Crystallization and preliminary crystallographic analysis of the DNA gyrase B protein from B-stearothermophilus

DNA gyrase B (GyrB) from B. stearothermophilus has been crystallized in the presence of the non-hydrolyzable ATP analogue, 5'-adenylpl-beta-gamma-imidodiphosphate (ADPNP), by the dialysis method. A complete native data set to 3.7 Angstrom has been collected from crystals which belonged to the cubic space group I23 with unit-cell dimension a = 250.6 Angstrom. Self-rotation function analysis indicates the position of a molecular twofold axis. Low-resolution data sets of a thimerosal and a selenomethionine derivative have also been analysed. The heavy-atom positions are consistent with one dimer in the asymmetric unit

On the Tapping Mode Measurement for Young’s Modulus of Nanocrystalline Metal Coatings

Young’s modulus of nanocrystalline metal coatings is measured using the oscillating, that is, tapping, mode of a cantilever with a diamond tip. The resonant frequency of the cantilever changes when the diamond tip comes in contact with a sample surface. A Hertz-contact-based model is further developed using higher-order terms in a Taylor series expansion to determine a relationship between the reduced elastic modulus and the shift in the resonant frequency of the cantilever during elastic contact between the diamond tip and sample surface. The tapping mode technique can be used to accurately determine Young’s modulus that corresponds with the crystalline orientation of the sample surface as demonstrated for nanocrystalline nickel, vanadium, and tantalum coatings

Flexible Lipid Bilayers in Implicit Solvent

A minimalist simulation model for lipid bilayers is presented. Each lipid is represented by a flexible chain of beads in implicit solvent. The hydrophobic effect is mimicked through an intermolecular pair potential localized at the ``water''/hydrocarbon tail interface. This potential guarantees realistic interfacial tensions for lipids in a bilayer geometry. Lipids self assemble into bilayer structures that display fluidity and elastic properties consistent with experimental model membrane systems. Varying molecular flexibility allows for tuning of elastic moduli and area/molecule over a range of values seen in experimental systems.Comment: 5 pages, 5 figure

Practising in a post-truth world: Pandemic ethics can inform patient autonomy and clinical communication

The COVID-19 pandemic posed an unprecedented challenge to modern bioethical frameworks in the clinical setting. Now, as the pandemic stabilises and we learn to ‘live with COVID’, the medical community has a duty to evaluate its response to the challenge, and reassess our ethical reasoning, considering how we practise in the future. This article considers a number of clinical and bioethical challenges encountered by the author team and colleagues during the most severe waves of the pandemic. We argue that the changed clinical context may require reframing our ethical thought in such a manner as to adequately accommodate all parties in the clinical interaction. We argue that clinicians have become relatively disempowered by the ‘infodemic’, and do not necessarily have adequate skills or training to assess the scientific literature being published at an unprecedented rate. Conversely, we acknowledge that patients and families are more empowered by the infodemic, and bring this empowerment to bear on the clinical consultation. Sometimes these interactions can be unpleasant and threatening, and involve inviting clinicians to practise against best evidence or even illegally. Generally, these requests are framed within ‘patient autonomy’ (which some patients or families perceive to be unlimited), and several factors may prevent clinicians from adequately navigating these requests. In this article, we conclude that embracing a framework of shared decision-making (SDM), which openly acknowledges clinical expertise and in which patient and family autonomy is carefully balanced against other bioethics principles, could serve us well going forward. One such principle is the recognition of clinician expertise as holding weight in the clinical encounter, when framed in terms of non-maleficence and beneficence. Such a framework incorporates much of our learning and experience from advising and treating patients during the pandemic

An analysis of the development and implementation of a smartphone application for the delivery of antimicrobial prescribing policy: lessons learnt

Objectives: Smartphone usage amongst clinicians is widespread. Yet smartphones are not widely used for the dissemination of policy or as clinical decision support systems. We report here on the development, adoption and implementation process of the Imperial Antimicrobial Prescribing Application across five teaching hospitals in London. Methods: Doctors and clinical pharmacists were recruited to this study, which employed a mixed methods indepth case-study design with focus groups, structured pre- and post-intervention survey questionnaires and live data on application uptake. The primary outcome measure was uptake of the application by doctors and its acceptability. The development and implementation processes were also mapped. Results: The application was downloaded by 40% (376) of junior doctors with smartphones (primary target user group) within the first month and by 100% within 12 months. There was an average of 1900 individual access sessions per month, compared with 221 hits on the Intranet version of the policy. Clinicians (71%) reported that using the application improved their antibiotic knowledge. Conclusions: Clinicians rapidly adopted the mobile application for antimicrobial prescribing at the point of care, enabling the policy to reach a much wider audience in comparison with paper- and desktop-based versions of the policy. Organizations seeking to optimize antimicrobial prescribing should consider utilizing mobile technology to deliver point-of-care decision support. The process revealed a series of barriers, which will need to be addressed at individual and organizational levels to ensure safe and high-quality delivery of local policy at the point of care

Probing the properties of Be star discs with spectroastrometry and NLTE radiative transfer modelling: beta CMi

While the presence of discs around classical Be stars is well established, their origin is still uncertain. To understand what processes result in the creation of these discs and how angular momentum is transported within them, their physical properties must be constrained. This requires comparing high spatial and spectral resolution data with detailed radiative transfer modelling. We present a high spectral resolution, R~80,000, sub milli-arcsecond precision, spectroastrometric study of the circumstellar disc around the Be star beta CMi. The data are confronted with three-dimensional, NLTE radiative transfer calculations to directly constrain the properties of the disc. Furthermore, we compare the data to disc models featuring two velocity laws; Keperian, the prediction of the viscous disc model, and angular momentum conserving rotation. It is shown that the observations of beta CMi can only be reproduced using Keplerian rotation. The agreement between the model and the observed SED, polarisation and spectroastrometric signature of beta CMi confirms that the discs around Be stars are well modelled as viscous decretion discs.Comment: Accepted for publication in MNRA

An ATP-binding cassette-type cysteine transporter in Campylobacter jejuni inferred from the structure of an extracytoplasmic solute receptor protein

Campylobacter jejuni is a Gram-negative food-borne pathogen associated with gastroenteritis in humans as well as cases of the autoimmune disease Guillain Barre syndrome. C. jejuni is asaccharolytic because it lacks an active glycolytic pathway for the use of sugars as a carbon source. This suggests an increased reliance on amino acids as nutrients and indeed the genome sequence of this organism indicates the presence of a number of amino acid uptake systems. Cj0982, also known as CjaA, is a putative extracytoplasmic solute receptor for one such uptake system as well as a major surface antigen and vaccine candidate. The crystal structure of Cj0982 reveals a two-domain protein with density in the enclosed cavity between the domains that clearly defines the presence of a bound cysteine ligand. Fluorescence titration experiments were used to demonstrate that Cj0982 binds cysteine tightly and specifically with a K-d of similar to 10(-7) M consistent with a role as a receptor for a high- affinity transporter. These data imply that Cj0982 is the binding protein component of an ABC-type cysteine transporter system and that cysteine uptake is important in the physiology of C. jejuni

The mass ratio and formation mechanisms of Herbig Ae/Be star binary systems

We present B and R band spectroastrometry of a sample of 45 Herbig Ae/Be stars in order to study their binary properties. All but one of the targets known to be binary systems with a separation of ~0.1-2.0 arcsec are detected by a distinctive spectroastrometric signature. Some objects in the sample exhibit spectroastrometric features that do not appear attributable to a binary system. We find that these may be due to light reflected from dusty halos or material entrained in winds. We present 8 new binary detections and 4 detections of an unknown component in previously discovered binary systems. The data confirm previous reports that Herbig Ae/Be stars have a high binary fraction, 74+/-6 per cent in the sample presented here. We use a spectroastrometric deconvolution technique to separate the spatially unresolved binary spectra into the individual constituent spectra. The separated spectra allow us to ascertain the spectral type of the individual binary components, which in turn allows the mass ratio of these systems to be determined. In addition, we appraise the method used and the effects of contaminant sources of flux. We find that the distribution of system mass ratios is inconsistent with random pairing from the Initial Mass Function, and that this appears robust despite a detection bias. Instead, the mass ratio distribution is broadly consistent with the scenario of binary formation via disk fragmentation.Comment: Accepted for publication in MNRAS, minor changes made in proof stag

Global Analysis of Protein N-Myristoylation and Exploration of N-Myristoyltransferase as a Drug Target in the Neglected Human Pathogen Leishmania donovani

N-Myristoyltransferase (NMT) modulates protein function through the attachment of the lipid myristate to the N terminus of target proteins, and is a promising drug target in eukaryotic parasites such as Leishmania donovani. Only a small number of NMT substrates have been characterized in Leishmania, and a global picture of N-myristoylation is lacking. Here, we use metabolic tagging with an alkyne-functionalized myristic acid mimetic in live parasites followed by downstream click chemistry and analysis to identify lipidated proteins in both the promastigote (extracellular) and amastigote (intracellular) life stages. Quantitative chemical proteomics is used to profile target engagement by NMT inhibitors, and to define the complement of N-myristoylated proteins. Our results provide new insight into the multiple pathways modulated by NMT and the pleiotropic effects of NMT inhibition. This work constitutes the first global experimental analysis of protein lipidation in Leishmania, and reveals the extent of NMT-related biology yet to be explored for this neglected human pathogen