The Ability of DNAJB6b to Suppress Amyloid Formation Depends on the Chaperone Aggregation State.

For many chaperones, a propensity to self-assemble correlates with function. The highly efficient amyloid suppressing chaperone DNAJB6b has been reported to oligomerize. A key question is whether the DNAJB6b self-assemblies or their subunits are active units in the suppression of amyloid formation. Here, we address this question using a nonmodified chaperone. We use the well-established aggregation kinetics of the amyloid ß 42 peptide (Aß42) as a readout of the amyloid suppression efficiency. The experimental setup relies on the slow dissociation of DNAJB6b assemblies upon dilution. We find that the dissociation of the chaperone assemblies correlates with its ability to suppress fibril formation. Thus, the data show that the subunits of DNAJB6b assemblies rather than the large oligomers are the active forms in amyloid suppression. Our results provide insights into how DNAJB6b operates as a chaperone and illustrate the importance of established assembly equilibria and dissociation rates for the design of kinetic experiments.

The C-terminal domain of the antiamyloid chaperone DNAJB6 binds to amyloid-ß peptide fibrils and inhibits secondary nucleation.

The DNAJB6 chaperone inhibits fibril formation of aggregation-prone client peptides through interaction with aggregated and oligomeric forms of the amyloid peptides. Here, we studied the role of its C-terminal domain (CTD) using constructs comprising either the entire CTD or the first two or all four of the CTD ß-strands grafted onto a scaffold protein. Each construct was expressed as WT and as a variant with alanines replacing five highly conserved and functionally important serine and threonine residues in the first ß-strand. We investigated the stability, oligomerization, antiamyloid activity, and affinity for amyloid-ß (Aß42) species using optical spectroscopy, native mass spectrometry, chemical crosslinking, and surface plasmon resonance technology. While DNAJB6 forms large and polydisperse oligomers, CTD was found to form only monomers, dimers, and tetramers of low affinity. Kinetic analyses showed a shift in inhibition mechanism. Whereas full-length DNAJB6 activity is dependent on the serine and threonine residues and efficiently inhibits primary and secondary nucleation, all CTD constructs inhibit secondary nucleation only, independently of the serine and threonine residues, although their dimerization and thermal stabilities are reduced by alanine substitution. While the full-length DNAJB6 inhibition of primary nucleation is related to its propensity to form coaggregates with Aß, the CTD constructs instead bind to Aß42 fibrils, which affects the nucleation events at the fibril surface. The retardation of secondary nucleation by DNAJB6 can thus be ascribed to the first two ß-strands of its CTD, whereas the inhibition of primary nucleation is dependent on the entire protein or regions outside the CTD.

On the micelle formation of DNAJB6b.

The human chaperone DNAJB6b increases the solubility of proteins involved in protein aggregation diseases and suppresses the nucleation of amyloid structures. Due to such favourable properties, DNAJB6b has gained increasing attention over the past decade. The understanding of how DNAJB6b operates on a molecular level may aid the design of inhibitors against amyloid formation. In this work, fundamental aspects of DNAJB6b self-assembly have been examined, providing a basis for future experimental designs and conclusions. The results imply the formation of large chaperone clusters in a concentration-dependent manner. Microfluidic diffusional sizing (MDS) was used to evaluate how DNAJB6b average hydrodynamic radius varies with concentration. We found that, in 20 mM sodium phosphate buffer, 0.2 mM EDTA, at pH 8.0 and room temperature, DNAJB6b displays a micellar behaviour, with a critical micelle concentration (CMC) of around 120 nM. The average hydrodynamic radius appears to be concentration independent between ∼10 µM and 100 µM, with a mean radius of about 12 nm. The CMC found by MDS is supported by native agarose gel electrophoresis and the size distribution appears bimodal in the DNAJB6b concentration range ∼100 nM to 4 µM.

Long-term risk of major adverse cardiovascular events following ischemic stroke or TIA.

Data are scarce on long-term outcomes after ischemic stroke (IS) or transient ischemic attack (TIA). In this prospective cohort study, we examined the cumulative incidence of major adverse cardiovascular events (MACE) after IS and TIA using a competing risk model and factors associated with new events using a Cox-proportional hazard regression model. All patients discharged alive from Östersund Hospital with IS or TIA between 2010 and 2013 (n = 1535) were followed until 31 December 2017. The primary endpoint was a composite of IS, type 1 acute myocardial infarction (AMI), and cardiovascular (CV) death. Secondary endpoints were the individual components of the primary endpoint, in all patients and separated in IS and TIA subgroups. The cumulative incidence of MACE (median follow-up: 4.4 years) was 12.8% (95% CI: 11.2-14.6) within 1 year after discharge and 35.6% (95% CI: 31.8-39.4) by the end of follow-up. The risk of MACE and CV death was significantly increased in IS compared to TIA (p-values < 0.05), but not the risk of IS or type 1 AMI. Age, kidney failure, prior IS, prior AMI, congestive heart failure, atrial fibrillation, and impaired functional status, were associated with an increased risk of MACE. The risk of recurring events after IS and TIA is high. IS patients have a higher risk of MACE and CV death than TIA patients.

Prediction of designer drugs: Synthesis and spectroscopic analysis of synthetic cathinone analogs that may appear on the Swedish drug market.

The use of hyphenated analytical techniques in forensic drug screening enables simultaneous identification of a wide range of different compounds. However, the appearance of drug seizures containing new substances, mainly new psychoactive substances (NPS), is steadily increasing. These new and other already known substances often possess structural similarities and consequently they exhibit spectral data with slight differences. This situation has made the criteria that ensure indubitable identification of compounds increasingly important. In this work, 6 new synthetic cathinones that have not yet appeared in any Swedish drug seizures were synthesized. Their chemical structures were similar to those of already known cathinone analogs of which 42 were also included in the study. Hence, a total of 48 synthetic cathinones making up sets of homologous and regioisomeric compounds were used to challenge the capabilities of various analytical techniques commonly applied in forensic drug screening, ie, gas chromatography-mass spectrometry (GC-MS), gas chromatography-Fourier transform infrared spectroscopy (GC-FTIR), nuclear magnetic resonance (NMR), and liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS). Special attention was paid to the capabilities of GC-MS and GC-FTIR to distinguish between the synthetic cathinones and the results showed that neither GC-MS nor GC-FTIR alone can successfully differentiate between all synthetic cathinones. However, the 2 techniques proved to be complementary and their combined use is therefore beneficial. For example, the structural homologs were better differentiated by GC-MS, while GC-FTIR performed better for the regioisomers. Further, new spectroscopic data of the synthesized cathinone analogs is hereby presented for the forensic community. The synthetic work also showed that cathinone reference compounds can be produced in few reaction steps.

Prediction of designer drugs: synthesis and spectroscopic analysis of synthetic cannabinoid analogues of 1H-indol-3-yl(2,2,3,3-tetramethylcyclopropyl)methanone and 1H-indol-3-yl(adamantan-1-yl)methanone.

In this work, emergence patterns of synthetic cannabinoids were utilized in an attempt to predict those that may appear on the drug market in the future. Based on this information, two base structures of the synthetic cannabinoid analogues - (1H-indol-3-yl(2,2,3,3-tetramethylcyclopropyl)methanone and 1H-indol-3-yl(adamantan-1-yl)methanone) - together with three substituents - butyl, 4-fluorobutyl and ethyl tetrahydropyran - were selected for synthesis. This resulted in a total of six synthetic cannabinoid analogues that to the authors' knowledge have not yet appeared on the drug market. Spectroscopic data, including nuclear magnetic resonance (NMR), mass spectrometry (MS), and Fourier transform infrared (FTIR) spectroscopy (solid and gas phase), are presented for the synthesized analogues and some additional related cannabinoids. In this context, the suitability of the employed techniques for the identification of unknowns is discussed and the use of GC-FTIR as a secondary complementary technique to GC-MS is addressed. Examples of compounds that are difficult to differentiate by their mass spectra, but can be distinguished based upon their gas phase FTIR spectra are presented. Conversely, structural homologues where mass spectra are more powerful than gas phase FTIR spectra for unambiguous assignments are also exemplified. This work further emphasizes that a combination of several techniques is the key to success in structural elucidations. Copyright © 2015 John Wiley & Sons, Ltd.

Second-harmonic generation in vortex-induced waveguides.

We study the second-harmonic generation and localization of light in a reconfigurable waveguide induced by an optical vortex soliton in a defocusing Kerr medium. We show that the vortex-induced waveguide greatly improves conversion efficiency from the fundamental to the second-harmonic field.