Abstract Around 110,000 people spend time in critical care units in England and Wales each year. The transition of care from the intensive care unit to the general ward exposes patients to potential ...harms from changes in healthcare providers and environment. Nurses working on general wards report anxiety and uncertainty when receiving patients from critical care. An innovative form of enhanced capability critical care outreach called ‘iMobile’ is being provided at King’s College Hospital (KCH). Part of the remit of iMobile is to review patients who have been transferred from critical care to general wards. The iMobile team wished to improve the quality of critical care discharge summaries. A collaborative evidence-based quality improvement project was therefore undertaken by the iMobile team at KCH in conjunction with researchers from King's Improvement Science (KIS). Plan, Do, Study, Act (PDSA) methodology was used. Three PDSA cycles were undertaken. Methods adopted comprised: a scoping literature review to identify relevant guidelines and research evidence to inform all aspects of the quality improvement project; a process mapping exercise; informal focus groups / interviews with staff; patient story-telling work with people who had experienced critical care and subsequent discharge to a general ward; and regular audits of the quality of both medical and nursing critical care discharge summaries. The following behaviour change interventions were adopted, taking into account evidence of effectiveness from published systematic reviews and considering the local context: regular audit and feedback of the quality of discharge summaries, feedback of patient experience, and championing and education delivered by local opinion leaders. The audit results were mixed across the trajectory of the project, demonstrating the difficulty of sustaining positive change. This was particularly important as critical care bed occupancy and through-put fluctuates which then impacts on work-load, with new cohorts of staff regularly passing through critical care. In addition to presenting the results of this quality improvement project, we also reflect on the lessons learned and make suggestions for future projects.
Headline Evaluating service innovations in health care and public health requires flexibility, collaboration and pragmatism; this collection identifies robust, innovative and mixed methods to inform ...such evaluations.
Contact-dependent growth inhibition (CDI) is a form of interbacterial competition mediated by CdiB-CdiA two-partner secretion systems. CdiA effector proteins carry polymorphic C-terminal toxin ...domains (CdiA-CT), which are neutralized by specific CdiI immunity proteins to prevent self-inhibition. Here, we present the crystal structures of CdiA-CT⋅CdiI complexes from Klebsiella pneumoniae 342 and Escherichia coli 3006. The toxins adopt related folds that resemble the ribonuclease domain of colicin D, and both are isoacceptor-specific tRNases that cleave the acceptor stem of deacylated tRNAGAUIle. Although the toxins are similar in structure and substrate specificity, CdiA-CTKp342 activity requires translation factors EF-Tu and EF-Ts, whereas CdiA-CTEC3006 is intrinsically active. Furthermore, the corresponding immunity proteins are unrelated in sequence and structure. CdiIKp342 forms a dimeric β sandwich, whereas CdiIEC3006 is an α-solenoid monomer. Given that toxin-immunity genes co-evolve as linked pairs, these observations suggest that the similarities in toxin structure and activity reflect functional convergence.
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•The crystal structures of two CDI toxin⋅immunity protein complexes are presented•Both toxins are isoacceptor-specific ribonucleases that cleave deacylated tRNAGAUIle•The immunity proteins do not share any sequence or structural homology•Similarities between these tRNase toxins likely arose through convergent evolution
Gucinski et al. present the structures of two CdiA toxin domains in complex with their cognate immunity proteins. These toxins adopt the same fold and exhibit similar tRNase activities. The immunity proteins are completely unrelated in sequence and structure, suggesting that similarities shared by the toxins arose through convergent evolution.
Many Gram-negative bacteria use CdiA effector proteins to inhibit the growth of neighboring competitors. CdiA transfers its toxic CdiA-CT region into the periplasm of target cells, where it is ...released through proteolytic cleavage. The N-terminal cytoplasm-entry domain of the CdiA-CT then mediates translocation across the inner membrane to deliver the C-terminal toxin domain into the cytosol. Here, we show that proteolysis not only liberates the CdiA-CT for delivery, but is also required to activate the entry domain for membrane translocation. Translocation function depends on precise cleavage after a conserved VENN peptide sequence, and the processed ∆VENN entry domain exhibits distinct biophysical and thermodynamic properties. By contrast, imprecisely processed CdiA-CT fragments do not undergo this transition and fail to translocate to the cytoplasm. These findings suggest that CdiA-CT processing induces a critical structural switch that converts the entry domain into a membrane-translocation competent conformation.
G-protein-coupled receptor 40 (GPR40) is known to play a role in the regulation of fatty acids, insulin secretion, and inflammation. However, the function of this receptor in human neutrophils, one ...of the first leukocytes to arrive at the site of infection, remains to be fully elucidated. In the present study, we demonstrate that GPR40 is upregulated on activated human neutrophils and investigated the functional effects upon treatment with a selective agonist; GW9508. Interestingly, GPR40 expression was up-regulated after neutrophil stimulation with platelet-activating factor (10 nM) or leukotriene B
(LTB
, 10 nM) suggesting potential regulatory roles for this receptor during inflammation. Indeed, GW9508 (1 and 10 μM) increased neutrophil chemotaxis in response to the chemokine IL-8 (30 ng/ml) and enhanced phagocytosis of
by approximately 50% when tested at 0.1 and 1 μM. These results were translated
whereby administration of GW9508 (10 mg/kg, i.p.) during
infections resulted in elevated peritoneal leukocyte infiltration with a higher phagocytic capacity. Importantly, GW9508 administration also modulated the lipid mediator profile, with increased levels of the pro-resolving mediators resolvin D3 and lipoxins. In conclusion, GPR40 is expressed by activated neutrophils and plays an important host protective role to aid clearance of bacterial infections.
Bacteria use several different secretion systems to deliver toxic EndoU ribonucleases into neighboring cells. Here, we present the first structure of a prokaryotic EndoU toxin in complex with its ...cognate immunity protein. The contact‐dependent growth inhibition toxin CdiA‐CTSTECO31 from Escherichia coli STEC_O31 adopts the eukaryotic EndoU fold and shares greatest structural homology with the nuclease domain of coronavirus Nsp15. The toxin contains a canonical His‐His‐Lys catalytic triad in the same arrangement as eukaryotic EndoU domains, but lacks the uridylate‐specific ribonuclease activity that characterizes the superfamily. Comparative sequence analysis indicates that bacterial EndoU domains segregate into at least three major clades based on structural variations in the N‐terminal subdomain. Representative EndoU nucleases from clades I and II degrade tRNA molecules with little specificity. In contrast, CdiA‐CTSTECO31 and other clade III toxins are specific anticodon nucleases that cleave tRNAGlu between nucleotides C37 and m2A38. These findings suggest that the EndoU fold is a versatile scaffold for the evolution of novel substrate specificities. Such functional plasticity may account for the widespread use of EndoU effectors by diverse inter‐bacterial toxin delivery systems.
EndoU ribonucleases are commonly used as weapons in inter‐bacterial conflict. We present the firststructure of a bacterial EndoU toxin domain. The CdiA‐CTSTECO31 toxin shares the same fold and catalytic triad as RNA processing enzymes from Xenopus laevis and human SARS virus. Unlike the eukaryotic EndoU ribonucleases, CdiA‐CTSTECO31 is exquisitely specific for the anticodon loop of tRNAGlu. These findings suggest that the EndoU fold is a versatile scaffold for the evolution of novel substrate specificities.
The present study determined the influence of initial moisture conditions on the production and consumption of nitrous oxide (N2O) during denitrification and on the isotopic fingerprint of ...soil-emitted N2O. Sieved arable soil was pre-incubated at two different moisture contents: pre-wet at 75% and pre-dry at 20% water-filled pore space. After wetting to 90% water-filled pore space the soils were amended with glucose (400 kg C ha−1) and KNO3 (80 kg N ha−1) and incubated for 10 days under a He/O2-atmosphere. Antecedent moisture conditions affected denitrification. N2 + N2O fluxes and the N2O-to-N2 ratio were higher in soils which were pre-incubated under dry conditions, probably because mobilization of organic C during the pre-treatment enhanced denitrification. Gaseous N fluxes showed similar time patterns of production and reduction of N2O in both treatments, where N2O fluxes were initially increasing and maximised 3–4 days after fertilizer application, and N2 fluxes were delayed by 1–2 days. Time courses of δ15Nbulk–N2O and δ18O–N2O exhibited in both treatments increasing trends until maximum N2 fluxes occurred, reflecting isotope fractionation during intense NO3− reduction. Later this trend slowed down in the pre-dry treatment, while δ18O–N2O was constant and δ15Nbulk–N2O decreased in the pre-wet treatment. We explain these time patterns by non-homogenous distribution of NO3− and denitrification activity, resulting from application of NO3− and glucose to the surface of the soil. We assume that several process zones were thus created, which affected differently the isotopic signature of N2O and the N2O and N2 fluxes during the different stages of the process. We modelled the δ15Nbulk–N2O using process rates and associated fractionation factors for the pre-treated soils, which confirmed our hypothesis. The site preference (SP) initially decreased while N2O reduction was absent, which we could not explain by the N-flux pattern. During the subsequent increase in N2 flux, SP and δ18O–N2O increased concurrently, confirming that this isotope pattern is indicative for N2O reduction to N2. The possible effect of the antecedent moisture conditions of the soil on N2O emissions was shown to be important.
► Antecedent soil moisture conditions affected denitrification. The N2+N2O fluxes and the N2/N2O ratios were higher in soils which were preincubated under dry conditions when compared to soils preincubated under wet conditions. ► The observed time patterns of N2O and N2 suggested a nonhomogeneous distribution of nitrate and denitrifier activity in the incubated soil. ► The time courses of δ15N–N2O, δ18O–N2O and Site Preference (SP) varied in line with the observed N2O and N2 emissions supporting the existence of multipools of nitrate. ► Isotopic fractionation modelling of the δ15N of emitted N2O, during the time series confirmed it being derived from multipools of nitrate. ► Manipulating the timing of fertiliser application according to antecedent moisture conditions could be a potential mitigation measure for soil N2O emissions.
Contact-dependent growth inhibition (CDI) is a mechanism of inter-cellular competition in which Gram-negative bacteria exchange polymorphic toxins using type V secretion systems. Here, we present ...structures of the CDI toxin from Escherichia coli NC101 in ternary complex with its cognate immunity protein and elongation factor Tu (EF-Tu). The toxin binds exclusively to domain 2 of EF-Tu, partially overlapping the site that interacts with the 3'-end of aminoacyl-tRNA (aa-tRNA). The toxin exerts a unique ribonuclease activity that cleaves the single-stranded 3'-end from tRNAs that contain guanine discriminator nucleotides. EF-Tu is required to support this tRNase activity in vitro, suggesting the toxin specifically cleaves substrate in the context of GTP·EF-Tu·aa-tRNA complexes. However, superimposition of the toxin domain onto previously solved GTP·EF-Tu·aa-tRNA structures reveals potential steric clashes with both aa-tRNA and the switch I region of EF-Tu. Further, the toxin induces conformational changes in EF-Tu, displacing a β-hairpin loop that forms a critical salt-bridge contact with the 3'-terminal adenylate of aa-tRNA. Together, these observations suggest that the toxin remodels GTP·EF-Tu·aa-tRNA complexes to free the 3'-end of aa-tRNA for entry into the nuclease active site.
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