B21-High Throughput SAXS
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Diamond Proposal Number(s):
[32486]
Open Access
Abstract: Lipoic acid is a biocompatible compound with antioxidant activity that is of considerable interest in cosmetic formulations, and the disulfide group in the N-terminal ring confers redox activity. Here, we study the self-assembly and aspects of the bioactivity of a lipopeptide (peptide amphiphile) comprising the KTTKS collagen-stimulating pentapeptide sequence conjugated to an N-terminal lipoic acid chain, lipoyl-KTTKS. Using SAXS, SANS and cryo-TEM, lipoyl-KTTKS is found to form a population of curly fibrils (wormlike micelles) above a critical aggregation concentration. Upon chemical reduction, the fibrils (and β-sheet structure) are disrupted because of the breaking of the disulfide bond, which produces dihydrolipoic acid. Lipoyl-KTTKS also undergoes photo-degradation in the presence of UV radiation. Through cell assays using fibroblasts, we found that lipoyl-KTTKS has excellent cytocompatibility across a wide concentration range, stimulates collagen production, and enhances the rate of cell coverage in a simple in vitro scratch assay of ‘wound healing’. Lipoyl-KTTKS thus has several notable properties that may be useful for the development of cosmetics, cell scaffolds or tissue engineering materials.
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Mar 2025
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I04-Macromolecular Crystallography
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Diamond Proposal Number(s):
[34438]
Open Access
Abstract: Ubiquitylation is critically implicated in the recognition and repair of DNA double-strand breaks. The adaptor protein MDC1 mediates the recruitment of the key DNA damage responsive E3 ubiquitin ligase RNF8 to the break sites. It does so by directly interacting with RNF8 in a phosphorylation-dependent manner that involves the RNF8 FHA domain, thus initiating targeted chromatin ubiquitylation at the break sites. Here, we report that MDC1 also directly binds to two additional E3 ubiquitin ligases, Pellino 1 and 2, which were recently implicated in the DNA damage response. Through a combination of biochemical, biophysical and X-ray crystallographic approaches, we reveal the molecular details of the MDC1-Pellino complexes. Furthermore, we show that in mammalian cells, MDC1 mediates Pellino recruitment to sites of DNA double-strand breaks by a direct phosphorylation-dependent interaction between the two proteins. Taken together, our findings provide new molecular insights into the ubiquitylation pathways that govern genome stability maintenance.
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Mar 2025
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I12-JEEP: Joint Engineering, Environmental and Processing
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Diamond Proposal Number(s):
[30413]
Abstract: The migration and deposition of fine particles in porous materials is critical in industries such as energy, pharmaceuticals, and environmental engineering. Using 3D time-lapse synchrotron X-ray imaging, we observe fine particles invading porous media, analyzing the effects of pore size and heterogeneity at both pore and macro scales. Glass beads model homogeneous and heterogeneous conditions, revealing a sequence of deposition processes: surface attachment, throat bridging, blocking, pore filling, compaction, and migration. A critical throat-to-particle size ratio of 1.7 governs deposition behavior. At the macro-scale, heterogeneities like beddings and flow pathways influence fines migration and deposition. Based on dynamic 3D imaging, we propose a mechanism for fines behavior in heterogeneous porous media. These findings enhance understanding of fines migration, offering a predictive framework for managing formation damage and optimizing filter cake design in drilling and clean energy applications.
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Mar 2025
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I14-Hard X-ray Nanoprobe
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Diamond Proposal Number(s):
[28688]
Open Access
Abstract: Understanding the interactions between metal-based nanoparticles and biological systems in complex environments (e.g., the human body, soils, and marine settings) remains challenging, especially at the single-cell and nanoscale levels. Capturing the dynamics of these interactions, such as metal distribution, nanoparticle growth, or degradation, in their native state (in vivo) is particularly difficult. Here, we demonstrate the direct measurement of iron content in hydrated, magnetite-biomineralizing magnetotactic bacteria using synchrotron-based nanobeam–scanning X-ray fluorescence microscopy combined with a liquid cell environment. In addition to X-ray fluorescence imaging, we collected iron chemical speciation information from individual bacteria in liquid using X-ray absorption spectroscopy. To follow biomineralization in situ, we developed a microfluidic device to track magnetite nanoparticle formation over several hours under the X-ray beam. This approach highlights the potential of X-ray fluorescence microscopy in liquid cell setups to provide elemental and chemical insights into biological processes at the single-cell level. Combining X-ray nanobeam techniques with liquid cell devices will enable more “on-chip” experiments on metals in biological contexts to be conducted at the synchrotron.
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Mar 2025
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B21-High Throughput SAXS
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Diamond Proposal Number(s):
[28659]
Abstract: In this research, we studied two novel lipopeptide sequences containing the amino acid serine (SPRWG) with one (compound 1) or two aliphatic tails (compound 2) to optimize the detection capabilities for organophosphate pesticides, specifically glyphosate. The study comprehensively explored how the incorporation of serine influences the physicochemical properties and supramolecular assembly of the lipopeptides, leading to enhanced interactions with glyphosate. Advanced analytical methods were employed to investigate these modifications, including fluorescence spectroscopy, circular dichroism, and small-angle X-ray scattering (SAXS). The results showed that serine significantly reduces the critical aggregation concentration, increases the hydrophilicity of the lipopeptides, and promotes the formation of distinct secondary structures—-turns in compound 1 and -sheets in compound 2. Moreover, isothermal titration calorimetry (ITC) and molecular dynamics confirmed the improved binding affinity with glyphosate strongly modulated by pH and pesticide load. Compound 1, with one alkyl chain, demonstrated notably higher catalytic activity and sensitivity linked to its pH equilibrium and structural features, marking it as particularly effective for acetylcholinesterase mimicry in pesticide detection. Density functional theory and molecular dynamics calculations showed that, when compared to the PRWG sequence, SPRWG has more unprotonated N-terminal sites due to a lower pKa, more beta-turn-like structures that improve stabilization, and more hydrogen bonds between N-(phosphonomethyl)glycine (PNG, commonly known as glyphosate) and aggregates across a wide pH range and P/L, which explains its enhanced reactivity in Ellman’s test and better inhibitory effects under the influence of PNG. Our results suggest that serine-functionalized lipopeptides have great potential as biomimetic sensors in environmental monitoring.
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Mar 2025
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I22-Small angle scattering & Diffraction
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Diamond Proposal Number(s):
[22237]
Open Access
Abstract: The effect of manufacturing conditions on the morphology of an industrially-processed 11-ply polyamide/glass fibre (PA66-GF60) laminate was investigated. Through-thickness temperature variation during the manufacturing process (pre-heating, stamp forming, demoulding) was revealed via eight inter-ply thermocouples. Thermal and X-ray analysis provided insights into process-induced polymer crystallinity and morphology through the laminate thickness. Cooling rates up to ∼ 2100 °C/min were observed in outer plies, compared to ∼ 420 °C/min for inner plies. A self-heating exothermal phenomenon was observed during crystallisation of the inner layers, leading to increased core crystallinity. X-ray diffraction revealed differences in preferred polymer orientation between the plies. For the inner plies, additional mobility from slower cooling leads to partially oriented crystallites along the glass fibre axis and a well-developed lamellar macromorphology. The rapidly cooled outer plies showed unoriented morphology, without long-range ordering. The work provides detailed understanding of polymer morphology for an industrially-relevant high-volume manufacturing process for thermoplastic matrix components.
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Feb 2025
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B18-Core EXAFS
I20-Scanning-X-ray spectroscopy (XAS/XES)
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Diamond Proposal Number(s):
[24074, 21441]
Open Access
Abstract: Operations at uranium (U)-mining and nuclear facilities have left a global legacy of significant radionuclide contamination in groundwaters which must be managed to minimize environmental harm. Uranium groundwater contamination is present at several sites globally, including Oak Ridge National Laboratory and Hanford, USA and Sellafield nuclear site, UK. In situ phosphate biomineralisation offers a promising method for radionuclide (including 90Sr and U) remediation at these sites. Typically, phosphate-generating amendments are injected into the subsurface to sequester select radionuclides in groundwaters by precipitation of poorly soluble Ca-phosphate phases and subsequent adsorption and/or incorporation of radionuclides to these poorly soluble phases, a remediation route being explored for both U and 90Sr. In this study, we investigate the mechanisms of U-phosphate precipitation in two phosphate-generating amendments (Ca-citrate/Na-phosphate and glycerol phosphate) under conditions relevant to Sellafield, UK. Using aerobic batch sediment experiments, we show both Ca-citrate/Na-phosphate and glycerol phosphate amendments are effective at enhancing removal of U(VI) from representative groundwaters (from 94% to >97%). Aqueous geochemical data coupled to speciation modelling highlighted that precipitation of U(VI) phosphate phases was the likely mechanism of U(VI) removal from groundwaters. Further X-ray absorption spectroscopy (XAS) analysis of solids confirmed U was present as a highly insoluble uranyl orthophosphate-like phase after treatment with both Ca-citrate/Na-phosphate and glycerol phosphate amendments. These data provide underpinning information on U-phosphate remediation in Sellafield relevant conditions thus expanding the range of treatment options for radionuclide contaminated groundwaters and defining the transport and fate of U during phosphate biomineralisation.
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Feb 2025
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I04-Macromolecular Crystallography
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Open Access
Abstract: We have recently demonstrated a novel anaerobic NADH-dependent haem breakdown reaction, which is carried out by a range of haemoproteins. The Yersinia enterocolitica protein, HemS, is the focus of further research presented in the current paper. Using conventional experimental methods, bioinformatics, and energy landscape theory (ELT), we provide new insight into the mechanism of the novel breakdown process. Of particular interest is the behavior of a double phenylalanine gate, which opens and closes according to the relative situations of haem and NADH within the protein pocket. This behavior suggests that the double phe-gate fulfills a regulatory role within the pocket, controlling the access of NADH to haem. Additionally, stopped-flow spectroscopy results provide kinetic comparisons between the wild-type and the selected mutants. We also present a fully resolved crystal structure for the F104AF199A HemS monomer, including its extensive loop, the first such structure to be completely resolved for HemS or any of its close homologues. The energy landscapes approach provided key information regarding the gating strategy employed by HemS, compensating for current limitations with conventional biophysical and molecular dynamics approaches. We propose that ELT become more widely used in the field, particularly in the investigation of the dynamics and interactions of weak-binding ligands, and for gating features, within protein cavities.
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Feb 2025
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I22-Small angle scattering & Diffraction
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Qian
Ma
,
Andri K.
Riau
,
Robert D.
Young
,
James S.
Bell
,
Olga
Shebanova
,
Nicholas J.
Terrill
,
Gary H. F.
Yam
,
Evelina
Han
,
Keith M.
Meek
,
Jodhbir S.
Mehta
,
Craig
Boote
Diamond Proposal Number(s):
[23514, 28285, 29862, 34903]
Open Access
Abstract: Purpose: Donor tissue shortfalls and postsurgical complications are driving novel corneal tissue regeneration approaches. Corneal stromal keratocytes (CSKs) have shown promise in promoting corneal repair and restoring transparency. We investigated the impact of intrastromal CSK injection on corneal ultrastructure and proteoglycan (PG) distribution in a rat injury model.
Methods: Rats were divided into four groups: normal (n = 12), injured (irregular phototherapeutic keratectomy centrally; n = 6), CSK (injured + human CSK intrastromal injection; n = 6), and PBS (injured + PBS injection; n = 6). Three weeks after treatment, corneas were examined by slit-lamp and optical coherence tomography. Corneal ultrastructure was analysed via small-angle x-ray scattering (collagen fibril diameter, interfibrillar spacing and matrix order), transmission electron microscopy with cuprolinic blue before and after chondroitinase digestion (CS/DS and KS PGs), and immunofluorescence staining (lumican and decorin).
Results: Irregular phototherapeutic keratectomy caused corneal opacity and significantly disrupted stromal ultrastructure, characterized by increased haze density (P < 0.0001), change in central corneal thickness (P = 0.0005), and interfibrillar spacing (P < 0.0001), along with decreased fibril diameter (P < 0.0001), matrix order (P < 0.0001), CS/DS (P < 0.0001) and KS (P < 0.0001) PGs, lumican, and decorin. CSK injection recovered corneal clarity and native stromal ultrastructure, with haze density (P = 0.8086), change in central corneal thickness (P = 0.9503), fibril diameter (P = 0.1139), interfibrillar spacing (P = 0.5879), matrix order (P = 0.9999), CS/DS (P = 0.9969) and KS (P = 0.2877) PGs, lumican, and decorin returning to normal. In contrast, the PBS group exhibited similar corneal injury responses to the injured group.
Conclusions: CSK injection resolved early stage corneal scarring by restoring stromal collagen arrangement and PG distribution, further endorsing its potential for treating corneal opacities.
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Feb 2025
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I09-Surface and Interface Structural Analysis
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Diamond Proposal Number(s):
[31918]
Open Access
Abstract: The discovery of ferroelectric phases in HfO2-based films has reignited interest in ferroelectrics and their application in resistive switching (RS) devices. This study investigates the pivotal role of electrodes in facilitating the Schottky-to-Ohmic transition (SOT) observed in devices consisting of ultrathin epitaxial ferroelectric Hf0.93Y0.07O2 (YHO) films deposited on La0.67Sr0.33MnO3-buffered Nb-doped SrTiO3 (NbSTO|LSMO) with Ti|Au top electrodes. These findings indicate combined filamentary RS and ferroelectric switching occurs in devices with designed electrodes, having an ON/OFF ratio of over 100 during about 105 cycles. Transport measurements of modified device stacks show no change in SOT when the ferroelectric YHO layer is replaced with an equivalent hafnia-based layer, Hf0.5Zr0.5O2 (HZO). However, incomplete SOT is observed for variations in the top electrode thickness or material, as well as LSMO electrode thickness. This underscores the importance of employing oxygen-reactive electrodes and a bottom electrode with reduced conductivity to stabilize SOT. These findings provide valuable insights for enhancing the performance of ferroelectric RS devices through integration with filamentary RS mechanism.
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Jan 2025
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