Structural basis of lipopolysaccharide maturation by the O-antigen ligase.

The outer membrane of Gram-negative bacteria has an external leaflet that is largely composed of lipopolysaccharide, which provides a selective permeation barrier, particularly against antimicrobials1. The final and crucial step in the biosynthesis of lipopolysaccharide is the addition of a species-dependent O-antigen to the lipid A core oligosaccharide, which is catalysed by the O-antigen ligase WaaL2. Here we present structures of WaaL from Cupriavidus metallidurans, both in the apo state and in complex with its lipid carrier undecaprenyl pyrophosphate, determined by single-particle cryo-electron microscopy. The structures reveal that WaaL comprises 12 transmembrane helices and a predominantly α-helical periplasmic region, which we show contains many of the conserved residues that are required for catalysis. We observe a conserved fold within the GT-C family of glycosyltransferases and hypothesize that they have a common mechanism for shuttling the undecaprenyl-based carrier to and from the active site. The structures, combined with genetic, biochemical, bioinformatics and molecular dynamics simulation experiments, offer molecular details on how the ligands come in apposition, and allows us to propose a mechanistic model for catalysis. Together, our work provides a structural basis for lipopolysaccharide maturation in a member of the GT-C superfamily of glycosyltransferases.

Establishment of a relationship between blastomere geometry and YAP localisation during compaction.

Precise patterning within the three-dimensional context of tissues, organs and embryos implies that cells can sense their relative position. During preimplantation development, outside and inside cells rely on apicobasal polarity and the Hippo pathway to choose their fate. Despite recent findings suggesting that mechanosensing might be central to this process, the relationship between blastomere geometry (i.e. shape and position) and the Hippo pathway effector YAP remains unknown. We used a highly quantitative approach to analyse information on the geometry and YAP localisation of individual blastomeres of mouse and human embryos. We identified the proportion of exposed cell surface area as most closely correlating with the nuclear localisation of YAP. To test this relationship, we developed several hydrogel-based approaches to alter blastomere geometry in cultured embryos. Unbiased clustering analyses of blastomeres from such embryos revealed that this relationship emerged during compaction. Our results therefore pinpoint the time during early embryogenesis when cells acquire the ability to sense changes in geometry and provide a new framework for how cells might integrate signals from different membrane domains to assess their relative position within the embryo.

Photonically-driven Schottky diode based 0.3 THz heterodyne receiver.

Photonics-based technologies are key players in a number of emerging applications in the terahertz (THz) field. These solutions exploit the well-known advantages of optical devices, such as ultra-wide tuneability and direct integration with fiber networks. However, THz receivers are mainly implemented by fully electronic solutions, where Schottky barrier diodes (SBD) are the preferred option as detectors and mixers due to their excellent response within the THz range at room temperature, and technological maturity. Here, we demonstrate an SBD-based subharmonic mixer (SHM) at 300 GHz pumped with a photonic local oscillator. The Schottky mixer is a prototype designed and manufactured by ACST GmbH, operating at 270-320 GHz. The local oscillator is generated by photomixing on a high-frequency and high-power uni-travelling-carrier photodiode (UTC-PD), providing enough power to saturate conversion loss. Minimum single-side-band conversion loss of 14.4 dB and a peak dynamic range of 130 dB have been measured. Finally, as a proof of concept we realize an all-photonics-based 5 Gbps wireless bridge, utilizing the optically-pumped SBD mixer. With this work, we prove the feasibility of high-performance hybrid Schottky-photonic THz receivers, incorporating the best of both worlds.

Using Twitter Comments to Understand People's Experiences of UK Health Care During the COVID-19 Pandemic: Thematic and Sentiment Analysis.

BACKGROUND: The COVID-19 pandemic has led to changes in health service utilization patterns and a rapid rise in care being delivered remotely. However, there has been little published research examining patients' experiences of accessing remote consultations since COVID-19. Such research is important as remote methods for delivering some care may be maintained in the future. OBJECTIVE: The aim of this study was to use content from Twitter to understand discourse around health and care delivery in the United Kingdom as a result of COVID-19, focusing on Twitter users' views on and attitudes toward care being delivered remotely. METHODS: Tweets posted from the United Kingdom between January 2018 and October 2020 were extracted using the Twitter application programming interface. A total of 1408 tweets across three search terms were extracted into Excel; 161 tweets were removed following deduplication and 610 were identified as irrelevant to the research question. The remaining relevant tweets (N=637) were coded into categories using NVivo software, and assigned a positive, neutral, or negative sentiment. To examine views of remote care over time, the coded data were imported back into Excel so that each tweet was associated with both a theme and sentiment. RESULTS: The volume of tweets on remote care delivery increased markedly following the COVID-19 outbreak. Five main themes were identified in the tweets: access to remote care (n=267), quality of remote care (n=130), anticipation of remote care (n=39), online booking and asynchronous communication (n=85), and publicizing changes to services or care delivery (n=160). Mixed public attitudes and experiences to the changes in service delivery were found. The proportion of positive tweets regarding access to, and quality of, remote care was higher in the immediate period following the COVID-19 outbreak (March-May 2020) when compared to the time before COVID-19 onset and the time when restrictions from the first lockdown eased (June-October 2020). CONCLUSIONS: Using Twitter data to address our research questions proved beneficial for providing rapid access to Twitter users' attitudes to remote care delivery at a time when it would have been difficult to conduct primary research due to COVID-19. This approach allowed us to examine the discourse on remote care over a relatively long period and to explore shifting attitudes of Twitter users at a time of rapid changes in care delivery. The mixed attitudes toward remote care highlight the importance for patients to have a choice over the type of consultation that best suits their needs, and to ensure that the increased use of technology for delivering care does not become a barrier for some. The finding that overall sentiment about remote care was more positive in the early stages of the pandemic but has since declined emphasizes the need for a continued examination of people's preference, particularly if remote appointments are likely to remain central to health care delivery.

A Dynamic Network of Proteins Facilitate Cell Envelope Biogenesis in Gram-Negative Bacteria.

Bacteria must maintain the ability to modify and repair the peptidoglycan layer without jeopardising its essential functions in cell shape, cellular integrity and intermolecular interactions. A range of new experimental techniques is bringing an advanced understanding of how bacteria regulate and achieve peptidoglycan synthesis, particularly in respect of the central role played by complexes of Sporulation, Elongation or Division (SEDs) and class B penicillin-binding proteins required for cell division, growth and shape. In this review we highlight relationships implicated by a bioinformatic approach between the outer membrane, cytoskeletal components, periplasmic control proteins, and cell elongation/division proteins to provide further perspective on the interactions of these cell division, growth and shape complexes. We detail the network of protein interactions that assist in the formation of peptidoglycan and highlight the increasingly dynamic and connected set of protein machinery and macrostructures that assist in creating the cell envelope layers in Gram-negative bacteria.

Psychometric evaluation of patient-reported experience measures: is it valid?

Multiple published studies and reviews have advocated the application of psychometric methods to the validation of patient experience measurement. Some such methods depend on measurement assumptions that may not be appropriate for patient experience. Rather than being the default approach for the validation of patient experience measurement, we argue that psychometric methods should be reviewed critically to determine their fit to the measurement application, and alternative approaches explored, so that the most appropriate validation methods can be identified.

High performance waveguide uni-travelling carrier photodiode grown by solid source molecular beam epitaxy.

The first waveguide coupled phosphide-based UTC photodiodes grown by Solid Source Molecular Beam Epitaxy (SSMBE) are reported in this paper. Metal Organic Vapour Phase Epitaxy (MOVPE) and Gas Source MBE (GSMBE) have long been the predominant growth techniques for the production of high quality InGaAsP materials. The use of SSMBE overcomes the major issue associated with the unintentional diffusion of zinc in MOVPE and gives the benefit of the superior control provided by MBE growth techniques without the costs and the risks of handling toxic gases of GSMBE. The UTC epitaxial structure contains a 300 nm n-InP collection layer and a 300 nm n++-InGaAsP waveguide layer. UTC-PDs integrated with Coplanar Waveguides (CPW) exhibit 3 dB bandwidth greater than 65 GHz and output RF power of 1.1 dBm at 100 GHz. We also demonstrate accurate prediction of the absolute level of power radiated by our antenna integrated UTCs, between 200 GHz and 260 GHz, using 3d full-wave modelling and taking the UTC-to-antenna impedance match into account. Further, we present the first optical 3d full-wave modelling of waveguide UTCs, which provides a detailed insight into the coupling between a lensed optical fibre and the UTC chip.

5 Gbps wireless transmission link with an optically pumped uni-traveling carrier photodiode mixer at the receiver.

We report the first demonstration of a uni-traveling carrier photodiode (UTC-PD) used as a 5 Gbps wireless receiver. In this experiment, a 35.1 GHz carrier was electrically modulated with 5 Gbps non-return with zero on-off keying (NRZ-OOK) data and transmitted wirelessly over a distance of 1.3 m. At the receiver, a UTC-PD was used as an optically pumped mixer (OPM) to down-convert the received radio frequency (RF) signal to an intermediate frequency (IF) of 11.7 GHz, before it was down-converted to the baseband using an electronic mixer. The recovered data show a clear eye diagram, and a bit error rate (BER) of less than 10-8 was measured. The conversion loss of the UTC-PD optoelectronic mixer has been measured at 22 dB. The frequency of the local oscillator (LO) used for the UTC-PD is defined by the frequency spacing between the two optical tones, which can be broadly tuneable offering the frequency agility of this photodiode-based receiver.

Interaction landscape of membrane-protein complexes in Saccharomyces cerevisiae.

Macromolecular assemblies involving membrane proteins (MPs) serve vital biological roles and are prime drug targets in a variety of diseases. Large-scale affinity purification studies of soluble-protein complexes have been accomplished for diverse model organisms, but no global characterization of MP-complex membership has been described so far. Here we report a complete survey of 1,590 putative integral, peripheral and lipid-anchored MPs from Saccharomyces cerevisiae, which were affinity purified in the presence of non-denaturing detergents. The identities of the co-purifying proteins were determined by tandem mass spectrometry and subsequently used to derive a high-confidence physical interaction map encompassing 1,726 membrane protein-protein interactions and 501 putative heteromeric complexes associated with the various cellular membrane systems. Our analysis reveals unexpected physical associations underlying the membrane biology of eukaryotes and delineates the global topological landscape of the membrane interactome.

Foundry fabricated photonic integrated circuit optical phase lock loop.

This paper describes the first foundry-based InP photonic integrated circuit (PIC) designed to work within a heterodyne optical phase locked loop (OPLL). The PIC and an external electronic circuit were used to phase-lock a single-line semiconductor laser diode to an incoming reference laser, with tuneable frequency offset from 4 GHz to 12 GHz. The PIC contains 33 active and passive components monolithically integrated on a single chip, fully demonstrating the capability of a generic foundry PIC fabrication model. The electronic part of the OPLL consists of commercially available RF components. This semi-packaged system stabilizes the phase and frequency of the integrated laser so that an absolute frequency, high-purity heterodyne signal can be generated when the OPLL is in operation, with phase noise lower than -100 dBc/Hz at 10 kHz offset from the carrier. This is the lowest phase noise level ever demonstrated by monolithically integrated OPLLs.

Modelling and measurement of the absolute level of power radiated by antenna integrated THz UTC photodiodes.

We determine the output impedance of uni-travelling carrier (UTC) photodiodes at frequencies up to 400 GHz by performing, for the first time, 3D full-wave modelling of detailed UTC photodiode structures. In addition, we demonstrate the importance of the UTC impedance evaluation, by using it in the prediction of the absolute power radiated by an antenna integrated UTC, over a broad frequency range and confirming the predictions by experimental measurements up to 185 GHz. This is done by means of 3D full-wave modelling and is only possible since the source (UTC) to antenna impedance match is properly taken into account. We also show that, when the UTC-to-antenna coupling efficiency is modelled using the classical junction-capacitance/series-resistance concept, calculated and measured levels of absolute radiated power are in substantial disagreement, and the maximum radiated power is overestimated by a factor of almost 7 dB. The ability to calculate the absolute emitted power correctly enables the radiated power to be maximised through optimisation of the UTC-to-antenna impedance match.

Accurate equivalent circuit model for millimetre-wave UTC photodiodes.

We present a comprehensive study of uni-travelling carrier photodiode impedance and frequency photo-response supported by measurements up to 110 GHz. The results of this investigation provide valuable new information for the optimisation of the coupling efficiency between UTC-PDs and THz antennas. We show that the measured impedance cannot be explained employing the standard junction-capacitance/series-resistance concept and propose a new model for the observed effects, which exhibits good agreement with the experimental data. The achieved knowledge of the photodiode impedance will allow the absolute level of power emitted by antenna integrated UTCs to be predicted and ultimately maximised.

Incidence and impact of proxy response in measuring patient experience: secondary analysis of a large postal survey using propensity score matching.

OBJECTIVE: To determine whether use of proxy respondents in a patient experience survey was related to patient characteristics, and to compare patient and proxy responses. DESIGN: Secondary analysis, using propensity score matching, of the NHS adult inpatient survey, a large cross-sectional survey. SETTING: Hospitals (n = 161) providing inpatient services in England in 2011. PARTICIPANTS: The survey received 70 863 responses: 10 661 (15.6%) involved proxy respondents in some way. INTERVENTION: None. MAIN OUTCOME MEASURES: Prevalence of proxy response was explored by patient demographic characteristics. Responses were compared using seven composite domains and one overall rating. Cases involving proxy responses were matched to similar independent responses via propensity score matching and mean scores compared using t-tests. RESULTS: Use of proxy respondents was common, with 15.7% of responses involving a proxy in some way: higher than in other similar collections internationally. Proxy response was more common for some patient groups, such as older people and those from black and minority ethnic groups. Reports made by or with the assistance of proxy respondents were markedly less positive than those from patients completing the survey unaided. This pattern was consistent across all tested variables, although the biggest differences were observed for a subjective 'overall rating' question. CONCLUSIONS: The prevalence of proxy response varied according to patient characteristics, but proxies were consistently less positive than patients responding unaided. Possible explanations include genuine differences in care, differential health outcomes or differences in perceptions. Patient experience surveys should collect information on use of proxy respondents to enable more refined analysis.

Mapping the functional yeast ABC transporter interactome.

ATP-binding cassette (ABC) transporters are a ubiquitous class of integral membrane proteins of immense clinical interest because of their strong association with human disease and pharmacology. To improve our understanding of these proteins, we used membrane yeast two-hybrid technology to map the protein interactome of all of the nonmitochondrial ABC transporters in the model organism Saccharomyces cerevisiae and combined this data with previously reported yeast ABC transporter interactions in the BioGRID database to generate a comprehensive, integrated 'interactome'. We show that ABC transporters physically associate with proteins involved in an unexpectedly diverse range of functions. We specifically examine the importance of the physical interactions of ABC transporters in both the regulation of one another and in the modulation of proteins involved in zinc homeostasis. The interaction network presented here will be a powerful resource for increasing our fundamental understanding of the cellular role and regulation of ABC transporters.

People with limiting long-term conditions report poorer experiences and more problems with hospital care.

BACKGROUND: Long-term conditions have a significant impact on individuals, their families, and the health service. As people with these conditions represent a high proportion of hospital admissions, investigating their experiences of inpatient care has become an important area of investigation. We conducted a secondary analysis of the NHS adult inpatient survey for England to compare the hospital experiences of three groups of patients: those without long-term conditions, those with a single long-term condition, and those with multiple long-term conditions. We were particularly interested in the extent to which these patients received self-management support from hospital staff, so we developed a brief summary tool drawn from salient questions in the survey to aid the comparison. METHODS: Analysis of data from the 2011 national adult inpatient survey (n = 65,134) to compare the experiences of three groups of patients: those with no limiting long-term conditions (No-LLTC), those with one limiting long-term condition (S-LLTC), and those with two or more limiting long-term conditions (M-LLTC). The main outcome measure was patients' self-reports of their experience of inpatient care, including staff-patient interactions, information provision, involvement in decisions and support for self-care and overall ratings of care. A short form scale, the Oxford Patient Involvement and Experience scale (OxPIE) was developed from the adult inpatient survey and used to compare the groups using logistic regression. RESULTS: There were significant differences between the No-LLTC group in comparison to both the S-LLTC and M-LLTC groups. Patients with limiting long-term conditions reported significantly worse hospital experiences than those without, as measured by OxPIE: S-LLTC odds ratio = 1.23, 95% CI 1.03-1.48; M-LLTC odds ratio = 1.64, 95% CI 1.19-2.26. Responses to a single global rating question were more positive but not strongly correlated with OxPIE. CONCLUSIONS: Patients with LLTCs were more critical of their inpatient care than those with no LLTCs. Those with more than one long-term condition reported worse experiences than those with a single limiting condition. Simple rating questions may not be sufficiently sensitive to reflect important aspects of patients' experience.

Genetic interaction maps in Escherichia coli reveal functional crosstalk among cell envelope biogenesis pathways.

As the interface between a microbe and its environment, the bacterial cell envelope has broad biological and clinical significance. While numerous biosynthesis genes and pathways have been identified and studied in isolation, how these intersect functionally to ensure envelope integrity during adaptive responses to environmental challenge remains unclear. To this end, we performed high-density synthetic genetic screens to generate quantitative functional association maps encompassing virtually the entire cell envelope biosynthetic machinery of Escherichia coli under both auxotrophic (rich medium) and prototrophic (minimal medium) culture conditions. The differential patterns of genetic interactions detected among > 235,000 digenic mutant combinations tested reveal unexpected condition-specific functional crosstalk and genetic backup mechanisms that ensure stress-resistant envelope assembly and maintenance. These networks also provide insights into the global systems connectivity and dynamic functional reorganization of a universal bacterial structure that is both broadly conserved among eubacteria (including pathogens) and an important target.

Thinking beyond the virus: perspective of patients on the quality of hospital care before and during the COVID-19 pandemic.

Objectives: The COVID-19 pandemic has a huge impact on the healthcare system and affects the normal delivery of routine healthcare services to hospitalized patients. This study aimed to examine the differences in patient experience of hospital service before and during COVID-19 among the discharged adult population. Methods: A territory-wide patient experience survey was conducted before and during COVID-19 (between October 2019 and April 2020) among patients discharged from the main acute and rehabilitation public hospitals in Hong Kong. A hierarchical ordinal logistic model was employed to examine the difference in multiple dimensions of patient experience, with adjustments of covariates. Results: In total, 9,800 participants were recruited. During the pandemic, there was a marginally significant increase in overall care rating (AOR: 1.12, 95% CI: 0.99-1.27), and an improvement in the timeliness of admission. However, significant reductions in patients' confidence in nurses were observed. Communication of information regarding medication side effects reduced significantly (AOR: 0.72, 95% CI: 0.64-0.82). Conclusion: The patients hospitalized during the pandemic reported worse responsiveness in communication in their patient journey than those admitted before the pandemic. These findings will help develop appropriate strategies to address patients' concerns in the new normal.

Education-based grant programmes for bottom-up distance learning and project catalysis: antimicrobial resistance in Sub-Saharan Africa.

International development and aid are often conducted through the allocation of funding determined by decisions of non-locals, especially in the west for those in the global south. In addition, such funding is often disassociated from local expertise, therefore providing little long-term developmental impact and generating distrust. This is particularly true for conservation, as well as environmental and educational programmes. We hypothesize that by granting local people the educational tools and the necessary funding to develop their own projects through the use of an applicant-driven peer-review approach, it is possible to relocalize the decision-making process to the programme participants, with the potential to generate and select more relevant projects with developmental outcomes of higher quality. Here we created an online curriculum for antimicrobial resistance (AMR) education that was followed by 89 participants across Ghana, Tanzania, Nigeria and Uganda. We then created an open research programme that facilitated the creation of eight de novo projects on AMR. Finally, we organized an applicant-driven grant round to allocate funding to the 'Neonatal Sepsis in Nigeria' project to conduct a pilot study and awareness campaign. This work opens perspectives for the design of frugal educational programmes and the funding of context-specific, community-driven projects aimed at empowering local stakeholders in the global South.

Structural basis of peptidoglycan synthesis by E. coli RodA-PBP2 complex.

Peptidoglycan (PG) is an essential structural component of the bacterial cell wall that is synthetized during cell division and elongation. PG forms an extracellular polymer crucial for cellular viability, the synthesis of which is the target of many antibiotics. PG assembly requires a glycosyltransferase (GT) to generate a glycan polymer using a Lipid II substrate, which is then crosslinked to the existing PG via a transpeptidase (TP) reaction. A Shape, Elongation, Division and Sporulation (SEDS) GT enzyme and a Class B Penicillin Binding Protein (PBP) form the core of the multi-protein complex required for PG assembly. Here we used single particle cryo-electron microscopy to determine the structure of a cell elongation-specific E. coli RodA-PBP2 complex. We combine this information with biochemical, genetic, spectroscopic, and computational analyses to identify the Lipid II binding sites and propose a mechanism for Lipid II polymerization. Our data suggest a hypothesis for the movement of the glycan strand from the Lipid II polymerization site of RodA towards the TP site of PBP2, functionally linking these two central enzymatic activities required for cell wall peptidoglycan biosynthesis.

A dual function of the CRISPR-Cas system in bacterial antivirus immunity and DNA repair.

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) and the associated proteins (Cas) comprise a system of adaptive immunity against viruses and plasmids in prokaryotes. Cas1 is a CRISPR-associated protein that is common to all CRISPR-containing prokaryotes but its function remains obscure. Here we show that the purified Cas1 protein of Escherichia coli (YgbT) exhibits nuclease activity against single-stranded and branched DNAs including Holliday junctions, replication forks and 5'-flaps. The crystal structure of YgbT and site-directed mutagenesis have revealed the potential active site. Genome-wide screens show that YgbT physically and genetically interacts with key components of DNA repair systems, including recB, recC and ruvB. Consistent with these findings, the ygbT deletion strain showed increased sensitivity to DNA damage and impaired chromosomal segregation. Similar phenotypes were observed in strains with deletion of CRISPR clusters, suggesting that the function of YgbT in repair involves interaction with the CRISPRs. These results show that YgbT belongs to a novel, structurally distinct family of nucleases acting on branched DNAs and suggest that, in addition to antiviral immunity, at least some components of the CRISPR-Cas system have a function in DNA repair.