Advanced microscopy analysis of the micro-nanoscale architecture of human menisci

AbstractThe complex inhomogeneous architecture of the human meniscal tissue at the micro and nano scale in the absence of artefacts introduced by sample treatments has not yet been fully revealed. The knowledge of the internal structure organization is essential to understand the mechanical functionality of the meniscus and its relationship with the tissue’s complex structure. In this work, we investigated human meniscal tissue structure using up-to-date non-invasive imaging techniques, based on multiphoton fluorescence and quantitative second harmonic generation microscopy complemented with Environmental Scanning Electron Microscopy measurements. Observations on 50 meniscal samples extract…

Amyloid-Like Superstructures: mechanisms of formation and morphologies

Characterization of protofibrillar aggregates of bovine serum albumin by tryptophans fluorescence lifetime

We report an experimental study on the thermally induced aggregation of Bovine Serum Albumin at basic pH. In these conditions, we observe the growth of simple protofibrillar structures via the formation of intermolecular beta-sheets promoted by the increased electrostatic repulsion. Here we present a study on the time resolved fluorescence of Tryptophans (Trp) along the aggregation kinetics in the above reported conditions. We use the lifetimes distribution approach as a useful tool for the interpretation of the fluorescence decay in terms of protein conformational substates and interconversion dynamics. Trp fluorescence lifetime depends from protein conformations, also in relation with sol…

Protein aggregates for water purification

Water pollution is the contamination of water bodies including lakes, rivers, oceans and it is a leading global risk factor for illness and death for people, plants and living organisms. A principal source of water pollution is industry, from which increasing amounts of toxic pollutants are released including heavy metals such as cobalt, lead and copper. Today various technologies for purifying contaminated water can be applied, a lot of them being typically expensive, ion specific and characterised by low efficiency. For these reason, the search of new biocompatible materials with increased capabilities is strongly needed.Protein aggregates have already revealed their potential as environm…

Oxidative alteration of Human Serum Albumin Amyloid Aggregation Pathway

Thermal aggregation of proteins in presence of metal ions.

The study of the aggregation processes in presence of metal ions is an essential step for understanding the key role of metals in protein-protein and protein-solvent interactions. Indeed, the presence of metal ions can radically change the main features of the standard denaturation/aggregation processes and such effects result to be strongly dependent on the kind of metal and on its concentration. Metal ions have an active role in thermal aggregation and cold set gelation processes. These processes are intrinsically different, but both are based on the proteins ability to form aggregates.

ThT influences Abeta(1-40) aggregation process

Improved Photocatalytic Activity of Polysiloxane TiO2 Composites by Thermally Induced Nanoparticle Bulk Clustering and Dye Adsorption

Fine control of nanoparticle clustering within polymeric matrices can be tuned to enhance the physicochemical properties of the resulting composites, which are governed by the interplay of nanoparticle surface segregation and bulk clustering. To this aim, out-of-equilibrium strategies can be leveraged to program the multiscale organization of such systems. Here, we present experimental results indicating that bulk assembly of highly photoactive clusters of titanium dioxide nanoparticles within an in situ synthesized polysiloxane matrix can be thermally tuned. Remarkably, the controlled nanoparticle clustering results in improved degradation photocatalytic performances of the material under …

Conformational changes involved in thermal aggregation processes of bovine serum albumin

We report a kinetic study on thermal aggregation process of the model protein bovine serum albumin (BSA) in low concentration regime. Aim of this study is to provide information on relationship between conformational changes and initial step of aggregation. The experimental approach is based on steady-state fluorescence spectra of the two tryptophans located in two different domains, in way to study conformational changes in the surrounding of these residues. We also follow emission spectra of Fluorescein-5-Maleimide dye bound to the single free cysteine of BSA. Complementary information on the extent of aggregation and on the structural changes is obtained by Rayleigh scattering and circul…

Thermal induced conformational changes involved in the aggregation pathways of beta-lactoglobulin.

Aggregation of proteins appears to be associated most often with conformational and structural changes that lead to exposure of some apolar residues. Depending on the native structure of the protein in exam, aggregation is a process that involves different mechanisms, whose time of occurrence and interplay can depend upon temperature. To single out information about the multistages of the aggregation pathway, here we investigate the thermally induced conformational and structural changes of the beta-lactoglobulin (BLG). The experimental approach consists in studying steady-state fluorescence spectra of intrinsic chromophores, two tryptophans, and Anylino-Naphthalene-Sulfonate dye (ANS) mole…

Back to the oligomeric state: pH-induced dissolution of concanavalin A amyloid-like fibrils into non-native oligomers

The subtle interplay between long range electrostatic forces, hydrophobic interactions and short range protein-protein interactions regulates the onset/evolution of protein aggregation processes as well as the stability of protein supramolecular structures. Using a combination of FTIR spectroscopy, light scattering and advanced imaging, we present evidence on the main role of electrostatic forces in the formation and stability of amyloid-like fibrils formed from concanavalin A (ConA), a protein showing structural homology with the human serum amyloid protein. At high protein concentration, where protein-protein interactions cannot be neglected, we highlight a thermal-induced aggregation pat…

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.

Detecting Protein Aggregation on Cells Surface: Concanavalin A Oligomers Formation

A number of neurodegenerative diseases involve protein aggregation and amyloid formation. Recently evidence has emerged indicating small-transient prefibrillar oligomers as the primary pathogenic agents. Noteworthy, strict analogies exist between the behaviour of cells in culture treated with misfolded non-pathogenic proteins and in pathologic conditions, this instance together with the observation that the oligomers and fibrils are characterised by common structural features suggest that common mechanisms for cytotoxicity could exists and have to be perused in common interactions involved in aggregation.We here report an experimental study on ConcanavalinA (ConA) aggregation and its effect…

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…

Confocal and Two-Photon Spectroscopy

The spectroscopic analysis of bulk samples provides useful information at the mesoscopic scale that can be fostered when coupled with microscopy, which allows to integrate the spatial information in the analysis, this being of utmost importance for non‐homogeneous samples. Technologies such as confocal and two‐photon microscopy quickly became mainstream methods as they provide the possibility of noninvasive real‐time analysis of specimens of different nature, eliciting specific features even in samples with non‐ergodic behaviour. The spatial resolution of optical microscopy has its roots in the diffraction of light on the objective lens of the imaging system. Fluorescence microscopy is an o…

CONCANAVALIN A AGGREGATION AND TOXICITY ON NEUROBLASTOMA LAN5 CELL CULTURES

Thermal aggregation of bovine serum albumin at different pH: comparison with human serum albumin.

We report here a study on thermal aggregation of BSA at two different pH values selected to be close to the isoelectric point (pI) of this protein. Our aim is to better understand the several steps and mechanisms accompanying the aggregation process. For this purpose we have performed kinetics of integrated intensity emission of intrinsic and extrinsic dyes, tryptophans and ANS respectively, kinetics of Rayleigh scattering and of turbidity. The results confirm the important role played by conformational changes in the tertiary structure, especially in the exposure of internal hydrophobic regions that promote intermolecular interactions. We also confirm that the absence of electrostatic repu…

Concanavalin A toxicity on Neuroblastoma LAN5 cell cultures

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…

Observation of the Early Structural Changes Leading to the Formation of Protein Superstructures.

Formation of superstructures in protein aggregation processes has been indicated as a general pathway for several proteins, possibly playing a role in human pathologies. There is a severe lack of knowledge on the origin of such species in terms of both mechanisms of formation and structural features. We use equine lysozyme as a model protein, and by combining spectroscopic techniques and microscopy with X-ray fiber diffraction and ab initio modeling of Small Angle X-ray Scattering data, we isolate the partially unfolded state from which one of these superstructures (i.e., particulate) originates. We reveal the low-resolution structure of the unfolded state and its mechanism of formation, hi…

Nucleation mechanisms and spatial hetereogeneity in insulin amyloid fibrils formation

Near-Infrared Light Responsive Folate Targeted Gold Nanorods for Combined Photothermal-Chemotherapy of Osteosarcoma.

Folate-targeted gold nanorods (GNRs) are proposed as selective theranostic agents for osteosarcoma treatment. An amphiphilic polysaccharide based graft-copolymer (INU-LA-PEG-FA) and an amino derivative of the α,β-poly(N-2-hydroxyethyl)-d,l-aspartamide functionalized with folic acid (PHEA-EDA-FA), have been synthesized to act as coating agents for GNRs. The obtained polymer-coated GNRs were characterized in terms of size, shape, zeta potential, chemical composition, and aqueous stability. They protected the anticancer drug nutlin-3 and were able to deliver it efficiently in different physiological media. The ability of the proposed systems to selectively kill tumor cells was tested on U2OS…

Microwave-assisted synthesis of anhydrous CdS nanoparticles in a water-oil microemulsion.

Abstract Microwave irradiation at a frequency of 2.45 GHz and a power ranging between 22 and 30 W was used, in a water–oil microemulsion at 35 ± 2 ° C , to obtain stable, small, crystalline, anhydrous CdS nanoparticles exhibiting enhanced luminescence properties. The process of nanoparticles growth at different irradiation times was followed by UV–vis spectroscopy. It was observed that irradiated nanoparticles grew faster and their size reached a constant value. The final mean nanoparticle diameter was 2.7 nm, smaller than that observed in a non-irradiated sample, in which particle dimensions slowly increased even after 10 h. This finding was confirmed by high resolution transmission electr…

pH effect on thermal aggregation of concanavalin A

Mast Cells and Th17 Cells Contribute to the Lymphoma-Associated Pro-Inflammatory Microenvironment of Angioimmunoblastic T-Cell Lymphoma

Reports focusing on the immunological microenvironment of peripheral T-cell lymphomas (PTCL) are rare. Here we studied the reciprocal contribution of regulatory (Treg) and interleukin-17-producing (Th17) T-cells to the composition of the lymphoma-associated microenvironment of angioimmunoblastic T-cell lymphoma (AITL) and PTCL not otherwise specified on tissue microarrays from 30 PTCLs not otherwise specified and 37 AITLs. We found that Th17 but not Treg cells were differently represented in the two lymphomas and correlated with the amount of mast cells (MCs) and granulocytes, which preferentially occurred in the cellular milieu of AITL cases. We observed that MCs directly synthesized inter…

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…

Autonomous molecularly crowded confinement in inkjet printed femtoliter-scale aqueous compartments

Natural evolution has chosen the localization of biomolecular processes into crowded sub-cellular femtoliter (fL) scale compartments for organizing complex biological processes. [1] Many synthetic biology platforms with life-like activities have been able to mimic these systems under different compartment sizes regimes. [2] However, the fabrication of crowded compartments down to sub-cellular scales is challenging, mainly because of high surface-volume ratio of these systems, finally compromising the stability of the encapsulated biomolecules. In this regard, we here bridge this gap by showing the possibility to produce femtoliter-scale aqueous droplets using a novel inkjet printing approac…

Bovine Serum Albumin protofibril-like aggregates formation: Solo but not simple mechanism

We report an experimental study on the model protein Bovine Serum Albumin (BSA), with the aim of elucidating the mechanisms by which a fully folded globular protein undergoes different aggregation pathways leading to the formation of amyloid fibrils or amorphous aggregates. We observe thermally induced formation of fibrillar structures at pH far from the protein isoelectric point. The increase of electrostatic repulsion results in protein destabilization and in modifications of inter and intra-molecular interactions leading to the growth of fibril-like aggregates stabilized by inter-molecular-β sheets. The aggregation kinetics is studied by means of fluorescence techniques, light scattering…

Thioflavin T templates amyloid β(1–40) conformation and aggregation pathway

Aβ(1-40) peptide supramolecular assembly and fibril formation processes are widely recognized to have direct implications in the progression of Alzheimer's disease. The molecular basis of this biological process is still unknown and there is a strong need of developing effective strategies to control the occurring events. To this purpose the exploitation of small molecules interacting with Aβ aggregation represents one of the possible routes. Moreover, the use specific labeling has represented so far one of the most common and effective methods to investigate such a process. This possibility in turn rests on the reliability of the probe/labels involved. Here we present evidences of the effe…

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…

Protein-membrane interaction: insights from advanced microscopy

The interaction between proteins and membranes is sub ject of renewed interest in biomedical and biotechnologi cal research for its implication in many functional and dys functional processes and for its pharmaceutical applications. It has been shown that the interaction between amyloido genic proteins and membranes results in mutually destruc tive structural perturbations. The study we present is focused on the interaction between synthetic model membranes and alpha-lactalbumin (α-La), widely studied for its biolog ical function since it can induce apoptosis in tumor cells. Upon α-La addition to giant vesicles (GVs) samples, the sys tem has been characterized by means of spectroscopy meth …

Thioflavin T Promotes Aβ(1−40) Amyloid Fibrils Formation

Fibrillogenesis of the small peptide Aβ(1-40) is considered to be the hallmark of Alzheimer's disease. Some evidence indicates small oligomers, rather than mature fibrils, as the key cytotoxic agents. The fluorescent dye Thioflavin T (ThT) is often used to detect amyloid deposits in both in vivo and in vitro experiments, and it is used to study kinetic measurements, under the fundamental hypothesis that this probe does not influence the aggregation processes. We report experimental data showing that ThT may promote the Aβ(1-40) peptide amyloid aggregation changing solvent-peptide interactions and stabilizing more ordered β-like conformation. This finding has a two-fold importance: It is a f…

Smart hydrogels for novel optical functions

Nanocomposites of inherently conductive polyaniline (PANT) within a highly hydrophilic polyvinyl alcohol (PVA) based hydrogel have been produced by coupling a conventional dispersion chemical oxidative polymerization to a subsequent high energy irradiation step, in order to convert the polymer stabilizing the aqueous dispersion, namely the PVA, into a highly water swollen hydrogel incorporating the PANT particles. The incorporation of the electroactive and "pH-sensitive" polymer into a transparent and highly permeable hydrogel matrix has been pursued as a route to the development of a novel class of potentially biocompatible, smart hydrogels that can respond to changes of the surrounding en…

Heterogeneity of aggregates in the fibrillation mechanisms of proteins probed by time resolved fluorescence

Under appropriate conditions almost all proteins are able to aggregate to form long, well-ordered and beta-sheet rich arrays known as amyloid fibrils. The formation of such structures involves complex intra and intermolecular interactions modulated by the structure and dynamics of the native protein, and by the physico-chemical properties of the solvent. Multiple interactions and cross-feedback during the aggregation pathway cause different ultimate aggregates’ morphologies and the possible simultaneous occurrence of multiple species. The structural definition of such assemblies is complicated by the polymorphism of the amyloid fibrils. Aim of this study is to inquire on the different natur…

Optical properties of biocompatible polyaniline nano-composites

Abstract Polyaniline (PANI) is an electro-active polymer of great interest thanks to its outstanding physical and chemical properties which make it suitable for various applications in optics, bioelectronics, biosensors, diagnostics and therapeutic devices. Unfortunately, PANI is infusible and insoluble in most common solvents and, thus, very difficult to process. In the attempt of improving processability, yet preserving its interesting properties, PANI has been synthesized in the form of particles and dispersed into a hydrogel matrix. The synthesis of PANI–hydrogel composites proceeds via γ-irradiation of PANI dispersions as obtained by ‘in situ’ polymerization of aniline in the presence …

Radiation processing: an alternative and environmentally friendly way to produce advanced Materials. Synthesis of PANI/hydrogels composites

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…

Molecular characterization of tunable microscale protein-based biomaterials

Monitoring few molecular binding events in scalable confined aqueous compartments by raster image correlation spectroscopy (CADRICS)

The assembly of scalable liquid compartments for binding assays in array formats constitutes a topic of fundamental importance in life sciences. This challenge can be addressed by mimicking the structure of cellular compartments with biological native conditions. Here, inkjet printing is employed to develop up to hundreds of picoliter aqueous droplet arrays stabilized by oil-confinement with mild surfactants (Tween-20). The aqueous environments constitute specialized compartments in which biomolecules may exploit their function and a wide range of molecular interactions can be quantitatively investigated. Raster Image Correlation Spectroscopy (RICS) is employed to monitor in each compartmen…

Effects of succinylation on thermal induced amyloid formation in Concanavalin A.

We have recently shown that upon slight thermal destabilization the legume lectin Concanavalin A may undergo two different aggregation processes, leading, respectively, to amyloid fibrils at high pH and amorphous aggregates at low pH. Here we present an experimental study on the amyloid aggregation of Succinyl Concanavalin A, which is a dimeric active variant of Concanavalin. The results show that, as for the native protein, the fibrillation process appears to be favoured by alkaline pH, far from the isoelectric point of the protein. Moreover, it strongly depends on temperature and requires large conformational changes both at secondary and tertiary structure level. With respect to the nati…

Fluctuation Methods To Study Protein Aggregation in Live Cells: Concanavalin A Oligomers Formation

Prefibrillar oligomers of proteins are suspected to be the primary pathogenic agents in several neurodegenerative diseases. A key approach for elucidating the pathogenic mechanisms is to probe the existence of oligomers directly in living cells. In this work, we were able to monitor the process of aggregation of Concanavalin A in live cells. We used number and brightness analysis, two-color cross number and brightness analysis, and Raster image correlation spectroscopy to obtain the number of molecules, aggregation state, and diffusion coefficient as a function of time and cell location. We observed that binding of Concanavalin A to the membrane and the formation of small aggregates paralle…

Towards bioarrays of cellular-like compartments for monitoring few molecular binding events

The aim of this work is to artificially reproduce scalable cellular-like compartments on a chip, thus realizing specialized small volume systems to study the behaviour of interacting biomolecules by few binding events. In particular, we show an unprecedented solution-based protein-binding assay based on arrays of oil-confined water droplets containing protein targets, labelled ligands and other compounds.

Cholesterol facilitates interactions between α‐synuclein oligomers and charge‐neutral membranes

AbstractOligomeric species formed during α-synuclein fibrillation are suggested to be membrane-disrupting agents, and have been associated with cytotoxicity in Parkinson’s disease. The majority of studies, however, have revealed that the effect of α-synuclein oligomers is only noticeable on systems composed of anionic lipids, while the more physiologically relevant zwitterionic lipids remain intact. We present experimental evidence for significant morphological changes in zwitterionic membranes containing cholesterol, induced by α-synuclein oligomers. Depending on the lipid composition, model membranes are either unperturbed, disrupt, or undergo dramatic morphological changes and segregate …

Aqueous Processed Biopolymer Interfaces for Single-Cell Microarrays

Single-cell microarrays are emerging tools to unravel intrinsic diversity within complex cell populations, opening up new approaches for the in-depth understanding of highly relevant diseases. However, most of the current methods for their fabrication are based on cumbersome patterning approaches, employing organic solvents and/or expensive materials. Here, we demonstrate an unprecedented green-chemistry strategy to produce single-cell capture biochips onto glass surfaces by all-aqueous inkjet printing. At first, a chitosan film is easily inkjet printed and immobilized onto hydroxyl-rich glass surfaces by electrostatic immobilization. In turn, poly(ethylene glycol) diglycidyl ether is graft…

THERMALLY INDUCED FIBRILLAR AGGREGATION OF BOVINE SERUM ALBUMIN

Blue autofluorescence in protein aggregates “lighted on” by UV induced oxidation

Oxidation of amino acid side chains in protein structure can be induced by UV irradiation leading to critical changes in molecular structure possibly modifying protein stability and bioactivity. Here we show, by using a combination of multiple spectroscopic techniques and Fluorescence Lifetime Imaging, that UV-light exposure induces irreversible oxidation processes in Ubiquitin structure. In particular, the growth of a new autofluorescence peak in the blue region is detected, that we attribute to tyrosine oxidation products. Blue autofluorescence intensity is found to progressively increase also during aggregation processes leading to the formation of aggregates of non-amyloid nature. Signi…

Thermal aggregation of two “beta-protein” models at different pH values.

Proinflammatory and amyloidogenic S100A9 induced by traumatic brain injury in mouse model.

Traumatic brain injury (TBI) represents a significant risk factor for development of neurodegenerative diseases such as Alzheimer’s and Parkinson’s. The S100A9-driven amyloid-neuroinflammatory cascade occurring during primary and secondary TBI events can serve as a mechanistic link between TBI and Alzheimer’s as demonstrated recently in the human brain tissues. Here by using immunohistochemistry in the controlled cortical impact TBI mouse model we have found pro-inflammatory S100A9 in the brain tissues of all mice on the first and third post-TBI days, while 70% of mice did not show any S100A9 presence on seventh post-TBI day similar to controls. This indicates that defensive mechanisms effe…

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-…

High-throughput screening at the picoliter scale by combining Dip Pen Lithography with Inkjet printing

Drug screening is a complex, expensive and time consuming field consisting of diseasebased target identification in conjunction with high-throughput screening of chemical and natural product libraries. Conventional drug screening technology is usually time and reagent consuming (micro-, nanoliter scale) and is based on complex liquid handling robotics. In this work, we show a low-cost and miniaturized drug screening methodology based on direct bio-printing methodologies like Inkjet Printing and Dip Pen Lithography. We show the possibility to precisely deliver femtoliter scale droplets of protein targets by Dip Pen Lithography by finely tuning deposition parameters. This allows obtaining mic…

Unlocked Concanavalin A Forms Amyloid-like Fibrils from Coagulation of Long-lived "Crinkled'' Intermediates

Understanding the early events during amyloid aggregation processes is crucial to single out the involved molecular mechanisms and for designing ad hoc strategies to prevent and reverse amyloidogenic disorders. Here, we show that, in conditions in which the protein is positively charged and its conformational flexibility is enhanced, Concanavalin A leads to fibril formation via a non-conventional aggregation pathway. Using a combination of light scattering, circular dichroism, small angle X-ray scattering, intrinsic (Tryptophan) and extrinsic (ANS) fluorescence and confocal and 2-photon fluorescence microscopy we characterize the aggregation process as a function of the temperature. We high…

Hydrogel Nanocomposites Containing Polyaniline With Different Morphologies for Possible Bioengineering Applications

Different aggregation pathways of human and bovine Serum Albumin.

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…

Highly tunable protein microspheres for drug delivery

It is well-known that protein amyloid aggregation has profound implications in several neurodegenerative diseases. In contrast, a natural role for amyloid structures as protection, adhesion and storage materials in living system is also reported, promoting protein aggregates as an interesting platform for the design of multifunctional biomaterials. Among the broad range of different amyloid structures protein particulates deserve special attention; they are spherical protein aggregates with radius ranging from hundreds of nm to few um which are readily formed in solution at pHs values near the isoelectric point of the protein they are made of. Interestingly, particulate appears to be a gene…

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…

Amyloid Fibrils Formation in Concanavalin A studied by Dynamic Light Scattering and Fluorescence techniques

Molecular mechanisms in thermally induced amyloid formation of Concanavalin A

Concanavalin A aggregation and toxicity on cell cultures

A number of neurodegenerative diseases are known to involve protein aggregation. Common mechanisms and structural properties of amyloids are thought to be involved in aggregation-related cytotoxicity. In this context we propose an experimental study on Concanavalin A (Con A) aggregation and use it as a model to study the relationship between cell toxicity and aggregation processes. Depending on solution conditions, Con A aggregation has been monitored by static and dynamic light scattering, Thioflavin T emission, and FTIR absorption. The morphology of different aggregate species was verified by means of Atomic Force Microscopy and Confocal Microscopy. During the aggregation pathway the nati…

Direct observation of alpha-lactalbumin, adsorption and incorporation into lipid membrane and formation of lipid/protein hybrid structures

The interaction between proteins and membranes is of great interest in biomedical and biotechnological research for its implication in many functional and dysfunctional processes. We present an experimental study on the interaction between model membranes and alpha-lactalbumin (alpha-La). alpha-La is widely studied for both its biological function and its anti-tumoral properties. We use advanced fluorescence microscopy and spectroscopy techniques to characterize alpha-La-membrane mechanisms of interaction and alpha-La-induced modifications of membranes when insertion of partially disordered regions of protein chains in the lipid bilayer is favored. Moreover, using fluorescence lifetime imag…

Polysorbate 80 controls Morphology, structure and stability of human insulin Amyloid-Like spherulites

AbstractAmyloid protein aggregates are not only associated with neurodegenerative diseases and may also occur as unwanted by-products in protein-based therapeutics. Surfactants are often employed to stabilize protein formulations and reduce the risk of aggregation. However, surfactants alter protein-protein interactions and may thus modulate the physicochemical characteristics of any aggregates formed. Human insulin aggregation was induced at low pH in the presence of varying concentrations of the surfactant polysorbate 80. Various spectroscopic and imaging methods were used to study the aggregation kinetics, as well as structure and morphology of the formed aggregates. Molecular dynamics s…

EARLY STAGES AND SPATIAL HETEROGENEITY IN INSULIN AMYLOID FIBRILS FORMATION

AMYLOID AGGREGATION IN CONCANAVALIN A AT HIGH PH STUDIED BY LIGHT SCATTERING, FLUORESCENCE AND CIRCULAR DICHROISM SPECTROSCOPY

Oxidation Enhances Human Serum Albumin Thermal Stability and Changes the Routes of Amyloid Fibril Formation

Oxidative damages are linked to several aging-related diseases and are among the chemical pathways determining protein degradation. Specifically, interplay of oxidative stress and protein aggregation is recognized to have a link to the loss of cellular function in pathologies like Alzheimer's and Parkinson's diseases. Interaction between protein and reactive oxygen species may indeed induce small changes in protein structure and lead to the inhibition/modification of protein aggregation process, potentially determining the formation of species with different inherent toxicity. Understanding the temperate relationship between these events can be of utmost importance in unraveling the molecul…

The route to protein aggregate superstructures: Particulates and amyloid-like spherulites.

AbstractDepending on external conditions, native proteins may change their structure and undergo different association routes leading to a large scale polymorphism of the aggregates. This feature has been widely observed but is not fully understood yet. This review focuses on morphologies, physico-chemical properties and mechanisms of formation of amyloid structures and protein superstructures. In particular, the main focus will be on protein particulates and amyloid-like spherulites, briefly summarizing possible experimental methods of analysis. Moreover, we will highlight the role of protein conformational changes and dominant forces in driving association together with their connection w…

Decoding vibrational states of Concanavalin A amyloid fibrils.

International audience; Amyloid and amyloid-like fibrils are a general class of protein aggregates and represent a central topic in life sciences for their involvement in several neurodegenerative disorders and their unique mechanical and supramolecular morphological properties. Both their biological role and their physical properties, including their high mechanical stability and thermodynamic inertia, are related to the structural arrangement of proteins in the aggregates at molecular level. Significant variations may exist in the supramolecular organization of the commonly termed cross-β structure that constitutes the amyloid core. In this context, a fine knowledge of the structural deta…

Modulation of α-Synuclein Aggregation Process and Fibril Stability by Co-solvents

Amyloid fibrils are involved in several amyloid-related pathologies such as Parkinson's disease, Alzheimer's disease and type II diabetes. As a result, scientific community is nowadays addressing considerable efforts towards the comprehension of fibrillation mechanisms, particularly focusing on how they are affected by environmental conditions, small molecules and/or membrane presence. In this scenario, alpha-synuclein (aSN), a small protein involved in Parkinson's disease, represents a challenging model system for studying aggregation phenomena, and understanding the pathogenesis at molecular level. Indeed, it is poorly understood how fibril formation is linked to the progressive neurodege…

Thioflavin T triggers β amyloid peptide (1-40) fibrils formation.

Introduction A general characteristic of aggregation is the multiple interaction and cross-feedback among distinct mechanisms occurring at different hierarchical levels. The comprehension of the different species interconversion during aggregation is very important since emerging evidences indicate intermediate oligomeric aggregates as primary toxic species. In this context, Aβ amyloid peptide provides a challenging model for studying aggregation phenomena both for the complexity of its association process and for the direct implications in Alzheimer’s Disease. Aggregates growth conditions strongly affect the final morphology, the fibrillar molecular structure as well as the aggregation pat…

Effects of confinement on insulin amyloid fibrils formation.

Insulin, a 51-residue protein universally used in diabetes treatment, is known to produce amyloid fibrils at high temperature and acidic conditions. As for other amyloidogenic proteins, the mechanisms leading to nucleation and growth of insulin fibrils are still poorly understood. We here report a study of the fibrillation process for insulin confined in a suitable polymeric hydrogel, with the aim of ascertain the effects of a reduced protein mobility on the various phases of the process. The results indicate that, with respect to standard aqueous solutions, the fibrillation process is considerably slowed down at moderately high concentrations and entirely suppressed at low concentration. M…

Thermal induced conformational changes involved in the aggregation of beta-lactoglobulin

Aggregation of proteins appears to be associated most often with conformational and structural changes that lead to exposure of some apolar residues. Depending on the native structure of the protein in exam, aggregation is a process that involves different mechanisms, whose time of occurrence and interplay can depend upon temperature. To single out information about the multistages of the aggregation pathway, here we investigate the thermally induced conformational and structural changes of the beta-lactoglobulin (BLG). The experimental approach consists in studying steady-state fluorescence spectra of intrinsic chromophores, two tryptophans, and Anylino-Naphthalene-Sulfonate dye (ANS) mole…

EFFECT OF SUCCINYLATION AND SUGAR BINDING ON THERMAL INDUCED AMYLOID FORMATION IN CONCANAVALIN A

HSA Oxidation ImprovesThermal Stability and InhibitsAmyloid Fibril Formation

Oxidation effects in antiaggregogenic properties of Epigallocatechingallate

Epigallocatechin-gallate (EGCG), the most abundant flavonoid in green tea, has been extensively studied for its potential in the treatment of amyloid related disorders. This molecule was found to modulate abnormal protein self-assembly, reducing resulting cellular toxicity. EGCG is known to suppress or to slow down the aggregation processes of several proteins, thus supporting the idea that general mechanisms regulate its anti-aggregogenic effects and, interestingly, in the oxidised form it demonstrated an higher efficiency in reducing protein aggregation with respect to intact molecule. We here investigate the effects of intact and oxidized EGCG the thermal aggregation pathway of Bovine Se…

Identification of circumferential regional heterogeneity of ascending thoracic aneurysmal aorta by biaxial mechanical testing

Abstract Ascending thoracic aortic aneurysm (ATAA) in patients with bicuspid aortic valve (BAV) can present an asymmetrical aortic dilatation compared with patients with tricuspid aortic valve (TAV). This pattern of aneurysm dilatation led us to hypothesize that biomechanical differences likely induced by regional heterogeneity of material properties can underlie the observed asymmetric enlargement discrepancies between BAV ATAA and TAV ATAA. This study aimed to characterize the mechanical properties and associated aortic tissue stiffness changes along the circumferential direction of aortic rings collected from surgically-repaired patients with ATAA. Biaxial material testing was performed …

On the Effect of Downscaling in Inkjet Printed Life-Inspired Compartments

The fabrication of size-scalable liquid compartments is a topic of fundamental importance in synthetic biology, aiming to mimic the structures and the functions of cellular compartments. Here, inkjet printing is demonstrated as a customizable approach to fabricate aqueous compartments at different size regimes (from nanoliter to femtoliter scale) revealing the crucial role of size in governing the emerging of new properties. At first, inkjet printing is shown to produce homogenous aqueous compartments stabilized by oil-confinement with mild surfactants down to the hundreds of picoliter scale [1]. Raster Image Correlation Spectroscopy allows to monitor few intermolecular events by the involv…

Molecular Confinement in Femtoliter scale aqueous Compartments

Molecular confinement is known to lead to acceleration of molecular dynamics along with surface interaction. Nature employs confinement in molecularly crowded, heterogeneous and, specialized femtoliter (fL) compartments inside living cells for spontaneously achieving higher reaction efficiency and spatial-programming of composite, multi-step biochemical processes. We here show the facile production of aqueous fL droplets for studying molecular confinement on a biochip. We prepare fL aqueous droplets in oil drops on solid substrates by a “field-free”- no external electric fields and electrolytes - piezoelectric inkjet printing in which a novel actuating waveform is employed by picoliter size…

Oxidation Processes in Sicilian Olive Oils Investigated by a Combination of Optical and EPR Spectroscopy

:  Extra virgin olive oil (EVOO) is recognized as one of the healthiest foods for its high content of antioxidants, which forestall and slow down radical formation. Free radical-initiated oxidation is considered one of the main causes of rancidity in fats and oils. As a consequence, reliable protocols for the investigation of oil oxidation based on selective, noninvasive, and fast methods are highly desirable. Here we report an experimental approach based on UV-Vis absorbance, steady-state fluorescence, and electron paramagnetic resonance (EPR) spectroscopy for studying oxidation processes induced by temperature for a period up to 35 d on Sicilian EVOO samples. We followed the decrease in β…

Donor–Acceptor Interfaces by Engineered Nanoparticles Assemblies for Enhanced Efficiency in Plastic Planar Heterojunction Solar Cells

Precisely positioning functionalized gold nanoparticles assemblies at planar donor-acceptor interfaces results in 14-fold enhancement of power conversion efficiency in P3HT/PCBM organic solar cells on plastic (ITO/PET) substrates. This result has been achieved by employing naphthalenethiol-capped gold nanoparticles (NT-Au-NPs) produced by laser ablation in liquid and size varied in the 10-30 nm range. Upon surface functionalization with the aromatic thiol, these particles self-assemble in submicrometer aggregates, which give increased light scattering. When these aggregates are deposited in the planar heterojunction between the donor and the acceptor systems, the localized scattering leads …

THz spectroscopy studies on proteins: exploring collective modes of amyloid fibrils

Ethanol Controls the Self-Assembly and Mesoscopic Properties of Human Insulin Amyloid Spherulites.

Protein self-assembly into amyloid fibrils or highly hierarchical superstructures is closely linked to neurodegenerative pathologies as Alzheimer's and Parkinson's diseases. Moreover, protein assemblies also emerged as building blocks for bioinspired nanostructured materials. In both the above mentioned fields, the main challenge is to control the growth and properties of the final protein structure. This relies on a more fundamental understanding of how interactions between proteins can determine structures and functions of biomolecular aggregates. Here, we identify a striking effect of the hydration of the single human insulin molecule and solvent properties in controlling hydrophobicity/…

Increased carrier peptide stability through ph adjustment improves insulin and pth(1-34) delivery in vitro and in vivo rather than by enforced carrier peptide-cargo complexation

Oral delivery of therapeutic peptides is hampered by their large molecular size and labile nature, thus limiting their permeation across the intestinal epithelium. Promising approaches to overcome the latter include co-administration with carrier peptides. In this study, the cell-penetrating peptide penetratin was employed to investigate effects of co-administration with insulin and the pharmacologically active part of parathyroid hormone (PTH(1-34)) at pH 5, 6.5, and 7.4 with respect to complexation, enzymatic stability, and transepithelial permeation of the therapeutic peptide in vitro and in vivo. Complex formation between insulin or PTH(1-34) and penetratin was pH-dependent. Micron-size…

Pulsatile protein release and protection using radiation-crosslinked polypeptide hydrogel delivery devices

Abstract In the recent years recombinant technology has identified numerous protein based therapeutics. Their effective delivery, though, can be challenging due to the poor stability of most proteins along their pathway to the target site in the body. Hydrogels have been identified as good candidates for protein encapsulation and delivery thanks to both material and manufacturing process advantages. In this work we propose high energy irradiation as a synthetic methodology of choice to engineer hydrogel-based delivery devices for encapsulation and pulsatile release of proteins, triggered by pH, and for prevention of their denaturation when encapsulated. In particular, maleic anhydride funct…

High-throughput drug screening by Printing Biology

Printing biology is our way to define a novel field employing material printing techniques generally used in plastic electronics to solve important issues of biology by miniaturized and high-throughput platforms. In this field, we already showed the possibility to use Dip Pen Lithography to fabricate single-cell biochips [1]. Also,we employed non-contact patterning methods such as inkjet printing methods to fabricate microarrays for drug screening at solid-liquid interfaces [2] or in picoliter-scale liquid droplets [3] so enabling high-throughput screening of chemical libraries onto disease-based targets. In this regard, printing methods would greatly reduce times and costs of standard drug…

Insights on amyloid spherulites structure at molecular level

Effects of Disaccarides On Thermal Aggregation of Bovine Serum Albumin.

Insulin-activated Akt rescues Aβ oxidative stress-induced cell death by orchestrating molecular trafficking

Increasing evidence indicates that Alzheimer's disease, one of the most diffused aging pathologies, and diabetes may be related. Here, we demonstrate that insulin signalling protects LAN5 cells by amyloid-β42 (Aβ)-induced toxicity. Aβ affects both activation of insulin receptors and the levels of phospho-Akt, a critical signalling molecule in this pathway. In contrast, oxidative stress induced by Aβ can be antagonized by active Akt that, in turn, inhibits Foxo3a, a pro-apoptotic transcription factor activated by reactive oxygen species generation. Insulin cascade protects against mitochondrial damage caused by Aβ treatment, restoring the mitochondrial membrane potential. Moreover, we show t…

Thioflavin T Hydroxylation at Basic pH and Its Effect on Amyloid Fibril Detection

The fluorescent dye thioflavin T (ThT) is commonly used for in situ amyloid fibril detection. In this work, we focused on the spectroscopic properties and chemical stability of ThT in aqueous solution as a function of pH, temperature, and dye concentration. A reversible hydroxylation process occurs in alkaline solutions, which was characterized using a combination of UV-vis absorption spectroscopy, proton NMR, and density functional theory (DFT). On the basis of these studies, we propose a chemical structure for the hydroxylated form. Finally, by means of fluorescence spectroscopy, ThT hydroxylation effects on in situ amyloid detection have been investigated, providing new insights on the e…

Printing Life-Inspired Subcellular Scale Compartments with Autonomous Molecularly Crowded Confinement.

A simple, rapid, and highly controlled platform to prepare life-inspired subcellular scale compartments by inkjet printing has been developed. These compartments consist of fL-scale aqueous droplets (few µm in diameter) incorporating biologically relevant molecular entities with programmed composition and concentration. These droplets are ink-jetted in nL mineral oil drop arrays allowing for lab-on-chip studies by fluorescence microscopy and fluorescence life time imaging. Once formed, fL-droplets are stable for several hours, thus giving the possibility of readily analyze molecular reactions and their kinetics and to verify molecular behavior and intermolecular interactions. Here, this pla…

Aggregation processes in beta-lactoglobulin studied by FTIR spectroscopy

A Long Journey into the Investigation of the Structure–Dynamics–Function Paradigm in Proteins through the Activities of the Palermo Biophysics Group

An overview of the biophysics activity at the Department of Physics and Chemistry Emilio Segrè of the University of Palermo is given. For forty years, the focus of the research has been on the protein structure–dynamics–function paradigm, with the aim of understanding the molecular basis of the relevant mechanisms and the key role of solvent. At least three research lines are identified; the main results obtained in collaboration with other groups in Italy and abroad are presented. This review is dedicated to the memory of Professors Massimo Ugo Palma, Maria Beatrice Palma Vittorelli, and Lorenzo Cordone, which were the founders of the Palermo School of Biophysics. We all have been, directl…

Sub-Cellular Scale Compartments: Printing Life-Inspired Subcellular Scale Compartments with Autonomous Molecularly Crowded Confinement (Adv. Biosys. 7/2019)

Glucagon fibril polymorphism reflects differences in protofilament backbone structure

Amyloid fibrils formed by the 29-residue peptide hormone glucagon at different concentrations have strikingly different morphologies when observed by transmission electron microscopy. Fibrils formed at low concentration (0.25 mg/mL) consist of two or more protofilaments with a regular twist, while fibrils at high concentration (8 mg/mL) consist of two straight protofilaments. Here, we explore the structural differences underlying glucagon polymorphism using proteolytic degradation, linear and circular dichroism, Fourier transform infrared spectroscopy (FTIR), and X-ray fiber diffraction. Morphological differences are perpetuated at all structural levels, indicating that the two fibril class…

Multiple aggregation mechanism in Abeta(1-40) fibril formation

A general characteristic of aggregation is the multiple interaction and cross-feedback among distinct mechanisms occurring at different hierarchical levels. The comprehension of the different species interconversion during aggregation is very important since emerging evidences indicate intermediate oligomeric aggregates as primary toxic species. In this context, Aβ amyloid peptide provides a challenging model for studying aggregation phenomena both for the complexity of its association process and for the direct implications in Alzheimer’s Disease. Indeed, aggregates growth conditions strongly affect their final morphology and their molecular structure as well as the time evolution of aggre…

Insulin Nanogel as New Strategy for the Treatment of Alzheimer’s Disease

A growing body of evidence shows that Insulin, Insulin Receptor (IR) and IR signaling are involved in brain cognitive functions and their dysfunction is implicated in neuronal degeneration associated with Alzheimer's disease (AD). Thus, the administration of insulin to the brain could be a strategy for the prevention and treatment of AD disease. With this aim, we have designed, synthesized and characterized a nanogel system (NG) that can be used as substrate for the conjugation of insulin and/or fluorescent molecules relevant for their characterization. In particular, a carboxyl-functionalized poly(N-vinyl pyrrolidone) nanogel system, has been produced by ionizing radiation starting from th…

Optically active and biocompatible polyaniline nanoparticles - hydrogel composites.

Blue light activated photodegradation of biomacromolecules by N-doped titanium dioxide in a chitosan hydrogel matrix

The use of photocatalysis activated by titanium dioxide nanostructured materials is a promising solution for many biomedical applications ranging from drug-free antibacterial to anticancer therapies, as well as for innovative hydrogel-supported phototherapies. This makes the effects of photocatalysis on the structure of biomolecules of a great relevance in order to define the applicability of photocatalytic materials in the biomedical fields. In this work, the effects of nitrogen-doped titanium dioxide (N-TiO2) dispersed in a biocompatible chitosan/PEG hydrogel on myoglobin and bovine serum albumin as target model proteins were investigated. The efficiency of this composite biocompatible ma…

Protein/lipid coaggregates are formed during α-synuclein-induced disruption of lipid bilayers.

Amyloid formation is associated with neurodegenerative diseases such as Parkinson's disease (PD). Significant α-synuclein (αSN) deposition in lipid-rich Lewy bodies is a hallmark of PD. Nonetheless, an unraveling of the connection between neurodegeneration and amyloid fibrils, including the molecular mechanisms behind potential amyloid-mediated toxic effects, is still missing. Interaction between amyloid aggregates and the lipid cell membrane is expected to play a key role in the disease progress. Here, we present experimental data based on hybrid analysis of two-photon-microscopy, solution small-angle X-ray scattering and circular dichroism data. Data show in real time changes in liposome …

Functional hydrogels containing polyaniline nanoparticles through e-beam irradiation

Interconnected mechanism in Abeta(1-40) peptide fibril formation

Trifluoroethanol modulates α-synuclein amyloid-like aggregate formation, stability and dissolution

The conversion of proteins into amyloid fibrils and other amyloid-like aggregates is closely connected to the onset of a series of age-related pathologies. Upon changes in environmental conditions, amyloid-like aggregates may also undergo disassembly into oligomeric aggregates, the latter being recognized as key effectors in toxicity. This indicates new possible routes for in vivo accumulation of toxic species. In the light of the recognized implication of α-Synuclein (αSN) in Parkinson's disease, we present an experimental study on supramolecular assembly of αSN with a focus on stability and disassembly paths of such supramolecular aggregate species. Using spectroscopic techniques, two-pho…

Internal properties of "ultra clean" hydrogels synthesized via e-beam irradiation through absorption and luminescence spectroscopy

Neutron Scattering Reveals Enhanced Protein Dynamics in Concanavalin A Amyloid Fibrils

Protein aggregation is one of the most challenging topics in life sciences, and it is implicated in several human pathologies. The nature and the role of toxic species is highly debated, with amyloid fibrils being among the most relevant species for their peculiar structural and functional properties. Protein dynamics and in particular the ability to fluctuate through a large number of conformational substates are closely related to protein function. This Letter focuses on amyloid fibril dynamics, and, to our knowledge, it is the first neutron scattering study on a protein (Concanavalin A) isolated in its fibril state. Our results reveal enhanced atomic fluctuations in amyloid fibrils and i…

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…

UV-induced modifications in collagen fibers molecular structure: a fluorescence spectroscopy and microscopy study

An overview on different processes in thermal aggregation of globular proteins

Multistage pathways involved in thermal aggregation processes of proteins.

Formation of covalent di-tyrosine dimers in recombinant α-synuclein

Parkinson's disease is associated with fibril deposition in the diseased brain. Misfolding events of the intrinsically disordered synaptic protein α-synuclein are suggested to lead to the formation of transient oligomeric and cytotoxic species. The etiology of Parkinson's disease is further associated with mitochondrial dysfunction and formation of reactive oxygen species. Oxidative stress causes chemical modification of native α-synuclein, plausibly further influencing misfolding events. Here, we present evidence for the spontaneous formation of covalent di-tyrosine α-synuclein dimers in standard recombinant protein preparations, induced without extrinsic oxidative or nitrative agents. The…

Secondary nucleation in stirring induced alpha-lactalbumin amyloid fibrils formation

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…

Solution structure of recombinant Pvfp-5β reveals insights into mussel adhesion

Solution structure of byssal plaque protein Pvfp-5 beta secreted by the Asian green mussel Perna viridis gives molecular insight into mussel adhesion on wet surfaces.Some marine organisms can resist to aqueous tidal environments and adhere tightly on wet surface. This behavior has raised increasing attention for potential applications in medicine, biomaterials, and tissue engineering. In mussels, adhesive forces to the rock are the resultant of proteinic fibrous formations called byssus. We present the solution structure of Pvfp-5 beta, one of the three byssal plaque proteins secreted by the Asian green mussel Perna viridis, and the component responsible for initiating interactions with the…

Electrostatics regulate Epigallocatechin-Gallate effects on Bovine Serum Albumin aggregation

Protein aggregation processes are complex phenomena often involved in the etiology of several pathologies. It is now assessed that all proteins, in suitable conditions, may undergo supramolecular assembly. Aggregation pathways are known to be controlled by solution conditions which regulate protein-protein and protein-solvent interactions affecting binding mechanisms, morphology and inherent toxicity of the aggregate species. In this context, the presence of small molecules was indicated as a promising method to modulate protein-protein interactions reducing pathogenic aggregation. In the light of the idea that common mechanisms regulate anti-aggregogenic properties of small molecules, we h…

Novel Gamma-crosslinked Hydrogels for Intelligent Release of Actives

Identification of microplastics using 4‐dimethylamino‐4′‐nitrostilbene solvatochromic fluorescence

In this work, we introduce the use of 4-dimethylamino-4'-nitrostilbene (DANS) fluorescent dye for applications in the detection and analysis of microplastics, an impendent source of pollution made of synthetic organic polymers with a size varying from less than 5 mm to nanometer scale. The use of this dye revealed itself as a versatile, fast and sensitive tool for readily discriminate microplastics in water environment. The experimental evidences herein presented demonstrate that DANS efficiently absorbs into a variety of polymers constituting microplastics, and its solvatochromic properties lead to a positive shift of the fluorescence emission spectrum according to the polarity of the poly…

Amyloid fibrils formation and amorphous aggregation in Concanavalin A

We here report an experimental study on the thermal aggregation process of concanavalin A, a protein belonging to the legume lectins family. The aggregation process and the involved conformational changes of the protein molecules were followed by means of fluorescence techniques, light scattering, circular dichroism, zeta potential measurements and atomic force microscopy. Our results show that the aggregation process of concanavalin A may evolve through two distinct pathways leading, respectively, to the formation of amyloids or amorphous aggregates. The relative extent of the two pathways is determined by pH, as amyloid aggregation is favored at high pH values ( approximately 9), while th…

Interconnected mechanisms in Abeta(1-40) peptide fibril formation

Review: "Thermal aggregation of proteins in the presence of metal ions"

Stirring effects in amyloid fibril formation

Development of a biosensor for copper detection in aqueous solutions using an Anemonia sulcata recombinant GFP.

Fluorescent proteins from marine organisms represent potential candidates for biosensor development. In this paper, we described the isolation of a native green fluorescent protein from Anemonia sulcata and the cloning and purification of its equivalent as a recombinant protein in Escherichia coli. Furthermore, the spectroscopic behaviours of the native and recombinant GFPs were investigated as a function of Cu2+, Cd2+, Pb 2+ and Ni2+ concentration. Our results suggest the high selectivity of both proteins at copper than the other metals and, for the recombinant protein, a great sensitivity at a very low concentration (0.1-1 μM). Moreover, starting from these data, using the combination of …

Disaccarides effects on thermal aggregation of Bovine Serum Albumin

Thermal aggregation of human and bovine serum albumin in the low concentration regime

NIR LASER-RESPONSIVE FOLATE-TARGETED GOLD NANORODS AS EFFICIENT THERANOSTIC TOOL FOR OSTEOSARCOMA TREATMENT

Folate-targeted gold nanorods (GNRs) are here proposed as selective theranostic agents for osteosarcoma treatment. Taking advantage of the attractive physiochemical and optical properties of GNRs they can be proposed as effective and selective platform to obtain a targeted intracellular drug release, photothermal therapy and cancer imaging, which may improve therapeutic outcomes of osteosarcoma. An amphiphilic polysaccharide graft-copolymer, named INU-LA-PEG-FA, and a folic acid functionalized α,β-poly(N-2-hydroxyethyl)-D,L-aspartamide (PHEA-FA), have been synthesized to act as coating agents for GNRs. The obtained polymer-coated GNRs were characterized in terms of size, shape, zeta potenti…

High Fluorescence of Thioflavin T Confined in Mesoporous Silica Xerogels

Trapping of organic molecules and dyes within nanoporous matrices is of great interest for the potential creation of new materials with tailored features and, thus, different possible applications ranging from nanomedicine to material science. The understanding of the physical basis of entrapment and the spectral properties of the guest molecules within the host matrix is an essential prerequisite for the design and control of the properties of these materials. In this work, we show that a mesoporous silica xerogel can efficiently trap the dye thioflavin T (ThT, a molecule used as a marker of amyloid fibrils and with potential drug benefits), sequestering it from an aqueous solution and pro…

Conformational Transitions upon Maturation Rule Surface and pH-Responsiveness of α-Lactalbumin Microparticulates

De novo designed protein supramolecular structures are nowadays attracting much interest as highly performing biomaterials. While a clear advantage is provided by the intrinsic biocompatibility and...

Determination of particle number and brightness using a laser scanning confocal microscope operating in the analog mode

We describe a method to obtain the brightness and number of molecules at each pixel of an image stack obtained with a laser scanning microscope. The method is based on intensity fluctuations due to the diffusion of molecules in a pixel. For a detector operating in the analog mode, the variance must be proportional to the intensity. Once this constant has been calibrated, we use the ratio between the variance and the intensity to derive the particle brightness. Then, from the ratio of the intensity to the brightness we obtain the average number of particles in the pixel. We show that the method works with molecules in solution and that the results are comparable to those obtained with fluctu…

Nile Red lifetime reveals microplastic identity

Microplastic pollution is recognized as a worldwide environmental problem. The increasing daily use and release of plastics into the environment have led to the accumulation of fragmented microplastics, with potentially awful consequences for the environment, and animal and human health. The detection and identification of microplastics are of utmost importance, but available methods are still limited. In this work, a new approach is presented for the analysis of microplastics based on hydrophobic fluorescence staining with Nile Red, using spectrally resolved confocal fluorescence microscopy and fluorescence lifetime imaging microscopy (FLIM). Significant differences were observed in the em…

PRINTING NANOBIOLOGY IN AQUEOUS SYSTEMS

Our studies in the field of printing nanobiology in aqueous solution are proposed to highlight the role of water in the processes of interaction between biomolecules in drug- screening devices fabricated by bioprinting technologies and to emphasize the influence of water evaporation on the diffusion of molecules in droplets of picoliter-scale.

Direct formation of highly tunable and biocompatible protein microparticles

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…

Photocatalytic activity of N-doped TiO2-based materials embedded with gold NPs for applications in antibacterial photodynamic therapy (aPDT)

Antibacterial photodynamic therapy (aPDT) is a potential treatment for antibiotic-resistant bacterial infections. It is based on the photosensitization of bacterial cells with exogenous agents that, when exposed to light, produce reactive oxygen species (ROS), such as OH-, O2-, H2O2. ROS can induce complex oxidative-reductive chains of reactions, resulting in damage of cellular components in target tissues1. Photocatalysts, like inorganic semiconductor oxides, represent an interesting class of materials to design new strategies for aPTD. As exposed to light of proper wavelengths, photocatalysts induce the formation of electron-hole pairs capable of producing a cascade of reactions suitable …

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…

The Boson Peak of Amyloid Fibrils: Probing the Softness of Protein Aggregates by Inelastic Neutron Scattering

Proteins and polypeptides are characterized by low-frequency vibrations in the terahertz regime responsible for the so-called "boson peak". The shape and position of this peak are related to the mechanical properties of peptide chains. Amyloid fibrils are ordered macromolecular assemblies, spontaneously formed in nature, characterized by unique biological and nanomechanical properties. In this work, we investigate the effects of the amyloid state and its polymorphism on the boson peak. We used inelastic neutron scattering to probe low-frequency vibrations of the glucagon polypeptide in the native state and in two different amyloid morphologies in both dry and hydrated sample states. The dat…

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 …

Synergies and compromises between charge and energy transfers in three-component organic solar cells

In this paper, we developed different three-component organic heterojunction structures supported by PET/ITO substrates with the aim to study the possible synergies and/or compromises between charge transfer (CT) and energy transfer (ET) processes in organic solar cells (OSCs). As components, we employed poly(3-hexylthiophene-2,5-diyl) (P3HT; donor), [6,6]-phenyl-C61-butyric acid methyl ester (PCBM; acceptor) and poly(9,9-dioctylfluorene-alt-benzothiadiazole) (F8BT) that is known to give good ET to P3HT. At first, we observed that in a planar heterojunction (PHJ) solar cell, F8BT has to be properly located in between P3HT and PCBM to get a cascade energy level configuration allowing for a b…

Insulin activated Akt rescues Aβ oxidative stress-induced cell death by orchestrating molecular trafficking.

Increasing evidence indicates that Alzheimer's disease, one of the most diffused aging pathologies, and diabetes may be related. Here, we demonstrate that insulin signalling protects LAN5 cells by amyloid-β42 (Aβ)-induced toxicity. Aβ affects both activation of insulin receptors and the levels of phospho-Akt, a critical signalling molecule in this pathway. In contrast, oxidative stress induced by Aβ can be antagonized by active Akt that, in turn, inhibits Foxo3a, a pro-apoptotic transcription factor activated by reactive oxygen species generation. Insulin cascade protects against mitochondrial damage caused by Aβ treatment, restoring the mitochondrial membrane potential. Moreover, we show t…

Protein materials as sustainable non- and minimally invasive strategies for biomedical applications

Protein-based materials have found applications in a wide range of biomedical fields because of their biocompatibility, biodegradability and great versatility. Materials of different physical forms including particles, hydrogels, films, fibers and microneedles have been fabricated e.g. as carriers for drug delivery, factors to promote wound healing and as structural support for the generation of new tissue. This review aims at providing an overview of the current scientific knowledge on protein-based materials, and selected preclinical and clinical studies will be reviewed in depth as examples of the latest progress within the field of protein-based materials, specifically focusing on non- …

Amyloid Fibrils Formation of Concanavalin A at Basic pH

Mechanisms of partial unfolding and aggregation of proteins are of extreme interest in view of the fact that several human pathologies are characterized by the formation and deposition of protein-insoluble material, mainly composed of amyloid fibrils. Here we report on an experimental study on the heat-induced aggregation mechanisms, at basic pH, of concanavalin A (ConA), used as a model system. Thioflavin T (ThT) fluorescence and multiangle light scattering allowed us to detect different intertwined steps in the formation of ConA aggregates. In particular, the ThT fluorescence increase, observed in the first phase of aggregation, reveals the formation of intermolecular β-sheet structure wh…

Ink depletion-free printing of phospholipids micropatches

Thermal aggregation of bovine serum albumin close to the isoelectric point.

Molecular Insights on alpha-synuclein mediated membrane disruption: protein-lipid co-aggregates formation

Different degrees in protein thermal aggregation processes in presence of trealose

Probing ensemble polymorphism and single aggregate structural heterogeneity in insulin amyloid self-assembly.

Ensembles of protein aggregates are characterized by a nano- and micro-scale heterogeneity of the species. This diversity translates into a variety of effects that protein aggregates may have in biological systems, both in connection to neurodegenerative diseases and immunogenic risk of protein drug products. Moreover, this naturally occurring variety offers unique opportunities in the field of protein-based biomaterials. In the above-mentioned fields, the isolation and structural analysis of the different amyloid types within the same ensemble remain a priority, still representing a significant experimental challenge. Here we address such complexity in the case of insulin for its relevance…

Internal properties of gamma-irradiated polymeric hydrogels studied by means of fluorescent probes