Phasor FLIM analysis of Thioflavin T fluorescence in protein amyloid aggregates: Mapping molecular interactions.

Thioflavin T (ThT) is a worldwide used dye to monitor protein aggregation as it stains with a certain specificity amyloid structures. The interactions between ThT and its hosts are largely studied suggesting that fluorescence properties of this dye critically depend both on the environment rigidity, electrostatic and hydrophobic properties as well as on molecular details binding site structure. Here FLIM and phasor approach analysis are used to exploit ThT amyloid interactions and, in turn, to address polymorphism and structural heterogeneity of amyloid species mapping aggregate-to-aggregate structural differences and revealing details of molecular architecture within the same aggregate.

Lead(II) ions adsorption onto amyloid particulates: An in depth study

The production of new cost-effective biocompatible sorbent sustainable materials, with natural origins, able to remove heavy metals from water resources is nowadays highly desirable in order to reduce pollution and increase clean water availability. In this context, self-assembled protein materials with amyloid structures seem to have a great potential as natural platform for a broader development of highly-tunable structures. In this work we show how protein particulates, a generic form of protein aggregates, with spherical micro sized shape can be used as adsorbents of Pb2+ ions from aqueous solution. The effect of pH, ionic medium, ionic strength and temperature of the metal ion solution…

3D MICROSIZED PROTEIN AMYLOID PARTICULATES AS EFFECTIVE BIOADSORBERS FOR Pb2+ IONS

The production of new cost-effective biocompatible sorbent sustainable materials, with natural origins, able to remove heavy metals from water resources is nowadays highly desirable in order to reduce pollution and increase clean water availability. In this context, self-assembled protein materials with highly ordered amyloid structures, stabilised by H-bonds, seems to have a great potential as natural platform for a broader development of highly-tunable structures. By suitably varying solution conditions it is possible to tune aggregate size, surface area and morphologies as well as their physicochemical (hydrophobicity, hydrophilicity, swelling/deswelling properties) and mechanical proper…

Phasor-FLIM analysis of Thioflavin T self-quenching in Concanavalin amyloid fibrils

The formation of amyloid structures has traditionally been related to human neurodegenerative pathologies and, in recent years, the interest in these highly stable nanostructures was extended to biomaterial sciences. A common method to monitor amyloid growth is the analysis of Thioflavin T fluorescence. The use of this highly selective dye, diffused worldwide, allows mechanistic studies of supramolecular assemblies also giving back important insight on the structure of these aggregates. Here we present experimental evidence of self-quenching effect of Thioflavin T in presence of amyloid fibrils. A significant reduction of fluorescence lifetime of this dye which is not related to the propert…

TRANSPORTAN 10 INTERACTION WITH GIANT VESICLES: INSERTION EFFECTS AND PORE FORMATION

Transportan 10 (TP10) is a 21 residues peptide that belongs to the family of the antimicrobial and cytolytic or cytotoxic amphipathic peptides. It contains a high proportion of positively charged amino acids (four lysines), no negative charges and the N-terminus that impart it a formal +5 charge at neutral pH.1 This large number of positive charges is an essential feature for the electrostatic interaction of TP10 with microbial and tumoral membranes, which are characherized by a net negative charge and also by a higher fluidity if compared with mammalin ones.2 Here, combining spectroscopic and fluorescence lifetime imaging techniques, we analyse the fate of the multifunctional3-4 TP10 and i…

α-casein micelles-membranes interaction: Flower-like lipid protein coaggregates formation

Background: Environmental conditions regulate the association/aggregation states of proteins and their action in cellular compartments. Analysing protein behaviour in presence of lipid membranes is fundamental for the comprehension of many functional and dysfunctional processes. Here, we present an experimental study on the interaction between model membranes and α-casein. α-casein is the major component of milk proteins and it is recognised to play a key role in performing biological functions. The conformational properties of this protein and its capability to form supramolecular structures, like micelles or irreversible aggregates, are key effectors in functional and pathological effects…

Ingestion of plastic and non-plastic microfibers by farmed gilthead sea bream (Sparus aurata) and common carp (Cyprinus carpio) at different life stages

Environmental pollution by plastic particles is of major global concern, as a potential threat to aquatic organisms and ecosystems. The accumulation of microplastics in freshwater and marine environments has strong ecological implications due to their long persistence, potential toxicity, and ability to adsorb other pollutants, acting as vectors of pathogens. Nevertheless, while the number of studies on the presence of microplastics in the wild fish increased, less attention has been paid to the farmed fish species. Here, we investigated the occurrence of microparticles in the digestive tracts of Sparus aurata and Cyprinus carpio at different life stages and reared by an intensive and semi-…

Nano-structured myelin: new nanovesicles for targeted delivery to white matter and microglia, from brain-to-brain

Neurodegenerative diseases affect millions of people worldwide and the presence of various physiological barriers limits the accessibility to the brain and reduces the efficacy of various therapies. Moreover, new carriers having targeting properties to specific brain regions and cells are needed in order to improve therapies for the brain disorder treatment. In this study, for the first time, Myelin nanoVesicles (hereafter defined MyVes) from brain-extracted myelin were produced. The MyVes have an average diameter of 100–150 ​nm, negative zeta potential, spheroidal morphology, and contain lipids and the key proteins of the myelin sheath. Furthermore, they exhibit good cytocompatibility. The…

Insight into mechanisms of creatinine optical sensing using fluorescein-gold complex

Abstract Creatinine level in biological fluids is a clinically relevant parameter to monitor vital functions and it is well assessed that measuring creatinine levels in the human body can be of great utility to evaluate renal, muscular, or thyroid dysfunctions. The accurate detection of creatinine levels may have a critical role in providing information on health status and represents a tool for the early diagnosis of severe pathologies. Among different methods for creatinine detection that have been introduced and that are evolving with increasing speed, fluorescence-based and colorimetric sensors represent one of the best alternatives, thanks to their affordability, sensitivity and easy r…

Phasor-FLIM for a direct investigation of Transportan 10 interactions with model membranes

Transportan 10 (TP10), a short and positive charged peptide, belonging to the family of the cell penetrating peptides has gained increasing attention for its antimicrobial and anticancer activity but also for its applications in drug delivery as it is able to translocate therapeutic molecules in cellular environment. Due to the complexity of the phenomena involved in cellular uptake and following processes, which strongly depend on the membrane lipid composition, structural details of the peptide (e.g., charge, hydrophobicity, steric hindrance) and environmental conditions, it is not easy to understand the general rules governing them. Here, we combine spectroscopic techniques and fluoresce…

Sustainable soy protein microsponges for efficient removal of lead (II) from aqueous environments

Protein-based materials recently emerged as good candidates for water cleaning applications, due to the large availability of the constituent material, their biocompatibility and the ease of preparation. In this work, new adsorbent biomaterials were created from Soy Protein Isolate (SPI) in aqueous solution using a simple environmentally friendly procedure. Protein microsponge-like structures were produced and characterized by means of spectroscopy and fluorescence microscopy methods. The efficiency of these structures in removing Pb2+ ions from aqueous solutions was evaluated by investigating the adsorption mechanisms. The molecular structure and, consequently, the physico-chemical propert…

Transportan 10 Induces Perturbation and Pores Formation in Giant Plasma Membrane Vesicles Derived from Cancer Liver Cells

Continuous progress has been made in the development of new molecules for therapeutic purposes. This is driven by the need to address several challenges such as molecular instability and biocompatibility, difficulties in crossing the plasma membrane, and the development of host resistance. In this context, cell-penetrating peptides (CPPs) constitute a promising tool for the development of new therapies due to their intrinsic ability to deliver therapeutic molecules to cells and tissues. These short peptides have gained increasing attention for applications in drug delivery as well as for their antimicrobial and anticancer activity but the general rules regulating the events involved in cell…

Different a-casein association states and their interaction with lipid vesicles to study antibacterial activity

The interactions between caseins and lipid membranes are fundamental for the physiological function of these proteins. Moreover, the understanding of the underlying molecular mechanisms is of great interest for the development of new casein derived antimicrobial peptides. Indeed, it was already shown that peptides derived from caseins possess antibacterial activity but their mechanisms of action is still debated. Here, we present an experimental study on the interaction between model lipid membranes and a-casein by means of spectroscopy and fluorescence microscopy techniques. a-casein is an unfolded protein that due to its amphiphilic nature is known to self-assembly into non-stable micella…

Effect of cholesterol on the interaction between amphyphylic peptides and liposomes

With the rise of antibiotic resistance, antimicrobial peptides (AMPs) have been proposed as an alternative novel class of therapeutic agents. They are polycationic, with a net positive charge of more than +2, and they are characterized by amphipathic structures, with both a hydrophobic and a hydrophilic domain. These characteristics allow them to selectively bind to negatively charged lipids (largely present in bacteria, not in mammalian cells), via hydrophobic and electrostatic interactions. Moreover, mammalian cells are characterized by a high content of cholesterol. For this reason, here we present an experimental study on the effect of the presence of cholesterol on the capability of am…

Peptide–Membrane Interactions Monitored by Fluorescence Lifetime Imaging: A Study Case of Transportan 10

The interest on detailed analysis of peptide-membrane interactions is of great interest in both fundamental and applied sciences as these may relate to both functional and pathogenic events. Such interactions are highly dynamic and spatially heterogeneous, making the investigation of the associated phenomena highly complex. The specific properties of membranes and peptide structural details, together with environmental conditions, may determine different events at the membrane interface, which will drive the fate of the peptide-membrane system. Here, we use an experimental approach based on the combination of spectroscopy and fluorescence microscopy methods to characterize the interactions …

Common mechanisms underlying membrane modifications induced by protein interactions