattering measurements are taken at the linear plasma generator Pilot-PSI, 40 mm downstream of the cascaded arc source. At this location, the ion temperature is about equal to the electron temperature in the bulk of the plasma beam

Radiocesium concentrations in wild mushrooms after the accident at the Fukushima Daiichi Nuclear Power Station: Follow-up study in Kawauchi village

Since the accident at the Chernobyl Nuclear Power Plant, it has become well known that radiocesium tends to concentrate in wild mushrooms. During the recovery process after the accident at the Fukushima Daiichi Nuclear Power Station (FDNPS), it is important to perform follow-up measurements of the activity concentrations of radiocesium in mushrooms. We evaluated the activity concentrations of the detected artificial radionuclides (radiocesium) in wild mushrooms collected from Kawauchi village, which is within 30?km of the FDNPS, in 2015, four years after the accident. We found that the radiocesium was determined in 147 of 159 mushroom samples (92.4%). Based on the average mushroom consumption of Japanese citizens (6.28?kg per year), we calculated committed effective doses ranging from <0.001 to 0.6?mSv. Although committed effective doses are relatively limited, even if residents have consumed mushrooms several times, continuous monitoring of the radiocesium in mushrooms in Fukushima is needed for sustained recovery from the nuclear disaster

Revision of the absolute configurations of chelocardin and amidochelocardin

Even with the aid of the available methods, the configurational assignment of natural products can be a challenging task that is prone to errors, and it sometimes needs to be corrected after total synthesis or single-crystal X-ray diffraction (XRD) analysis. Herein, the absolute configuration of amidochelocardin is revised using a combination of XRD, NMR spectroscopy, experimental ECD spectra, and time-dependent density-functional theory (TDDFT)-ECD calculations. As amidochelocardin was obtained via biosynthetic engineering of chelocardin, we propose the same absolute configuration for chelocardin based on the similar biosynthetic origins of the two compounds and result of TDDFT-ECD calculations. The evaluation of spectral data of two closely related analogues, 6-desmethyl-chelocardin and its semisynthetic derivative 1, also supports this conclusion