Abstract
Treatment of methicillin-resistant
Staphylococcus aureus
infections is dependent on the efficacy of last-line antibiotics including vancomycin. Treatment failure is commonly linked to ...isolates with intermediate vancomycin resistance (termed VISA). These isolates have accumulated point mutations that collectively reduce vancomycin sensitivity, often by thickening the cell wall. Changes in regulatory small RNA expression have been correlated with antibiotic stress in VISA isolates however the functions of most RNA regulators is unknown. Here we capture RNA–RNA interactions associated with RNase III using CLASH. RNase III-CLASH uncovers hundreds of novel RNA–RNA interactions in vivo allowing functional characterisation of many sRNAs for the first time. Surprisingly, many mRNA–mRNA interactions are recovered and we find that an mRNA encoding a long 3′ untranslated region (UTR) (termed
vigR
3′UTR) functions as a regulatory ‘hub’ within the RNA–RNA interaction network. We demonstrate that the
vigR
3′UTR promotes expression of
folD
and the cell wall lytic transglycosylase
isaA
through direct mRNA–mRNA base-pairing. Deletion of the
vigR
3′UTR re-sensitised VISA to glycopeptide treatment and both
isaA
and
vigR
3′UTR deletions impact cell wall thickness. Our results demonstrate the utility of RNase III-CLASH and indicate that
S. aureus
uses mRNA-mRNA interactions to co-ordinate gene expression more widely than previously appreciated.
Resolution and sensitivity improvements in mass spectrometry technology have enabled renewed attempts at solving challenging analytical issues. One such issue involves the analysis of energetic ionic ...species. Energetic ionic species make up an important class of chemical materials, and a more robust and versatile analytical platform would provide tremendous value to the analytical community. Initial attempts at quantification of energetic ionic species employed high-resolution time-of-flight measurements with crown ether (CE) complexation and flow injection analysis (FIA). In this investigation, ammonium nitrate (AN) and urea nitrate (UN) in the presence of a crown ether complexation agent were explored by using high-resolution orbitrap mass spectrometry. Product ion scans of these signature complexes reveal positive identification of these energetic ionic species. Finally, quantification was demonstrated for both flow injection and liquid chromatography–mass spectrometry (LC-MS) analysis, suggesting the capability for routine and rapid analysis of these energetic ionic materials.
Smoke dyes are complex molecular systems that have the potential to form many molecular derivatives and fragments when deployed. The chemical analysis of smoke samples is challenging due to the ...adiabatic temperature of the pyrotechnic combustion and the molecular complexity of the physically dispersed reaction products. Presented here is the characterization of the reaction byproducts of a simulant Mk124 smoke signal on a multigram scale, which contain the dye disperse red 9 (1‐(methylamino)anthraquinone), by ambient ionization mass spectrometry. Our previous work has examined the thermal decomposition of a simplified smoke system consisting of disperse red 9, potassium chlorate, and sucrose by anaerobic pyrolysis gas chromatography mass spectrometry performed at the laboratory milligram scale. The results from the lab scale test were compared with a fully functioned Mk124 in the field. To achieve this, Mk124 smokes were functioned in the presence of sampling swabs that collected byproduct residues from the smoke plume in the ambient environment. These swabs were then analyzed using ambient ionization mass spectrometry to identify the expended pyrotechnic residues, with particular interest in halogenated species. Previous work determined the toxicity of unforeseen byproducts identified on the laboratory scale, which were also detected in the field demonstrating the correlation of the laboratory testing to the fielded systems. By understanding the chemical composition of smokes and their reaction products, potential toxicity effects can be easily assessed, leading to safer formulations with improved performance. These results can help assess how smoke byproducts may impact Warfighter performance, personnel health, and the environment.
“Green” pyrotechnics seek to remove known environmental pollutants and health hazards from their formulations. This chemical engineering approach often focuses on maintaining performance effects upon ...replacement of objectionable ingredients, yet neglects the chemical products formed by the exothermic reaction. In this work, milligram quantities of a lab-scale pyrotechnic red smoke composition were functioned within a thermal probe for product identification by pyrolysis-gas chromatography-mass spectrometry. Thermally decomposed ingredients and new side product derivatives were identified at lower relative abundances to the intact organic dye (as the engineered sublimation product). Side products included chlorination of the organic dye donated by the chlorate oxidizer. Machine learning quantitative structure-activity relationship models computed impacts to health and environmental hazards. High to very high toxicities were predicted for inhalation, mutagenicity, developmental, and endocrine disruption for common military pyrotechnic dyes and their analogous chlorinated side products. These results underscore the need to revise objectives of “green” pyrotechnic engineering.
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•Lab-scale analytical characterization of a red smoke pyrotechnic reaction via pyrolysis-gas chromatography-mass spectrometry.•Discovery of unforeseen side product derivatives onto the colored dye to include chlorination from the potassium oxidizer.•High toxicities predicted for inhalation, mutagenicity, and developmental endocrine disruption for reacted dye byproducts.•Evidence to augment future sustainable pyrotechnic engineering to consider all formed reaction products.
GABABreceptors are heterodimeric G protein-coupled receptors composed of R1 and R2 subunits that mediate slow synaptic inhibition in the brain by activating inwardly rectifying K+channels (GIRKs) and ...inhibiting Ca2+channels. We demonstrate here that GABABreceptors are intimately associated with 5'AMP-dependent protein kinase (AMPK). AMPK acts as a metabolic sensor that is potently activated by increases in 5'AMP concentration that are caused by enhanced metabolic activity, anoxia, or ischemia. AMPK binds the R1 subunit and directly phosphorylates S783 in the R2 subunit to enhance GABABreceptor activation of GIRKs. Phosphorylation of S783 is evident in many brain regions, and is increased dramatically after ischemic injury. Finally, we also reveal that S783 plays a critical role in enhancing neuronal survival after ischemia. Together our results provide evidence of a neuroprotective mechanism, which, under conditions of metabolic stress or after ischemia, increases GABABreceptor function to reduce excitotoxicity and thereby promotes neuronal survival.
GABA(B) receptors are heterodimeric G protein-coupled receptors composed of R1 and R2 subunits that mediate slow synaptic inhibition in the brain by activating inwardly rectifying K(+) channels ...(GIRKs) and inhibiting Ca(2+) channels. We demonstrate here that GABA(B) receptors are intimately associated with 5'AMP-dependent protein kinase (AMPK). AMPK acts as a metabolic sensor that is potently activated by increases in 5'AMP concentration that are caused by enhanced metabolic activity, anoxia, or ischemia. AMPK binds the R1 subunit and directly phosphorylates S783 in the R2 subunit to enhance GABA(B) receptor activation of GIRKs. Phosphorylation of S783 is evident in many brain regions, and is increased dramatically after ischemic injury. Finally, we also reveal that S783 plays a critical role in enhancing neuronal survival after ischemia. Together our results provide evidence of a neuroprotective mechanism, which, under conditions of metabolic stress or after ischemia, increases GABA(B) receptor function to reduce excitotoxicity and thereby promotes neuronal survival.
Even in the setting of optimal resuscitation in high-income countries severe sepsis and septic shock have a mortality of 20-40%, with antibiotic resistance dramatically increasing this mortality ...risk. To develop a reference dataset enabling the identification of common bacterial targets for therapeutic intervention, we applied a standardized genomic, transcriptomic, proteomic and metabolomic technological framework to multiple clinical isolates of four sepsis-causing pathogens: Escherichia coli, Klebsiella pneumoniae species complex, Staphylococcus aureus and Streptococcus pyogenes. Exposure to human serum generated a sepsis molecular signature containing global increases in fatty acid and lipid biosynthesis and metabolism, consistent with cell envelope remodelling and nutrient adaptation for osmoprotection. In addition, acquisition of cholesterol was identified across the bacterial species. This detailed reference dataset has been established as an open resource to support discovery and translational research.
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•Relative humidity affects the amount of dimethyl methylphosphonate (DMMP) sorbed on office materials.•Cardboard retains captured DMMP longer than paint layer and polyurethane ...foam.•DMMP, a nerve agent simulant, was retained after 6 weeks on office materials.
Dimethyl methylphosphonate (DMMP) was used as a simulant to investigate the sorption and desorption of G-series nerve agents such as sarin (GB) released into an office space. DMMP was disseminated systematically as an aerosol and vapor on three common office materials, cardboard, polyurethane foam, and cured acrylic-based paint. The amount of DMMP initially captured on the office materials and the amount retained over a period of 10 h was tracked gravimetrically and by solvent extraction and gas chromatography/mass spectrometry (GC/MS). Physicochemical properties of the office media including the surface area per mass, polarity, and Henry’s constant were determined to help explain the capture and release of DMMP that was observed. Polyurethane foam was found to have the largest sorption capacity for DMMP in a low-humidity setting, however, cardboard was found to have the largest sorption capacity at higher humidity. A fraction of the collected DMMP desorbed with first-order kinetics from all of the office materials in an open-air atmosphere over 8 h. However, importantly for forensic purposes relevant to an indoor attack, a significant quantity (most notably on cardboard) of the nerve agent simulant was retained after 6 weeks and could be analyzed efficiently using solvent extraction followed by GC/MS.
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