Thioflavin T (ThT) has been widely used to investigate amyloid formation since 1989. While concerns have recently been raised about its use as a probe specific for amyloid, ThT still continues to be a very valuable tool for studying kinetic aspects of fibrillation and associated inhibition mechanisms. This review aims to provide a conceptual instruction manual, covering appropriate considerations and pitfalls related to the use of ThT. We start by giving a brief introduction to amyloid formation with focus on the morphology of different aggregate species, followed by a discussion of the quality of protein needed to obtain reliable fibrillation data. After an overview of the photochemical basis for ThT's amyloid binding properties and artifacts that may arise from this, we describe how to plan and analyze ThT assays. We conclude with recommendations for complementary techniques to address shortcomings in the ThT assay.

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Glycosaminoglycans (GAGs) bind all known amyloid plaques and help store protein hormones in (acidic) granular vesicles, but the molecular mechanisms underlying these important effects are unclear. Here we investigate GAG interactions with the peptide hormone salmon calcitonin (sCT). GAGs induce fast sCT fibrillation at acidic pH and only bind monomeric sCT at acidic pH, inducing sCT helicity. Increasing GAG sulfation expands the pH range for binding. Heparin, the most highly sulfated GAG, binds sCT in the pH interval 3-7. Small angle x-ray scattering indicates that sCT monomers densely decorate and pack single heparin chains, possibly via hydrophobic patches on helical sCT. sCT fibrillates without GAGs, but heparin binding accelerates the process by decreasing the otherwise long fibrillation lag times at low pH and accelerates fibril growth rates at neutral pH. sCT⅐heparin complexes form ␤-sheet-rich heparin-covered fibrils. Solid-state NMR reveals that heparin does not alter the sCT fibrillary core around Lys 11 but makes changes to Val 8 on the exterior side of the ␤-strand, possibly through contacts to Lys 18. Thus GAGs significantly modulate sCT fibrillation in a pH-dependent manner by interacting with both monomeric and aggregated sCT.

1. Temperature is one of the primary environmental drivers of the distribution of species, and particularly high temperatures challenge physiological processes by disruption of cellular homoeostasis. This exerts selection on organisms to maintain cellular homoeostasis by adaptive physiological and/or behavioural responses. The social spider Stegodyphus dumicola occurs across several climate zones inSouthern Africa, and populations experience high and variable temperatures, suggesting a wide temperature niche, or alternatively that populations respond with plastic or locally adapted responses to temperature. Using a common garden design, we investigated complementary adaptive heat responses (behavioural thermoregulation and cuticle wax composition) in individuals from warmer and cooler locations.3. The spiders exhibited higher temperature tolerance than most ectotherms (CTmax almost 49℃), with the individuals from warmer locations showing the highest tolerance. Analyses of cuticle wax revealed chemical compositions consistent with a higher melting temperature (e.g. increased chain length and lower occurrence of branching) and therefore improved waterproofing in spiders originating from warmer locations and acclimated at a higher temperature, as expected if local temperature drives changes in the cuticle composition to improve waterproofing.4. The spiders exhibited a clear behavioural escape response from increasing temperature, with individuals from warmer locations and kept at higher acclimation temperature showing a lower threshold temperature at which this behaviour was triggered, suggesting that this threshold is under natural selection. Our study provides evidence of both local adaptation and phenotypic plasticity inphysiological and behavioural traits relating to temperature tolerance. Population differences in trait expression suggest local adaptation to different thermal environments, and individuals plastically adjust cuticle wax composition and cooling behaviour in response to temperature changes. 6. The Bogert effect predicts that behavioural thermoregulation may relax selection for physiological adaptations. Our study instead suggests that synergistic effects | 2729 Functional Ecology MALMOS et AL.

We investigate the potential of 31 P NMR with simple, maintenance-free benchtop spectrometers to probe phospholipids in complex mixtures. 31 P NMR-based lipidomics has become an important topic in a wide range of applications in food-and health-sciences, and the continuous improvements of compact, maintenance-and cryogen-free instruments opens new opportunities for NMR routine analyses. A prior milestone is the evaluation of the analytical performance provided by 31 P NMR at low magnetic field. To address this, we assess the ability of state-ofthe-art benchtop NMR spectrometers to detect, identify, and quantify several types of phospholipids in mixtures. Relying on heteronuclear crosspolarization experiments, phospholipids can be detected in 2 h with a limit of detection of 0.5 mM at 1 T and 0.2 mM at 2 T, while the headgroups of phosphatidylcholine (PC), phosphatidyl-ethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), and phosphatidyl-glycerol (PG) can be unambiguously assigned based on 2D 1 H− 31 P total correlated spectroscopy (TOCSY) spectra. Furthermore, two quantitative methods to obtain absolute concentrations are proposed and discussed, and the performance is evaluated regarding precision and accuracy.