Rapid determination of levels of the main constituents in e-liquids by near infrared spectroscopy.

Use of e-cigarettes is increasing, alongside an expanding variety of devices and e-liquids. To match this growth and in line with the expanding legal and regulatory requirements applicable to manufacturers of e-cigarettes (e.g. disclosure of list of ingredients and quantities thereof in a product), rapid methods for determining levels of the main e-liquid constituents-namely, propylene glycol (PG), vegetable glycerol (VG), water and nicotine-are needed. We have assessed the ability of near infrared (NIR) spectroscopy, coupled with partial least squares (PLS) regression, to predict the levels of these constituents in e-liquid formulations. Using NIR spectral data from a large set of reference e-liquids incorporating working concentration ranges, flavourings, and other ingredients, linear calibration models were established for PG, VG, water and nicotine (predicted vs theoretical values, all R2 > 0.995). The performance of these models was then evaluated on commercial e-liquids using NIR and compared to results obtained by gas chromatography (GC). A strong correlation was observed between NIR-predicted values and measured values for PG, VG and nicotine (all R2 > 0.955). There was less consistency between predicted and GC measured values for water due to the relatively high limit of quantification (LOQ) of the GC method (2.6% w/w) versus the e-liquid content (0-18% w/w). The LOQ of the NIR method for water was 0.6% w/w, suggesting that NIR may be a more accurate method than GC to predict water concentration in e-liquids, especially at low levels (< 2.6% w/w). Collectively, although limitations of the technique have been identified, specifically for e-liquids containing compounds that might interfere with the set calibrations, our findings suggest that NIR combined with PLS regression is a suitable tool for rapid, simultaneous and high-throughput measurement of PG, VG, water and nicotine levels in most commercial e-liquids.

A single mutation on the human amyloid polypeptide modulates fibril growth and affects the mechanism of amyloid-induced membrane damage.

Amyloid fibril formation has been implicated in a wide range of human diseases and the interactions of amyloidogenic proteins with cell membranes are considered to be important in the aetiology of these pathologies. In type 2 diabetes mellitus (T2DM), the human islet amyloid polypeptide (hIAPP) forms amyloid fibrils which impair the functionality and viability of pancreatic ß cells. The mechanisms of hIAPP cytotoxicity are linked to the ability of the peptide to self-aggregate and to interact with membranes. Previous studies have shown that the N-terminal part of hIAPP from residues 1 to 19 is the membrane binding domain. The non-amyloidogenic and nontoxic mouse IAPP differs from hIAPP by six residues out of 37, among which a single one, residue 18, lies in the membrane binding region. To gain more insight into hIAPP-membrane interactions we herein performed comprehensive biophysical studies on four analogues (H18R-IAPP, H18K-IAPP, H18E-IAPP and H18A-IAPP). Our data reveal that all peptides are able to insert efficiently in the membrane, indicating that residue 18 is not essential for hIAPP membrane binding and insertion. However, only wild-type hIAPP and H18K-IAPP are able to form fibrils at the membrane. Importantly, all peptides induce membrane damage; wild-type hIAPP and H18K-IAPP presumably cause membrane disruption mainly by fibril growth at the membrane, while for H18R-IAPP, H18E-IAPP and H18A-IAPP, membrane leakage is most likely due to high molecular weight oligomeric species. These results highlight the importance of the residue at position 18 in IAPP for modulating fibril formation at the membrane and the mechanisms of membrane leakage.

Time dependence of NMR observables reveals salient differences in the accumulation of early aggregated species between human islet amyloid polypeptide and amyloid-ß.

Type 2 diabetes mellitus and Alzheimer's disease are characterized by the accumulation of fibrillar amyloid deposits consisting mainly of islet amyloid polypeptide (IAPP) and amyloid-ß (Aß), respectively. Fibril formation is a multi-step nucleation process that involves the transient build-up of oligomeric species that are thought to be the most toxic components. To gain more insight into the molecular mechanism of early IAPP aggregated species formation, we performed a combination of direct and indirect biophysical approaches on IAPP and also on Aß42 for the sake of comparison. Thioflavin T fluorescence kinetics measurements revealed a stronger autocatalytic behaviour of IAPP and a weaker concentration dependence of fibrillization half-time t1/2, as compared to Aß42. Our NMR experiments highlight the absence of micelle reservoir or supercritical regime in the studied concentration range, indicating that the low concentration dependence of IAPP fibril formation can be ascribed to saturable pathways. IAPP and Aß42 displayed marked differences in formation of oligomeric species, as observed by 1D 1H, pulsed-field gradient (PFG) diffusion and saturation transfer difference (STD) NMR experiments. A fast equilibrium between monomer and oligomeric species was detected in the case of Aß42 but not IAPP, with a significant build-up of aggregated species, as shown by the time dependence of diffusion coefficient and STD magnetization transfer efficiency during the aggregation process. Altogether our data show significant differences between IAPP and Aß42 regarding the microscopic events of amyloid species formation.

Designed Glycopeptidomimetics Disrupt Protein-Protein Interactions Mediating Amyloid ß-Peptide Aggregation and Restore Neuroblastoma Cell Viability.

How anti-Alzheimer's drug candidates that reduce amyloid 1-42 peptide fibrillization interact with the most neurotoxic species is far from being understood. We report herein the capacity of sugar-based peptidomimetics to inhibit both Aß1-42 early oligomerization and fibrillization. A wide range of bio- and physicochemical techniques, such as a new capillary electrophoresis method, nuclear magnetic resonance, and surface plasmon resonance, were used to identify how these new molecules can delay the aggregation of Aß1-42. We demonstrate that these molecules interact with soluble oligomers in order to maintain the presence of nontoxic monomers and to prevent fibrillization. These compounds totally suppress the toxicity of Aß1-42 toward SH-SY5Y neuroblastoma cells, even at substoichiometric concentrations. Furthermore, demonstration that the best molecule combines hydrophobic moieties, hydrogen bond donors and acceptors, ammonium groups, and a hydrophilic ß-sheet breaker element provides valuable insight for the future structure-based design of inhibitors of Aß1-42 aggregation.

Molecular Structure, Membrane Interactions, and Toxicity of the Islet Amyloid Polypeptide in Type 2 Diabetes Mellitus.

Human islet amyloid polypeptide (hIAPP) is the major component of the amyloid deposits found in the pancreatic islets of patients with type 2 diabetes mellitus (T2DM). Mature hIAPP, a 37-aa peptide, is natively unfolded in its monomeric state but forms islet amyloid in T2DM. In common with other misfolded and aggregated proteins, amyloid formation involves aggregation of monomers of hIAPP into oligomers, fibrils, and ultimately mature amyloid deposits. hIAPP is coproduced and stored with insulin by the pancreatic islet ß-cells and is released in response to the stimuli that lead to insulin secretion. Accumulating evidence suggests that hIAPP amyloid deposits that accompany T2DM are not just an insignificant phenomenon derived from the disease progression but that hIAPP aggregation induces processes that impair the functionality and the viability of ß-cells. In this review, we particularly focus on hIAPP structure, hIAPP aggregation, and hIAPP-membrane interactions. We will also discuss recent findings on the mechanism of hIAPP-membrane damage and on hIAPP-induced cell death. Finally, the development of successful antiamyloidogenic agents that prevent hIAPP fibril formation will be examined.

(R)-α-trifluoromethylalanine containing short peptide in the inhibition of amyloid peptide fibrillation.

The extracellular deposition of insoluble amyloid fibrils resulting from the aggregation of the amyloid-ß (Aß) is a pathological feature of neuronal loss in Alzheimer's disease (AD). Numerous small molecules have been reported to interfere with the process of Aß aggregation. Compounds containing aromatic structures, hydrophobic amino acids and/or the α-aminoisobutyric acid (Aib) as ß-sheet breaker elements have been reported to be effective inhibitors of Aß aggregation. We synthesized two peptides, one containing the Aib amino acid and the other including its trifluoromethylated analog (R)-α-Trifluoromethylalanine ((R)-Tfm-Alanine) and we evaluated the impact of these peptides on Aß amyloid formation. The compounds were tested by standard methods such as thioflavin-T fluorescence spectroscopy and transmission electron microscopy but also by circular dichroism, liquid state nuclear magnetic resonance (NMR) and NMR saturation transfer difference (STD) experiments to further characterize the effect of the two molecules on Aß structure and on the kinetics of depletion of monomeric, soluble Aß. Our results demonstrate that the peptide containing Aib reduces the quantity of aggregates containing ß-sheet structure but slightly inhibits Aß fibril formation, while the molecule including the trifluoromethyl (Tfm) group slows down the kinetics of Aß fibril formation, delays the random coil to ß-sheet structure transition and induces a change in the oligomerization pathway. These results suggest that the hydrophobic Tfm group has a better affinity with Aß than the methyl groups of the Aib and that this Tfm group is effective and important in preventing the Aß aggregation.