X-ray free-electron lasers (XFELs) provide very intense X-ray pulses suitable for macromolecular crystallography. Each X-ray pulse typically lasts for tens of femtoseconds and the interval between ...pulses is many orders of magnitude longer. Here we describe two novel acoustic injection systems that use focused sound waves to eject picoliter to nanoliter crystal-containing droplets out of microplates and into the X-ray pulse from which diffraction data are collected. The on-demand droplet delivery is synchronized to the XFEL pulse scheme, resulting in X-ray pulses intersecting up to 88% of the droplets. We tested several types of samples in a range of crystallization conditions, wherein the overall crystal hit ratio (e.g., fraction of images with observable diffraction patterns) is a function of the microcrystal slurry concentration. We report crystal structures from lysozyme, thermolysin, and stachydrine demethylase (Stc2). Additional samples were screened to demonstrate that these methods can be applied to rare samples.
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•Acoustic methods inject crystal-containing droplets directly from microplate wells•On-demand acoustic injection uses crystals efficiently without orifices or clogging•Diffraction patterns from crystals measuring several tens of μm are of high quality•Complete datasets can be obtained from fewer than 50,000 crystals
Acoustic droplet ejection provides an automated tool for efficient use of protein crystals in SFX experiments. Roessler et al. used this method to deliver crystal-containing droplets into the XFEL beam to coincide with each X-ray pulse.
Botulinum neurotoxins are one of the most poisonous biological substances known to humans and present a potential bioterrorism threat. There are no therapeutic interventions developed so far. Here, ...we report the first small molecule non-peptide inhibitor for botulinum neurotoxin serotype E discovered by structure-based virtual screening and propose a mechanism for its inhibitory activity.
Although trunk compensation during stroke rehabilitation is widely studied, the proposed solutions primarily include a trunk constraint, which has several disadvantages. In this study, we have ...proposed a haptic feedback-based system for postural training during upper-limb motor rehabilitation. We have tested the proposed system on six healthy people in this preliminary study. Participants performed a simple 1-dimensional reaching task while their posture was being monitored. They received haptic feedback based on their trunk posture. Preliminary results revealed a significant decline in postural error (p<0.05) after the haptic-based training. The reduction in error was maintained even after haptic feedback was turned off. This study shows that haptic feedback could be a viable alternative to the traditional constraint-based methods for postural adaptation. Additional studies need to be conducted to further evaluate the influence of using such feedback strategies.
•Interaction between titanium dioxide nanoparticles (TNP) and aldrin in zebrafish.•Combined exposure showed significant reversal of LPO levels in liver and brain.•Combined exposure does not show ...significant changes in aldrin accumulation.•Combined exposure fails to protect other toxic responses.•Combined exposure does not effective for providing safeguard in aldrin toxicity.
Aldrin (ALD), a persistent-organic-pollutant (POP), an organochlorine-cyclodiene-pesticide is highly toxic in nature. Titanium dioxide nanoparticles (TNP) are widely used for various industrial applications. Despite the remarkable research on pesticide toxicity, the work with impact of nanoparticles on POP has been dealt with marginally. Chemicals co-exist in the environment and exhibit interactive effects. An investigation was carried out to evaluate the individual and combined effects of ALD (6 ppm) and TNP (60 ppm) exposure at sub-lethal concentration for 24 h in zebrafish. Significant reversal of lipid peroxidation level in liver and brain tissues and restoration in enhanced catalase activity in all examined tissues were observed in combined group. For other parameters, combined exposure of ALD and TNP does not show significant reversal action on ALD toxicity. Further studies are inline to understand combined effects of both to achieve significant reversal of ALD toxicity by TNP nanoparticles with threshold concentration of aldrin.
Botulinum neurotoxins are one of the most poisonous biological substances known to humans and present a potential bioterrorism threat. There are no therapeutic interventions developed so far. Here, ...we report the first small molecule non-peptide inhibitor for botulinum neurotoxin serotype E discovered by structure-based virtual screening and propose a mechanism for its inhibitory activity.
NSC-77053, the first small molecule non-peptide inhibitor of BoNT/E with an inhibition constant of 1.29 M is identified and its mechanism of action elucidated.
Abstract We have examined the effect of both pre- and post-treatment of vitamin E on mercury induced acute toxicity in rats. Mercury (12 μmol/kg b.w., single intraperitoneal injection) resulted in ...oxidative injury and metallothionein mRNA expression together with alterations in tissue histology and accumulation of mercury in the body organs. The ameliorating potential of vitamin E (24 μmol/kg b.w., single intraperitoneal injection) was observed in mercury administered rats. Our findings indicate that vitamin E provides complete protection from mercury toxicity in the liver with both pre- and post-treatments. As mercury is nephrotoxic and neurotoxic, it is interesting to note that post-treatment of vitamin E showed more protection in the kidney compared to pre-treatment. In brain tissue, partial protection was observed on oxidative stress parameters. Our results thus suggest that post-treatment with vitamin E could be more beneficial than pre- treatment in mercury intoxication.
Acoustic droplet ejection (ADE) is a powerful technology that supports crystallographic applications such as growing, improving and manipulating protein crystals. A fragment‐screening strategy is ...described that uses ADE to co‐crystallize proteins with fragment libraries directly on MiTeGen MicroMeshes. Co‐crystallization trials can be prepared rapidly and economically. The high speed of specimen preparation and the low consumption of fragment and protein allow the use of individual rather than pooled fragments. The Echo 550 liquid‐handling instrument (Labcyte Inc., Sunnyvale, California, USA) generates droplets with accurate trajectories, which allows multiple co‐crystallization experiments to be discretely positioned on a single data‐collection micromesh. This accuracy also allows all components to be transferred through small apertures. Consequently, the crystallization tray is in equilibrium with the reservoir before, during and after the transfer of protein, precipitant and fragment to the micromesh on which crystallization will occur. This strict control of the specimen environment means that the crystallography experiments remain identical as the working volumes are decreased from the few microlitres level to the few nanolitres level. Using this system, lysozyme, thermolysin, trypsin and stachydrine demethylase crystals were co‐crystallized with a small 33‐compound mini‐library to search for fragment hits. This technology pushes towards a much faster, more automated and more flexible strategy for structure‐based drug discovery using as little as 2.5 nl of each major component.
Clostridium botulinum neurotoxins (BoNTs) cleave neuronal proteins responsible for neurotransmitter release, causing the neuroparalytic disease botulism. BoNT serotypes B, D, F and G cleave and ...inactivate vesicle-associated membrane protein (VAMP), each at a unique peptide bond. The specificity of BoNTs depends on the mode of substrate recognition. We have investigated the mechanism of substrate recognition of BoNT F by determining the crystal structures of its complex with two substrate-based inhibitors, VAMP 22-58/Gln58D-cysteine and 27-58/Gln58D-cysteine. The inhibitors bind to BoNT F in the canonical direction (as seen for BoNTs A and E substrates) but are positioned specifically via three major exosites away from the active site. The cysteine sulfur of the inhibitors interacts with the zinc and exists as sulfinic acid in the inhibitor VAMP 27-58/Gln58D-cysteine. Arg133 and Arg171, which form part of two separate exosites, are crucial for substrate binding and catalysis.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
The seven serologically distinct Clostridium botulinum neurotoxins (BoNTs A−G) are zinc endopeptidases which block the neurotransmitter release by cleaving one of the three proteins of the soluble ...N-ethylmaleimide-sensitive-factor attachment protein receptor complex (SNARE complex) essential for the fusion of vesicles containing neurotransmitters with target membranes. These metallopeptidases exhibit unique specificity for the substrates and peptide bonds they cleave. Development of countermeasures and therapeutics for BoNTs is a priority because of their extreme toxicity and potential misuse as biowarfare agents. Though they share sequence homology and structural similarity, the structural information on each one of them is required to understand the mechanism of action of all of them because of their specificity. Unraveling the mechanism will help in the ultimate goal of developing inhibitors as antibotulinum drugs for the toxins. Here, we report the high-resolution structure of active BoNT/F catalytic domain in two crystal forms. The structure was exploited for modeling the substrate binding and identifying the S1‘ subsite and the putative exosites which are different from BoNT/A or BoNT/B. The orientation of docking of the substrate at the active site is consistent with the experimental BoNT/A-LC:SNAP-25 peptide model and our proposed model for BoNT/E-LC:SNAP-25.
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