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Abstract The main protease (Mpro) of SARS-CoV-2 is critical for viral function and a key drug target. Mpro is only active when reduced; turnover ceases upon oxidation but is restored by re-reduction. This suggests the system has evolved to survive periods in an oxidative environment, but the mechanism of this protection has not been confirmed. Here, we report a crystal structure of oxidized Mpro showing a disulfide bond between the active site cysteine, C145, and a distal cysteine, C117. Previous work proposed this disulfide provides the mechanism of protection from irreversible oxidation. Mpro forms an obligate homodimer, and the C117-C145 structure shows disruption of interactions bridging the dimer interface, implying a correlation between oxidation and dimerization. We confirm dimer stability is weakened in solution upon oxidation. Finally, we observe the protein’s crystallization behavior is linked to its redox state. Oxidized Mpro spontaneously forms a distinct, more loosely packed lattice. Seeding with crystals of this lattice yields a structure with an oxidation pattern incorporating one cysteine-lysine-cysteine (SONOS) and two lysine-cysteine (NOS) bridges. These structures further our understanding of the oxidative regulation of Mpro and the crystallization conditions necessary to study this structurally.

Artificial intelligence (AI) has witnessed rapid advances in the healthcare domain in recent years, especially in the emergency field, where AI is likely to radically reshape medical service delivery. Although AI has substantial potential to enhance diagnostic accuracy and operational efficiency in hospitals, research on its applications in Emergency Department building design remains relatively scarce. Therefore, this study aims to investigate Emergency Department facility design by identifying the challenges and opportunities of using AI. Two systematic literature reviews are combined, one in AI and the other in sensors, to explore their potential application to support decision-making, resource optimisation and patient monitoring. These reviews have then informed a discussion on integrating AI sensors in contemporary Emergency Department designs for use in China to support the evidence base on resuscitation units, emergency operating rooms and Emergency Department Intensive Care Unit (ED-ICU) design. We hope to inform the strategic implementation of AI sensors and how they might transform Emergency Department design to support medical staff and enhance the patient experience.

Abstract The strategies deployed by antibiotic‐resistant bacteria to counteract host defences are poorly understood. Here, we elucidate a novel host–pathogen interaction resulting in skewing lung macrophage polarisation by the human pathogen Klebsiella pneumoniae. We identify interstitial macrophages (IMs) as the main population of lung macrophages associated with Klebsiella. Single‐cell transcriptomics and trajectory analysis of cells reveal type I IFN and IL10 signalling, and macrophage polarisation are characteristic of infected IMs, whereas Toll‐like receptor (TLR) and Nod‐like receptor signalling are features of infected alveolar macrophages. Klebsiella‐induced macrophage polarisation is a singular M2‐type we termed M(Kp). To rewire macrophages, Klebsiella hijacks a TLR‐type I IFN‐IL10‐STAT6 axis. Absence of STAT6 limits Klebsiella intracellular survival and facilitates the clearance of the pathogen in vivo. Glycolysis characterises M(Kp) metabolism, and inhibition of glycolysis results in clearance of intracellular Klebsiella. Capsule polysaccharide governs M(Kp). Klebsiella also skews human macrophage polarisation towards M(Kp) in a type I IFN‐IL10‐STAT6‐dependent manner. Klebsiella induction of M(Kp) represents a novel strategy to overcome host restriction, and identifies STAT6 as target to boost defences against Klebsiella.

Pump–probe experiments at X-ray free-electron laser (XFEL) facilities are a powerful tool for studying dynamics at ultrafast and longer timescales. Observing the dynamics in diverse scientific cases requires optical laser systems with a wide range of wavelength, flexible pulse sequences and different pulse durations, especially in the pump source. Here, the pump–probe instrumentation available for measurements at the Single Particles, Clusters, and Biomolecules and Serial Femtosecond Crystallography (SPB/SFX) instrument of the European XFEL is reported. The temporal and spatial stability of this instrumentation is also presented.

Free-electron lasers are capable of high repetition rates and it is assumed that protein crystals often do not survive the first X-ray pulse. Here the authors address these issues with a demonstration of multi-hit serial crystallography in which multiple FEL pulses interact with the sample without destroying it.

Measurement(s) lysozyme measurement Technology Type(s) X-ray crystallography

The Sample Environment and Characterization (SEC) group of the European X-ray Free-Electron Laser (EuXFEL) develops sample delivery systems for the various scientific instruments, including systems for the injection of liquid samples that enable serial femtosecond X-ray crystallography (SFX) and single-particle imaging (SPI) experiments, among others. For rapid prototyping of various device types and materials, sub-micrometre precision 3D printers are used to address the specific experimental conditions of SFX and SPI by providing a large number of devices with reliable performance. This work presents the current pool of 3D printed liquid sample delivery devices, based on the two-photon polymerization (2PP) technique. These devices encompass gas dynamic virtual nozzles (GDVNs), mixing-GDVNs, high-viscosity extruders (HVEs) and electrospray conical capillary tips (CCTs) with highly reproducible geometric features that are suitable for time-resolved SFX and SPI experiments at XFEL facilities. Liquid sample injection setups and infrastructure on the Single Particles, Clusters, and Biomolecules and Serial Femtosecond Crystallography (SPB/SFX) instrument are described, this being the instrument which is designated for biological structure determination at the EuXFEL.

Here, we illustrate what happens inside the catalytic cleft of an enzyme when substrate or ligand binds on single-millisecond timescales. The initial phase of the enzymatic cycle is observed with near-atomic resolution using the most advanced X-ray source currently available: the European XFEL (EuXFEL). The high repetition rate of the EuXFEL combined with our mix-and-inject technology enables the initial phase of ceftriaxone binding to the Mycobacterium tuberculosis β-lactamase to be followed using time-resolved crystallography in real time. It is shown how a diffusion coefficient in enzyme crystals can be derived directly from the X-ray data, enabling the determination of ligand and enzyme–ligand concentrations at any position in the crystal volume as a function of time. In addition, the structure of the irreversible inhibitor sulbactam bound to the enzyme at a 66 ms time delay after mixing is described. This demonstrates that the EuXFEL can be used as an important tool for biomedically relevant research.

Many soft-matter systems are composed of macromolecules or nanoparticles suspended in water. The characteristic times at intrinsic length scales of a few nanometres fall therefore in the microsecond and sub-microsecond time regimes. With the development of free-electron lasers (FELs) and fourth-generation synchrotron light-sources, time-resolved experiments in such time and length ranges will become routinely accessible in the near future. In the present work we report our findings on prototypical soft-matter systems, composed of charge-stabilized silica nanoparticles dispersed in water, with radii between 12 and 15 nm and volume fractions between 0.005 and 0.2. The sample dynamics were probed by means of X-ray photon correlation spectroscopy, employing the megahertz pulse repetition rate of the European XFEL and the Adaptive Gain Integrating Pixel Detector. We show that it is possible to correctly identify the dynamical properties that determine the diffusion constant, both for stationary samples and for systems driven by XFEL pulses. Remarkably, despite the high photon density the only observable induced effect is the heating of the scattering volume, meaning that all other X-ray induced effects do not influence the structure and the dynamics on the probed timescales. This work also illustrates the potential to control such induced heating and it can be predicted with thermodynamic models.

Due to the pulsed nature of X-ray free electron laser (XFEL) instruments the majority of protein crystals, which are injected using continuous jet injection techniques are wasted. Here, the authors present a microfluidic device to deliver aqueous protein crystal laden droplets segmented with an immiscible oil and demonstrate that with this device an approx. 60% reduction in sample waste was achieved for data collection of 3-deoxy-D-manno-octulosonate 8-phosphate synthase crystals at the EuXFEL.

The European X-ray free-electron laser (EuXFEL) in Hamburg is the first XFEL with a megahertz repetition rate. Here the authors present the 2.9 Å structure of the large membrane protein complex Photosystem I from T. elongatus that was determined at the EuXFEL.

The new European X-Ray Free-Electron Laser (EuXFEL) is the first XFEL that generates X-ray pulses with a megahertz inter-pulse spacing. Here the authors demonstrate that high-quality and damage-free protein structures can be obtained with the currently available 1.1 MHz repetition rate pulses using lysozyme as a test case and furthermore present a β-lactamase structure.

The European X-ray free-electron laser (EuXFEL) in Hamburg is the first megahertz (MHz) repetition rate XFEL. Here the authors use lysozyme crystals and microcrystals from jack bean proteins and demonstrate that damage-free high quality data can be collected at a MHz repetition rate.

Abstract Klebsiella pneumoniae is an important cause of multidrug‐resistant infections worldwide. Recent studies highlight the emergence of multidrug‐resistant K. pneumoniae strains which show resistance to colistin, a last‐line antibiotic, arising from mutational inactivation of the mgrB regulatory gene. However, the precise molecular resistance mechanisms of mgrB‐associated colistin resistance and its impact on virulence remain unclear. Here, we constructed an mgrB gene K. pneumoniae mutant and performed characterisation of its lipid A structure, polymyxin and antimicrobial peptide resistance, virulence and inflammatory responses upon infection. Our data reveal that mgrB mutation induces PhoPQ‐governed lipid A remodelling which confers not only resistance to polymyxins, but also enhances K. pneumoniae virulence by decreasing antimicrobial peptide susceptibility and attenuating early host defence response activation. Overall, our findings have important implications for patient management and antimicrobial stewardship, while also stressing antibiotic resistance development is not inexorably linked with subdued bacterial fitness and virulence.

Ultrabright pulses produced in X-ray free-electron lasers (XFELs) offer new possibilities for industry and research, particularly for biochemistry and pharmaceuticals. The unprecedented brilliance of these next-generation sources enables structure determination from sub-micron crystals as well as radiation-sensitive proteins. The European X-Ray Free-Electron Laser (EuXFEL), with its first light in 2017, ushered in a new era for ultrabright X-ray sources by providing an unparalleled megahertz-pulse repetition rate, with orders of magnitude more pulses per second than previous XFEL sources. This rapid pulse frequency has significant implications for structure determination; not only will data collection be faster (resulting in more structures per unit time), but experiments requiring large quantities of data, such as time-resolved structures, become feasible in a reasonable amount of experimental time. Early experiments at the SPB/SFX instrument of the EuXFEL demonstrate how such closely-spaced pulses can be successfully implemented in otherwise challenging experiments, such as time-resolved studies.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Bioluminescent reporter genes, such as those from fireflies and bacteria, let researchers use light production as a non-invasive and non-destructive surrogate measure of microbial numbers in a wide variety of environments. As bioluminescence needs microbial metabolites, tagging microorganisms with luciferases means only live metabolically active cells are detected. Despite the wide use of bioluminescent reporter genes, very little is known about the impact of continuous (also called constitutive) light expression on tagged bacteria. We have previously made a bioluminescent strain of Citrobacter rodentium, a bacterium which infects laboratory mice in a similar way to how enteropathogenic Escherichia coli (EPEC) and enterohaemorrhagic E. coli (EHEC) infect humans. In this study, we compared the growth of the bioluminescent C. rodentium strain ICC180 with its non-bioluminescent parent (strain ICC169) in a wide variety of environments. To understand more about the metabolic burden of expressing light, we also compared the growth profiles of the two strains under approximately 2,000 different conditions. We found that constitutive light expression in ICC180 was near-neutral in almost every non-toxic environment tested. However, we also found that the non-bioluminescent parent strain has a competitive advantage over ICC180 during infection of adult mice, although this was not enough for ICC180 to be completely outcompeted. In conclusion, our data suggest that constitutive light expression is not metabolically costly to C. rodentium and supports the view that bioluminescent versions of microbes can be used as a substitute for their non-bioluminescent parents to study bacterial behaviour in a wide variety of environments.

A case of a 19-year-old female patient is presented to a private practice dental clinician with swelling of the lower lip and inflammation of the anterior dorsal tongue. The patient presented with moderate oral pain as well as abdominal pain. The lesions were biopsied and noted for a granulomatous histopathologic appearance. The patient reported a history of using cinnamon as a flavoring agent. The lesions resolved within two weeks after the biopsy procedures and topical steroid therapy. The lesions were diagnosed as cheilitis granulomatosa/orofacial granulomatosis. The patient has remained lesion free as of the three-year follow-up. Etiologic, diagnostic and therapeutic issues related to this relatively rare condition of cheilitis granulomatosa/orofacial granulomatosis are discussed. [ABSTRACT FROM AUTHOR]

In this chapter we describe the institutional and policy‐level strategies that dental schools in the Pipeline, Profession, and Practice: Community‐Based Dental Education program used to modify their admissions practices to increase the diversity of their student bodies. Schools developed and used clear statements recognizing the value of diversity. They incorporated recent U.S. Supreme Court rulings regarding educational diversity into their revised admissions practices; these rulings cited diversity as both a "compelling interest" and its use in only "narrowly tailored" circumstances. We make a case for admissions decisions based on a comprehensive evaluation that balances the quantitative and qualitative qualities of a candidate. It refutes the practice of overreliance on standardized tests by detailing the whole‐file review process to measure merit and professional promise. Also described is a range of noncognitive variables (e.g., leadership, ability to sustain academic achievement with competing priorities, volunteerism, communication, social background, and disadvantaged status) that schools can take into consideration in admissions decisions. Admissions committees can tie this comprehensive review of candidates into the case for promoting cross‐cultural understanding and enhanced competence to provide care to patients from diverse backgrounds. In addition, the chapter reviews the challenges schools face in developing admissions policies and procedures that reflect the university's mission for diversity. It addresses the importance of a diverse composition of the admissions committee. It also describes how tailored workshops and technical assistance for admissions committees can help schools improve their student diversity and how admissions committees can engage in a process of periodic review of their diversity objectives in relationship to the school's mission. [ABSTRACT FROM AUTHOR]

Purpose: The purpose of this study was to examine the association between sugar-sweetened beverage (SSB) consumption and dental caries prevalence among underserved Black adolescents. Methods: This was a cross-sectional study of 545 Black adolescents, ages 12 to 17 years, who participated in the Howard Meharry Adolescent Caries Study (HMACS). The outcome was dental caries prevalence, measured using the decayed, missing, and filled permanent tooth surfaces (DMFS) index. Participants were recruited from middle and high schools in Washington, D.C., USA, and Nashville, Tenn., USA. Questionnaires were used to assess beverage intake, demographic, and health-related behavioral characteristics. The multivariable analysis used marginalized zero-inflated Poisson regression (MZIP) stratified by toothbrushing frequency to estimate adjusted mean caries ratios (MRs), adjusted odds ratios (ORs), and 95 percent confidence intervals (95 percent CIs). Results: The mean age of the participants was 14.1 years. Participants in the highest quartile for SSB consumption had a higher caries ratio than those in the lowest quartile [MR equals (=) 1.59, 95 percent CI equals 1.15 to 2.20] and a lower odds of not being at risk for caries (OR = 0.24, 95 percent CI = 0.09 to 0.61). These findings were only observed among those brushing once a day or less (n=202). Conclusions: Among Black adolescents in this study who brushed once a day or less, high levels of sugar-sweetened beverage consumption were associated with greater caries prevalence and a reduced likelihood of remaining caries-free than those with lower levels of SSB consumption. Future studies will focus on interventions to reduce SSB consumption. [ABSTRACT FROM AUTHOR]

The development and use of tailored interventions in overcoming barriers to optimum health in long-term care facility residents is of the highest importance. One successful approach to improving health outcomes is the use of a checklist by health care providers. Despite the evidence of the success of such checklists in nursing and medicine, there is little evidence on their use in improving dental outcomes. This study investigated whether an intervention comprising the daily use of a checklist for oral care by nursing staff supplemented by random inspections by a charge nurse resulted in lower dental plaque scores in patient participants at a long-term care facility (n=19) as compared with in those at another long-term care facility that did not receive the intervention (n=13). All participants received a dental cleaning at baseline. At a follow-up examination at 6 to 8 weeks post-baseline, significant differences were observed in the plaque scores between the participants at each location, with the median plaque scores in those undergoing the intervention being less than half of those in the patients that did not (20.8% vs. 52.8%, p<0.001). After adjusting for age in a linear regression model, this difference remained significant. The use of a daily checklist for oral care supplemented by random inspections by a charge nurse was associated with lower plaque scores (p<0.001). These results warrant further research, including prospective studies aimed at establishing how use of both clinical supervision and a checklist for oral health may influence plaque scores in geriatric patients in long-term care facilities over time.

Unlabelled: OBJECTIVE; The aim of this international study was to develop a valid and reliable psychometric measure to examine the extent to which parents' attitudes about engaging in twice-daily tooth brushing and controlling sugar snacking predict these respective behaviours in their children. A supplementary objective was to assess whether ethnic group, culture, level of deprivation or children's caries experience impact upon the relationships between oral health related behaviours, attitudes to these respective behaviours and to dental caries., Clinical Setting: Nurseries, health centres and dental clinics in 17 countries., Participants: 2822 children aged 3 to 4 years and their parents., Main Outcome Measures: Dental examination of children and questionnaire to parents., Results: Factor analysis identified 8 coherent attitudes towards toothbrushing, sugar snacking and childhood caries. Attitudes were significantly different in families from deprived and non-deprived backgrounds and in families of children with and without caries. Parents perception of their ability to control their children's toothbrushing and sugar snacking habits were the most significant predictor of whether or not favourable habits were reported. Some differences were found by site and ethnic group., Conclusions: This study supports the hypothesis that parental attitudes significantly impact on the establishment of habits favourable to oral health. An appreciation of the impact of cultural and ethnic diversity is important in understanding how parental attitudes to oral health vary. Further research should examine in a prospective intervention whether enhancing parenting skills is an effective route to preventing childhood caries.

Objective: The aim of this formative international collaborative research on childhood dental caries was to undertake an initial investigation comparing the dental plaque of young children from diverse ethnic and socioeconomic backgrounds with and without dental caries., Basic Research Design: The following four null hypotheses were investigated. There were no differences in numbers of individual taxa when comparing plaque samples from: 1) caries-free children from deprived and non-deprived backgrounds; 2) children from deprived and non-deprived backgrounds with at least 3 decayed teeth; 3) children from non-deprived backgrounds who are caries free with those from similar backgrounds with at least 3 decayed teeth; and, 4) children from deprived backgrounds who are caries free with those from similar backgrounds with at least 3 decayed teeth., Participants: 277 children aged 3-4 years from 5 countries., Main Outcome Measures: A sample of interproximal plaque from anterior teeth was collected using sterile dental floss, and cultured according to accepted international standards., Results: Analysis of the data found that the first null hypothesis was accepted and that the fourth was rejected. Unexpectedly, the second null hypothesis was rejected as the children with caries from deprived and non-deprived backgrounds had a different caries-associated flora. In particular, children living in deprivation harbored more caries-associated bacteria [mutans streptococci and lactobacilli]. This greater microbial challenge was associated with a higher level of cavitated carious lesions and with more frequent consumption of confectionery. Conclusions Children from deprived backgrounds with caries may be further disadvantaged by having higher levels of caries-associated microflora.