99 research outputs found
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 one-dimensional Hubbard model with open ends: Universal divergent contributions to the magnetic susceptibility
The magnetic susceptibility of the one-dimensional Hubbard model with open
boundary conditions at arbitrary filling is obtained from field theory at low
temperatures and small magnetic fields, including leading and next-leading
orders. Logarithmic contributions to the bulk part are identified as well as
algebraic-logarithmic divergences in the boundary contribution. As a
manifestation of spin-charge separation, the result for the boundary part at
low energies turns out to be independent of filling and interaction strength
and identical to the result for the Heisenberg model. For the bulk part at zero
temperature, the scale in the logarithms is determined exactly from the Bethe
ansatz. At finite temperature, the susceptibility profile as well as the
Friedel oscillations in the magnetisation are obtained numerically from the
density-matrix renormalisation group applied to transfer matrices. Agreement is
found with an exact asymptotic expansion of the relevant correlation function.Comment: 30 pages, 8 figures, reference adde
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 . 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
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 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
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
Structure of PatF from Prochloron didemni
Patellamides are macrocyclic peptides with potent biological effects and are a subset of the cyanobactins. Cyanobactins are natural products that are produced by a series of enzymatic transformations and a common modification is the addition of a prenyl group. Puzzlingly, the pathway for patellamides in Prochloron didemni contains a gene, patF, with homology to prenylases, but patellamides are not themselves prenylated. The structure of the protein PatF was cloned, expressed, purified and determined. Prenylase activity could not be demonstrated for the protein, and examination of the structure revealed changes in side-chain identity at the active site. It is suggested that these changes have inactivated the protein. Attempts to mutate these residues led to unfolded protein
The structure of the cyanobactin domain of unknown function from PatG in the patellamide gene cluster
Patellamides are members of the cyanobactin family of ribosomally synthesized and post-translationally modified cyclic peptide natural products, many of which, including some patellamides, are biologically active. A detailed mechanistic understanding of the biosynthetic pathway would enable the construction of a biotechnological `toolkit' to make novel analogues of patellamides that are not found in nature. All but two of the protein domains involved in patellamide biosynthesis have been characterized. The two domains of unknown function (DUFs) are homologous to each other and are found at the C-termini of the multi-domain proteins PatA and PatG. The domain sequence is found in all cyanobactin-biosynthetic pathways characterized to date, implying a functional role in cyanobactin biosynthesis. Here, the crystal structure of the PatG DUF domain is reported and its binding interactions with plausible substrates are investigated
Long-term effects of chronic light pollution on seasonal functions of European blackbirds (turdus merula)
Light pollution is known to affect important biological functions of wild animals, including daily and annual cycles. However, knowledge about long-term effects of chronic exposure to artificial light at night is still very limited. Here we present data on reproductive physiology, molt and locomotor activity during two-year cycles of European blackbirds (Turdus merula) exposed to either dark nights or 0.3 lux at night. As expected, control birds kept under dark nights exhibited two regular testicular and testosterone cycles during the two-year experiment. Control urban birds developed testes faster than their control rural conspecifics. Conversely, while in the first year blackbirds exposed to light at night showed a normal but earlier gonadal cycle compared to control birds, during the second year the reproductive system did not develop at all: both testicular size and testosterone concentration were at baseline levels in all birds. In addition, molt sequence in light-treated birds was more irregular than in control birds in both years. Analysis of locomotor activity showed that birds were still synchronized to the underlying light-dark cycle. We suggest that the lack of reproductive activity and irregular molt progression were possibly the results of i) birds being stuck in a photorefractory state and/or ii) chronic stress. Our data show that chronic low intensities of light at night can dramatically affect the reproductive system. Future studies are needed in order to investigate if and how urban animals avoid such negative impact and to elucidate the physiological mechanisms behind these profound long-term effects of artificial light at night. Finally we call for collaboration between scientists and policy makers to limit the impact of light pollution on animals and ecosystems
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
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