Evaluation of Dynamic Structural Disorders in the Upper Airways and Applied Rein Tension in Healthy Dressage Horses During Riding in Different Gaits and Head-Neck Positions.

Flexion of the horse's head and neck during dressage riding reduces the pharyngeal lumen with the risk of increased upper airway resistance and upper airway obstructions. According to the Fédération Equestre Internationale, hyperflexion is achieved through force, whereas the position low-deep-round is nonforced. The objectives of this study were to evaluate (1) applied rein tension and (2) dynamic structural disorders in the upper airways in dressage horses in different gaits and different head-neck positions (HNPs). Overground endoscopy (OGE) and rein tension were evaluated in 13 clinically healthy and high-performance Warmblood dressage horses while being ridden in a standardized program comprised of four different gaits (halt, walk, trot, and canter) and in four HNPs (unrestrained, competition frame, hyperflexion, and low-deep-round). All included horses were able to achieve the desired HNPs. The HNP low-deep-round showed significantly lower rein tension than competition frame (P < .001) and hyperflexion (P < .001). An association was found between dynamic structural disorders in the upper airway tract evaluated by OGE and head-neck flexion, but this association was not linked to the degree of flexion. The HNP hyperflexion was neither associated with greater rein tension nor severe dynamic structural disorders than the HNP competition frame. This study confirms that low-deep-round is a nonforced position, in contrast to hyperflexion. Further studies are needed to evaluate whether dynamic structural disorders are a result of flexion or if the degree of flexion has an impact.

Direct Correlation Between Ligand-Induced α-Synuclein Oligomers and Amyloid-like Fibril Growth.

Aggregation of proteins into amyloid deposits is the hallmark of several neurodegenerative diseases such as Alzheimer's and Parkinson's disease. The suggestion that intermediate oligomeric species may be cytotoxic has led to intensified investigations of pre-fibrillar oligomers, which are complicated by their transient nature and low population. Here we investigate alpha-synuclein oligomers, enriched by a 2-pyridone molecule (FN075), and the conversion of oligomers into fibrils. As probed by leakage assays, the FN075 induced oligomers potently disrupt vesicles in vitro, suggesting a potential link to disease related degenerative activity. Fibrils formed in the presence and absence of FN075 are indistinguishable on microscopic and macroscopic levels. Using small angle X-ray scattering, we reveal that FN075 induced oligomers are similar, but not identical, to oligomers previously observed during alpha-synuclein fibrillation. Since the levels of FN075 induced oligomers correlate with the amounts of fibrils among different FN075:protein ratios, the oligomers appear to be on-pathway and modeling supports an 'oligomer stacking model' for alpha-synuclein fibril elongation.

Small angle X-ray scattering-based elucidation of the self-association mechanism of human insulin analogue lys(B29)(N(ε)ω-carboxyheptadecanoyl) des(B30).

Lys(B29)(N(ε)ω-carboxyheptadecanoyl) des(B30) human insulin is an insulin analogue belonging to a class of analogues designed to form soluble depots in subcutis by self-association, aiming at a protracted action. On the basis of small angle X-ray scattering (SAXS) supplemented by a range of biophysical and structural methods (field flow fractionation, dynamic and multiangle light scattering, circular dichroism, size exclusion chromatography, and crystallography), we propose a mechanism for the self-association expected to occur upon subcutaneous injection of this insulin analogue. SAXS data provide evidence of the in solution structure of the self-associated oligomer, which is a long straight rod composed of "tense" state insulin hexamers (T(6)-hexamers) as the smallest repeating unit. The smallest oligomer building block in the process is a T(6)T(6)-dihexamer. This tense dihexamer is formed by the allosteric change of the initial equilibrium between a proposed "relaxed" state R(6)-hexamer and an R(3)T(3)T(3)R(3)-dihexamer. The allosteric change from relaxed to tense is triggered by removal of phenol, mimicking subcutaneous injection. The data hence provide the first unequivocal evidence of the mechanism of self-association for this type of insulin analogue.

Time-resolved SAXS measurements facilitated by online HPLC buffer exchange.

Small-angle X-ray scattering (SAXS) is a powerful technique to structurally characterize biological macromolecules in solution. Heterogeneous solutions are inherently challenging to study. However, since SAXS data from ideal solutions are additive, with careful computational analysis it may be possible to separate contributions from individual species present in solution. Hence, time-resolved SAXS (TR-SAXS) data of processes in development can be analyzed. Many reported TR-SAXS results are initialized by a sudden change in buffer conditions facilitated by rapid mixing combined with either continuous or stopped flow. In this paper a method for obtaining TR-SAXS data from systems where the reaction is triggered by removal of a species is presented. This method is based on fast buffer exchange over a short desalting column facilitated by an online HPLC (high-performance liquid chromatography) connected to the SAXS sample cell. The sample is stopped in the sample cell and the evolving reaction is followed. In this specific system the removal of phenol initiates a self-association process of long-acting insulin analogues. For this experiment, data were collected in time series while varying concentrations. The method can be generally applied to other systems where removal of a species or other changes in experimental conditions trigger a process.

Small angle X-ray scattering study of calreticulin reveals conformational plasticity.

Calreticulin plays a central role in vital cell processes such as protein folding, Ca(2+) homeostasis and immunogenicity. Even so, only limited three-dimensional structural information is presently available. We present a series of Small-Angle X-ray Scattering data on human placenta calreticulin. The data from the calreticulin monomer reveal the shape of calreticulin in solution: The previously structurally un-described C-terminal is seen as a globular domain, and the P-domain beta-hairpin extends from the N-domain in a spiral like conformation. In the calreticulin solution dimer, the N-, C-, and P-domains are easily identified, and the P-domain is in an extended conformation connecting to the second calreticulin molecule. The SAXS solution data enables the construction of a medium-resolution model of calreticulin. In the light of the unresolved chaperone mechanism of calreticulin and calnexin, we discuss the functional consequences of the conformational plasticity of the calreticulin P-domain.