Search

The outer membrane of Gram-negative bacteria presents a robust physicochemical barrier protecting the cell from both the natural environment and acting as the first line of defense against antimicrobial materials. The proteins situated within the outer membrane are responsible for a range of biological functions including controlling influx and efflux. These outer membrane proteins (OMPs) are ultimately inserted and folded within the membrane by the β-barrel assembly machine (Bam) complex. The precise mechanism by which the Bam complex folds and inserts OMPs remains unclear. Here, we have developed a platform for investigating Bam-mediated OMP insertion. By derivatizing a gold surface with a copper-chelating self-assembled monolayer, we were able to assemble a planar system containing the complete Bam complex reconstituted within a phospholipid bilayer. Structural characterization of this interfacial protein-tethered bilayer by polarized neutron reflectometry (PNR) revealed distinct regions consistent with known high-resolution models of the Bam complex. Additionally, by monitoring changes of mass associated with OMP insertion by quartz crystal microbalance with dissipation monitoring (QCM-D), we were able to demonstrate the functionality of this system by inserting two diverse OMPs within the membrane, pertactin and OmpT. This platform has promising application in investigating the mechanism of Bam-mediated OMP insertion, in addition to OMP function and activity within a phospholipid bilayer environment.

The Mla pathway is believed to be involved in maintaining the asymmetrical Gram-negative outer membrane via retrograde phospholipid transport. The pathway is composed of three components: the outer membrane MlaA-OmpC/F complex, a soluble periplasmic protein, MlaC, and the inner membrane ATPase, MlaFEDB complex. Here, we solve the crystal structure of MlaC in its phospholipid-free closed apo conformation, revealing a pivoting β-sheet mechanism that functions to open and close the phospholipid-binding pocket. Using the apo form of MlaC, we provide evidence that the inner-membrane MlaFEDB machinery exports phospholipids to MlaC in the periplasm. Furthermore, we confirm that the phospholipid export process occurs through the MlaD component of the MlaFEDB complex and that this process is independent of ATP. Our data provide evidence of an apparatus for lipid export away from the inner membrane and suggest that the Mla pathway may have a role in anterograde phospholipid transport.

The polymeric mucin MUC5B provides the structural and functional framework of respiratory mucus, conferring both viscoelastic and antimicrobial properties onto this vital protective barrier. Whilst it is established that MUC5B forms disulfide-linked linear polymers, how this relates to their packaging in secretory granules, and their molecular form in mucus remain to be fully elucidated. Moreover, the role of the central heavily O-glycosylated mucin domains in MUC5B conformation is incompletely described. Here we have completed a detailed structural analysis on native MUC5B polymers purified from saliva and subsequently investigated how MUC5B conformation is affected by changes in calcium concentration and pH, factors important for mucin intragranular packaging and post-secretory expansion. The results identify that MUC5B has a beaded structure repeating along the polymer axis and suggest that these repeating motifs arise from distinct glycosylation patterns. Moreover, we demonstrate that the conformation of these highly entangled linear polymers is sensitive to calcium concentration and changes in pH. In the presence of calcium (Ca2+, 10 mM) at pH 5.0, MUC5B adopted a compact conformation which was lost either upon removal of calcium with EGTA, or by increasing the pH to 7.4. These results suggest a pathway of mucin collapse to enable intracellular packaging and mechanisms driving mucin expansion following secretion. They also point to the importance of the tight control of calcium and pH during different stages of mucin biosynthesis and secretion, and in the generation of correct mucus barrier properties.

Bacterial infection remains a major challenge to healthcare and is responsible for significant morbidity and mortality. This situation is becoming complicated by an increasingly ageing and susceptible population and large numbers of bacterial isolates which have developed resistance to antibiotics. Bacteria which form biofilms and colonise or infect medical devices or wounds are particularly hard to treat as biofilms are inherently highly antibiotic resistant. Most infections have a component where bacteria exist as a biofilm and as a result prevention or treatment of biofilm associated infections is highly important. A number of novel strategies to kill biofilms have been in development, these include the use of weak organic acids, photo irradiation and the application of bacteriophage. All have promise and are able to effectively kill biofilms in model systems but for each there are still unanswered questions. This review summarizes the main features of biofilm infections, each of these novel approaches and the evidence that is still lacking before these potential treatments can be incorporated into clinical usage.

Cystic fibrosis (CF) is characterized by increased mucus viscosity and delayed mucociliary clearance that contributes to progressive decline of lung function. Mucus in the respiratory and GI tract is excessively adhesive in the presence of airway dehydration and excess extracellular Ca(2+) upon mucin release, promoting hyperviscous, densely packed mucins characteristic of CF. Therapies that target mucins directly through ionic interactions remain unexploited. Here we show that poly (acetyl, arginyl) glucosamine (PAAG), a polycationic biopolymer suitable for human use, interacts directly with mucins in a Ca(2+)-sensitive manner to reduce CF mucus viscoelasticity and improve its transport. Notably, PAAG induced a linear structure of purified MUC5B and altered its sedimentation profile and viscosity, indicative of proper mucin expansion. In vivo, PAAG nebulization improved mucociliary transport in CF rats with delayed mucus clearance, and cleared mucus plugging in CF ferrets. This study demonstrates the potential use of a synthetic glycopolymer PAAG as a molecular agent that could benefit patients with a broad array of mucus diseases.

SecA is an evolutionarily conserved and essential ATPase that is required for the translocation of a subset of proteins across the cytoplasmic membrane in bacteria. SecA can recognise proteins that are destined for translocation as they are still being synthesised in order to deliver them to the membrane-bound Sec machinery. However, the mechanism of cotranslational substrate recognition is not well defined. In E. coli, SecA contains a relatively long C-terminal tail (CTT), which consists of a small metal-binding domain (MBD) that is attached to the C-terminus of the catalytic core by a flexible linker (FLD). In this study, we investigated the role of the CTT in the interaction of SecA with the ribosome and nascent polypeptides. Previous work indicates that the CTT is required for interaction with the molecular chaperone SecB. However, phylogenetic analysis suggested that the CTT (and the MBD in particular) has an additional function. Binding experiments indicated that the CTT interacts with 70S ribosomes, and disruption of the entire CTT moderately reduced the affinity of SecA for ribosomes. However, disruption of the MBD alone significantly increased the affinity of SecA for ribosomes and inhibited the interaction of SecA with substrate protein, suggesting that the FLD affects the conformation of SecA. Photocrosslinking and mass spectrometry indicated that the FLD is bound at the site where SecA binds to substrate proteins. Structural analysis by x-ray crystallography and small-angle x-ray scattering (SAXS) provided insight into how the CTT influences the structure of SecA in solution. Finally, site-specific crosslinking experiments indicated that binding to nascent substrate protein affects the conformation of SecA. Taken together, our results suggest that the CTT regulates the ability of SecA to interact with substrate proteins.

The Mla pathway is believed to be involved in maintaining the asymmetrical Gram-negative outer membrane via retrograde phospholipid transport. The pathway is composed of three components: the outer membrane MlaA-OmpC/F complex, a soluble periplasmic protein, MlaC, and the inner membrane ATPase, MlaFEDB complex. Here, we solve the crystal structure of MlaC in its phospholipid-free closed apo conformation, revealing a pivoting β-sheet mechanism that functions to open and close the phospholipid-binding pocket. Using the apo form of MlaC, we provide evidence that the inner-membrane MlaFEDB machinery exports phospholipids to MlaC in the periplasm. Furthermore, we confirm that the phospholipid export process occurs through the MlaD component of the MlaFEDB complex and that this process is independent of ATP. Our data provide evidence of an apparatus for lipid export away from the inner membrane and suggest that the Mla pathway may have a role in anterograde phospholipid transport.

The Mla pathway is believed to be involved in maintaining the asymmetrical Gram-negative outer membrane via retrograde phospholipid transport. The pathway is composed of 3 components: the outer membrane MlaA-OmpC/F complex, a soluble periplasmic protein, MlaC, and the inner membrane ATPase, MlaFEDB complex. Here we solve the crystal structure of MlaC in its phospholipid free closed apo conformation, revealing a novel pivoting β-sheet mechanism which functions to open and close the phospholipid-binding pocket. Using the apo form of MlaC we provide evidence that the Mla pathway functions in an anterograde rather than a retrograde direction by showing the inner membrane MlaFEDB machinery exports phospholipids and transfers them to MlaC in the periplasm. We confirm that the lipid export process occurs through the MlaD component of the MlaFEDB complex. This lipid export process is shown to be independent of ATP. Our data provides, for the first time, evidence of an apparatus for lipid export to the outer membrane.

Background Nintedanib has been shown to slow disease progression in patients with idiopathic pulmonary fibrosis (IPF). It was approved by the National Institute for Health and Care Excellence (NICE) in January 2016 for IPF patients with a forced vital capacity (FVC) of 50–80% in the United Kingdom (UK). Aim To report real world data about our early clinical experience using nintedanib in 187 patients with a multi-disciplinary (MDT) diagnosis of IPF in a manufacturer funded patient in need scheme (three UK centres) prior to NICE approval. Methods All patients with a MDT diagnosis of IPF from December 2014 to January 2016 commenced on nintedanib were included. Demographic details, adverse events (AEs) and where available lung function results were retrospectively collected from clinical letters. Results 187 patients (76% males) with a median age of 72 years (49–89) were treated with nintedanib. The average pre-treatment FVC was 81.1 ± 19.8% and diffusion capacity of the lungs for carbon monoxide was 43.9 ± 15% (n = 82). Fifty percent of patients started nintedanib because they were ineligible for pirfenidone due to an FVC > 80%. The median treatment course was 8 ± 4 months. The majority of patients experienced 1–3 AEs with nintedanib (52%, n = 97). The most frequent AEs were diarrhoea (50%), nausea (36%), reduced appetite (24%), tiredness (20%) and gastro-oesophageal reflux (18%). The majority of AEs resulted in no change in treatment (64%, n = 461). 21% (n = 150) of AEs resulted in a dose reduction and 13% (n = 94) necessitated discontinuation of treatment. 1 in 5 patients discontinued treatment either temporarily or on a permanent basis during the monitoring period. In a select cohort of patients, a statistically significant greater proportion of patients remained stable or improved and a lower proportion declined, as depicted by FVC changes of > 5% after nintedanib commencement (P

A variety of simulation-based strategies have been developed to facilitate technical and non-technical learning in simulated airway scenarios, some of which follow predetermined algorithms to guide the responders.1 2 However, simulation can also be used to devise the responses to emergency scenarios, particularly useful for relatively rare events. We describe this novel use of in-situ simulation to iteratively test and refine practice guidelines during simulated tracheostomy emergencies. These methods can challenge established expert peer review processes for developing airway algorithms. The UK National Tracheostomy Safety Project (NTSP) was tasked with improving emergency tracheostomy management. We recognised that feedback from simulated scenarios could be used in the development of emergency algorithms (figure 1). A Working Party was established comprising medical, nursing and allied health professionals with experience of simulation training and tracheostomy care, supported by representatives of key organisations with a stated interest in airway management.2 Figure 1 Timeline detailing the key steps in developing the emergency algorithms. Four distinct simulated scenarios were developed from clinical reports.3 Manikins deteriorated physiologically (eg, progressive hypoxia), intended to prompt specific actions from participants. Scenarios were tested for validity by four members of the Working Party (BAM, DA, LB and JAM) prior to consenting multidisciplinary participants attempting them. Formal ethical approval was not required but local research governance procedures were followed. Simulators used were SimMan Essential (Laerdal, Stavanger, Norway) and MetiMan …

In this paper a solar sail magnetotail mission concept was examined. The 43-m square solar sail is used to providethe required propulsion for continuous sun-synchronous apse-line precession. The main driver in this mission was found to be the reduction of launch mass and mission cost while enabling a nominal duration of 2 years within the framework of a demonstration mission. It was found that the mission concept provided an excellent solar sail technology demonstration option. The baseline science objectives and engineering goals were addressed, and mission analysis for solar sail, electric, and chemical propulsion performed. Detailed subsystems were defined for each propulsion system and it was found that the optimum propulsion system is solar sailing. A detailed tradeoff as to the effect of spacecraft and sail technology levels, and requirements, on sail size is presented for the first time. The effect of, for example, data acquisition rate and RF output power on sail size is presented, in which it is found that neither have a significant effect. The key sail technology requirements have been identified through a parametric analysis.

A conventional Mercury sample return mission requires significant launch mass due to the large AV required for the outbound and return trips and the large mass of a planetary lander and ascent vehicle. It is shown that solar sail spacecraft can be used to reduce lander mass allocation by delivering the lander to a low, thermally safe orbit close to the planetary terminator. In addition, the ascending node of the solar sail spacecraft parking orbit plane can be artificially forced to avoid out-of-plane maneuvers during ascent from the planetary surface. Propellant mass is not an issue for spacecraft with solar sails, and so a sample can be returned relatively easily without resorting to lengthy, multiple gravity assists. A 275-m2 solar sail with a sail assembly loading of 5.9 g/m2 is used to deliver a lander, cruise stage, and science payload to a forced sun-synchronous orbit at Mercury in 2.85 years. The lander acquires samples and conducts limited surface exploration. An ascent vehicle delivers a small cold-gas rendezvous vehicle containing the samples for transfer to the solar sail spacecraft. The solar sail spacecraft then spirals back to Earth in 1 year. The total mission launch mass is 2353 kg, launched using a Japanese H2 class launch vehicle, C3 = 0. Extensive launch date scans have revealed an optimal launch date in April 2014 with sample return to Earth 4.4 years later. Solar sailing reduces launch mass by 60% and trip time by 40%, relative to conventional mission concepts. In comparison, mission analysis has demonstrated that solar-sail-powered Mars and Venus sample returns appear to have only modest benefits in terms of reduced launch mass, at the expense of longer mission durations, than do conventional propulsion systems.

The use of solar sail propulsion is investigated for both Mercury orbiter (MO) and Mercury sample return missions (MeSR). It will be demonstrated that solar sail propulsion can significantly reduce launch mass and enhance payload mass fractions for MO missions, while MeSR missions are enabled, again with a relatively low launch mass. Previous investigations of MeSR type missions using solar electric propulsion have identified a requirement for an Ariane V launcher to deliver a lander and sample return vehicle. The analysis presented in this paper demonstrates that, in principle, a MeSR mission can be enabled using a single Soyuz-Fregat launch vehicle, leading to significant reductions in launch mass and mission costs. Similarly, it will be demonstrated that the full payload of the ESA Bepi Colombo orbiter mission can be delivered to Mercury using a Soyuz-Fregat launch vehicle, rather than Ariane V, again leading to a reduction in mission costs.

SOLAR sailing offers the potential to reduce the required initial mass in low Earth orbit for future piloted Mars missions.1i3 Athough solar sailing does not appear to be suitable for crew transport, it can be an extremely efŽ cient mode of propulsion for the transport of logistics in support of a human crew. This may include premission caching of logistics and/or resupply missions to support long-duration surface stays. Because solar sails do not require reaction mmass, a single solar sail may, in principle, be used for multiple Earth-Mars-Earth round trips. The limit to the number of round trips that can be made by a single solar sail will be dictated largely by the lifetime of the sail Ž lm in the space environment. Previous studies of the solar sail cargo mission problem have considered either point designs1 or have considered speciŽ c launch opportunities.2 However, a key question that arises when considering the use of solar sails for round-trip logistic supply missions is the optimum payload mass fraction of the solar sail. As the payload mass fraction of the solar sail is increased, a greater payload mass is delivered, but the trip time will also increase. Similarly, as the payload mass fraction of the solar sail is decreased, a smaller payload is delivered, but with a shorter trip time. The payload mass fractionthat is selectedshould, therefore,be chosento balancethese two effects and maximize the mean rate of payload mass transfer to Mars.

SOLAR sails have long been seen as an attractive concept for low-thrust propulsion.They transcend reliance on reaction mass and have the ability to provide a small, but continuous, acceleration. Because propellant mass is not an issue, high-performance sails can enable new exotic non-Keplerianor bits (NKOs)1 that are not feasible for conventional chemical or electric propulsion.A constant out-of plane sail force is utilized to raise the spacecraft's orbit high above the ecliptic plane in two- or three-body systems. Potential beneŽfits to the science community are large. Circular, displaced orbits can be used to provide continuous observation of the solar poles or to provide a unique vantage point for infrared astronomy. (There is much less resolution-limiting dust out of the ecliptic plane enabling smaller telescope mirror dimensions for equivalent performance.) Very high performance sails can even levitate, in equilibrium, at any point in space.

Cometary Rendezvous and Flybys have large V requirements, which impose almost unattainable, and sometimes prohibitive, demands on the propellant budget of conventional, chemical propulsion. Ion Propulsion is a viable alternative, but as the number and difficulty of target objectives increases then the potential of this technology becomes rapidly less attractive. Solar sails exhibit an extremely high effective specific impulse over long mission durations. No propellant is required so that large changes in V could be realised without necessitating the introduction of complex gravity assists, which prolong mission duration and restrict launch opportunities. The endurance of the structures and materials are thus the only limiting factors dictating the number and range of bodies with which the solar-sail propelled vehicle can encounter throughout its lifetime. In this paper we have analysed a number of high-energy, small-body mission scenarios using a parameterised approach to sail control representation. The sail cone and clock angle histories were characterised by linear interpolation across a set of discrete nodes. The optimal control problem was thus transcribed to a Non-Linear Programming problem to select the optimal controls at the nodes that minimised the transfer time while enforcing the cartesian end-point boundary constraints (6 states for rendezvous, 3 for flypast). The Fortran77 optimisation package NPSOL 5.0 was used for this purpose with the variational equations of motion formulated in modified equinoctial orbital elements and integrated using a variable-order, adaptive step-size Adams-Moulton-Bashforth method. We present optimal rendezvous trajectories to Short-Period Comets such as 46P/Wirtanen in 484 days with a sail characteristic acceleration of 1.9 mms-2, and with 2P/Encke in 574 days with a characteristic acceleration of 1.0 mms-2. An analysis using high-performance sails has been conducted to permit fast flyby intercepts of newly discovered Long Period Comets (LPCs). Previous examples adopted were C/1995 O1/Hale- Bopp, C/1995 Y1/Hyakutake, C/1999 T1/McNaught-Hartley, C/1999 F1/Catalina, C/1999 N2/Lynn and C/1999 H1/Lee, to demonstrate the feasibility of a late launch to quickly intercept a new LPC using a solar sail. Since the time between discovery of a new LPC such as Hale-Bopp and perihelion passage was less then 2 years, this then leaves a very short time-span for orbit determination, preparation, planning and operational phases. Preliminary mission analysis shows that a Hale-Bopp perihelion flypast could have been achieved, with a sail characteristic acceleration of 5.0 mms-2, by launching just 209 days before comet perihelion passage. With a characteristic acceleration of 2.0 mms-2 Hale-Bopp could also have been intercepted at its descending node by launching 270 days before nodal descent. The sail could then have returned to rendezvous with the Earth 261 days later, giving a minimum total mission turn-around time of 531 days. An alternative, dual flyby scenario has been investigated, to continue on to C/1997 D1/Mueller, after which solar system escape was reached and arrival at Heliopause would occur in 12 years. Solar Electric Propulsion has been adopted as the primary propulsion system for the DAWN dual asteroid rendezvous mission scheduled for launch in 2006. The objective of this mission is to rendezvous with inner main-belt asteroids, Vesta and Ceres. We have also investigated solar sail adaptation to this mission, for the same launch date and 11 month orbiter stay-times. We have extended the mission objectives to two further asteroids, Lucina and Lutetia, with the aim of demonstrating a Mainbelt Asteroid Survey scenario.

Various alternatives to psychiatric in-patient care have recently been reviewed (Boardman & Hodgson, 2000). The Accredited Accommodation Scheme in north Powys represents a novel, local alternative to in-patient care, for a targeted group of patients with enduring forms of mental illness.

Of all the decisions taken by psychiatrists with or on behalf of their patients, few are as potentially far-reaching as the decision to repatriate a mentally ill person to his or her country of origin. Although many psychiatrists have anecdotal experience of individual cases, published research on repatriation is surprisingly sparse. Burke (1973) reported 66 persons repatriated from Britain to Jamaica over a four year period, and Asuni (1968) found 82 returning via Aro Hospital, Nigeria, over a similar period. Although it is difficult to extrapolate an estimate of the number leaving the UK each year, these figures suggest that the practice is not uncommon and that significant, possibly increasing, numbers of people are affected.

An assessment of terrestrial planet sample return missions utilising solar sail propulsion is presented, in addition to sample return from a high inclination near-Earth asteroid, 2001 QP153. Payloads have been sized based on existing studies or have been custom designed and sized. Heliocentric and planetocentric trajectory analysis has been conducted to assess the sail performance level required to return samples within a reasonable timescale, whilst maintaining manageable sail dimensions. Sail technology is based on projected data from existing development programmes. Solar sailing appears to offer modest benefits in returning a sample from Mars or Venus, but significant benefits for Mercury Sample Return. In addition, sample return from high-energy targets such as asteroid 2001 QP153 appears to be only possible when using the solar sail as the enabling propulsion technology.

The 1959 Mental Health Act has provided the legal framework for psychiatric practice in the UK for the past two decades. The Mental Health (Amendment) Bill (DHSS, 1981) currently before Parliament proposes to update the Act by improving the safeguards for detained patients, clarifying the position of staff looking after them, and by the removal of uncertainties in the law. The Bill incorporates changes relating to the compulsory care of Mentally Disordered patients in the community whereby a person may be accepted into Guardianship on the grounds that he or she is suffering from a Mental Disorder. Once accepted into Guardianship, the person or body named as Guardian has the power to exercise control over the person as if he or they were the father of the patient, and the patient was under 14 years of age.