Direct LiF imaging diagnostics on refractive X-ray focusing at the EuXFEL High Energy Density instrument.

The application of fluorescent crystal media in wide-range X-ray detectors provides an opportunity to directly image the spatial distribution of ultra-intense X-ray beams including investigation of the focal spot of free-electron lasers. Here the capabilities of the micro- and nano-focusing X-ray refractive optics available at the High Energy Density instrument of the European XFEL are reported, as measured in situ by means of a LiF fluorescent detector placed into and around the beam caustic. The intensity distribution of the beam focused down to several hundred nanometers was imaged at 9 keV photon energy. A deviation from the parabolic surface in a stack of nanofocusing Be compound refractive lenses (CRLs) was found to affect the resulting intensity distribution within the beam. Comparison of experimental patterns in the far field with patterns calculated for different CRL lens imperfections allowed the overall inhomogeneity in the CRL stack to be estimated. The precise determination of the focal spot size and shape on a sub-micrometer level is essential for a number of high energy density studies requiring either a pin-size backlighting spot or extreme intensities for X-ray heating.

A von Hámos spectrometer for diamond anvil cell experiments at the High Energy Density Instrument of the European X-ray Free-Electron Laser.

A von Hámos spectrometer has been implemented in the vacuum interaction chamber 1 of the High Energy Density instrument at the European X-ray Free-Electron Laser facility. This setup is dedicated, but not necessarily limited, to X-ray spectroscopy measurements of samples exposed to static compression using a diamond anvil cell. Si and Ge analyser crystals with different orientations are available for this setup, covering the hard X-ray energy regime with a sub-eV energy resolution. The setup was commissioned by measuring various emission spectra of free-standing metal foils and oxide samples in the energy range between 6 and 11 keV as well as low momentum-transfer inelastic X-ray scattering from a diamond sample. Its capabilities to study samples at extreme pressures and temperatures have been demonstrated by measuring the electronic spin-state changes of (Fe0.5Mg0.5)O, contained in a diamond anvil cell and pressurized to 100 GPa, via monitoring the Fe Kß fluorescence with a set of four Si(531) analyser crystals at close to melting temperatures. The efficiency and signal-to-noise ratio of the spectrometer enables valence-to-core emission signals to be studied and single pulse X-ray emission from samples in a diamond anvil cell to be measured, opening new perspectives for spectroscopy in extreme conditions research.

A MHz X-ray diffraction set-up for dynamic compression experiments in the diamond anvil cell.

An experimental platform for dynamic diamond anvil cell (dDAC) research has been developed at the High Energy Density (HED) Instrument at the European X-ray Free Electron Laser (European XFEL). Advantage was taken of the high repetition rate of the European XFEL (up to 4.5 MHz) to collect pulse-resolved MHz X-ray diffraction data from samples as they are dynamically compressed at intermediate strain rates (≤103 s-1), where up to 352 diffraction images can be collected from a single pulse train. The set-up employs piezo-driven dDACs capable of compressing samples in ≥340 µs, compatible with the maximum length of the pulse train (550 µs). Results from rapid compression experiments on a wide range of sample systems with different X-ray scattering powers are presented. A maximum compression rate of 87 TPa s-1 was observed during the fast compression of Au, while a strain rate of ∼1100 s-1 was achieved during the rapid compression of N2 at 23 TPa s-1.

Damage threshold of LiF crystal irradiated by femtosecond hard XFEL pulse sequence.

Here we demonstrate the results of investigating the damage threshold of a LiF crystal after irradiating it with a sequence of coherent femtosecond pulses using the European X-ray Free Electron Laser (EuXFEL). The laser fluxes on the crystal surface varied in the range ∼ 0.015-13 kJ/cm2 per pulse when irradiated with a sequence of 1-100 pulses (tpulse ∼ 20 fs, Eph = 9 keV). Analysis of the surface of the irradiated crystal using different reading systems allowed the damage areas and the topology of the craters formed to be accurately determined. It was found that the ablation threshold decreases with increasing number of X-ray pulses, while the depth of the formed craters increases non-linearly and reaches several hundred nanometers. The obtained results have been compared with data already available in the literature for nano- and picosecond pulses from lasers in the soft X-ray/VUV and optical ranges. A failure model of lithium fluoride is developed and verified with simulation of material damage under single-pulse irradiation. The obtained damage threshold is in reasonably good agreement with the experimentally measured one.

The High Energy Density Scientific Instrument at the European XFEL.

The European XFEL delivers up to 27000 intense (>1012 photons) pulses per second, of ultrashort (≤50 fs) and transversely coherent X-ray radiation, at a maximum repetition rate of 4.5 MHz. Its unique X-ray beam parameters enable groundbreaking experiments in matter at extreme conditions at the High Energy Density (HED) scientific instrument. The performance of the HED instrument during its first two years of operation, its scientific remit, as well as ongoing installations towards full operation are presented. Scientific goals of HED include the investigation of extreme states of matter created by intense laser pulses, diamond anvil cells, or pulsed magnets, and ultrafast X-ray methods that allow their diagnosis using self-amplified spontaneous emission between 5 and 25 keV, coupled with X-ray monochromators and optional seeded beam operation. The HED instrument provides two target chambers, X-ray spectrometers for emission and scattering, X-ray detectors, and a timing tool to correct for residual timing jitter between laser and X-ray pulses.

Ariadne's Thread in the Analytical Labyrinth of Membrane Proteins: Integration of Targeted and Shotgun Proteomics for Global Absolute Quantification of Membrane Proteins.

The field of systems biology has been rapidly developing in the past decade. However, the data produced by "omics" approaches is lagging behind the requirements of this field, especially when it comes to absolute abundances of membrane proteins. In the present study, a novel approach for large-scale absolute quantification of this challenging subset of proteins has been established and evaluated using osmotic stress management in the Gram-positive model bacterium Bacillus subtilis as proof-of-principle precedent. Selected membrane proteins were labeled using a SNAP-tag, which allowed us to visually inspect the enrichment of the membrane fraction by immunoassays. Absolute membrane protein concentrations were determined via shotgun proteomics by spiking crude membrane extracts of chromosomally SNAP-tagged and wild-type B. subtilis strains with protein standards of known concentration. Shotgun data was subsequently calibrated by targeted mass spectrometry using SNAP as an anchor protein, and an enrichment factor was calculated in order to obtain membrane protein copy numbers per square micrometer. The presented approach enabled the accurate determination of physiological changes resulting from imposed hyperosmotic stress, thereby offering a clear visualization of alterations in membrane protein arrangements and shedding light on putative membrane complexes. This straightforward and cost-effective methodology for quantitative proteome studies can be implemented by any research group with mass spectrometry expertise. Importantly, it can be applied to the full spectrum of physiologically relevant conditions, ranging from environmental stresses to the biotechnological production of small molecules and proteins, a field heavily relying on B. subtilis secretion capabilities.

Identification and optimization of PrsA in Bacillus subtilis for improved yield of amylase.

BACKGROUND: PrsA is an extracytoplasmic folding catalyst essential in Bacillus subtilis. Overexpression of the native PrsA from B. subtilis has repeatedly lead to increased amylase yields. Nevertheless, little is known about how the overexpression of heterologous PrsAs can affect amylase secretion. RESULTS: In this study, the final yield of five extracellular alpha-amylases was increased by heterologous PrsA co-expression up to 2.5 fold. The effect of the overexpression of heterologous PrsAs on alpha-amylase secretion is specific to the co-expressed alpha-amylase. Co-expression of a heterologous PrsA can significantly reduce the secretion stress response. Engineering of the B. licheniformis PrsA lead to a further increase in amylase secretion and reduced secretion stress. CONCLUSIONS: In this work we show how heterologous PrsA overexpression can give a better result on heterologous amylase secretion than the native PrsA, and that PrsA homologs show a variety of specificity towards different alpha-amylases. We also demonstrate that on top of increasing amylase yield, a good PrsA-amylase pairing can lower the secretion stress response of B. subtilis. Finally, we present a new recombinant PrsA variant with increased performance in both supporting amylase secretion and lowering secretion stress.

Study of the distribution of actinides in human tissues using synchrotron radiation micro X-ray fluorescence spectrometry.

This study aims at evaluating the capabilities of synchrotron radiation micro X-ray fluorescence spectrometry (SR micro-XRF) for qualitative and semi-quantitative elemental mapping of the distribution of actinides in human tissues originating from individuals with documented occupational exposure. The investigated lymph node tissues were provided by the United States Transuranium and Uranium Registries (USTUR) and were analyzed following appropriate sample pre-treatment. Semi-quantitative results were obtained via calibration by external standards and demonstrated that the uranium concentration level in the detected actinide hot spots reaches more than 100 µg/g. For the plutonium hot spots, concentration levels up to 31 µg/g were found. As illustrated by this case study on these unique samples, SR micro-XRF has a high potential for this type of elemental bio-imaging owing to its high sensitivity, high spatial resolution, and non-destructive character.

The combined effect of dissolved organic carbon and salinity on the bioaccumulation of copper in marine mussel larvae.

Larvae of Mytilus spp. are among the most Cu sensitive marine species. In this study we assessed the combined effect of salinity and dissolved organic carbon (DOC) on Cu accumulation on mussel larvae. Larvae were exposed for 48 h to three Cu concentrations in each of nine salinity/DOC treatments. Synchrotron radiation X-ray fluorescence was used to determine the Cu concentration in 36 individual larvae with a spatial resolution of 10 × 10 µm. Cu body burden concentrations varied between 1.1 and 27.6 µg/g DW larvae across all treatments and Cu was homogeneously distributed at this spatial resolution level. Our results indicate decreasing Cu accumulation with increasing DOC concentrations which can be explained by an increase in Cu complexation. In contrast, salinity had a nonlinear effect on Cu. This cannot be explained by copper speciation or competition processes and suggests a salinity-induced alteration in physiology.

Three-dimensional Fe speciation of an inclusion cloud within an ultradeep diamond by confocal µ-X-ray absorption near edge structure: evidence for late stage overprint.

A stream of 1-20 µm sized mineral inclusions having the negative crystal shape of its host within an "ultra-deep" diamond from Rio Soriso (Juina area, Mato Grosso State, Brazil) has been studied with confocal µ-X-ray absorption near edge structure (µXANES) at the Fe K and Mn K edges. This technique allows the three-dimensional nondestructive speciation of the Fe and Mn containing minerals within the inclusion cloud. The observed Fe-rich inclusions were identified to be ferropericlase (Fe,Mg)O, hematite and a mixture of these two minerals. Confocal µ-X-ray fluorescence (µXRF) further showed that Ca-rich inclusions were present as well, which are spatially separated from or in close contact with the Fe-rich inclusions. The inclusions are aligned along a plane, which most likely represents a primary growth zone. In the close vicinity of the inclusions, carbon coated planar features are visible. The three-dimensional distribution indicates a likely fluid overprint along an open crack. Our results imply that an imposed negative diamond shape of an inclusion alone does not exclude epigenetic formation or intense late stage overprint.

X-ray Free Electron Laser-Induced Synthesis of ε-Iron Nitride at High Pressures.

The ultrafast synthesis of ε-Fe3N1+x in a diamond-anvil cell (DAC) from Fe and N2 under pressure was observed using serial exposures of an X-ray free electron laser (XFEL). When the sample at 5 GPa was irradiated by a pulse train separated by 443 ns, the estimated sample temperature at the delay time was above 1400 K, confirmed by in situ transformation of α- to γ-iron. Ultimately, the Fe and N2 reacted uniformly throughout the beam path to form Fe3N1.33, as deduced from its established equation of state (EOS). We thus demonstrate that the activation energy provided by intense X-ray exposures in an XFEL can be coupled with the source time structure to enable exploration of the time-dependence of reactions under high-pressure conditions.

A confocal set-up for micro-XRF and XAFS experiments using diamond-anvil cells.

A confocal set-up is presented that improves micro-XRF and XAFS experiments with high-pressure diamond-anvil cells (DACs). In this experiment a probing volume is defined by the focus of the incoming synchrotron radiation beam and that of a polycapillary X-ray half-lens with a very long working distance, which is placed in front of the fluorescence detector. This set-up enhances the quality of the fluorescence and XAFS spectra, and thus the sensitivity for detecting elements at low concentrations. It efficiently suppresses signal from outside the sample chamber, which stems from elastic and inelastic scattering of the incoming beam by the diamond anvils as well as from excitation of fluorescence from the body of the DAC.

Evidence of shock-compressed stishovite above 300 GPa.

SiO2 is one of the most fundamental constituents in planetary bodies, being an essential building block of major mineral phases in the crust and mantle of terrestrial planets (1-10 ME). Silica at depths greater than 300 km may be present in the form of the rutile-type, high pressure polymorph stishovite (P42/mnm) and its thermodynamic stability is of great interest for understanding the seismic and dynamic structure of planetary interiors. Previous studies on stishovite via static and dynamic (shock) compression techniques are contradictory and the observed differences in the lattice-level response is still not clearly understood. Here, laser-induced shock compression experiments at the LCLS- and SACLA XFEL light-sources elucidate the high-pressure behavior of stishovite on the lattice-level under in situ conditions on the Hugoniot to pressures above 300 GPa. We find stishovite is still (meta-)stable at these conditions, and does not undergo any phase transitions. This contradicts static experiments showing structural transformations to the CaCl2, α-PbO2 and pyrite-type structures. However, rate-limited kinetic hindrance may explain our observations. These results are important to our understanding into the validity of EOS data from nanosecond experiments for geophysical applications.

Prescribing and testing by primary care providers to assess adherence to the Choosing Wisely Canada recommendations: a retrospective cohort study.

BACKGROUND: Choosing Wisely Canada is an initiative to reduce overprescribing and overtesting. This study assessed adherence to 4 Choosing Wisely Canada recommendations for interventions commonly performed in primary care: (a) antibiotic prescriptions for infections that are probably viral in origin, (b) routine vitamin D tests in low-risk adults, (c) annual screening blood tests and (d) prescriptions of antipsychotic medication to treat symptoms of dementia. METHODS: We conducted a retrospective cohort study of data from the electronic medical records of patients who had an encounter between 2014 and 2016 with a participating Manitoba Primary Care Research Network primary care provider in Manitoba, Canada. Patient encounter data were reviewed for prescribing and testing practices. Descriptive statistics and multivariable models assessed associations between patient and provider characteristics and rates of prescribing and testing. RESULTS: Data for 164 195 patients from 230 providers were included in the study. Sixteen percent (n = 25 629) of patients had an encounter that involved potentially unnecessary diagnostic testing and treatment. A minority of providers contributed to above-average rates of prescribing and testing: 29% (n = 69) of providers prescribed antibiotics for a viral indication,11% (n = 24) prescribed an antipsychotic to a patient diagnosed with dementia, 9% (n = 24) ordered prostate-specific antigen tests and 14% (n = 34) ordered vitamin D tests at above-average rates, respectively. Patient and provider characteristics were associated with each of the prescribing and testing practices assessed. INTERPRETATION: This study demonstrated that fewer than 30% of primary care providers contributed to interventions in direct contradiction to Choosing Wisely Canada recommendations. Improvement strategies specific to each prescription or testing recommendation should target specific providers to prevent patient harm and reduce unnecessary health care spending.

Distribution of selected elements in calcific human aortic valves studied by microscopy combined with SR-µXRF: influence of lipids on progression of calcification.

Calcified heart valves display a significant imbalance in tissue content of trace and essential elements. The valvular calcification is an age-related process and there are data suggesting involvement of lipids. We studied elemental composition and lipid distribution in three distinct regions of calcified human aortic valves, representing successive stages of the calcific degeneration: normal, thickened (early lesion) and calcified (late lesion), using SR-µXRF (Synchrotron Radiation Micro X-Ray Fluorescence) for elemental composition and Oil Red O (ORO) staining for demonstration of lipids. Two-dimensional SR-µXRF maps and precise point spectra were compared with histological stainings on consecutive valve sections to prove topographical localization and colocalization of the examined elements and lipids. In calcified valve areas, accumulation of calcium and phosphorus was accompanied by enhanced concentrations of strontium and zinc. Calcifications preferentially developed in lipid-rich areas of the valves. Calcium concentration ratio between lipid-rich and lipid-free areas was not age-dependent in early lesions, but showed a significant increase with age in late lesions, indicating age-dependent intensification of lipid involvement in calcification process. The results suggest that mechanisms of calcification change with progression of valve degeneration and with age.

Characterisation of the Mucor circinelloides regulated promoter gpd1P.

The promoter of the Mucor circinelloides gpd1 gene encoding glyceraldehyde-3-phosphate dehydrogenase (gpd1P) was recently cloned and used for the production of recombinant proteins, such as the Aspergillus niger glucose oxidase 1 (GOX). This represents the first example of the application of a strong and regulated promoter from this fungus for recombinant protein production. The original 741-bp gpd1P promoter fragment conferred hexose-dependent expression of GOX in M. circinelloides. To understand the regulatory mechanisms involved in gpd1P-driven expression and to develop improved promoter fragments, deletion derivatives of gpd1P were constructed. These derivatives were fused to the A. niger gox1 gene and used to construct strains containing a single copy of the expression cassette. GOX activity was detected in strains containing the full-length gpd1P and also in strains containing a 713-bp or a 361-bp derivative. Expression levels for the 361-bp derivative were high and comparable, regardless of the carbon source used. This promoter represents a useful derivative for constitutive heterologous gene expression in M. circinelloides.

Identification and analysis of genes involved in the control of dimorphism in Mucor circinelloides (syn. racemosus).

Mucor circinelloides (syn. racemosus) is a non-pathogenic dimorphic fungus belonging to the class of zygomycetes. We are developing a novel system for heterologous protein production exploiting the dimorphic growth characteristics of M. circinelloides. In order to identify potential genetic regulators of morphology we have initiated a characterisation of key genes involved in signal transduction in Mucor. We have cloned and characterised pkaR and pkaC encoding the regulatory subunit (PKAR) and the catalytic subunit (PKAC), respectively, of the cAMP-dependent protein kinase A (PKA) of M. circinelloides. In anaerobically grown yeast cells, the levels of expression of both pkaR and pkaC were significantly higher than the levels of expression in aerobically grown mycelium. However, during the dimorphic shift, i.e. during the transition from anaerobic yeast growth to aerobic filamentous growth, the expression of pkaR was found to increase approximately two-fold. These results indicate that regulation of PKA activity is conferred at different levels according to growth and environmental conditions. Overexpression of pkaR resulted in a multi-branched colony phenotype on solid medium indicating that PKAR plays a role in filamentation and branching. Fragments of genes encoding factors of the mitogen-activated protein (MAP) kinase (MAPK) pathway have also been cloned: mpk1 (mitogen-activated protein kinase 1) encoding a MAPK homologue and ste12 encoding a transcription factor.