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Blood clots are essential biomaterials that prevent blood loss and provide a temporary scaffold for tissue repair. In their function, these materials must be capable of resisting mechanical forces from hemodynamic shear and contractile tension without rupture. Fibrin networks, the primary load-bearing element in blood clots, have unique nonlinear mechanical properties resulting from fibrin's hierarchical structure. This structure provides multiscale load bearing from fiber deformation to protein unfolding. Here, we study the fiber and molecular scale response of fibrin under shear and tensile loads in situ using a combination of fluorescence and vibrational (molecular) microscopy. Imaging protein fiber orientation and molecular vibrations, we find that fiber alignment and molecular unfolding in fibrin appear at much larger strains under shear compared to uniaxial tension. Alignment levels reached at 150% shear strain were reached already at 60% tensile strain, and molecular unfolding of fibrin was only detected at shear strains above 300%, whereas fibrin unfolding began already at 20% tensile strain. Moreover, shear deformation caused progressive changes in vibrational modes consistent with increased protofibril and fiber packing that were already present even at very low tensile deformation. Together with a bioinformatic analysis of the primary fibrinogen structure, we propose a scheme for the molecular response of fibrin from low to high deformation, which may relate to the teleological origin of fibrin's resistance to shear and tensile forces. Image, graphical abstract [ABSTRACT FROM AUTHOR]

Abstract: The phase behavior of bilayers of binary mixtures of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and cholesterol has been studied using Raman spectroscopy. It is observed that the shape of the cholesterol vibrational spectrum in lipid–cholesterol binary mixtures does not vary significantly with either the cholesterol concentration or the temperature. This permits determination of the lipid vibrational signatures of the liquid-disordered (ld), solid-ordered (so) and liquid-ordered (lo) phases. Within the phase coexistence region, the measured spectra are described very well by a linear combination of the different spectral components, which permits a quantitative analysis of the phase diagram. In contrast to earlier findings, our experiments provide no indication of a phase boundary at low cholesterol concentration. The upper boundary of the phase coexistence region is found at ∼27 and ∼22mol% for ld–lo and so–lo coexistence region, respectively. Within these phase coexistence regions, the partitioning of cholesterol between the cholesterol-poor and the cholesterol-rich phases is in close agreement with the lever rule. [Copyright &y& Elsevier]

Cell-penetrating peptides (CPPs) are short membrane-permeating amino acid sequences that can be used to deliver cargoes, e.g. drugs, into cells. The mechanism for CPP internalization is still subject of ongoing research. An interesting family of CPPs is the sweet arrow peptides – SAP(E) – which are known to adopt a polyproline II helical secondary structure. SAP(E) peptides stand out among CPPs because they carry a net negative charge while most CPPs are positively charged, the latter being conducive to electrostatic interaction with generally negatively charged membranes. For SAP(E)s, an internalization mechanism has been proposed, based on polypeptide aggregation on the cell surface, followed by an endocytic uptake. However, this process has not yet been observed directly – since peptide–membrane interactions are inherently difficult to monitor on a molecular scale. Here, we use sum frequency generation (SFG) vibrational spectroscopy to investigate molecular interactions of SAP(E) with differently charged model membranes, in both mono- and bi-layer configurations. The data suggest that the initial binding mechanism is accompanied by structural changes of the peptide. Also, the peptide-model membrane interaction depends on the charge of the lipid headgroup with phosphocholine being a favorable binding site. Moreover, while direct penetration has also been observed for some CPPs, the spectroscopy reveals that for SAP(E), its interaction with model membranes remains limited to the headgroup region, and insertion into the hydrophobic core of the lipid layer does not occur. [ABSTRACT FROM AUTHOR]

Abstract: Many proteins of diverse sequence, structure and function self-assemble into morphologically similar fibrillar aggregates known as amyloids. Amyloids are remarkable polymers in several respects. First of all, amyloids can be formed from proteins with very different amino acid sequences; the common denominator is that the individual proteins constituting the amyloid fold predominantly into a β-sheet structure. Secondly, the formation of the fibril occurs through non-covalent interactions between primarily the β-sheets, causing the monomers to stack into fibrils. The fibrils are remarkably robust, considering that the monomers are bound non-covalently. Finally, a common characteristic of fibrils is their unbranched, straight, fiber-like structure arising from the intertwining of the multiple β-sheet filaments. These remarkably ordered and stable nanofibrils can be useful as building blocks for protein-based functional materials, but they are also implicated in severe neurodegenerative diseases. The overall aim of this article is to highlight recent efforts aimed at obtaining insights into amyloid proteins on different length scales. Starting from molecular information on amyloids, single fibril properties and mechanical properties of networks of fibrils are described. Specifically, we focus on the self-assembly of amyloid protein fibrils composed of peptides and denatured model proteins, as well as the influence of inhibitors of fibril formation. Additionally, we will demonstrate how the application of recently developed vibrational spectroscopic techniques has emerged as a powerful approach to gain spatially resolved information on the structure–function relation of amyloids. While spectroscopy provides information on local molecular conformations and protein secondary structure, information on the single fibril level has been developed by diverse microscopic techniques. The approaches to reveal basic mechanical properties of single fibrils like bending rigidity, shear modulus, ultimate tensile strength and fracture behavior are illustrated. Lastly, mechanics of networks of amyloid fibrils, typically forming viscoelastic gels are outlined, with a focus on (micro-) rheological properties. The resulting fundamental insights are essential for the rational design of novel edible and biodegradable protein-based polymers, but also to devise therapeutic strategies to combat amyloid assembly and accumulation during pathogenic disorders. [Copyright &y& Elsevier]

We present an experimental and theoretical study of sum-frequency generation of two infrared photons and one visible photon in a lithium iodate crystal. The sum-frequency light can be generated directly in a third-order process as well as in two subsequent second-order processes. By comparing experimental results to numerical and analytical calculations, we demonstrate that the three possible cascading processes, i.e. two second-order cascading processes with phase matching in either the first or the second step and a cascading process with overall phase matching with both steps equally mismatched, are comparable in intensity to the direct third-order process. Polarization and angular-dependent measurements are used to assign the different processes. By using a spectrally broad infrared pulse and a spectrally narrow visible pulse we investigate the possibility of using the spectrally resolved nonlinear signals as a tool to unravel their origin. For the cascading processes, phase matching of the first step results in a spectrally broad sum-frequency signal while overall and second step phase matching give rise to a narrow signal. [Copyright &y& Elsevier]

Elastomer-based nanocomposites, mixtures of elastomeric rubber with hard nanoparticles, generally exhibit strain stiffening under large strains while also showing strain softening at low strain during large cyclic deformations due to the Mullins Effect. Numerous studies have documented the Mullins effect over the last 50 years, and many models have been developed to explain (and predict) the Mullins effect as it is closely related to material failure. Even with this rich history, relatively few studies have connected experimentally measured changes in nanocomposite chain structure with nonlinear strain hardening and nanofiller dispersion. In this work, we quantify the strain hardening mechanics, and chain anisotropy in situ in silica-filled acrylonitrile butadiene rubber (NBR) nanocomposites under cyclic tensile loading. Macroscopic strain hardening and molecular chain anisotropy increased after the first loading cycle, as a strong Mullins effect appeared, for nanocomposites with sufficient filler volume fraction. In composites with low filler content, we observed little increase in strain hardening or chain anisotropy and a barely-visible Mullins effect. Our results provide strong experimental support that chain delamination from the filler surface is related to the Mullins effect in nanocomposite materials. Image 1 • In situ mechano-chemical (Raman) spectroscopy allows quantification of mechanics and chain anisotropy in nanocomposites. • Nanocomposites with sufficient fillers show strain hardening and chain anisotropy that increase after the first loading cycle. • Cyclic loading of silica/NBR nanocomposites with non-specific filler-matrix interaction does not affect filler dispersion. • Chain slippage from filler surfaces and subsequent alignment with cyclic loading coincides with the Mullins effect. [ABSTRACT FROM AUTHOR]