H-1, N-15 and C-13 assignment of the amyloidogenic protein medin using fast-pulsing NMR techniques

Thirty-one proteins are known to form extracellular fibrillar amyloid in humans. Molecular information about many of these proteins in their monomeric, intermediate or fibrillar form and how they aggregate and interact to form the insoluble fibrils is sparse. This is because amyloid proteins are notoriously difficult to study in their soluble forms, due to their inherent propensity to aggregate. Using recent developments in fast NMR techniques, band-selective excitation short transient and band-selective optimized flip-angle short-transient heteronuclear multiple quantum coherence we have been able to assign a 5 kDa full-length amyloidogenic protein called medin. Medin is the key protein component of the most common form of localised amyloid with a proposed role in aortic aneurysm and dissection. This assignment will now enable the study of the early interactions that could influence initiation and progression of medin aggregation. The chemical shifts have been deposited in the BioMagRes-Bank accession Nos. 25399 and 26576

19F labelled glycosaminoglycan probes for solution NMR and non-linear (CARS) microscopy

Studying polysaccharide-protein interactions under physiological conditions by conventional techniques is challenging. Ideally, macromolecules could be followed by both in vitro spectroscopy experiments as well as in tissues using microscopy, to enable a proper comparison of results over these different scales but, often, this is not feasible. The cell surface and extracellular matrix polysaccharides, glycosaminoglycans (GAGs) lack groups that can be detected selectively in the biological milieu. The introduction of 19F labels into GAG polysaccharides is explored and the interaction of a labelled GAG with the heparin-binding protein, antithrombin, employing 19F NMR spectroscopy is followed. Furthermore, the ability of 19F labelled GAGs to be imaged using CARS microscopy is demonstrated. 19F labelled GAGs enable both 19F NMR protein-GAG binding studies in solution at the molecular level and non-linear microscopy at a microscopic scale to be conducted on the same material, essentially free of background signals

Photoionisation loading of large Sr+ ion clouds with ultrafast pulses

This paper reports on photoionisation loading based on ultrafast pulses of singly-ionised strontium ions in a linear Paul trap. We take advantage of an autoionising resonance of Sr neutral atoms to form Sr+ by two-photon absorption of femtosecond pulses at a wavelength of 431nm. We compare this technique to electron-bombardment ionisation and observe several advantages of photoionisation. It actually allows the loading of a pure Sr+ ion cloud in a low radio-frequency voltage amplitude regime. In these conditions up to 4x10^4 laser-cooled Sr+ ions were trapped

Monitoring changes of paramagnetically-shifted 31P signals in phospholipid vesicles

Phospholipid vesicles are commonly used as biomimetics in the investigation of the interaction of various species with cell membranes. In this paper we present a 31P NMR investigation of a simple vesicle system using a paramagnetic shift reagent to probe the inner and outer layers of the lipid bilayer. Time-dependent changes in the 31P NMR signal are observed, which differ whether the paramagnetic species is inside or outside the vesicle, and on the choice of buffer solution used. An interpretation of these results is given in terms of the interaction of the paramagnetic shift reagent with the lipids

Macrocyclic β-Sheet Peptides That Inhibit the Aggregation of a Tau-Protein-Derived Hexapeptide

This paper describes studies of a series of macrocyclic β-sheet peptides 1 that inhibit the aggregation of a tau-protein-derived peptide. The macrocyclic β-sheet peptides comprise a pentapeptide "upper" strand, two δ-linked ornithine turn units, and a "lower" strand comprising two additional residues and the β-sheet peptidomimetic template "Hao". The tau-derived peptide Ac-VQIVYK-NH(2) (AcPHF6) aggregates in solution through β-sheet interactions to form straight and twisted filaments similar to those formed by tau protein in Alzheimer's neurofibrillary tangles. Macrocycles 1 containing the pentapeptide VQIVY in the "upper" strand delay and suppress the onset of aggregation of the AcPHF6 peptide. Inhibition is particularly pronounced in macrocycles 1a, 1d, and 1f, in which the two residues in the "lower" strand provide a pattern of hydrophobicity and hydrophilicity that matches that of the pentapeptide "upper" strand. Inhibition varies strongly with the concentration of these macrocycles, suggesting that it is cooperative. Macrocycle 1b containing the pentapeptide QIVYK shows little inhibition, suggesting the possibility of a preferred direction of growth of AcPHF6 β-sheets. On the basis of these studies, a model is proposed in which the AcPHF6 amyloid grows as a layered pair of β-sheets and in which growth is blocked by a pair of macrocycles that cap the growing paired hydrogen-bonding edges. This model provides a provocative and appealing target for future inhibitor design

The role of copper(II) in the aggregation of human amylin

Amylin is the 37-residue peptide hormone produced by the islet β-cells in the pancreas and the formation of amylin aggregates is strongly associated with β-cells degeneration in type 2 diabetes, as demonstrated by more than 95% of patients exhibiting amylin amyloid upon autopsy. It is widely recognized that metal ions such as copper(II) have been implicated in the aggregation process of amyloidogenic peptides such as Aβ and α-synuclein and there is evidence that also amylin self-assembly is largely affected by copper(II). For this reason, in this work, the role of copper(II) in the aggregation of amylin has been investigated by several different experimental approaches. Mass spectrometric investigations show that copper(II) induces significant changes in the amylin structure which decrease the protein fibrillogenesis as observed by ThT measurements. Accordingly, solid-state NMR experiments together with computational analysis carried out on a model amylin fragment confirmed the non fibrillogenic nature of the copper(II) induced aggregated structure. Finally, the presence of copper(II) is also shown to have a major influence on amylin proneness to be degraded by proteases and cytotoxicity studies on different cell cultures are reported

Thoracic aortic aneurysm and atrial fibrillation: clinical associations with the risk of stroke from a global federated health network analysis

Background: An association with aortic aneurysm has been reported among patients with atrial fibrillation (AF). The aims of this study were to investigate the prevalence of thoracic aorta aneurysm (TAA) among patients with AF and to assess whether the co-presence of TAA is associated with a higher risk of adverse clinical outcomes. Methods and results: Using TriNetX, a global federated health research network of anonymised electronic medical records, all adult patients with AF, were categorised into two groups based on the presence of AF and TAA or AF alone. Between 1 January 2017 and 1 January 2019, 874,212 people aged ≥ 18 years with AF were identified. Of these 17,806 (2.04%) had a TAA. After propensity score matching (PSM), 17,805 patients were included in each of the two cohorts. During the 3 years of follow-up, 3079 (17.3%) AF patients with TAA and 2772 (15.6%) patients with AF alone, developed an ischemic stroke or transient ischemic attack (TIA). The risk of ischemic stroke/TIA was significantly higher in patients with AF and TAA (HR 1.09, 95% CI 1.04–1.15; log-rank p value < 0.001) The risk of major bleeding was higher in patients with AF and TAA (OR 1.07, 95% CI 1.01–1.14), but not significant in time-dependent analysis (HR 1.04, 95% CI 0.98–1.10; log-rank p value = 0.187), Conclusion: This retrospective analysis reports a clinical concomitance of the two medical conditions, and shows in a PSM analysis an increased risk of ischemic events in patients affected by TAA and AF compared to AF alone

Progressive Neurodegeneration or Endogenous Compensation in an Animal Model of Parkinson's Disease Produced by Decreasing Doses of Alpha-Synuclein

The pathological hallmarks of Parkinson's disease (PD) are degeneration of dopamine (DA) neurons of the substantia nigra (SN) and the presence of alpha-synuclein (α-syn)-rich Lewy bodies in DA cells that remain. To model these aspects of the disease, we previously showed that high titer (5.1×10exp12 gp/ml) AAV1/2 driven expression of A53T α-syn in the SN of rats caused nigrostriatal pathology including a loss of DA neurons, but also with toxicity in the GFP control group. In the current study, we evaluate the effects of two lower titers by dilution of the vector (1∶3 [1.7×10exp12] and 1∶10 [5.1×10exp11]) to define a concentration that produced pathology specific for α-syn. In GFP and empty vector groups there were no behavioural or post-mortem changes at 3 or 6 weeks post-administration at either vector dose. Dilution of the AAV1/2 A53T α-syn (1∶3) produced significant paw use asymmetry, reductions in striatal tyrosine hydroxylase (TH), and increases in DA turnover at 3 weeks in the absence of overt pathology. By 6 weeks greater evidence of pathology was observed and included, reductions in SN DA neurons, striatal DA, TH and DA-transporter, along with a sustained behavioural deficit. In contrast, the 1∶10 AAV1/2 A53T α-syn treated animals showed normalization between 3 and 6 weeks in paw use asymmetry, reductions in striatal TH, and increased DA turnover. Progression of dopaminergic deficits using the 1∶3 titer of AAV1/2 A53Tα-syn provides a platform for evaluating treatments directed at preventing and/or reversing synucleinopathy. Use of the 1∶10 titer of AAV1/2 A53T α-syn provides an opportunity to study mechanisms of endogenous compensation. Furthermore, these data highlight the need to characterize the titer of vector being utilized, when using AAV to express pathogenic proteins and model disease process, to avoid producing non-specific effects