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Nature Communications 11(1), 4511 (2020). doi:10.1038/s41467-020-18156-7, Serial femtosecond crystallography (SFX) with X-ray free electron lasers (XFELs) allows structure determination of membrane proteins and time-resolved crystallography. Common liquid sample delivery continuously jets the protein crystal suspension into the path of the XFEL, wasting a vast amount of sample due to the pulsed nature of all current XFEL sources. The European XFEL (EuXFEL) delivers femtosecond (fs) X-ray pulses in trains spaced 100 ms apart whereas pulses within trains are currently separated by 889 ns. Therefore, continuous sample delivery via fast jets wastes >99% of sample. Here, we introduce a microfluidic device delivering crystal laden droplets segmented with an immiscible oil reducing sample waste and demonstrate droplet injection at the EuXFEL compatible with high pressure liquid delivery of an SFX experiment. While achieving ~60% reduction in sample waste, we determine the structure of the enzyme 3-deoxy-D-manno-octulosonate-8-phosphate synthase from microcrystals delivered in droplets revealing distinct structural features not previously reported., Published by Nature Publishing Group UK, [London]

This study identified the specific factors which influence the treatment of gum disease. Specifically, the aims are to examine i) the differences in the prevalence of self-reported periodontitis treatment in 2011–2012 and 2017–2018, ii) the association between the background characteristics of respondents and treatment of gum disease, iii) the effects of the background characteristics on the treatment of gum disease and iv) reasons for the lack of access to dental care in 2011–2012 and 2017–2018. Data from the National Health an Nutrition Examination Survery (NHANES) oral health questionnaires 2011-2012 and 2017-2018 were used. NHANES is a periodic survey conducted by the National Center for Health Statistics of the Centers for Disease Control and Prevention. This data represents a stratified, multistage probability sample of the civilian noninstitutionalized population in the 50 United States U.S) and the District of Columbia. Binary multivariable logistic regression was used to examine the socioeconomic predictors of self-reported therapy for gum disease. Compared to 2011-2012, more participants accessed oral health care services during 2017-2018, resulting in fewer people seeking treatment for gum disease. Affordability and lack of dental health insurance were the primary reasons for not receiving treatment for gum disease. Compared with Whites, Asians were more likely to report having had treatment, followed by Hispanics and Blacks. Increasing oral health services for the elderly could improve access to care related to periodontal disease in this cohort of patients. In addition, the expansion of dental coverage with reduced out-of-pocket expenditure could improve access to dental services and overall health.

This article focuses on the challenges of hit finding and data reduction in serial crystallography (SX). An effective and reliable Bragg-peak-finding method, called robust peak finder (RPF), has been developed. RPF is based on the principle of robust statistics and can be used for SX data analysis., A peak-finding algorithm for serial crystallography (SX) data analysis based on the principle of ‘robust statistics’ has been developed. Methods which are statistically robust are generally more insensitive to any departures from model assumptions and are particularly effective when analysing mixtures of probability distributions. For example, these methods enable the discretization of data into a group comprising inliers (i.e. the background noise) and another group comprising outliers (i.e. Bragg peaks). Our robust statistics algorithm has two key advantages, which are demonstrated through testing using multiple SX data sets. First, it is relatively insensitive to the exact value of the input parameters and hence requires minimal optimization. This is critical for the algorithm to be able to run unsupervised, allowing for automated selection or ‘vetoing’ of SX diffraction data. Secondly, the processing of individual diffraction patterns can be easily parallelized. This means that it can analyse data from multiple detector modules simultaneously, making it ideally suited to real-time data processing. These characteristics mean that the robust peak finder (RPF) algorithm will be particularly beneficial for the new class of MHz X-ray free-electron laser sources, which generate large amounts of data in a short period of time.

Proceedings of the National Academy of Sciences of the United States of America 117(39), 1-7 (2020). doi:10.1073/pnas.2003337117, Dynamics and kinetics in soft matter physics, biology, and nanoscience frequently occur on fast (sub)microsecond but not ultrafast timescales which are difficult to probe experimentally. The European X-ray Free-Electron Laser (European XFEL), a megahertz hard X-ray Free-Electron Laser source, enables such experiments via taking series of diffraction patterns at repetition rates of up to 4.5 MHz. Here, we demonstrate X-ray photon correlation spectroscopy (XPCS) with submicrosecond time resolution of soft matter samples at the European XFEL. We show that the XFEL driven by a superconducting accelerator provides unprecedented beam stability within a pulse train. We performed microsecond sequential XPCS experiments probing equilibrium and nonequilibrium diffusion dynamics in water. We find nonlinear heating on microsecond timescales with dynamics beyond hot Brownian motion and superheated water states persisting up to 100 μs at high fluences. At short times up to 20 μs we observe that the dynamics do not obey the Stokes–Einstein predictions., Published by National Acad. of Sciences, Washington, DC

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.

Journal of applied crystallography 55, 1549 - 1561 (2022). doi:10.1107/S1600576722009815, X-ray crystallography has witnessed a massive development over the past decade, driven by large increases in the intensity and brightness of X-ray sources and enabled by employing high-frame-rate X-ray detectors. The analysis of large data sets is done via automatic algorithms that are vulnerable to imperfections in the detector and noise inherent with the detection process. By improving the model of the behaviour of the detector, data can be analysed more reliably and data storage costs can be significantly reduced. One major requirement is a software mask that identifies defective pixels in diffraction frames. This paper introduces a methodology and program based upon concepts of machine learning, called robust mask maker (RMM), for the generation of bad-pixel masks for large-area X-ray pixel detectors based on modern robust statistics. It is proposed to discriminate normally behaving pixels from abnormal pixels by analysing routine measurements made with and without X-ray illumination. Analysis software typically uses a Bragg peak finder to detect Bragg peaks and an indexing method to detect crystal lattices among those peaks. Without proper masking of the bad pixels, peak finding methods often confuse the abnormal values of bad pixels in a pattern with true Bragg peaks and flag such patterns as useful regardless, leading to storage of enormous uninformative data sets. Also, it is computationally very expensive for indexing methods to search for crystal lattices among false peaks and the solution may be biased. This paper shows how RMM vastly improves peak finders and prevents them from labelling bad pixels as Bragg peaks, by demonstrating its effectiveness on several serial crystallography data sets., Published by Wiley-Blackwell, [S.l.]

Tpp49Aa1 fromLysinibacillus sphaericusis a Toxin_10 family protein that – in combination with Cry48Aa1, a 3-domain crystal protein - has potent mosquitocidal activity, specifically againstCulex quinquefasciatusmosquitoes. MHz serial femtosecond crystallography at a nano-focused X-ray free electron laser, allowed rapid and high-quality data collection to determine the Tpp49Aa1 structure at 1.62 Å resolution from native nanocrystals. This revealed the packing of Tpp49Aa1 within these nanocrystals, isolated from sporulated bacteria, as a homodimer with a large intermolecular interface, shedding light on natural crystallization. Complementary experiments conducted at varied pH also enabled investigations of the early structural events leading up to the dissolution of natural Tpp49Aa1 crystals. Using modelling, we propose a potential interaction between Tpp49Aa1 and Cry48Aa1 that may play a role in their codependency and broaden our understanding of this two-component system. We expand the known target range, demonstrating Tpp49Aa1/Cry48Aa1 susceptibility of larvae fromAnopheles stephensi, Aedes albopictusandCulex tarsalis– substantially increasing the potential use of this toxin pair in mosquito control. Further functional insights are gained usingCulexcell lines to characterise cellular models for future investigations into Cry48Aa1/Tpp49Aa1 mechanism of action and to demonstrate transient detrimental effects of individual toxin components.Significance StatementThe Tpp49Aa1/Cry48Aa1 protein pair kills mosquito larvae. Innovative use of nano-focused X-ray free electron laser to match the size of natural Tpp49Aa1 nanocrystals and the highest beam intensity available in any XFEL for high-throughput data collection, allowed structural resolution to 1.62 Å.Tpp proteins show a range of interactions with different partners to elicit toxicity. To gain insight into Tpp49Aa1, its interaction with Cry48Aa1 was modelled. We also establish cell-based assays of Tpp49Aa1/Cry48Aa1 activity.We expand the known target range to include three more mosquito species:Anopheles stephensi, Aedes albopictusandCulex tarsalis. This study will underpin future Tpp mode of action investigations and aid insecticide optimization against mosquito vectors of emerging diseases such as West Nile Virus and malaria.

The interdisciplinary evidence to support healthcare building is insufficient. New innovation processes are needed that enable clinical, research evidence and practice-based design teams to apply research-informed and evidence based design. This article aims to challenge the basis for prioritizing the reconfiguration of eyecare to respond to patient backlog and develop an innovative di-agnostics hub. Ophthalmology, the busiest NHS outpatient specialty (7.9 million episodes; 2018-19), is perfectly suited to providing a testbed for rapid, research-driven innovation and to show how research in the built environment can better inform clinical and technological advancement. A clinically-led case study is described which reports the approach taken to bring together the de-sign, engineering and modelling expertise of multiple experts in the built environment. Various disciplines contributed to three clinical trials which saw three unique building configurations, which involved 3,000 patients across three iterations of the building layout design. Circa ~ 30 staff were involved in the interdisciplinary co-design process to encourage an innovative approach to equipment configuration, layout design and an emerging scientific evidence-base. There is a significant need to address the methodological, interdisciplinary and theoretical implications of evidence-informed healthcare building. We offer up a conceptualization of an evidence-based co-production process that optimized safety, efficiency of patient movement and staff satisfaction through iterative dialogues.

The strategies deployed by antibiotic resistant bacteria to counteract host defences are poorly understood. Here, we elucidate a novel host-pathogen interaction that results in the control of 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 uncover that type I IFN and IL10 signalling, and macrophage polarization are characteristic of infected IMs, whereas Toll-like receptor (TLR) and Nod-like receptor signalling are features of infected alveolar macrophages. Klebsiella-induced macrophage polarization is a singular M2-type we termed M(Kp). To rewire macrophages towards M(Kp), K. pneumoniae hijacks a hitherto unknown TLR-type I IFN-IL10-STAT6 innate axis. Absence of STAT6 limits the intracellular survival of Klebsiella whereas the inhibition of STAT6 facilitates the clearance of the pathogen in vivo. Glycolysis characterises M(Kp) metabolism, and inhibition of glycolysis results in clearance of intracellular Klebsiella. We demonstrate the capsule polysaccharide is the Klebsiella factor governing M(Kp). Klebsiella also skews human macrophage polarization towards M(Kp) in a type I IFN-IL10-STAT6-dependent manner. Altogether, our work demonstrates that Klebsiella induction of M(Kp) represents a hitherto unknown strategy to overcome host restriction during pneumonia.

Use of community-based participatory research (CBPR) principles can help identify strategies for development and implementation of studies that can address oral health disparities disfavoring African American youth. This paper summarizes approaches of the Howard Meharry Adolescent Caries Study (HMACS) to provide sustained oral health services beyond the life of a research study.

OBJECTIVE Associations between food insecurity, meal patterns, beverage intake, and body mass index (BMI) were investigated using data from the Howard Meharry Adolescent Caries Study. METHODS Secondary analyses of food security status used the Wilcoxon rank sum, chi-square, and Fisher's exact tests. RESULTS The group of adolescents (n=627) was 42.1% male, 14.2±1.9 years, 86.9% African American, and 19.9% food-insecure. Meal frequency, meal structure, most beverage intake, and BMI did not differ by food-security status. Adolescents from Washington, DC were more likely to be food insecure than adolescents from Nashville, TN (P=0.003). Most had unstructured meal patterns and irregular breakfast intake. Median milk intake was below and sugar-sweetened beverage intake above dietary recommendations. CONCLUSIONS This study extends our knowledge concerning food insecurity in urban African American adolescents and suggests public health initiatives designed to encourage meal structure, increase milk intake, and reduce sugar-sweetened beverage intake can improve diet quality of underserved youth.

The increasing prevalence of multidrug-resistant Klebsiella pneumoniae has led to a resurgence in the use of colistin as a last-resort drug. Colistin is a cationic antibiotic that selectively acts on Gram-negative bacteria through electrostatic interactions with anionic phosphate groups of the lipid A moiety of lipopolysaccharides (LPSs). Colistin resistance in K. pneumoniae is mediated through loss of these phosphate groups, their modification by cationic groups, and by the hydroxylation of acyl groups of lipid A. Here, we study the in vitro evolutionary trajectories toward colistin resistance in four clinical K. pneumoniae complex strains and their impact on fitness and virulence characteristics. Through population sequencing during in vitro evolution, we found that colistin resistance develops through a combination of single nucleotide polymorphisms, insertions and deletions, and the integration of insertion sequence elements, affecting genes associated with LPS biosynthesis and modification and capsule structures. Colistin resistance decreased the maximum growth rate of one K. pneumoniae sensu stricto strain, but not those of the other three K. pneumoniae complex strains. Colistin-resistant strains had lipid A modified through hydroxylation, palmitoylation, and l-Ara4N addition. K. pneumoniae sensu stricto strains exhibited cross-resistance to LL-37, in contrast to the Klebsiella variicola subsp. variicola strain. Virulence, as determined in a Caenorhabditis elegans survival assay, was increased in two colistin-resistant strains. Our study suggests that nosocomial K. pneumoniae complex strains can rapidly develop colistin resistance through diverse evolutionary trajectories upon exposure to colistin. This effectively shortens the life span of this last-resort antibiotic for the treatment of infections with multidrug-resistant Klebsiella.

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.

Optica 6(9), 1106 - 1109 (2019). doi:10.1364/OPTICA.6.001106, Modern emerging technologies, such as additive manufacturing, bioprinting, and new material production, require novel metrology tools to probe fundamental high-speed dynamics happening in such systems. Here we demonstrate the application of the megahertz (MHz) European X-ray Free-Electron Laser (EuXFEL) to image the fast stochastic processes induced by a laser on water-filled capillaries with micrometer-scale spatial resolution. The EuXFEL provides superior contrast and spatial resolution compared to equivalent state-of-the-art synchrotron experiments. This work opens up new possibilities for the characterization of MHz stochastic processes on the nanosecond to microsecond time scales with object velocities up to a few kilometers per second using XFEL sources., Published by OSA, Washington, DC

Exhaust gas recirculation (EGR) cooler fouling occurs when particulate matter (PM) and hydrocarbons (HC) in diesel exhaust form a deposit on the walls of the EGR cooler through thermophoresis and condensation. To better understand the mechanisms controlling deposit formation and removal and how operating conditions can affect cooler performance, 20 identical tube-in-shell EGR coolers with sinusoidal fins were fouled using a 5-factor, 3-level experimental design. The deposit thickness was measured using two methods: (1) epoxy-mounting and polishing cooler cross-sections and comparing deposit thicknesses on the primary (outer tube) to the secondary (fins) heat transfer surfaces, and (2) milling tube sections such that the surface of a fin could be observed and measuring the deposit thickness across the fin using a 3D profilometer. Near the cooler inlet, high inlet gas temperatures reduced deposit thickness by promoting mud-cracking and spallation. Near the middle of the cooler, the flow rate had the largest impact on the deposit thickness through the effect on residence time of the PM. The HC concentration along with flow rate had the largest effects near the cooler outlet where the lower temperatures allows for more HC condensation. These insights into how engine operating conditions influence the development of fouling layers in EGR coolers learned through this study will aid in the development of more fouling resistant coolers in the future.

A spectroscopic examination of six galactonoamidines with inhibition constants and efficacy in the low nanomolar concentration range (Ki = 6–11 nM, IC50 = 12–36 nM) suggested only two of them as putative transition state analogs for the hydrolysis of β-galactosides by β-galactosidase (A. oryzae). A rationale for the experimental results was elaborated using docking and molecular dynamics studies. An analysis of the combined observations reveals several common factors of the compounds suggested as transition state analogs (TSAs): the putative TSAs have a similar orientation in the active site; show conserved positioning of the glycon; display a large number of H-bond interactions toward the catalytically active amino acid residues via their glycon; and exhibit hydrophobic interactions at the outer rim of the active site with small changes of the position and orientation of their respective aglycons.

The increasing prevalence of multidrug-resistant Gram-negative opportunistic pathogens, includingKlebsiella pneumoniae, has led to a resurgence in the use of colistin as a last-resort drug. Colistin is a cationic lipopeptide antibiotic that selectively acts on Gram-negative bacteria through electrostatic interactions with anionic phosphate groups of the lipid A moiety of lipopolysaccharides (LPS). Colistin resistance inK. pneumoniaeis mediated through loss of these phosphate groups, or modification with cationic groups (e.g. 4-amino-4-deoxy-L-arabinose (L-Ara4N), or phosphoethanolamine), but also hydroxylation of acyl-groups of lipid A. Here, we study thein vitroevolutionary trajectories towards colistin resistance in clinicalK. pneumoniaecomplex strains (threeK. pneumoniae sensu strictostrains and oneK. variicolasubsp.variicolastrain) and their impact on fitness and virulence characteristics.Through population sequencing during thein vitroevolution experiment, we found that resistance develops through a combination of single nucleotide polymorphisms (SNPs), insertion and deletions (indels), and the integration of insertion sequence (IS) elements, affecting genes associated with LPS biosynthesis and modification, and capsule structures. The development of colistin resistance decreased the maximum growth rate of oneK. pneumoniae sensu strictostrain, but not in the other threeK. pneumoniae sensu latostrains. Colistin-resistant strains had lipid A modified through hydroxylation, palmitoylation, and L-Ara4N addition. Colistin-resistantK. pneumoniae sensu strictostrains exhibited cross-resistance to LL-37, in contrast to theK. variicolasubsp.variicolastrain that did not change in susceptibility to LL-37. Virulence, as determined in aCaenorhabditis eleganssurvival assay, was higher in two colistin-resistant strains.Our study suggests that nosocomialK. pneumoniaecomplex strains can rapidly develop colistin resistancede novothrough diverse evolutionary trajectories upon exposure to colistin. This effectively shortens the lifespan of this last-resort antibiotic for the treatment of infections with multidrug-resistantKlebsiella.Author summaryBacteria that frequently cause infections in hospitalised patients are becoming increasingly resistant to antibiotics. Colistin is a positively charged antibiotic that is used for the treatment of infections with multidrug-resistant Gram-negative bacteria. Colistin acts by specifically interacting with the negatively charged LPS molecule in the outer membrane of Gram-negative bacteria. Colistin resistance is mostly mediated through modification of LPS to reduce its negative charge. Here, we use a laboratory evolution experiment to show that strains belonging to theKlebsiella pneumoniaecomplex, a common cause of multidrug-resistant hospital-acquired infections, can rapidly accumulate mutations that reduce the negative charge of LPS without an appreciable loss of fitness. Colistin resistance can lead to cross-resistance to an antimicrobial peptide of the human innate immune system, but can increase susceptibility to serum, and virulence in a nematode model. These findings show that extensively resistantK. pneumoniaecomplex strains may rapidly develop resistance to the last-resort antibiotic colistin via different evolutionary trajectories, while retaining their ability to cause infections.

Optica 7(6), 716 - (2020). doi:10.1364/OPTICA.396728, A critical challenge of pump-probe experiments with x-ray free-electron lasers (XFELs) is accurate synchronization of x-ray and optical pulses. At the European XFEL we observed megahertz rate timing jitter of 24.0±12.4fs., Published by OSA, Washington, DC

Scientific data 6(1), 18 (2019). doi:10.1038/s41597-019-0010-0, We provide a detailed description of a serial femtosecond crystallography (SFX) dataset collected at the European X-ray free-electron laser facility (EuXFEL). The EuXFEL is the first high repetition rate XFEL delivering MHz X-ray pulse trains at 10 Hz. The short spacing (, Published by Nature Publ. Group, London

Using computational simulations, we examine heat transfer enhancement in laminar flows through asymmetric wavy channels with sinusoidal walls. Specifically, we probe the influence of the amplitude and period of the wall waviness as well as the driving pressure on the enhancement of heat transfer for both steady and unsteady flows. Our simulations reveal that for the steady flow occurring at lower flow rates, the heat transfer enhancement is defined by the amplitude of the wall waviness. When the flow rate is increased and the flow transitions to unsteady, the heat transfer enhancement exhibits a stronger dependence on the pressure drop than the amplitude. We find that the increase in heat transfer achieved in the unsteady regime is significantly higher than that in the steady regime. Furthermore, we use a thermal–hydraulic performance factor to investigate if this enhancement outweighs the increased frictional losses the geometry induces. Our data indicates that wavy walled channels with small wave amplitudes outperform straight channels at Reynolds numbers as low as 300 and exhibit greater than 50% improvement at Re ≈ 600 . These results are important for designing compact heat exchangers capable of high performance in the laminar regime.