Interaction analysis of RNA G-quadruplex with ligands and in situ imaging application.

RNA G4, as an integral branch of G4 structure, possesses distinct interactions with ligands compared to the common DNA G4, thus the investigation of RNA G4/ligand interactions might be considered as a fresh breakthrough to improve the biosensing performance of G4/ligand system. In this study, we comparatively explored the structural and functional mechanisms of RNA G4 and DNA G4 in the interaction with ligands, hemin and thioflavin T (ThT), utilizing the classical PS2.M sequence as a model. We found that although the catalytic performance of RNA G4/hemin system was lower than DNA G4/hemin, RNA G4/ThT fluorescence system exhibited a significant improvement (2∼3-fold) compared to DNA G4/ThT, and adenine modification could further enhance the signaling. Further, by exploring the interaction between RNA G4 and ThT, we deemed that RNA G4 and ThT were stacked in a bimolecular mode compared to single-molecule binding of DNA G4/ThT, thus more strongly limiting the structural spin in ThT excited state. Further, RNA G4/ThT displayed higher environmental tolerance and lower ion dependence than DNA G4/ThT. Finally, we employed RNA G4/ThT as a highly sensitive label-free fluorescent signal output system for in situ imaging of isoforms BCR-ABL e13a2 and e14a2. Overall, this study successfully screened a high-performance RNA G4 biosensing system through systematic RNA G4/ligands interaction studies, which was expected to provide a promising reference for subsequent G4/ligand research.

Exploitation of the nitro- and/or 4-Trifluoromethyl-decorated phenyl fragment to develop small inhibitors of Alpha-Syn fibril aggregation.

Here, we report new 2-nitro and/or 4-trifluoromethylphenyl-based small molecules developed as inhibitors of alpha-Syn fibril formation. The set of eighteen compounds was inspired by well-known alpha-Syn aggregation modulators retrieved from literature. The preliminary biochemical data suggested that the two molecules out of eighteen compounds exerted activity comparable to that of reference compound SynuClean-D (SC-D, 5-nitro-6-(3-nitrophenyl)-2-oxo-4-(trifluoromethyl)-1H-pyridine-3-carbonitrile), according to Thioflavin T kinetics. Pharmacophore modelling deciphered the main structural requirements for alpha-Syn aggregation modulators. Moreover, docking and molecular dynamics simulations depicted the binding mode with the targeted alpha-Syn fibrils. The structural data of these new potential α-Syn binders might furnish additional information for understanding the mechanism of action of the ligands that specifically target the NAC domain as theranostic agents for α-synucleopathies.

Sensitive and reliable analysis of endometrial cancer related microRNA using ternary hybridization hairpin probe.

MicroRNA (miRNA), as a kind of small non-coding ribonucleic acid (RNA) that plays a crucial role in regulating transcriptional activities, is a potential biomarker for EC diagnosis. However, reliable detection of miRNA remains a huge challenge, especially for these methods that require multiple probes for signal amplifications, due to the detective deviation caused by variation of probe concentrations. Herein, we present a novel approach for miRNA-205 identification and quantification by employing simply a ternary hairpin probe (TH probe). The ternary hybridization of three sequences results in the construction of the TH probe, which combines high-efficient signal amplification and specific target recognition. A significant number of G-rich sequences have been produced as a result of the enzymes assisted signal amplification process. The G-rich sequences can fold into G-quadruplexes, which can then be detected in a label-free manner by a common fluorescent dye (thioflavin T). Eventually, the approach exhibits a low limit of detection of 278 aM with a wide detection range of 7 orders of magnitude. In summary, the proposed approach possesses a great potential for both clinical diagnosis of EC and fundamental biomedical researches.

Combinatorial screening for therapeutics in ATTRv amyloidosis identifies naphthoquinone analogues as TTR-selective amyloid disruptors.

Hereditary ATTR amyloidosis is caused by the point mutation in serum protein transthyretin (TTR) that destabilizes its tetrameric structure to dissociate into monomer. The monomers form amyloid fibrils, which are deposited in peripheral nerves and organs, resulting in dysfunction. Therefore, a drug that dissolves amyloid after it has formed, termed amyloid disruptor, is needed as a new therapeutic drug. Here, we first established a high throughput screening system to find TTR interactors from the LOPAC1280 compound library. Among the hit compounds, thioflavin T-based post-treatment assay determined lead compounds for TTR amyloid disruptors, NSC95397 and Gossypol, designated as B and R, respectively. Because these compounds have naphthoquinone-naphthalene structures, we tested 100 naphthoquinone derivatives, and found 10 candidate compounds that disrupted TTR amyloid. Furthermore, to determine whether these 10 compounds are selective for TTR amyloid, we evaluated them against beta-amyloid (Aß1-42). We found two compounds that were selective for TTR and did not disrupt Aß-derived amyloid. Therefore, we succeeded in identifying TTR-selective amyloid disruptors, and demonstrated that naphthoquinone compounds are useful structures as amyloid disruptors. These findings contribute to the on-going efforts to discover new therapeutic tools for TTR amyloidosis.

Self-assembly of a peptide sequence, EKKE, composed of exclusively charged amino acids: Role of charge in morphology and lead binding.

The self-assembly of peptides is influenced by their amino acid sequence and other factors including pH, charge, temperature, and solvent. Herein, we explore whether a four-residue sequence, EKKE, consisting of exclusively charged amino acids shows the propensity to form self-assembled ordered nanostructures and whether the overall charge plays any role in morphological and functional properties. From a combination of experimental data provided by Thioflavin T fluorescence, Congo red absorbance, circular dichroism spectroscopy, dynamic light scattering, field emission-scanning electron microscopy, atomic force microscopy, and confocal microscopy, it is clear that the all-polar peptide and charged EKKE sequence shows a pH-dependent tendency to form amyloid-like structures, and the self-assembled entities under acidic, basic and neutral conditions exhibit morphological variation. Additionally, the ability of the self-assembled amyloid nanostructures to bind to the toxic metal, lead (Pb2+ ), was demonstrated from the analysis of the ultraviolet absorbance and X-ray photoelectron spectroscopy data. The modulation at the sequence level for the amyloid-forming EKKE scaffold can further extend its potential role not only in the remediation of other toxic metals but also towards biomedical applications.

Effect of Ovocystatin on Amyloid ß 1-42 Aggregation-In Vitro Studies.

Amyloid ß peptides (Aß) aggregating in the brain have a potential neurotoxic effect and are believed to be a major cause of Alzheimer's disease (AD) development. Thus, inhibiting amyloid polypeptide aggregation seems to be a promising approach to the therapy and prevention of this neurodegenerative disease. The research presented here is directed at the determination of the inhibitory activity of ovocystatin, the cysteine protease inhibitor isolated from egg white, on Aß42 fibril genesis in vitro. Thioflavin-T (ThT) assays, which determine the degree of aggregation of amyloid peptides based on fluorescence measurement, circular dichroism spectroscopy (CD), and transmission electron microscopy (TEM) have been used to assess the inhibition of amyloid fibril formation by ovocystatin. Amyloid beta 42 oligomer toxicity was measured using the MTT test. The results have shown that ovocystatin possesses Aß42 anti-aggregation activity and inhibits Aß42 oligomer toxicity in PC12 cells. The results of this work may help in the development of potential substances able to prevent or delay the process of beta-amyloid aggregation-one of the main reasons for Alzheimer's disease.

Extent of N-Terminus Folding of Semenogelin 1 Cleavage Product Determines Tendency to Amyloid Formation.

It is known that four peptide fragments of predominant protein in human semen Semenogelin 1 (SEM1) (SEM1(86-107), SEM1(68-107), SEM1(49-107) and SEM1(45-107)) are involved in fertilization and amyloid formation processes. In this work, the structure and dynamic behavior of SEM1(45-107) and SEM1(49-107) peptides and their N-domains were described. According to ThT fluorescence spectroscopy data, it was shown that the amyloid formation of SEM1(45-107) starts immediately after purification, which is not observed for SEM1(49-107). Seeing that the peptide amino acid sequence of SEM1(45-107) differs from SEM1(49-107) only by the presence of four additional amino acid residues in the N domain, these domains of both peptides were obtained via solid-phase synthesis and the difference in their dynamics and structure was investigated. SEM1(45-67) and SEM1(49-67) showed no principal difference in dynamic behavior in water solution. Furthermore, we obtained mostly disordered structures of SEM1(45-67) and SEM1(49-67). However, SEM1(45-67) contains a helix (E58-K60) and helix-like (S49-Q51) fragments. These helical fragments may rearrange into ß-strands during amyloid formation process. Thus, the difference in full-length peptides' (SEM1(45-107) and SEM1(49-107)) amyloid-forming behavior may be explained by the presence of a structured helix at the SEM1(45-107) N-terminus, which contributes to an increased rate of amyloid formation.

Molecular Docking Insight into the Label-Free Fluorescence Aptasensor for Ochratoxin A Detection.

Ochratoxin A (OTA) is the most common mycotoxin and can be found in wheat, corn and other grain products. As OTA pollution in these grain products is gaining prominence as a global issue, the demand to develop OTA detection technology has attracted increasing attention. Recently, a variety of label-free fluorescence biosensors based on aptamer have been established. However, the binding mechanisms of some aptasensors are still unclear. Herein, a label-free fluorescent aptasensor employing Thioflavin T (ThT) as donor for OTA detection was constructed based on the G-quadruplex aptamer of the OTA aptamer itself. The key binding region of aptamer was revealed by using molecular docking technology. In the absence of the OTA target, ThT fluorescent dye binds with the OTA aptamer to form an aptamer/ThT complex, and results in the fluorescence intensity being obviously enhanced. In the presence of OTA, the OTA aptamer binds to OTA because of its high affinity and specificity to form an aptamer/OTA complex, and the ThT fluorescent dye is released from the OTA aptamer into the solution. Therefore, the fluorescence intensity is significantly decreased. Molecular docking results revealed that OTA is binding to the pocket-like structure and surrounded by the A29-T3 base pair and C4, T30, G6 and G7 of the aptamer. Meanwhile, this aptasensor shows good selectivity, sensitivity and an excellent recovery rate of the wheat flour spiked experiment.

Inhibitory mechanism of an antifungal drug, caspofungin against amyloid ß peptide aggregation: Repurposing via neuroinformatics and an experimental approach.

The multifactorial neurological condition called Alzheimer's disease (AD) primarily affects elderly individuals. Despite the calamitous consequences of AD, curative strategies for a regimen to apply remain inadequate as several factors contribute to AD etiology. Drug repurposing is an advance strategy prior to drug discovery as various effective drugs perform through alteration of multiple targets, and the present "poly-pharmacology" can be a curative approach to complex disorders. AD's multifactorial behavior actively encourages the hypothesis for a drug design approach focused on drug repurposing. In this study, we discovered that an antifungal drug, Caspofungin (CAS) is a potent Aß aggregation inhibitor that displays significantly reduced toxicity associated with AD. Drug reprofiling and REMD simulations demonstrated that CAS interacts with the ß-sheet section, known as Aß amyloid fibrils hotspot. CAS leads to destabilization of ß-sheet and, conclusively, in its devaluation. Later, in vitro experiments were acquired in which the fibrillar volume was reduced for CAS-treated Aß peptide. For the first time ever, this study has determined an antifungal agent as the Aß amyloid aggregation's potent inhibitor. Several efficient sequence-reliant potent inhibitors can be developed in future against the amyloid aggregation for different amyloid peptide by the processing and conformational optimization of CAS.

Insights into Network of Hot Spots of Aggregation in Nucleophosmin 1.

In a protein, point mutations associated with diseases can alter the native structure and provide loss or alteration of functional levels, and an internal structural network defines the connectivity among domains, as well as aggregate/soluble states' equilibria. Nucleophosmin (NPM)1 is an abundant nucleolar protein, which becomes mutated in acute myeloid leukemia (AML) patients. NPM1-dependent leukemogenesis, which leads to its aggregation in the cytoplasm (NPMc+), is still obscure, but the investigations have outlined a direct link between AML mutations and amyloid aggregation. Protein aggregation can be due to the cooperation among several hot spots located within the aggregation-prone regions (APR), often predictable with bioinformatic tools. In the present study, we investigated potential APRs in the entire NPM1 not yet investigated. On the basis of bioinformatic predictions and experimental structures, we designed several protein fragments and analyzed them through typical aggrsegation experiments, such as Thioflavin T (ThT), fluorescence and scanning electron microscopy (SEM) experiments, carried out at different times; in addition, their biocompatibility in SHSY5 cells was also evaluated. The presented data clearly demonstrate the existence of hot spots of aggregation located in different regions, mostly in the N-terminal domain (NTD) of the entire NPM1 protein, and provide a more comprehensive view of the molecular details potentially at the basis of NPMc+-dependent AML.

Ligand-Based Discovery of a Small Molecule as Inhibitor of α-Synuclein Amyloid Formation.

α-Synuclein (α-Syn) aggregates are implicated in Parkinson's disease (PD), so inhibitors of α-Syn aggregation have been intensively explored. It has been demonstrated that small molecules might be able to reduce α-Syn aggregation in fibrils, thus exerting neuroprotective effects in models of PD. To expand our knowledge about the structural requirements for blocking the recognition process into the oligomeric assembly of α-Syn aggregates, we performed a ligand-based virtual screening procedure using two well-known α-Syn aggregation inhibitors, SynuClean-D and ZPD-2, as query compounds. A collection of thirty-four compounds bearing distinct chemical functionalities and mutual chemical features were studied in a Th-T fluorescence test, thus identifying 5-(2,6-dinitro-4-(trifluoromethyl)benzyl)-1-methyl-1H-tetrazole (named MeSC-04) as a potent α-Syn amyloid formation inhibitor that demonstrated similar behavior when compared to SynuClean-D in the thioflavin-T-monitored kinetic assays, with both molecules reducing the number and size of amyloid fibrils, as evidenced by electron microscopy. Molecular modeling studies suggested the binding mode of MeSC-04 through the identification of putative druggable pockets on α-syn fibrils and a subsequent consensus docking methodology. Overall, this work could furnish new insights in the development of α-Syn amyloid inhibitors from synthetic sources.

Influence of Cortisol on the Fibril Formation Kinetics of Aß42 Peptide: A Multi-Technical Approach.

Amyloid-ß peptide (Aß) aggregates are known to be correlated with pathological neurodegenerative diseases. The fibril formation process of such peptides in solution is influenced by several factors, such as the ionic strength of the buffer, concentration, pH, and presence of other molecules, just to mention a few. In this paper, we report a detailed analysis of in vitro Aß42 fibril formation in the presence of cortisol at different relative concentrations. The thioflavin T fluorescence assay allowed us to monitor the fibril formation kinetics, while a morphological characterization of the aggregates was obtained by atomic force microscopy. Moreover, infrared absorption spectroscopy was exploited to investigate the secondary structure changes along the fibril formation path. Molecular dynamics calculations allowed us to understand the intermolecular interactions with cortisol. The combined results demonstrated the influence of cortisol on the fibril formation process: indeed, at cortisol-Aß42 concentration ratio (ρ) close to 0.1 a faster organization of Aß42 fragments into fibrils is promoted, while for ρ = 1 the formation of fibrils is completely inhibited.

Interactions between S100A9 and Alpha-Synuclein: Insight from NMR Spectroscopy.

S100A9 is a pro-inflammatory protein that co-aggregates with other proteins in amyloid fibril plaques. S100A9 can influence the aggregation kinetics and amyloid fibril structure of alpha-synuclein (α-syn), which is involved in Parkinson's disease. Currently, there are limited data regarding their cross-interaction and how it influences the aggregation process. In this work, we analyzed this interaction using solution 19F and 2D 15N-1H HSQC NMR spectroscopy and studied the aggregation properties of these two proteins. Here, we show that α-syn interacts with S100A9 at specific regions, which are also essential in the first step of aggregation. We also demonstrate that the 4-fluorophenylalanine label in alpha-synuclein is a sensitive probe to study interaction and aggregation using 19F NMR spectroscopy.

Investigating Chaperone like Activity of Green Silver Nanoparticles: Possible Implications in Drug Development.

Protein aggregation and amyloidogenesis have been associated with several neurodegenerative disorders like Alzheimer's, Parkinson's etc. Unfortunately, there are still no proper drugs and no effective treatment available. Due to the unique properties of noble metallic nanoparticles, they have been used in diverse fields of biomedicine like drug designing, drug delivery, tumour targeting, bio-sensing, tissue engineering etc. Small-sized silver nanoparticles have been reported to have anti-biotic, anti-cancer and anti-viral activities apart from their cytotoxic effects. The current study was carried out in a carefully designed in-vitro to observe the anti-amyloidogenic and inhibitory effects of biologically synthesized green silver nanoparticles (B-AgNPs) on human serum albumin (HSA) aggregation taken as a model protein. We have used different biophysical assays like thioflavin T (ThT), 8-Anilino-1-naphthalene-sulphonic acid (ANS), Far-UV CD etc. to analyze protein aggregation and aggregation inhibition in vitro. It has been observed that the synthesized fluorescent B-AgNPs showed inhibitory effects on protein aggregation in a concentration-dependent manner reaching a plateau, after which the effect of aggregation inhibition was significantly declined. We also observed meaningful chaperone-like aggregation-inhibition activities of as-synthesized florescent B-AgNPs in astrocytes.

Evaluation of ThT augmentation and RLS inner filter effect caused by highly fluorescent coumarin derivative and establishing it as true inhibitor of amyloid fibrillation.

Screening of inhibitors that slow down or suppress amyloid fibrils formation relies on some simple but sensitive spectroscopy techniques. Thioflavin T (ThT) fluorescence assay is one of the most common, amyloid specific and sensitive method. However, if an inhibitor is itself fluorescent in the ThT fluorescence range, its screening becomes complicated and require complementary assays. One of such molecules, 6, 7-dihydroxycoumarin (6, 7-DHC, also known as aesculetin, esculetin, and cichorigenin) is fluorescent in the ThT emission range and absorbs in the ThT excitation range. Therefore, it can produce a subtractive effect attributed to primary inner filter effect and/or additive effect due to its self-fluorescence in ThT assay. Our study shows that 6, 7-DHC produces an additive effect in ThT fluorescence, which is minimized at high concentration of ThT and decrease in ThT fluorescence is solely due to its inhibitory effect against HSA fibrillation. These ThT fluorescence-based results are verified through other complementary assays, such as Rayleigh and dynamic light scattering and amyloid-specific Congo red binding assay. Furthermore, hydrophobicity reduction is studied through Nile red (NR) and kinetics through far-UV circular dichroism (far-UV CD) in place of the most commonly employed ThT assay owing to extremely high fluorescence of 6, 7-DHC during initial incubation period.

Fluorescence method for kanamycin detection based on the conversion of G-triplex and G-quadruplex.

A versatile fluorescence scaffold was constructed by connecting a G-triplex sequence (G31) with G-rich DNA (aptamer of kanamycin) and using thioflavin T (ThT) as the fluorescent molecule. With the assistance of an aptamer, the G-quadruplex DNA structure was fabricated using G31 as three strands and the aptamer as the fourth strand. Due to the parallel planar morphology of the final products, which was favorable for ThT binding and which restricted the rotation of the aromatic rings of ThT, the fluorescence signal intensity of ThT was significantly enhanced. Because of the specific interaction of aptamer and kanamycin, in addition to the greater ability for kanamycin to bind with G-triplex than ThT, the conformation of G-quadruplex DNA was changed; in addition, ThT was dissociated from the aptamer-G31, and therefore a 'turn-on' to 'turn-off' detection principle was applied for kanamycin detection, which yielded reasonable sensitivity and selectivity. The detection range was 50-2000 nM, with a limit of detection of 1.05 nM. Our proposed method was thus successfully applied for kanamycin determination in pork, chicken, and beef samples, and satisfactory results were obtained.

Prospective observational study of abdominal wall reconstruction with THT technique in primary midline defects with diastasis recti: clinical and functional outcomes in 110 consecutive patients.

INTRODUCTION: Surgical treatment of diastasis recti is still a matter of debate. Open approaches such as abdominoplasty, which offer the possibility to combine reparation of the diastasis with abdominal cosmetic surgery, are challenged by the emerging less-invasive laparoscopic or robotic techniques that offer shorter recovery for patients. However, evidence in favour of one of the two approaches concerning both short- and long-term complications and functional results is still lacking. In this paper, we analysed clinical and functional results of a new endo-laparoscopic technique for midline reconstruction (THT technique) in patients with primary abdominal wall defects associated with diastasis recti. METHODS: Prospective observational study on 110 consecutive patients was submitted to endo-laparoscopic reconstruction of the abdominal wall with linear staplers. Morbidity and relapse rates with clinical and radiological follow-up were recorded at 1, 6, 12, and 24 months after the operation. Data regarding the impact of surgery on patients' quality of life (EuraHSQol) on chronic low back pain (Oswestry Disability Index, ODI) and urinary stress incontinence (Incontinence Severity Index, ISI) were gathered. RESULTS: After a mean follow-up of 14 months, the morbidity rate was 9.1% and no recurrences were recorded. 6-month follow-up ultrasound showed a rectus muscles mean distance of 6.7 mm; EuraHSQol, ODI, and ISI scores significantly improved in 93%, 77%, and 63% of the cases, respectively. CONCLUSIONS: The THT technique proved to be a feasible, safe, and effective alternative for corrective surgery of primary midline hernias associated with diastasis recti. Short- and mid-term results are encouraging but need to be confirmed by further studies with longer follow-up. The achieved midline reconstruction offers a significant improvement of patients' perceived quality of life through reduction of abdominal wall pain, bulging, low back pain, and urinary stress incontinence.

Ions in the Deep Subsurface of Earth, Mars, and Icy Moons: Their Effects in Combination with Temperature and Pressure on tRNA-Ligand Binding.

The interactions of ligands with nucleic acids are central to numerous reactions in the biological cell. How such reactions are affected by harsh environmental conditions such as low temperatures, high pressures, and high concentrations of destructive ions is still largely unknown. To elucidate the ions' role in shaping habitability in extraterrestrial environments and the deep subsurface of Earth with respect to fundamental biochemical processes, we investigated the effect of selected salts (MgCl2, MgSO4, and Mg(ClO4)2) and high hydrostatic pressure (relevant for the subsurface of that planet) on the complex formation between tRNA and the ligand ThT. The results show that Mg2+ salts reduce the binding tendency of ThT to tRNA. This effect is largely due to the interaction of ThT with the salt anions, which leads to a strong decrease in the activity of the ligand. However, at mM concentrations, binding is still favored. The ions alter the thermodynamics of binding, rendering complex formation that is more entropy driven. Remarkably, the pressure favors ligand binding regardless of the type of salt. Although the binding constant is reduced, the harsh conditions in the subsurface of Earth, Mars, and icy moons do not necessarily preclude nucleic acid-ligand interactions of the type studied here.

Trypsin Induced Degradation of Amyloid Fibrils.

Proteolytic enzymes are known to be involved in the formation and degradation of various monomeric proteins, but the effect of proteases on the ordered protein aggregates, amyloid fibrils, which are considered to be extremely stable, remains poorly understood. In this work we study resistance to proteolytic degradation of lysozyme amyloid fibrils with two different types of morphology and beta-2-microglobulun amyloids. We showed that the proteolytic enzyme of the pancreas, trypsin, induced degradation of amyloid fibrils, and the mechanism of this process was qualitatively the same for all investigated amyloids. At the same time, we found a dependence of efficiency and rate of fibril degradation on the structure of the amyloid-forming protein as well as on the morphology and clustering of amyloid fibrils. It was assumed that the discovered relationship between fibrils structure and the efficiency of their degradation by trypsin can become the basis of a new express method for the analysis of amyloids polymorphism. Unexpectedly lower resistance of both types of lysozyme amyloids to trypsin exposure compared to the native monomeric protein (which is not susceptible to hydrolysis) was attributed to the higher availability of cleavage sites in studied fibrils. Another intriguing result of the work is that the cytotoxicity of amyloids treated with trypsin was not only failing to decline, but even increasing in the case of beta-2-microglobulin fibrils.

Evidence for different in vitro oligomerization behaviors of synthetic hIAPP obtained from different sources.

Type 2 diabetes is characterized by the aggregation of human islet amyloid polypeptide (hIAPP), from monomer to amyloid deposits that are made of insoluble fibrils. Discrepancies concerning the nature of formed species or oligomerization kinetics among reported in vitro studies on hIAPP aggregation process have been highlighted. In this work, we investigated if the sample itself could be at the origin of those observed differences. To this aim, four hIAPP samples obtained from three different sources or suppliers have been analyzed and compared by ThT fluorescence spectroscopy and by two recently developed techniques, capillary electrophoresis (CE), and ESI-IMS-QToF-MS. Lots provided by the same supplier were shown to be very similar whatever the analytical technique used to characterize them. In contrast, several critical differences could be pointed out for hIAPP provided by different suppliers. We demonstrated that in several samples, some oligomerized peptides (e.g., dimer) were already present upon reception. Purity was also different, and the proneness of the peptide solution to form fibrils in vitro within 24 h could vary considerably from one sample source to another but not from lot to lot of the same source. All those results demonstrate that the initial state of conformation, oligomerization, and quality of the hIAPP can greatly impact the aggregation kinetics, and thus the information provided by these in vitro tests. Finally, a careful selection of the peptide batch and source is mandatory to perform relevant in vitro studies on hIAPP oligomerization and to screen new molecules modulating this pathological process. Graphical abstract.