Explore the vast range of titles available.

In this paper, we demonstrate the coupling of synchrotron small angle X-ray scattering (SAXS) to asymmetrical flow-field flow fractionation (AF4) for protein characterization. To the best of our knowledge, this is the first time AF4 is successfully coupled to a synchrotron for on-line measurements on proteins. This coupling has potentially high impact, as it opens the possibility to characterize individual constituents of sensitive and/or complex samples, not suited for separation using other techniques, and for low electron density samples where high X-ray flux is required, e.g., biomolecules and biologics. AF4 fractionates complex samples in native or close to native environment, with low shear forces and system surface area. Many orders of magnitude in size can be fractionated in one measurement, without having to reconfigure the experimental setup. We report AF4 fractionations with correlated UV and statistically adequate SAXS data of bovine serum albumin and a monoclonal antibody and evaluate SAXS data recorded for the two protein systems. Graphical Abstract

The cadherin–catenin adhesion complex is the central component of the cell–cell adhesion adherens junctions that transmit mechanical stress from cell to cell. We have determined the nanoscale structure of the adherens junction complex formed by the α-catenin•β-catenin•epithelial cadherin cytoplasmic domain (ABE) using negative stain electron microscopy, small-angle X-ray scattering, and selective deuteration/small-angle neutron scattering. The ABE complex is highly pliable and displays a wide spectrum of flexible structures that are facilitated by protein-domain motions in α- and β-catenin. Moreover, the 107-residue intrinsically disordered N-terminal segment of β-catenin forms a flexible “tongue” that is inserted into α-catenin and participates in the assembly of the ABE complex. The unanticipated ensemble of flexible conformations of the ABE complex suggests a dynamic mechanism for sensitivity and reversibility when transducing mechanical signals, in addition to the catch/slip bond behavior displayed by the ABE complex under mechanical tension. Our results provide mechanistic insight into the structural dynamics for the cadherin–catenin adhesion complex in mechanotransduction.

Aluminum-based adjuvants are widely used in vaccine formulations due to their immunostimulatory properties and strong safety profile. Despite their effectiveness and safety, the exact mechanisms by which they enhance vaccine efficacy remain unclear. One proposed mechanism is that aluminum adjuvants form a depot that gradually releases antigens, thereby improving antigen uptake by antigen-presenting cells. This study investigates the porous structures of two commonly used aluminum adjuvants, aluminum hydroxide (AH) and aluminum phosphate (AP), using small-angle X-ray scattering (SAXS) and small-angle neutron scattering (SANS). Our measurements reveal that AH nanoparticles, with their needle-like morphology, form smaller, interconnected pores within the aggregated architecture. In contrast, AP nanoparticles, with a plate-like shape, form more discrete, isolated porous structures. Both adjuvants have pore sizes within the range of commonly used vaccine antigens, supporting the depot theory. Our findings also reveal that antigen retention is prolonged when the antigen size is comparable to the average pore size of the adjuvant. This study highlights the utility of SAXS and SANS for in-situ characterization of adjuvant porosity and provides insights into how nanoparticle morphology affects antigen retention and release. By elucidating these structural details, our research underscores the importance of porous structure in adjuvant function and offers potential pathways for improving vaccine formulations through tailored adjuvant design.

Scattering techniques represent non-invasive experimental approaches and powerful tools for the investigation of structure and conformation of biomaterial systems in a wide range of distances, ranging from the nanometric to micrometric scale. More specifically, small-angle X-rays and neutron scattering and light scattering techniques represent well-established experimental techniques for the investigation of the structural properties of biomaterials and, through the use of suitable models, they allow to study and mimic various biological systems under physiologically relevant conditions. They provide the ensemble averaged (and then statistically relevant) information under in situ and operando conditions, and represent useful tools complementary to the various traditional imaging techniques that, on the contrary, reveal more local structural information. Together with the classical structure characterization approaches, we introduce the basic concepts that make it possible to examine inter-particles interactions, and to study the growth processes and conformational changes in nanostructures, which have become increasingly relevant for an accurate understanding and prediction of various mechanisms in the fields of biotechnology and nanotechnology. The upgrade of the various scattering techniques, such as the contrast variation or time resolved experiments, offers unique opportunities to study the nano- and mesoscopic structure and their evolution with time in a way not accessible by other techniques. For this reason, highly performant instruments are installed at most of the facility research centers worldwide. These new insights allow to largely ameliorate the control of (chemico-physical and biologic) processes of complex (bio-)materials at the molecular length scales, and open a full potential for the development and engineering of a variety of nano-scale biomaterials for advanced applications.

As part of its Extremely Brilliant Source (EBS) upgrade project, the ESRF's BM29 BioSAXS beamline was subject to a significant upgrade and refurbishment. In addition to the replacement of the beamline's original bending magnet source by a two-pole wiggler, leading to an increase in brilliance by a factor of 60, the sample environment of the beamline was almost completely refurbished: a vacuum-compatible Pilatus3 X 2M with a sensitive area of 253.7 mm × 288 mm and frame rates up to 250 Hz was installed, increasing the active area available and thus the q -scaling of scattering images taken; the sample changer was replaced with an upgraded version, allowing more space for customizable sample environments and the installation of two new sample exposure units; the software associated with the beamline was also renewed. In addition, the layout and functionality of the BSXCuBE3 ( BioSAXS Customized Beamline Environment ) data acquisition software was redesigned, providing an intuitive `user first' approach for inexperienced users, while at the same time maintaining more powerful options for experienced users and beamline staff. Additional features of BSXCuBE3 are queuing of samples; either consecutive sample changer and/or SEC-SAXS (size-exclusion chromatography small-angle X-ray scattering) experiments, including column equilibration were also implemented. Automatic data processing and analysis are now managed via Dahu , an online server with upstream data reduction, data scaling and azimuthal integration built around PyFAI ( Python Fast Azimuthal Integration ), and data analysis performed using the open source FreeSAS . The results of this automated data analysis pipeline are displayed in ISPyB/ExiSAXS . The upgraded BM29 has been in operation since the post-EBS restart in September 2020, and here a full description of its new hardware and software characteristics together with examples of data obtained are provided.

The present review summarizes the use of differential scanning calorimetry (DSC) and scattering techniques in the context of protein formulation design and characterization. The scattering techniques include wide angle X-ray diffractometry (XRD), small-angle neutron scattering (SANS) and small-angle X-ray scattering (SAXS). While DSC is valuable for understanding thermal behavior of the excipients, XRD provides critical information about physical state of solutes during freezing, annealing and in the final lyophile. However, as these techniques lack the sensitivity to detect biomolecule-related transitions, complementary characterization techniques such as small-angle scattering can provide valuable insights.

X-ray computed tomography (CT) is one of the most commonly used three-dimensional medical imaging modalities today. It has been refined over several decades, with the most recent innovations including dual-energy and spectral photon-counting technologies. Nevertheless, it has been discovered that wave-optical contrast mechanisms—beyond the presently used X-ray attenuation—offer the potential of complementary information, particularly on otherwise unresolved tissue microstructure. One such approach is dark-field imaging, which has recently been introduced and already demonstrated significantly improved radiological benefit in small-animal models, especially for lung diseases. Until now, however, dark-field CT could not yet be translated to the human scale and has been restricted to benchtop and small-animal systems, with scan durations of several minutes or more. This is mainly because the adaption and upscaling to the mechanical complexity, speed, and size of a human CT scanner so far remained an unsolved challenge. Here, we now report the successful integration of a Talbot–Lau interferometer into a clinical CT gantry and present dark-field CT results of a human-sized anthropomorphic body phantom, reconstructed from a single rotation scan performed in 1 s. Moreover, we present our key hardware and software solutions to the previously unsolved road-blocks, which so far have kept dark-field CT from being translated from the optical bench into a rapidly rotating CT gantry, with all its associated challenges like vibrations, continuous rotation, and large field of view. This development enables clinical dark-field CT studies with human patients in the near future.

Scanning small‐angle X‐ray scattering (sSAXS) has found multiple applications as a technique to probe the nanostructure in soft tissues and pathologies thereof. However, fresh tissue is fragile and prone to the quick onset of decomposition and autolysis. It lacks the firmness required for uniform and thin sectioning, resulting in the loss of 2D resolution offered by focused X‐ray beams, because the signal would be integrated through thick and/or irregular sections. Tissue processing, that includes fixation and embedding, is used to mitigate these issues but can by itself introduce structural changes in the tissues and impede the correct interpretation of sSAXS data. Here the extent of these structural changes in the SAXS signal caused by common tissue preservation methods on the example of skeletal muscle tissue, consisting of both muscle and surrounding connective tissue, was studied. This can guide an informed choice of preservation method tailored for specific experimental requirements. While some techniques performed better than others, all tissue‐processing methods induced structural changes to a certain degree. The choice of preservation method is therefore a balance between sectioning requirements and type of tissue used, as well as targeted structural information. The effect of tissue‐processing techniques, including fixation and embedding, on the structural information obtained from scanning small‐angle X‐ray scattering of muscle and connective tissue has been studied. This provides a framework for sSAXS users working with soft tissues to make an informed choice on the sample preservation method, tailored to their own requirements.

Solution-based interrogation of the physical nature of nucleosomes has its roots in X-ray and neutron scattering experiments, including those that provided the initial observation that DNA wraps around core histones. In this study, we performed a comprehensive small-angle scattering study to compare canonical nucleosomes with variant centromeric nucleosomes harboring the histone variant, CENP-A. We used nucleosome core particles (NCPs) assembled on an artificial positioning sequence (Widom 601) and compared these to those assembled on a natural α-satellite DNA from human centromeres. We establish the native solution properties of octameric H3 and CENP-A NCPs using analytical ultracentrifugation (AUC), small-angle X-ray scattering (SAXS), and contrast variation small-angle neutron scattering (CV-SANS). Using high-pressure SAXS (HP-SAXS), we discovered that both histone and DNA sequence have an impact on the stability of octameric nucleosomes in solution under high pressure (300 MPa), with evidence of reversible unwrapping in these experimental conditions. Both canonical nucleosomes harboring conventional histone H3 and their centromeric counterparts harboring CENP-A have a substantial increase in their radius of gyration, but this increase is much less prominent for centromeric nucleosomes. More broadly for chromosome-related research, we note that as HP-SAXS methodologies expand in their utility, we anticipate this will provide a powerful solution-based approach to study nucleosomes and higher-order chromatin complexes.

The second phase of the ESRF upgrade program did not only provide a new storage ring (Extremely Brilliant Source, EBS) but also allowed several beamlines to be refurbished. The BioSAXS beamline (located on port BM29) was upgraded with a new wiggler source and a larger detector. All analysis software has been rewritten to cope with the increased data flux and continues to provide beamline users with reduced and pre‐processed data in real time. This article describes FreeSAS, an open‐source collection of various small‐angle scattering analysis algorithms needed to reduce and analyze BioSAXS data, and Dahu, the tool used to interface data analysis with beamline control. It further presents the data‐processing pipelines for the different data acquisitions modes of the beamline, using either a sample changer for individual homogeneous samples or an inline size‐exclusion chromatography setup. A detailed presentation of the automatic data analysis pipelines for the BioSAXS beamline at the European synchrotron.