B21-High Throughput SAXS
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Diamond Proposal Number(s):
[29767]
Open Access
Abstract: Sterilisation of implantable biomaterials such as hydrogels remains a key step towards their clinical translation. Standard sterilisation methods can significantly alter hydrogels' physicochemical and biological performance. Previously, we developed composite hydrogels based on ε-polylysine (ε-PL) and hyaluronic acid (HA). The developed hydrogels demonstrated promising antibacterial activity and in vitro cell viability and their variable properties depending on the chosen ε-PL to HA ratio. In this study, we fabricated a series of chemically cross-linked ԑ-PL/HA hydrogels with expanded ԑ-PL to HA mass ratios.
Using small-angle X-ray scattering (SAXS), we unravelled the topological differences between physically and chemically crosslinked hydrogels. We then selected the chemically crosslinked hydrogel ԑ-PL/HA series of 60:40 wt%, 70:30 wt%, and 80:20 wt% ratios, with similar network topologies, to evaluate the impact of steam sterilisation on their physicochemical and viscoelastic properties. The antibacterial activity of the sterilized hydrogels was also evaluated against Gram-negative and Gram-positive bacteria. Our results showed that steam sterilisation minimally affects structure and physicochemical properties of ԑ-PL/HA hydrogels. Furthermore, the developed hydrogel ԑ-PL/HA series of 60:40 wt%, 70:30 wt%, and 80:20 wt% ratios showed pronounced antibacterial activity against Gram-negative and Gram-positive pathogenic bacteria. We expect our results will contribute to the growing understanding of using sterilisation methods for antibacterial hydrogels that have the potential for wider tissue engineering applications.
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Dec 2023
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I22-Small angle scattering & Diffraction
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Diamond Proposal Number(s):
[14126]
Open Access
Abstract: Echinoderms, such as sea cucumbers, have the remarkable property of changing the stiffness of their dermis according to the surrounding chemical environments. When sea cucumber dermal specimens are constantly strained, stress decays exponentially with time. Such stress relaxation is a hallmark of visco-elastic mechanical behavior. In this paper, in contrast, we attempted to interpret stress relaxation from the chemoelasticity viewpoint. We used a finite element model for the microstructure of the sea cucumber dermis. We varied stiffness over time and framed such changes against the first-order reactions of the interfibrillar matrix. Within this hypothetical scenario, we found that stress relaxation would then occur primarily due to fast crosslink splitting between the chains and a much slower macro-chain scission, with characteristic reaction times compatible with relaxation times measured experimentally. A byproduct of the model is that the concentration of undamaged macro-chains in the softened state is low, less than 10%
, which tallies with physical intuition. Although this study is far from being conclusive, we believe it opens an alternative route worthy of further investigation.
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Dec 2023
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B21-High Throughput SAXS
Krios III-Titan Krios III at Diamond
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Amaia
González-Magaña
,
Igor
Tascon
,
Jon
Altuna-Alvarez
,
María
Queralt-Martín
,
Jake
Colautti
,
Carmen
Velázquez
,
Maialen
Zabala
,
Jessica
Rojas-Palomino
,
Marité
Cárdenas
,
Antonio
Alcaraz
,
John C.
Whitney
,
Iban
Ubarretxena-Belandia
,
David
Albesa-Jove
Diamond Proposal Number(s):
[23872, 28248]
Open Access
Abstract: Bacterial competition is a significant driver of toxin polymorphism, which allows continual compensatory evolution between toxins and the resistance developed to overcome their activity. Bacterial Rearrangement hot spot (Rhs) proteins represent a widespread example of toxin polymorphism. Here, we present the 2.45 Å cryo-electron microscopy structure of Tse5, an Rhs protein central to Pseudomonas aeruginosa type VI secretion system-mediated bacterial competition. This structural insight, coupled with an extensive array of biophysical and genetic investigations, unravels the multifaceted functional mechanisms of Tse5. The data suggest that interfacial Tse5-membrane binding delivers its encapsulated pore-forming toxin fragment to the target bacterial membrane, where it assembles pores that cause cell depolarisation and, ultimately, bacterial death.
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Dec 2023
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Krios III-Titan Krios III at Diamond
Krios IV-Titan Krios IV at Diamond
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Diamond Proposal Number(s):
[29812]
Abstract: Our cells use an ensemble of histone proteins to fold and package the DNA genome into the nucleus. Histones also determine whether to expose DNA to enzymes to allow processes like gene expression, replication, and repair to occur. Although many in vitro studies have explored the mechanism histones use to fold and package DNA into higher-order structures called chromatin, less is known about chromatin organisation inside the nucleus of intact cells, and understanding this phenomenon could be key to understanding multiple DNA-associated processes. Recent advances in cryo-electron tomography have enabled scientists to observe these structures within the nucleus of rapidly cryopreserved cells. Reporting in Nature Communications, scientists at the University of Oxford collaborated with the electron Bio-Imaging Centre (eBIC) at the Diamond Light Source to capture chromatin in the nucleus of immune T cells, revealing that DNA is folded into more flexible and heterogenous fibres than previously modelled. Their experiments lay the groundwork for future studies into the roles of chromatin in health and disease.
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Dec 2023
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Open Access
Abstract: Simulations of cryo-electron microscopy (cryo-EM) images of biological samples can be used to produce test datasets to support the development of instrumentation, methods, and software, as well as to assess data acquisition and analysis strategies. To be useful, these simulations need to be based on physically realistic models which include large volumes of amorphous ice. The gold standard model for EM image simulation is a physical atom-based ice model produced using molecular dynamics simulations. Although practical for small sample volumes; for simulation of cryo-EM data from large sample volumes, this can be too computationally expensive. We have evaluated a Gaussian Random Field (GRF) ice model which is shown to be more computationally efficient for large sample volumes. The simulated EM images are compared with the gold standard atom-based ice model approach and shown to be directly comparable. Comparison with experimentally acquired data shows the Gaussian random field ice model produces realistic simulations. The software required has been implemented in the Parakeet software package and the underlying atomic models are available online for use by the wider community.
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Nov 2023
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B23-Circular Dichroism
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Maria
Dalla Pozza
,
Pierre
Mesdom
,
Ahmad
Abdullrahman
,
Tayler D.
Prieto Otoya
,
Philippe
Arnoux
,
Céline
Frochot
,
Germain
Niogret
,
Bruno
Saubaméa
,
Pierre
Burckel
,
James
Hall
,
Marcel
Hollenstein
,
Christine J
Cardin
,
Gilles
Gasser
Diamond Proposal Number(s):
[30390]
Abstract: Lack of selectivity is one of the main issues with currently used chemotherapies, causing damage not only to altered cells but also to healthy cells. Over the last decades, photodynamic therapy (PDT) has increased as a promising therapeutic tool due to its potential to treat diseases like cancer or bacterial infections with a high spatiotemporal control. Ruthenium(II) polypyridyl compounds are gaining attention for their application as photosensitizers (PSs) since they are generally nontoxic in dark conditions, while they show remarkable toxicity after light irradiation. In this work, four Ru(II) polypyridyl compounds with sterically expansive ligands were studied as PDT agents. The Ru(II) complexes were synthesized using an alternative route to those described in the literature, which resulted in an improvement of the synthesis yields. Solid-state structures of compounds [Ru(DIP)2phen]Cl2 and [Ru(dppz)2phen](PF6)2 have also been obtained. It is well-known that compound [Ru(dppz)(phen)2]Cl2 binds to DNA by intercalation. Therefore, we used [Ru(dppz)2phen]Cl2 as a model for DNA interaction studies, showing that it stabilized two different sequences of duplex DNA. Most of the synthesized Ru(II) derivatives showed very promising singlet oxygen quantum yields, together with noteworthy photocytotoxic properties against two different cancer cell lines, with IC50 in the micro- or even nanomolar range (0.06–7 μM). Confocal microscopy studies showed that [Ru(DIP)2phen]Cl2 and [Ru(DIP)2TAP]Cl2 accumulate preferentially in mitochondria, while no mitochondrial internalization was observed for the other compounds. Although [Ru(dppn)2phen](PF6)2 did not accumulate in mitochondria, it interestingly triggered an impairment in mitochondrial respiration after light irradiation. Among others, [Ru(dppn)2phen](PF6)2 stands out for its very good IC50 values, correlated with a very high singlet oxygen quantum yield and mitochondrial respiration disruption.
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Nov 2023
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I04-Macromolecular Crystallography
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George H.
Hutchins
,
Sebastian
Kiehstaller
,
Pascal
Poc
,
Abigail H.
Lewis
,
Jisun
Oh
,
Raya
Sadighi
,
Nicholas M.
Pearce
,
Mohamed
Ibrahim
,
Ivana
Drienovská
,
Anouk M.
Rijs
,
Saskia
Neubacher
,
Sven
Hennig
,
Tom N.
Grossmann
Diamond Proposal Number(s):
[25413]
Open Access
Abstract: Proteins are essential biomolecules and central to biotechnological applications. In many cases, assembly into higher-order structures is a prerequisite for protein function. Under conditions relevant for applications, protein integrity is often challenged, resulting in disassembly, aggregation, and loss of function. The stabilization of quaternary structure has proven challenging, particularly for trimeric and higher-order complexes, given the complexity of involved inter- and intramolecular interaction networks. Here, we describe the chemical bicyclization of homotrimeric protein complexes, thereby increasing protein resistance toward thermal and chemical stress. This approach involves the structure-based selection of cross-linking sites, their variation to cysteine, and a subsequent reaction with a triselectrophilic agent to form a protein assembly with bicyclic topology. Besides overall increased stability, we observe resistance toward aggregation and greatly prolonged shelf life. This bicyclization strategy gives rise to unprecedented protein chain topologies and can enable new biotechnological and biomedical applications.
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Nov 2023
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B18-Core EXAFS
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Diamond Proposal Number(s):
[15859]
Abstract: Metabolisms that evolved in the Archaean era (4.0–2.5 billion years ago) preferentially selected iron, manganese and molybdenum to form metalloproteins, whereas the majority of zinc-, copper- and vanadium-binding proteins emerged much later. The initial preference for these elements is commonly interpreted to reflect their availability in anoxic seawater, with free sulfide proposed as a key influence. While sulfidic waters reduce the availability of zinc and copper, they also remove molybdenum and leave behind vanadium. Furthermore, current geochemical data reflect predominantly ferruginous (Fe2+-rich), rather than sulfidic, conditions. Consistent with this, recent sedimentological work has uncovered abundant iron silicate minerals in Archaean rocks. Here we quantify metal partitioning during the formation and subsequent diagenesis of an Fe(II) silicate mineral, a precursor to crystalline greenalite, in both seawater and hot hydrothermal fluids. Our data show that Fe(II) silicates could have precipitated rapidly in Archaean hydrothermal plumes, severely attenuating hydrothermal delivery of key nutrients, in particular copper, zinc and vanadium. These results provide a mechanistic explanation for metal availability patterns in Archaean oceans that is consistent with temporal patterns of metal utilization predicted from protein structures and comparative genomics. Further, our data suggest natural greenalite may provide an archive of metal availability in deep time.
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Nov 2023
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I22-Small angle scattering & Diffraction
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Diamond Proposal Number(s):
[29072]
Open Access
Abstract: Synthetic cells, like their biological counterparts, require internal compartments with distinct chemical and physical properties where different functionalities can be localized. Inspired by membrane-less compartmentalization in biological cells, here, we demonstrate how microphase separation can be used to engineer heterogeneous cell-like architectures with programmable morphology and compartment-targeted activity. The synthetic cells self-assemble from amphiphilic DNA nanostructures, producing core-shell condensates due to size-induced de-mixing. Lipid deposition and phase-selective etching are then used to generate a porous pseudo-membrane, a cytoplasm analog, and membrane-less organelles. The synthetic cells can sustain RNA synthesis via in vitro transcription, leading to cytoplasm and pseudo-membrane expansion caused by an accumulation of the transcript. Our approach exemplifies how architectural and functional complexity can emerge from a limited number of distinct building blocks, if molecular-scale programmability, emergent biophysical phenomena, and biochemical activity are coupled to mimic those observed in live cells.
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Nov 2023
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I04-1-Macromolecular Crystallography (fixed wavelength)
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Diamond Proposal Number(s):
[24948, 18598]
Open Access
Abstract: Bacteria and yeasts grow on biomass polysaccharides by expressing and excreting a complex array of glycoside hydrolase (GH) enzymes. Identification and annotation of such GH pools, which are valuable commodities for sustainable energy and chemistries, by conventional means (genomics, proteomics) are complicated, as primary sequence or secondary structure alignment with known active enzymes is not always predictive for new ones. Here we report a “low-tech”, easy-to-use, and sensitive multiplexing activity-based protein-profiling platform to characterize the xyloglucan-degrading GH system excreted by the soil saprophyte, Cellvibrio japonicus, when grown on xyloglucan. A suite of activity-based probes bearing orthogonal fluorophores allows for the visualization of accessory exo-acting glycosidases, which are then identified using biotin-bearing probes. Substrate specificity of xyloglucanases is directly revealed by imbuing xyloglucan structural elements into bespoke activity-based probes. Our ABPP platform provides a highly useful tool to dissect xyloglucan-degrading systems from various sources and to rapidly select potentially useful ones. The observed specificity of the probes moreover bodes well for the study of other biomass polysaccharide-degrading systems, by modeling probe structures to those of desired substrates.
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Nov 2023
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