I02-Macromolecular Crystallography
I22-Small angle scattering & Diffraction
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S.
Hayes
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N.
Aldahlawi
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A. L.
Marcovich
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J.
Brekelmans
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A.
Goz
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A.
Scherz
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R. D.
Young
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J. S.
Bell
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D. P.
O’brart
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R. M. M. A.
Nuijts
,
K. M.
Meek
Diamond Proposal Number(s):
[8458, 11316]
Open Access
Abstract: A cross-linking technique involving application of Bacteriochlorophyll Derivative WST-11 mixed with dextran (WST-D) to the epithelium-debrided cornea and illumination with Near Infrared (NIR), has been identified as a promising therapy for stiffening pathologically weakened corneas. To investigate its effect on corneal collagen architecture, x-ray scattering and electron microscopy data were collected from paired WST-D/NIR treated and untreated rabbit corneas. The treated eye received 2.5 mg/mL WST-D and was illuminated by a NIR diode laser (755 nm, 10 mW/cm2). An increase in corneal thickness (caused by corneal oedema) occurred at 1-day post-treatment but resolved in the majority of cases within 4 days. The epithelium was fully healed after 6–8 days. X-ray scattering revealed no difference in average collagen interfibrillar spacing, fibril diameter, D-periodicity or intermolecular spacing between treated and untreated specimens. Similarly, electron microscopy images of the anterior and posterior stroma in healed WST-D/NIR corneas and untreated controls revealed no obvious differences in collagen organisation or fibril diameter. As the size and organisation of stromal collagen is closely associated with the optical properties of the cornea, the absence of any large-scale changes following treatment confirms the potential of WST-D/NIR therapy as a means of safely stiffening the cornea.
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Jun 2020
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I22-Small angle scattering & Diffraction
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A.
Doekhie
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R.
Dattani
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Y-c.
Chen
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Y.
Yang
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A.
Smith
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A. P.
Silve
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F.
Koumanov
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S. A.
Wells
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K. J.
Edler
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K. J.
Marchbank
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J. M. H.
Van Den Elsen
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A.
Sartbaeva
Diamond Proposal Number(s):
[14148]
Open Access
Abstract: Our recently developed ensilication approach can physically stabilize proteins in silica without use of a pre-formed particle matrix. Stabilisation is done by tailor fitting individual proteins with a silica coat using a modified sol-gel process. Biopharmaceuticals, e.g. liquid-formulated vaccines with adjuvants, frequently have poor thermal stability; heating and/or freezing impairs their potency. As a result, there is an increase in the prevalence of vaccine-preventable diseases in low-income countries even when there are means to combat them. One of the root causes lies in the problematic vaccine ‘cold chain’ distribution. We believe that ensilication can improve vaccine availability by enabling transportation without refrigeration. Here, we show that ensilication stabilizes tetanus toxin C fragment (TTCF), a component of the tetanus toxoid present in the diphtheria, tetanus and pertussis (DTP) vaccine. Experimental in vivo immunization data show that the ensilicated material can be stored, transported at ambient temperatures, and even heat-treated without compromising the immunogenic properties of TTCF. To further our understanding of the ensilication process and its protective effect on proteins, we have also studied the formation of TTCF-silica nanoparticles via time-resolved Small Angle X-ray Scattering (SAXS). Our results reveal ensilication to be a staged diffusion-limited cluster aggregation (DLCA) type reaction. An early stage (tens of seconds) in which individual proteins are coated with silica is followed by a subsequent stage (several minutes) in which the protein-containing silica nanoparticles aggregate into larger clusters. Our results suggest that we could utilize this technology for vaccines, therapeutics or other biopharmaceuticals that are not compatible with lyophilization.
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Jun 2020
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I03-Macromolecular Crystallography
I04-Macromolecular Crystallography
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Stefania
Digiovanni
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Cristina
Visentin
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Genny
Degani
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Alberto
Barbiroli
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Matteo
Chiara
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Luca
Regazzoni
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Flavio
Di Pisa
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Andrew J.
Borchert
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Diana M.
Downs
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Stefano
Ricagno
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Maria Antonietta
Vanoni
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Laura
Popolo
Diamond Proposal Number(s):
[20221]
Open Access
Abstract: Reactive Intermediate Deaminase (Rid) protein superfamily includes eight families among which the RidA is conserved in all domains of life. RidA proteins accelerate the deamination of the reactive 2-aminoacrylate (2AA), an enamine produced by some pyridoxal phosphate (PLP)-dependent enzymes. 2AA accumulation inhibits target enzymes with a detrimental impact on fitness. As a consequence of whole genome duplication, teleost fish have two ridA paralogs, while other extant vertebrates contain a single-copy gene. We investigated the biochemical properties of the products of two paralogs, identified in Salmo salar. SsRidA-1 and SsRidA-2 complemented the growth defect of a Salmonella enterica ridA mutant, an in vivo model of 2AA stress. In vitro, both proteins hydrolyzed 2-imino acids (IA) to keto-acids and ammonia. SsRidA-1 was active on IA derived from nonpolar amino acids and poorly active or inactive on IA derived from other amino acids tested. In contrast, SsRidA-2 had a generally low catalytic efficiency, but showed a relatively higher activity with IA derived from L-Glu and aromatic amino acids. The crystal structures of SsRidA-1 and SsRidA-2 provided hints of the remarkably different conformational stability and substrate specificity. Overall, SsRidA-1 is similar to the mammalian orthologs whereas SsRidA-2 displays unique properties likely generated by functional specialization of a duplicated ancestral gene.
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Jun 2020
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Open Access
Abstract: To travel safely behind screens that can protect us from stones and hail, we must understand the response of glass to impact. However, without a means to observe the mechanisms that fail different silicate architectures, engineering has relied on external sensors, post-impact examination and best-guess to glaze our vehicles. We have used single and multi-bunch, X-ray imaging to differentiate distinct phases of failure in two silicates. We identified distinct micromechanisms, operating in tandem and leading to failure in borosilicate glass and Z-cut quartz. A surface zone in the amorphous glass densifies before bulk fracture occurs and then fails the block, whilst in quartz, fast cracks, driven down cleavage planes, fails the bulk. Varying the rate at which ejecta escapes by using different indenter tip geometries controls the failed target’s bulk strength. This opens the way to more physically based constitutive descriptions for the glasses allowing design of safer, composite panels by controlling the impulses felt by protective screens.
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Jun 2020
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I08-Scanning X-ray Microscopy beamline (SXM)
I10-Beamline for Advanced Dichroism
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Open Access
Abstract: Atypical low-oxidation-state iron phases in Alzheimer’s disease (AD) pathology are implicated in disease pathogenesis, as they may promote elevated redox activity and convey toxicity. However, the origin of low-oxidation-state iron and the pathways responsible for its formation and evolution remain unresolved. Here we investigate the interaction of the AD peptide β-amyloid (Aβ) with the iron storage protein ferritin, to establish whether interactions between these two species are a potential source of low-oxidation-state iron in AD. Using X-ray spectromicroscopy and electron microscopy we found that the co-aggregation of Aβ and ferritin resulted in the conversion of ferritin’s inert ferric core into more reactive low-oxidation-states. Such findings strongly implicate Aβ in the altered iron handling and increased oxidative stress observed in AD pathogenesis. These amyloid-associated iron phases have biomarker potential to assist with disease diagnosis and staging, and may act as targets for therapies designed to lower oxidative stress in AD tissue.
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Jun 2020
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B16-Test Beamline
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Diamond Proposal Number(s):
[20103]
Open Access
Abstract: Highly efficient scintillation crystals with short decay times are indispensable for improving the performance of numerous detection and imaging instruments that use- X-rays, gamma-quanta, ionising particles or neutrons. Halide perovskites emerged recently as very promising materials for detection of ionising radiation that motivated further exploration of the materials. In this work, we report on excellent scintillation properties of CsPbBr3 crystals when cooled to cryogenic temperatures. The temperature dependence of luminescence spectra, decay kinetics and light yield under excitation with X-rays and α-particles was investigated. It is shown that the observed changes of spectral and kinetic characteristics of the crystal with temperature can be consistently explained by radiative decay of free excitons, bound and trapped excitons as well as electron-hole pairs originating from their disintegration. It has been found that the crystal exhibits a fast decay time constant of 1 ns at 7 K. The scintillation light yield of CsPbBr3 at 7 K is assessed to be 50,000 ± 10,000 ph/MeV at excitation with 12 keV X-rays and 109,000 ± 22,000 ph/MeV at excitation with α-particles of 241Am. This finding places CsPbBr3 in an excellent position for the development of a new generation of cryogenic, efficient scintillation detectors with nanosecond response time, marking a step-change in opportunities for scintillator-based applications.
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May 2020
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I04-Macromolecular Crystallography
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Open Access
Abstract: The human 2-oxoglutarate dependent oxygenase aspartate/asparagine-β-hydroxylase (AspH) catalyses the hydroxylation of Asp/Asn-residues in epidermal growth factor-like domains (EGFDs). AspH is upregulated on the surface of malign cancer cells; increased AspH levels correlate with tumour invasiveness. Due to a lack of efficient assays to monitor the activity of isolated AspH, there are few reports of studies aimed at identifying small-molecule AspH inhibitors. Recently, it was reported that AspH substrates have a non-canonical EGFD disulfide pattern. Here we report that a stable synthetic thioether mimic of AspH substrates can be employed in solid phase extraction mass spectrometry based high-throughput AspH inhibition assays which are of excellent robustness, as indicated by high Z’-factors and good signal-to-noise/background ratios. The AspH inhibition assay was applied to screen approximately 1500 bioactive small-molecules, including natural products and active pharmaceutical ingredients of approved human therapeutics. Potent AspH inhibitors were identified from both compound classes. Our AspH inhibition assay should enable the development of potent and selective small-molecule AspH inhibitors and contribute towards the development of safer inhibitors for other 2OG oxygenases, e.g. screens of the hypoxia-inducible factor prolyl-hydroxylase inhibitors revealed that vadadustat inhibits AspH with moderate potency.
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May 2020
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B16-Test Beamline
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Anna
Turyanskaya
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Mirjam
Rauwolf
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Vanessa
Pichler
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Rolf
Simon
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Manfred
Burghammer
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Oliver J. L.
Fox
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Kawal
Sawhney
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Jochen G.
Hofstaetter
,
Andreas
Roschger
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Paul
Roschger
,
Peter
Wobrauschek
,
Christina
Streli
Diamond Proposal Number(s):
[16260]
Open Access
Abstract: Gadolinium-based contrast agents (GBCAs) are frequently used in patients undergoing magnetic resonance imaging. In GBCAs gadolinium (Gd) is present in a bound chelated form. Gadolinium is a rare-earth element, which is normally not present in human body. Though the blood elimination half-life of contrast agents is about 90 minutes, recent studies demonstrated that some tissues retain gadolinium, which might further pose a health threat due to toxic effects of free gadolinium. It is known that the bone tissue can serve as a gadolinium depot, but so far only bulk measurements were performed. Here we present a summary of experiments in which for the first time we mapped gadolinium in bone biopsy from a male patient with idiopathic osteoporosis (without indication of renal impairment), who received MRI 8 months prior to biopsy. In our studies performed by means of synchrotron radiation induced micro- and submicro-X-ray fluorescence spectroscopy (SR-XRF), gadolinium was detected in human cortical bone tissue. The distribution of gadolinium displays a specific accumulation pattern. Correlation of elemental maps obtained at ANKA synchrotron with qBEI images (quantitative backscattered electron imaging) allowed assignment of Gd structures to the histological bone structures. Follow-up beamtimes at ESRF and Diamond Light Source using submicro-SR-XRF allowed resolving thin Gd structures in cortical bone, as well as correlating them with calcium and zinc.
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Apr 2020
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Open Access
Abstract: In Pseudomonas aeruginosa, the transition between planktonic and biofilm lifestyles is modulated by the intracellular secondary messenger cyclic dimeric-GMP (c-di-GMP) in response to environmental conditions. Here, we used gene deletions to investigate how the environmental stimulus nitric oxide (NO) is linked to biofilm dispersal, focusing on biofilm dispersal phenotype from proteins containing putative c-di-GMP turnover and Per-Arnt-Sim (PAS) sensory domains. We document opposed physiological roles for the genes ΔrbdA and Δpa2072 that encode proteins with identical domain structure: while ΔrbdA showed elevated c-di-GMP levels, restricted motility and promoted biofilm formation, c-di-GMP levels were decreased in Δpa2072, and biofilm formation was inhibited, compared to wild type. A second pair of genes, ΔfimX and ΔdipA, were selected on the basis of predicted impaired c-di-GMP turnover function: ΔfimX showed increased, ΔdipA decreased NO induced biofilm dispersal, and the genes effected different types of motility, with reduced twitching for ΔfimX and reduced swimming for ΔdipA. For all four deletion mutants we find that NO-induced biomass reduction correlates with increased NO-driven swarming, underlining a significant role for this motility in biofilm dispersal. Hence P. aeruginosa is able to differentiate c-di-GMP output using structurally highly related proteins that can contain degenerate c-di-GMP turnover domains.
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Apr 2020
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I13-1-Coherence
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Diamond Proposal Number(s):
[21555]
Open Access
Abstract: We present here a new method of performing X-ray edge-subtraction ptychographic imaging by combining multiple harmonics from an undulator synchtrotron source and an energy discriminating photon counting detector. Conventionally, monochromatic far-field X-ray ptychography is used to perform edge subtraction through the use of multiple monochromatic energy scans to obtain spectral information for a variety of applications. Here, we use directly the undulator spectrum from a synchrotron source, selecting two separate harmonics post sample using the Pixirad-1/Pixie-III detector. The result is two monochromatic images, above and below an absorption edge of interest. The proposed method is applied to obtain Au L-edge subtraction imaging of a Au-Ni grid test sample. The Au L-edge subtraction is particularly relevant for the identification of gold nanoparticles for biomedical applications. Switching the energy scan mechanism from a mechanical monochromator to an electronic detector threshold allows for faster spectral data collection with improved stability.
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Apr 2020
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