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Cluster Model Study into the Catalytic Mechanism of α-Ketoglutarate Biodegradation by the Ethylene-Forming-Enzyme Reveals Structural Differences with Nonheme Iron Hydroxylases
No full textEthylene is an important signaling molecule in plants that triggers the growth of leaves, flowers and fruits. One of the enzymes involved in the biosynthesis of ethylene is the ethylene-forming enzyme (EFE), which is an usual nonheme iron enzyme that biodegrades α-ketoglutarate to three CO2 molecules and ethylene. As the detailed mechanism of EFE and its biosynthesis of ethylene remains controversial and particularly the function of the co-substrate L-arginine, we decided to pursue a density functional theory study on possible pathways of the enzyme leading to its ethylene biosyn-thesis and test many possible pathways and mechanisms. A large active site cluster model of 322 atoms was created that contains all features of the first- and second-coordination sphere of the active site and substrate (α-ketoglutarate) binding pockets. The calculations identify a persuccinate intermediate that triggers a bifurcation pathway in the enzyme and either reacts with a molecule of CO2 to form carbonate or forms a high-valent iron(IV)-oxo species through heterolytic dioxygen bond cleavage. Our studies show that both bifurcation pathways converge to the same intermediate again and can lead to ethylene products, although the two pathways have different kinetics. Interestingly, our studies also show that the iron(IV)-oxo itself can form carbonate and ethylene but through much higher barriers. As a matter of fact, these barriers are higher in energy than typical aliphatic hydroxylation barriers and may not be competitive with arginine hydroxylation. Inclusion of L-arginine co-substrate into the model leads to minor changes in the structure and fold and its charge and dipole moment does not seem to affect the first stage of the catalytic cycle. Moreover, key activation barriers appear little affected by the inclusion of L-arginine into the model. We, therefore, believe that L-arginine’s role is to lock α-ketoglutarate and its products into a tight binding pocket to enable its degradation and to prevent early release of CO2. Our studies show that due to distinct differences in α-ketoglutarate positioning between different arginine activating nonheme iron dioxygenases in the co-substrate binding pocket and its tighter binding in EFE, we predict that the release of CO2 is prevented in the first stage of the oxygen activation mechanism. This enables attack of CO2 on a persuccinate complex to form carbonate products leading to ethylene formation. The work gives suggestions on the engineering of EFE into a hydroxylase or improve the ethylene biosynthesis
Synthesis and characterisation of an integratively self-sorted [Fe<sub>4</sub>L<sub>6</sub>]<sup>8+</sup> tetrahedron
No full textIsolating metal-organic cage structures which incorporate more than one distinct ligand has been challenging due to competing pressures from narcissistic and social sorting phenomena. Here we report the first example of exclusive formation of a single tetrahedral product from a reaction mixture containing two different bidentate ligands. Exclusive formation of the tetrahedron, which incorporates one unique metal vertex, relies on a triamine to orientate the heteroditopic ligand. Inclusion of perchlorate counterions during the self-assembly process is also found to be a requirement if social sorting is to be avoided. The C3-symmetric structure is characterised by HR-MS, NMR spectroscopy and X-ray crystallography, and provides proof of principle for use of heteroditopic ligands in classical M4L6 supramolecular structures, opening exciting possibilities for their use in separation, storage and catalysis applications
Implications of the changes to patient online records access in English primary care
No full textNHS England have announced that patients in England registered for online services such as the NHS App will soon be able to see all new entries in their primary care record by default. This includes free text, hospital letters, test results, and new data added to the detailed coded record. Despite documented benefits of online records access for patients, primary care staff have raised concerns which can be grouped into issues around: 1) workload, 2) safeguarding, 3) patient confusion or distress, and 4) health inequities. This editorial examines each of these in turn, and considers what the future might hold for patient online records access.<br/
Analysing single cell secretions by ‘shadow imaging’
No full textHere, we describe a method, which we term ‘shadow imaging’, to analyse the secretions of individual cells at immune synapses, or other cell contacts. Following immune synapse formation and cellular activation on ligand-rich slides the position of each cell is recorded using a pulsed immunofluorescence stain against the proteins on the ligand-rich slide surface. The pulsed stain does not penetrate the synaptic cleft, resulting in an unlabelled region or ‘shadow’ beneath cells that is retained following cellular detachment. The secreted components, such as perforin, exosomes or other types of extracellular vesicles are retained on the slide and can be analysed on a single-cell basis using immunofluorescence. The ability to identify single cells secreting different combinations of particles, proteins and vesicles, enables us to better understand the heterogeneity in immune cell secretions, and can be used as a novel approach for phenotyping cell populations
Position Paper: Towards a Hybrid Approach to Protect Against Memory Safety Vulnerabilities
No full textMemory corruption bugs continue to plague lowlevel systems software, generally written in unsafe programming languages. In order to detect and protect against such exploits, many pre- and post-deployment techniques exist. In this position paper, we propose and motivate the need for a hybrid approach for the protection against memory safety vulnerabilities, combining techniques that can identify the presence (and absence) of vulnerabilities pre-deployment with those that can detect and mitigate such vulnerabilities post-deployment. Our proposed hybrid approach involves three layers: hardware runtime protection provided by capability hardware, software runtime protection provided by compiler instrumentation, and static analysis provided by bounded model checking and symbolic execution. The key aspect of the proposed hybrid approach is that the protection offered is greater than the sum of its parts – the expense of postdeployment runtime checks is potentially reduced via information obtained during pre-deployment analysis. During pre-deployment analysis, static checking can be guided by runtime information
Unintended consequences of patient online access to health records: a qualitative study in UK primary care
No full textEvolution of the Xenon (e,2e) Differential Cross Section from a Coplanar Geometry to the Perpendicular Plane in the Intermediate Energy Regime
No full textNew (e,2e) differential cross section measurements from xenon are presented as the scattering geometry changes from coplanar to the perpendicular plane. Measurements were taken at energies of 60 eV, 80 eV, and 100 eV above the ionization potential. These new data are compared to measurements collected for the first four noble gases and to that from xenon at lower energies. A new technique is used to measure the ratio of relative cross-sections between xenon and helium using a xenon-helium mixture. With this data absolute differential cross sections of xenon can be determined once the helium cross sections have been calculated
Host-guest interactions and confinement effects in HZSM-5 and chabazite zeolites studied by low-field NMR spin relaxation
No full textCharacterisation of fluid/solid interactions in porous materials is crucial for their design and optimisation, most notably in applications such as adsorption and catalysis. Yet, probing interfacial phenomena of fluids confined in porous systems is particularly challenging. NMR spin relaxation has emerged in recent years as a rapid, non-invasive experimental technique to probe adsorbate/adsorbent interactions in mesoporous catalytic materials. More recently, NMR relaxation measurements performed on high-field (300 MHz) superconducting magnets have been successfully validated as a robust method to characterise acidity in HZSM-5 zeolites. Expanding such techniques in the context of low-field, bench-top NMR instruments would be highly beneficial as it would make NMR relaxation a much more appealing and accessible tool for non-invasive, rapid characterisation of adsorbate/adsorbent interactions in zeolites and microporous materials. Herein, we validate the use of low-field, bench-top NMR spin relaxation as an indicator for characterising host-guest interactions in microporous zeolitic materials, using water as guest molecules confined within two different zeolite frameworks, HZSM-5 and chabazite, with varying silica/alumina ratio (SAR). The results reported here demonstrate the robustness and sensitivity of low-field NMR relaxation measurements as a rapid screening tool for characterising adsorption and molecular dynamics in microporous materials, with important implications for both academics and industrialists in terms of making the method more widely accessible, hence expanding the set of tools for material chemistry and characterisation