The Prenucleation Equilibrium of the Parathyroid Hormone Determines the Critical Aggregation Concentration and Amyloid Fibril Nucleation.

Nucleation and growth of amyloid fibrils were found to only occur in supersaturated solutions above a critical concentration (ccrit ). The biophysical meaning of ccrit remained mostly obscure, since typical low values of ccrit in the sub-µM range hamper investigations of potential oligomeric states and their structure. Here, we investigate the parathyroid hormone PTH84 as an example of a functional amyloid fibril forming peptide with a comparably high ccrit of 67±21 µM. We describe a complex concentration dependent prenucleation ensemble of oligomers of different sizes and secondary structure compositions and highlight the occurrence of a trimer and tetramer at ccrit as possible precursors for primary fibril nucleation. Furthermore, the soluble state found in equilibrium with fibrils adopts to the prenucleation state present at ccrit . Our study sheds light onto early events of amyloid formation directly related to the critical concentration and underlines oligomer formation as a key feature of fibril nucleation. Our results contribute to a deeper understanding of the determinants of supersaturated peptide solutions. In the current study we present a biophysical approach to investigate ccrit of amyloid fibril formation of PTH84 in terms of secondary structure, cluster size and residue resolved intermolecular interactions during oligomer formation. Throughout the investigated range of concentrations (1 µM to 500 µM) we found different states of oligomerization with varying ability to contribute to primary fibril nucleation and with a concentration dependent equilibrium. In this context, we identified the previously described ccrit of PTH84 to mark a minimum concentration for the formation of homo-trimers/tetramers. These investigations allowed us to characterize molecular interactions of various oligomeric states that are further converted into elongation competent fibril nuclei during the lag phase of a functional amyloid forming peptide.

Probing Polymer Chain Conformation and Fibril Formation of Peptide Conjugates.

Covalent conjugates between a synthetic polymer and a peptide hormone were used to probe the molecular extension of these macromolecules and how the polymer modifies the fibril formation of the hormone. NMR spectroscopy of 15 N labeled parathyroid hormone (PTH) was employed to visualize the conformation of the conjugated synthetic polymer, triggered by small temperature changes via its lower critical solution temperature. A shroud-like polymer conformation dominated the molecular architecture of the conjugated chimeras. PTH readily forms amyloid fibrils, which is probably the physiological storage form of the hormone. The polyacrylate based polymers stimulated the nucleation processes of the peptide.

Inhibition of Aß(1-40) fibril formation by cyclophilins.

Cyclophilins interact directly with the Alzheimer's disease peptide Aß (amyloid ß-peptide) and are therefore involved in the early stages of Alzheimer's disease. Aß binding to CypD (cyclophilin D) induces dysfunction of human mitochondria. We found that both CypD and CypA suppress in vitro fibril formation of Aß(1-40) at substoichiometric concentrations when present early in the aggregation process. The prototypic inhibitor CsA (cyclosporin A) of both cyclophilins as well as the new water-soluble MM258 derivative prevented this suppression. A SPOT peptide array approach and NMR titration experiments confirmed binding of Aß(1-40) to the catalytic site of CypD mainly via residues Lys(16)-Glu(22) The peptide Aß(16-20) representing this section showed submicromolar IC50 values for the peptidyl prolyl cis-trans isomerase activity of CypD and CypA and low-micromolar KD values in ITC experiments. Chemical cross-linking and NMR-detected hydrogen-deuterium exchange experiments revealed a shift in the populations of small Aß(1-40) oligomers towards the monomeric species, which we investigated in the present study as being the main process of prevention of Aß fibril formation by cyclophilins.

Structural characterization of amyloid fibrils from the human parathyroid hormone.

Amyloid deposits are common in various tissues as a consequence of misfolded proteins. However, secretory protein and peptides are often stored in membrane coated granules as functional amyloids. In this article, we present a detailed characterization of in vitro generated amyloid fibrils from human parathyroid hormone (hPTH(1-84)). Fully mature fibrils could be obtained after a short lag phase within less than one hour at 65°C. These fibrils showed all characteristic of a cross-ß structure. Protease cleavage combined with mass spectrometry identified the central region of the peptide hormone involved in the fibril core formation. EGCG, an inhibitor of amyloid fibril formation, showed binding to residues in the peptide monomers corresponding to the later fibril core and thus explaining the inhibition of the fibril growth. Conformational and dynamic studies by solid-state NMR further corroborated the cross-ß core of the fibrils, but also identified highly mobile segments with a random coil structure not belonging to the rigid fibril core.

A Detailed Analysis of the Morphology of Fibrils of Selectively Mutated Amyloid ß (1-40).

A small library of rationally designed amyloid ß [Aß(1-40)] peptide variants is generated, and the morphology of their fibrils is studied. In these molecules, the structurally important hydrophobic contact between phenylalanine 19 (F19) and leucine 34 (L34) is systematically mutated to introduce defined physical forces to act as specific internal constraints on amyloid formation. This Aß(1-40) peptide library is used to study the fibril morphology of these variants by employing a comprehensive set of biophysical techniques including solution and solid-state NMR spectroscopy, AFM, fluorescence correlation spectroscopy, and XRD. Overall, the findings demonstrate that the introduction of significant local physical perturbations of a crucial early folding contact of Aß(1-40) only results in minor alterations of the fibrillar morphology. The thermodynamically stable structure of mature Aß fibrils proves to be relatively robust against the introduction of significantly altered molecular interaction patterns due to point mutations. This underlines that amyloid fibril formation is a highly generic process in protein misfolding that results in the formation of the thermodynamically most stable cross-ß structure.

Enhanced Fibril Fragmentation of N-Terminally Truncated and Pyroglutamyl-Modified Aß Peptides.

N-terminal truncation and pyroglutamyl (pE) formation are naturally occurring chemical modifications of the Aß peptide in Alzheimer's disease. We show herein that these two modifications significantly reduce the fibril length and the transition midpoint of thermal unfolding of the fibrils, but they do not substantially perturb the fibrillary peptide conformation. This observation implies that the N terminus of the unmodified peptide protects Aß fibrils against mechanical stress and fragmentation and explains the high propensity of pE-modified peptides to form small and particularly toxic aggregates.

Molecular basis of ß-amyloid oligomer recognition with a conformational antibody fragment.

Oligomers are intermediates of the ß-amyloid (Aß) peptide fibrillogenic pathway and are putative pathogenic culprits in Alzheimer's disease (AD). Here we report the biotechnological generation and biochemical characterization of an oligomer-specific antibody fragment, KW1. KW1 not only discriminates between oligomers and other Aß conformations, such as fibrils or disaggregated peptide; it also differentiates between different types of Aß oligomers, such as those formed by Aß (1-40) and Aß (1-42) peptide. This high selectivity of binding contrasts sharply with many other conformational antibodies that interact with a large number of structurally analogous but sequentially different antigens. X-ray crystallography, NMR spectroscopy, and peptide array measurements imply that KW1 recognizes oligomers through a hydrophobic and significantly aromatic surface motif that includes Aß residues 18-20. KW1-positive oligomers occur in human AD brain samples and induce synaptic dysfunctions in living brain tissues. Bivalent KW1 potently neutralizes this effect and interferes with Aß assembly. By altering a specific step of the fibrillogenic cascade, it prevents the formation of mature Aß fibrils and induces the accumulation of nonfibrillar aggregates. Our data illuminate significant mechanistic differences in oligomeric and fibril recognition and suggest the considerable potential of KW1 in future studies to detect or inhibit specific types of Aß conformers.

Listen Carefully protocol: an exploratory case-control study of the association between listening effort and cognitive function.

INTRODUCTION: A growing body of evidence suggests that hearing loss is a significant and potentially modifiable risk factor for cognitive impairment. Although the mechanisms underlying the associations between cognitive decline and hearing loss are unclear, listening effort has been posited as one of the mechanisms involved with cognitive decline in older age. To date, there has been a lack of research investigating this association, particularly among adults with mild cognitive impairment (MCI). METHODS AND ANALYSIS: 15-25 cognitively healthy participants and 15-25 patients with MCI (age 40-85 years) will be recruited to participate in an exploratory study investigating the association between cognitive functioning and listening effort. Both behavioural and objective measures of listening effort will be investigated. The sentence-final word identification and recall (SWIR) test will be administered with single talker non-intelligible speech background noise while monitoring pupil dilation. Evaluation of cognitive function will be carried out in a clinical setting using a battery of neuropsychological tests. This study is considered exploratory and proof of concept, with information taken to help decide the validity of larger-scale trials. ETHICS AND DISSEMINATION: Written approval exemption was obtained by the Scientific Ethics Committee in the central region of Denmark (De Videnskabsetiske Komiteer i Region Hovedstaden), reference 19042404, and the project is registered pre-results at clinicaltrials.gov, reference NCT04593290, Protocol ID 19042404. Study results will be disseminated in peer-reviewed journals and conferences.

Towards Auditory Profile-Based Hearing-Aid Fittings: BEAR Rationale and Clinical Implementation.

(1) Background: To improve hearing-aid rehabilitation, the Danish 'Better hEAring Rehabilitation' (BEAR) project recently developed methods for individual hearing loss characterization and hearing-aid fitting. Four auditory profiles differing in terms of audiometric hearing loss and supra-threshold hearing abilities were identified. To enable auditory profile-based hearing-aid treatment, a fitting rationale leveraging differences in gain prescription and signal-to-noise (SNR) improvement was developed. This report describes the translation of this rationale to clinical devices supplied by three industrial partners. (2) Methods: Regarding the SNR improvement, advanced feature settings were proposed and verified based on free-field measurements made with an acoustic mannikin fitted with the different hearing aids. Regarding the gain prescription, a clinically feasible fitting tool and procedure based on real-ear gain adjustments were developed. (3) Results: Analyses of the collected real-ear gain and SNR improvement data confirmed the feasibility of the clinical implementation. Differences between the auditory profile-based fitting strategy and a current 'best practice' procedure based on the NAL-NL2 fitting rule were verified and are discussed in terms of limitations and future perspectives. (4) Conclusion: Based on a joint effort from academic and industrial partners, the BEAR fitting rationale was transferred to commercially available hearing aids.

The Effect of Heated CO2 Insufflation in Minimising Surgical Wound Contamination During Open Surgery.

The primary source of infections in open surgeries has been found to be bacteria and viruses carried into the surgical wound on the surfaces of skin particles shed by patients and surgical staff. In open cardiac surgeries, insufflation of the wound with carbon dioxide is used to limit the quantity of air able to enter into the heart, avoiding air embolisms when the heart is restarted. This surgical technique has been evaluated as a method of limiting the number of skin particles able to enter into the wound, using computational fluid dynamics (CFD) simulations and experimental testing. Spherical particles of 5.0 and 13.5 µm in diameter were used to simulate skin particles falling above a wound, travelling in air ventilation velocities of either 0.2 or 0.4 m/s, and with or without CO2 insufflation. The CFD simulations with CO2 included a diffuser placed in the wound and supplied with CO2 at a rate of 10 L/min. Experimental testing was completed under similar conditions. The results of CFD simulations and experimental testing showed CO2 insufflation can significantly limit the number of particles able to enter into the wound.

Small Molecule Inhibited Parathyroid Hormone Mediated cAMP Response by N-Terminal Peptide Binding.

Ligand binding to certain classes of G protein coupled receptors (GPCRs) stimulates the rapid synthesis of cAMP through G protein. Human parathyroid hormone (PTH), a member of class B GPCRs, binds to its receptor via its N-terminal domain, thereby activating the pathway to this secondary messenger inside cells. Presently, GPCRs are the target of many pharmaceuticals however, these drugs target only a small fraction of structurally known GPCRs (about 10%). Coordination complexes are gaining interest due to their wide applications in the medicinal field. In the present studies we explored the potential of a coordination complex of Zn(II) and anthracenyl-terpyridine as a modulator of the parathyroid hormone response. Preferential interactions at the N-terminal domain of the peptide hormone were manifested by suppressed cAMP generation inside the cells. These observations contribute a regulatory component to the current GPCR-cAMP paradigm, where not the receptor itself, but the activating hormone is a target. To our knowledge, this is the first report about a coordination complex modulating GPCR activity at the level of deactivating its agonist. Developing such molecules might help in the control of pathogenic PTH function such as hyperparathyroidism, where control of excess hormonal activity is essentially required.

Molecular architecture of Aß fibrils grown in cerebrospinal fluid solution and in a cell culture model of Aß plaque formation.

OBJECTIVES: The detailed structure of brain-derived Aß amyloid fibrils is unknown. To approach this issue, we investigate the molecular architecture of Aß(1-40) fibrils grown in either human cerebrospinal fluid solution, in chemically simple phosphate buffer in vitro or extracted from a cell culture model of Aß amyloid plaque formation. METHODS: We have used hydrogen-deuterium exchange (HX) combined with nuclear magnetic resonance, transmission electron microscopy, seeding experiments both in vitro and in cell culture as well as several other spectroscopic measurements to compare the morphology and residue-specific conformation of these different Aß fibrils. RESULTS AND CONCLUSIONS: Our data reveal that, despite considerable variations in morphology, the spectroscopic properties and the pattern of slowly exchanging backbone amides are closely similar in the fibrils investigated. This finding implies that a fundamentally conserved molecular architecture of Aß peptide fold is common to Aß fibrils.