Extracellular chaperones and proteostasis.

There exists a family of currently untreatable, serious human diseases that arise from the inappropriate misfolding and aggregation of extracellular proteins. At present our understanding of mechanisms that operate to maintain proteostasis in extracellular body fluids is limited, but it has significantly advanced with the discovery of a small but growing family of constitutively secreted extracellular chaperones. The available evidence strongly suggests that these chaperones act as both sensors and disposal mediators of misfolded proteins in extracellular fluids, thereby normally protecting us from disease pathologies. It is critically important to further increase our understanding of the mechanisms that operate to effect extracellular proteostasis, as this is essential knowledge upon which to base the development of effective therapies for some of the world's most debilitating, costly, and intractable diseases.

The Dual Roles of Clusterin in Extracellular and Intracellular Proteostasis.

Clusterin (CLU) was the first reported secreted mammalian chaperone and impacts on serious diseases associated with inappropriate extracellular protein aggregation. Many studies have described intracellular CLU in locations outside the secretory system and recent work has shown that CLU can be released into the cytosol during cell stress. In this article, we critically evaluate evidence relevant to the proposed origins of cellular CLU found outside the secretory system, and advance the hypothesis that the cytosolic release of CLU induced by stress serves to facilitate the trafficking of misfolded proteins to the proteasome and autophagy for degradation. We also propose future research directions that could help establish CLU as a unique chaperone performing critical and synergic roles in both intracellular and extracellular proteostasis.

Unravelling Guest Dynamics in Crystalline Molecular Organics Using 2H Solid-State NMR and Molecular Dynamics Simulation.

2H solid-state NMR and atomistic molecular dynamics (MD) simulations are used to understand the disorder of guest solvent molecules in two cocrystal solvates of the pharmaceutical furosemide. Traditional approaches to interpreting the NMR data fail to provide a coherent model of molecular behavior and indeed give misleading kinetic data. In contrast, the direct prediction of the NMR properties from MD simulation trajectories allows the NMR data to be correctly interpreted in terms of combined jump-type and libration-type motions. Time-independent component analysis of the MD trajectories provides additional insights, particularly for motions that are invisible to NMR. This allows a coherent picture of the dynamics of molecules restricted in molecular-sized cavities to be determined.

Computational predictions of interfacial tension, surface tension, and surfactant adsorption isotherms.

All-atom (AA) molecular dynamics (MD) simulations are employed to predict interfacial tensions (IFT) and surface tensions (ST) of both ionic and non-ionic surfactants. The general AMBER force field (GAFF) and variants are examined in terms of their performance in predicting accurate IFT/ST, γ, values for chosen water models, together with the hydration free energy, ΔGhyd, and density, ρ, predictions for organic bulk phases. A strong correlation is observed between the quality of ρ and γ predictions. Based on the results, the GAFF-LIPID force field, which provides improved ρ predictions is selected for simulating surfactant tail groups. Good γ predictions are obtained with GAFF/GAFF-LIPID parameters and the TIP3P water model for IFT simulations at a water-triolein interface, and for GAFF/GAFF-LIPID parameters together with the OPC4 water model for ST simulations at a water-vacuum interface. Using a combined molecular dynamics-molecular thermodynamics theory (MD-MTT) framework, a mole fraction of C12E6 molecule of 1.477 × 10-6 (from the experimental critical micelle concentration, CMC) gives a simulated surface excess concentration, ΓMAX, of 76 C12E6 molecules at a 36 nm2 water-vacuum surface (3.5 × 10-10 mol cm-2), which corresponds to a simulated ST of 35 mN m-1. The results compare favourably with an experimental ΓMAX of C12E6 of 3.7 × 10-10 mol cm-2 (80 surfactants for a 36 nm2 surface) and experimental ST of C12E6 of 32 mN m-1 at the CMC.

Clusterin Neutralizes the Inflammatory and Cytotoxic Properties of Extracellular Histones in Sepsis.

Rationale: Extracellular histones, released into the surrounding environment during extensive cell death, promote inflammation and cell death, and these deleterious roles have been well documented in sepsis. Clusterin (CLU) is a ubiquitous extracellular protein that chaperones misfolded proteins and promotes their removal. Objectives: We investigated whether CLU could protect against the deleterious properties of histones. Methods: We assessed CLU and histone expression in patients with sepsis and evaluated the protective role of CLU against histones in in vitro assays and in vivo models of experimental sepsis. Measurements and Main Results: We show that CLU binds to circulating histones and reduces their inflammatory, thrombotic, and cytotoxic properties. We observed that plasma CLU levels decreased in patients with sepsis and that the decrease was greater and more durable in nonsurvivors than in survivors. Accordingly, CLU deficiency was associated with increased mortality in mouse models of sepsis and endotoxemia. Finally, CLU supplementation improved mouse survival in a sepsis model. Conclusions: This study identifies CLU as a central endogenous histone-neutralizing molecule and suggests that, in pathologies with extensive cell death, CLU supplementation may improve disease tolerance and host survival.

A many-body dissipative particle dynamics parametrisation scheme to study behaviour at air-water interfaces.

In this article, we present a general parametrisation scheme for many-body dissipative particle dynamics (MDPD). The scheme is based on matching model components to experimental surface tensions and chemical potentials. This allows us to obtain the correct surface and mixing behaviours of complex, multicomponent systems. The methodology is tested by modelling the behaviour of nonionic polyoxyethylene alkyl ether surfactants at an air/water interface. In particular, the influence of the number of ethylene oxide units in the surfactant head group is investigated. We find good agreement with many experimentally obtained parameters, such as minimum surface area per molecule; and a decrease in the surface tension with increasing surfactant surface density. Moreover, we observe an orientational transition, from surfactants lying directly on the water surface at low surface coverage, to surfactants lying parallel or tilted with respect to the surface normal at high surface coverage. The parametrisation scheme is also extended to cover the zwitterionic surfactant lauryldimethylamine oxide (LDAO), where we provide good predictions for the surface tension at maximum surface coverage. Here, if we exceed this coverage, we are able to demonstrate the spontaneous production of micelles from the surface surfactant layer.

Investigating anionic surfactant phase diagrams using dissipative particle dynamics: development of a transferable model.

Dissipative particle dynamics (DPD) provides a powerful coarse-grained simulation technique for the study of a wide range of soft matter systems. Here, we investigate the transferability of DPD models to the prediction of anionic surfactant phase diagrams, taking advantage of fast parameter sweeps to optimise the choice of DPD parameters for these systems. Parameters are developed which provide a good representation of the phase diagrams of SDS (sodium dodecyl sulfate) and three different isomeric forms of LAS (linear alkylbenzene sulfonates) across an extensive concentration range. A high degree of transferability is seen, with parameters readily transferable to other systems, such as AES (alkyl ether sulfates). Excellent agreement is obtained with experimentally measured quantities, such as the lamellar layer spacing. Isosurfaces are produced from the surfactant head group, from which the second moment M of the isosurface normal distribution is calculated for different phase structures. Lyotropic liquid crystalline phases are characterised by a combination of the eigenvalues of M, radial distribution functions, and visual inspections.

The perceived control model of falling: developing a unified framework to understand and assess maladaptive fear of falling.

BACKGROUND: fear of falling is common in older adults and can have a profound influence on a variety of behaviours that increase fall risk. However, fear of falling can also have potentially positive outcomes for certain individuals. Without progressing our understanding of mechanisms underlying these contrasting outcomes, it is difficult to clinically manage fear of falling. METHODS: this paper first summarises recent findings on the topic of fear of falling, balance and fall risk-including work highlighting the protective effects of fear. Specific focus is placed on describing how fear of falling influences perceptual, cognitive and motor process in ways that might either increase or reduce fall risk. Finally, it reports the development and validation of a new clinical tool that can be used to assess the maladaptive components of fear of falling. RESULTS: we present a new conceptual framework-the Perceived Control Model of Falling-that describes specific mechanisms through which fear of falling can influence fall risk. The key conceptual advance is the identification of perceived control over situations that threaten one's balance as the crucial factor mediating the relationship between fear and increased fall risk. The new 4-item scale that we develop-the Updated Perceived Control over Falling Scale (UP-COF)-is a valid and reliable tool to clinically assess perceived control. CONCLUSION: this new conceptualisation and tool (UP-COF) allows clinicians to identify individuals for whom fear of falling is likely to increase fall risk, and target specific underlying maladaptive processes such as low perceived control.

Recombinant Human Clusterin Seals Damage to the Ocular Surface Barrier in a Mouse Model of Ophthalmic Preservative-Induced Epitheliopathy.

There is a significant unmet need for therapeutics to treat ocular surface barrier damage, also called epitheliopathy, due to dry eye and related diseases. We recently reported that the natural tear glycoprotein CLU (clusterin), a molecular chaperone and matrix metalloproteinase inhibitor, seals and heals epitheliopathy in mice subjected to desiccating stress in a model of aqueous-deficient/evaporative dry eye. Here we investigated CLU sealing using a second model with features of ophthalmic preservative-induced dry eye. The ocular surface was stressed by topical application of the ophthalmic preservative benzalkonium chloride (BAC). Then eyes were treated with CLU and sealing was evaluated immediately by quantification of clinical dye uptake. A commercial recombinant form of human CLU (rhCLU), as well as an rhCLU form produced in our laboratory, designed to be compatible with U.S. Food and Drug Administration guidelines on current Good Manufacturing Practices (cGMP), were as effective as natural plasma-derived human CLU (pCLU) in sealing the damaged ocular surface barrier. In contrast, two other proteins found in tears: TIMP1 and LCN1 (tear lipocalin), exhibited no sealing activity. The efficacy and selectivity of rhCLU for sealing of the damaged ocular surface epithelial barrier suggests that it could be of therapeutic value in treating BAC-induced epitheliopathy and related diseases.

N-Terminal Acetylation of α-Synuclein Slows down Its Aggregation Process and Alters the Morphology of the Resulting Aggregates.

Parkinson's disease is associated with the aberrant aggregation of α-synuclein. Although the causes of this process are still unclear, post-translational modifications of α-synuclein are likely to play a modulatory role. Since α-synuclein is constitutively N-terminally acetylated, we investigated how this post-translational modification alters the aggregation behavior of this protein. By applying a three-pronged aggregation kinetics approach, we observed that N-terminal acetylation results in a reduced rate of lipid-induced aggregation and slows down both elongation and fibril-catalyzed aggregate proliferation. An analysis of the amyloid fibrils produced by the aggregation process revealed different morphologies for the acetylated and non-acetylated forms in both lipid-induced aggregation and seed-induced aggregation assays. In addition, we found that fibrils formed by acetylated α-synuclein exhibit a lower ß-sheet content. These findings indicate that N-terminal acetylation of α-synuclein alters its lipid-dependent aggregation behavior, reduces its rate of in vitro aggregation, and affects the structural properties of its fibrillar aggregates.

Identifying new molecular players in extracellular proteostasis.

Proteostasis refers to a delicately tuned balance between the processes of protein synthesis, folding, localization, and the degradation of proteins found inside and outside cells. Our understanding of extracellular proteostasis is rather limited and largely restricted to knowledge of 11 currently established extracellular chaperones (ECs). This review will briefly outline what is known of the established ECs, before moving on to discuss experimental strategies used to identify new members of this growing family, and an examination of a group of putative new ECs identified using one of these approaches. An observation that emerges from an analysis of the expanding number of ECs is that all of these proteins are multifunctional. Strikingly, the armory of activities each possess uniquely suit them as a group to act together at sites of tissue damage, infection, and inflammation to restore homeostasis. Lastly, we highlight outstanding questions to guide future research in this field.

Protective or harmful? A qualitative exploration of older people's perceptions of worries about falling.

BACKGROUND: worries about falling are common in older people. It has been suggested that these worries can reduce balance safety by acting as a distracting dual-task. However, it is also possible that worries may serve a protective purpose. The present work adopted a qualitative approach to conduct an in-depth exploration of older people's experiences of worries about falling. METHODS: semi-structured interviews were conducted with 17 community-dwelling older people (mean age = 79 years; males = 5/17) who reported experiencing worries about falling. Reflexive thematic analysis was used to analyse the data. RESULTS: experiencing a fall-or otherwise recognising one's balance limitations-brought the physical realities of participants' ageing bodies to the forefront of their awareness. This led to the recognition of their susceptibility for an injurious fall, which triggered worries about falling in situations that threatened their balance. When preventing the subject of their worries (i.e. an injurious fall) was perceived to be within the individual's locus of control, worries led to protective adaptations to behaviour. In contrast, when the subject of their worries was perceived to be outside their control, worries triggered feelings of panic-leading to unhelpful changes in behaviour. CONCLUSION: these findings provide novel insight into the development and consequences of worries about falling in older people. They highlight the importance of considering an individual's perception of control before deciding to clinically intervene to reduce worries about falling.

Human pregnancy zone protein stabilizes misfolded proteins including preeclampsia- and Alzheimer's-associated amyloid beta peptide.

Protein misfolding underlies the pathology of a large number of human disorders, many of which are age-related. An exception to this is preeclampsia, a leading cause of pregnancy-associated morbidity and mortality in which misfolded proteins accumulate in body fluids and the placenta. We demonstrate that pregnancy zone protein (PZP), which is dramatically elevated in maternal plasma during pregnancy, efficiently inhibits in vitro the aggregation of misfolded proteins, including the amyloid beta peptide (Aß) that is implicated in preeclampsia as well as with Alzheimer's disease. The mechanism by which this inhibition occurs involves the formation of stable complexes between PZP and monomeric Aß or small soluble Aß oligomers formed early in the aggregation pathway. The chaperone activity of PZP is more efficient than that of the closely related protein alpha-2-macroglobulin (α2M), although the chaperone activity of α2M is enhanced by inducing its dissociation into PZP-like dimers. By immunohistochemistry analysis, PZP is found primarily in extravillous trophoblasts in the placenta. In severe preeclampsia, PZP-positive extravillous trophoblasts are adjacent to extracellular plaques containing Aß, but PZP is not abundant within extracellular plaques. Our data support the conclusion that the up-regulation of PZP during pregnancy represents a major maternal adaptation that helps to maintain extracellular proteostasis during gestation in humans. We propose that overwhelming or disrupting the chaperone function of PZP could underlie the accumulation of misfolded proteins in vivo. Attempts to characterize extracellular proteostasis in pregnancy will potentially have broad-reaching significance for understanding disease-related protein misfolding.

Atomistic simulation studies of ionic cyanine dyes: self-assembly and aggregate formation in aqueous solution.

Cyanine dyes are known to form large-scale aggregates of various morphologies via spontaneous self-assembly in aqueous solution, akin to chromonic liquid crystals. Atomistic molecular dynamics simulations have been performed on four cyanine dyes: pseudoisocyanine chloride (PIC), pinacyanol chloride (PCYN), 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolylcarbocyanine chloride (TTBC) and 1,1'-disulfopropyl-3,3'-diethyl-5,5',6,6'-tetrachloro-benzimidazolylcarbocyanine sodium salt (BIC). Simulations employed an optimised general AMBER force field and demonstrate the organisation of the dyes into stacked structures at dilute concentrations. The thermodynamics of self-assembly was studied by calculating potentials of mean force for n-mers (n = 2, 3 or 4), from which the free energies of association are determined. We report binding free energies in the range of 8 to 15kBT for dimerisation, concordant with typical values for ionic chromonics (7 to 14kBT), and examine the enthalpic and entropic contributions to the aggregation process. The self-assembly of these dyes yields two distinct classes of structures. We observe the formation of H-aggregate stacks for PCYN, with further complexity in these assemblies for PIC; where the aggregates contain shift and Y junction defects. TTBC and BIC associate into a J-aggregate sheet structure of unimolecular thickness, and is composed of a brickwork arrangement between molecules. These sheet structures are characteristic of the smectic chromonic mesophase, and such assemblies provide a route to the emergence of nanoscale tubular architectures.

A multidisciplinary investigation into "playing-up" in academy football according to age phase.

In an attempt to facilitate more appropriate levels of challenge, a common practice in academy football is to play-up talented youth players with chronologically older peers. However, the context of playing-up in academy football is yet to be empirically explored. Thus, the purpose of this study was to examine the multidimensional factors that differentiated players who play-up from those who do not. Ninety-eight participants from a single football academy were examined within their age phase: Foundation Development Phase (FDP; under-9 to under-11; n = 40) and Youth Development Phase (YDP; under-12 to under-16; n = 58). Drawing upon the FA Four Corner Model, 27 factors relating to Technical/Tactical, Physical, Psychological, and Social development were assessed. Following MANOVA analysis within both the FDP and YDP, significant differences were observed for Technical/Tactical and Social sub-components (P < 0.05). Further differences were observed for Physical and Psychological sub-components (P < 0.05) within the YDP. In sum, Technical/Tactical and Social characteristics appeared to differentiate those who play-up compared to those who do not within the FDP. In the YDP however, there were measures representing all sub-components from the FA Four Corner Model. Subsequently, it is suggested coaches and practitioners consider these holistic factors when playing-up youth football players within relevant age-phases.

Neurophysiological evidence of how quiet eye supports motor performance.

Prolonged quiet eye (QE) duration is associated with greater performance in various types of targeting and interceptive tasks. However, the mechanism by which QE affects performance remains debatable. This study aimed to test the validity of the pre-programming and online control hypotheses using electromyography (EMG), electrooculography (EOG) and electroencephalography (EEG) during a golf putting task. Twenty-one college students were recruited for this study. Each participant performed 100 golf putting trials during which the putting performance, EMG, EOG, and EEG signals were recorded. The QE duration including the pre- and post-movement initiation components, and movement-related cortical potentials (MRCPs) were analyzed off-line. We found that successful putts were associated with longer QEtotal (the total QE duration from QE onset to QE offset), QEpre (QE occurring before movement initiation), and QEpost (QE occurring after movement initiation) durations than failed putts. Greater cortical activation in the MRCPs was observed within the prefrontal, premotor, and parietal cortices during successful putts compared with failed putts. These findings suggest that QE serves both pre-programming and online control roles in supporting golf putting performance.

Missing Link between Helical Nano- and Microfilaments in B4 Phase Bent-Core Liquid Crystals, and Deciphering which Chiral Center Controls the Filament Handedness.

The range of possible morphologies for bent-core B4 phase liquid crystals has recently expanded from helical nanofilaments (HNFs) and modulated HNFs to dual modulated HNFs, helical microfilaments, and heliconical-layered nanocylinders. These new morphologies are observed when one or both aliphatic side chains contain a chiral center. Here, the following questions are addressed: which of these two chiral centers controls the handedness (helicity) and which morphology of the nanofilaments is formed by bent-core liquid crystals with tris-biphenyl diester core flanked by two chiral 2-octyloxy side chains? The combined results reveal that the longer arm of these nonsymmetric bent-core liquid crystals controls the handedness of the resulting dual modulated HNFs. These derivatives with opposite configuration of the two chiral side chains now feature twice as large dimensions compared to the homochiral derivatives with identical configuration. These results are supported by density functional theory calculations and stochastic dynamic atomistic simulations, which reveal that the relative difference between the para- and meta-sides of the described series of compounds drives the variation in morphology. Finally, X-ray diffraction, scanning electron microscopy (SEM), transmission electron microscopy (TEM), and atomic force microscopy (AFM) data also uncover the new morphology for B4 phases featuring p2/m symmetry within the filaments and less pronounced crystalline character.

Vitellogenin offsets oxidative costs of reproduction in female painted dragon lizards.

Vitellogenesis ('yolking' of follicles) is a bioenergetically costly stage of reproduction requiring enlargement of the liver to produce vitellogenin (VTG) yolk precursor proteins, which are transported and deposited at the ovary. VTG may, however, serve non-nutritive anti-oxidant functions, a hypothesis supported by empirical work on aging and other life-history transitions in several taxa. We test this hypothesis in female painted dragon lizards (Ctenophorus pictus) by examining covariation in VTG with the ovarian cycle, and relative to reactive oxygen species (ROS) including baseline superoxide (bSO). Plasma VTG decreased prior to ovulation, when VTG is deposited into follicles. VTG, however, remained elevated post-ovulation when no longer necessary for yolk provisioning and was unrelated to reproductive investment. Instead, VTG was strongly and positively predicted by prior bSO. ROS, in turn, was negatively predicted by prior VTG, while simultaneously sampled VTG was a positive predictor. These findings are consistent with the hypothesis that VTG functions as an anti-oxidant to counteract oxidative stress associated with vitellogenesis. The relationship between bSO and VTG was strongest in post-ovulatory females, indicating that its function may be largely anti-oxidant at this time. In conclusion, VTG may be under selection to offset oxidative costs of reproduction in egg-producing species.

Self-assembly and mesophase formation in a non-ionic chromonic liquid crystal: insights from bottom-up and top-down coarse-grained simulation models.

New coarse-grained models are introduced for a non-ionic chromonic molecule, TP6EO2M, in aqueous solution. The multiscale coarse-graining (MS-CG) approach is used, in the form of hybrid force matching (HFM), to produce a bottom-up CG model that demonstrates self-assembly in water and the formation of a chromonic stack. However, the high strength of binding in stacks is found to limit the transferability of the HFM model at higher concentrations. The MARTINI 3 framework is also tested. Here, a top-down CG model is produced which shows self-assembly in solution in good agreement with atomistic studies and transfers well to higher concentrations, allowing the full phase diagram of TP6EO2M to be studied. At high concentration, both self-assembly of molecules into chromonic stacks and self-organisation of stacks into mesophases occurs, with the formation of nematic (N) and hexagonal (M) chromonic phases. This CG-framework is suggested as a suitable way of studying a range of chromonic-type drug and dye molecules that exhibit complex self-assembly and solubility behaviour in solution.

Spinal motor neuron protein supersaturation patterns are associated with inclusion body formation in ALS.

Amyotrophic lateral sclerosis (ALS) is a heterogeneous degenerative motor neuron disease linked to numerous genetic mutations in apparently unrelated proteins. These proteins, including SOD1, TDP-43, and FUS, are highly aggregation-prone and form a variety of intracellular inclusion bodies that are characteristic of different neuropathological subtypes of the disease. Contained within these inclusions are a variety of proteins that do not share obvious characteristics other than coaggregation. However, recent evidence from other neurodegenerative disorders suggests that disease-affected biochemical pathways can be characterized by the presence of proteins that are supersaturated, with cellular concentrations significantly greater than their solubilities. Here, we show that the proteins that form inclusions of mutant SOD1, TDP-43, and FUS are not merely a subset of the native interaction partners of these three proteins, which are themselves supersaturated. To explain the presence of coaggregating proteins in inclusions in the brain and spinal cord, we observe that they have an average supersaturation even greater than the average supersaturation of the native interaction partners in motor neurons, but not when scores are generated from an average of other human tissues. These results suggest that inclusion bodies in various forms of ALS result from a set of proteins that are metastable in motor neurons, and thus prone to aggregation upon a disease-related progressive collapse of protein homeostasis in this specific setting.