Molecular Crowding: The History and Development of a Scientific Paradigm.

It is now generally accepted that macromolecules do not act in isolation but "live" in a crowded environment, that is, an environment populated by numerous different molecules. The field of molecular crowding has its origins in the far 80s but became accepted only by the end of the 90s. In the present issue, we discuss various aspects that are influenced by crowding and need to consider its effects. This Review is meant as an introduction to the theme and an analysis of the evolution of the crowding concept through time from colloidal and polymer physics to a more biological perspective. We introduce themes that will be more thoroughly treated in other Reviews of the present issue. In our intentions, each Review may stand by itself, but the complete collection has the aspiration to provide different but complementary perspectives to propose a more holistic view of molecular crowding.

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 of the multifunctional amphiphilic peptide transportan 10 with model membranes. Our approach, based on the use of suitable fluorescence reporters, exploits the advantages of phasor plot analysis of fluorescence lifetime imaging microscopy measurements to highlight the molecular details of occurring membrane alterations in terms of rigidity and hydration. Simultaneously, it allows following dynamic events in real time without sample manipulation distinguishing, with high spatial resolution, whether the peptide is adsorbed to or inserted in the membrane.

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 1 sun toward methylene blue. The resulting coatings, in particular the 35 wt % TiO2-loaded composites, show a photocatalytic degradation of about 80%, which was comparable to the equivalent amount of bare TiO2 and two-fold higher with respect to the corresponding composites not subjected to thermal treatment. These findings highlight the role of thermally induced bulk clustering in enhancing photoactive nanoparticle/polymer composite properties.

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 better CT and power conversion efficiency. Then, we observed that by producing a P3HT:F8BT blend, the energy transfer process can be improved in the P3HT:F8BT/PCBM active layer. This may enable decreasing the thickness of the active layer while maintaining a similar PCE that is very interesting for the development of transparent OSCs. However, the P3HT:F8BT blend limits the P3HT-PCBM CT with respect to a P3HT/F8BT/PCBM PHJ, showing that a compromise between CT and ET is needed to get a higher PCE or higher transparency. By the above approach, in this paper, we developed highly transparent heterojunction structures for solar cell devices with PCEs comparable to those observed from the colorful reference P3HT/PCBM PHJ solar cells on PET/ITO substrates.

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

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 tissue specimens extrapolated from all aortic quadrants (i.e. anterior, posterior, major and minor curvature of the aorta), and then the tissue stiffness was quantified at both physiological and supra-physiological stress levels (i.e. 142 kPa and 242 kPa, respectively). Tissue stiffness revealed that the major curvature of BAV ATAA is statistically less stiff than the anterior quadrant (276.6 ±â€¯137.1 kPa for BAV ATAA and 830.1 ±â€¯557.1 kPa for BAV ATAA, p = .024, at 142 kPa) and to that of major curvature of TAV ATAA (276.6 ±â€¯137.0 kPa for BAV ATAA and 733.2 ±â€¯391.1 kPa for TAV ATAA, p = .001, at 142 kPa), suggesting local weakening of bicuspid aortic wall. Multiphoton imaging revealed local changes on elastic fiber networks. The recovered material parameters for the Fung-type constitutive model are crucial for reliable stress predictions while the information on regional tissue stiffness changes are fundamental to develop risk stratification strategies not based on aortic size.

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 morphology and stability upon protein addition and reveal that membrane disruption mediated by amyloidogenic αSN is associated with dehydration of anionic lipid membranes and stimulation of protein secondary structure. As a result of membrane fragmentation, soluble αSN:-lipid coaggregates are formed, hence, suggesting a novel molecular mechanism behind PD amyloid cytotoxicity.

Deciphering amyloid fibril molecular maturation through FLIM-phasor analysis of thioflavin T.

The investigation of amyloid fibril formation is paramount for advancing our understanding of neurodegenerative diseases and for exploring potential correlated therapeutic strategies. Moreover, the self-assembling properties of amyloid fibrils show promise for the development of advanced protein-based biomaterials. Among the methods employed to monitor protein aggregation processes, fluorescence has emerged as a powerful tool. Its exceptional sensitivity enables the detection of early-stage aggregation events that are otherwise challenging to observe. This research underscores the pivotal role of fluorescence analysis, particularly in investigating the aggregation processes of hen egg white lysozyme, a model protein extensively studied for insights into amyloid fibril formation. By combining classical spectroscopies with fluorescence microscopy and by exploiting the fluorescence properties (intensity and lifetime) of the thioflavin T, we were able to noninvasively monitor key and complex molecular aspects of the process. Intriguingly, the fluorescence lifetime imaging-phasor analysis of thioflavin T fluorescence lifetime on structures at different stages of aggregation allowed to decipher the complex fluorescence decay behavior, highlighting that their changes rise from the combination of specific binding to amyloid typical cross-ß structures and of the rigidity of the molecular environment.

Phasor-FLIM analysis of cellulose paper ageing mechanism with carbotrace 680 dye.

Ageing of paper is a complex process of great relevance for application purposes because of its widespread use as support for information storage in books and documents, and as common low-cost and green packaging material, to name a few. A key factor in paper ageing is the oxidation of cellulose, a macromolecule of natural origin that constitutes the main chemical component of paper. Such a complex process results in changes in the cellulose polymeric chains in chemical and structural properties. The scope of this work is to explore the effects of oxidation of cellulose as one of the principal mechanisms of ageing of paper using a fluorescence-based approach. To this aim, fluorescence-lifetime imaging microscopy (FLIM) measurements on pure cellulose samples stained using Carbotrace 680 dye were performed, and data were analyzed by phasor approach. The comparison with results from conventional techniques allowed to map paper microstructure as a function of the sample oxidation degree correlating the fluorescence-lifetime changes to cellulose oxidation. A two-step oxidation kinetics that produced specific modification in paper organization was highlighted indicating that FLIM measurements using Carbotrace 680 dye may provide a simple tool to obtain information on the oxidation process also adding spatial information at sub-micrometric scale.

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 producing a highly fluorescent material with a ThT quantum yield 1500 times greater than that of the free molecule. The study of spectroscopical properties of this system and the comparison with fluorescence of an uncharged analogue of ThT give indications about the mechanism responsible for the fluorescence switching-on of ThT molecules during their uptaking into the glass. Diffusion and nanocapillarity are responsible for ThT absorption, whereas electrostatic interaction between positive ThT molecules and negative dangling ≡SiO groups covering the pore surfaces causes the immobilization of ThT molecules inside the pores and the enhancement of its fluorescence, in line with the molecular rotor model proposed for this dye. We also show that entrapment efficiency and kinetics can be tuned by varying the electrostatic properties of the dye and/or the matrix.

Carrier peptide interactions with liposome membranes induce reversible clustering by surface adsorption and shape deformation.

The cell-penetrating peptide penetratin and its analogues shuffle and penetramax have been used as carrier peptides for oral delivery of therapeutic peptides such as insulin. Their mechanism of action for this purpose is not fully understood but is believed to depend on the interactions of the peptide with the cell membrane. In the present study, peptide-liposome interactions were investigated using advanced biophysical techniques including small-angle neutron scattering and fluorescence lifetime imaging microscopy. Liposomes were used as a model system for the cell membrane. All the investigated carrier peptides induced liposome clustering at a specific peptide/lipid ratio. However, distinctively different types of membrane interactions were observed, as the liposome clustering was irreversible for penetratin, but fully or partly reversible for shuffle and penetramax, respectively. All three peptides were found to adsorb to the surface of the lipid bilayers, while only shuffle and penetramax led to shape deformation of the liposomes. Importantly, the peptide interactions did not disrupt the liposomes under any of the investigated conditions, which is advantageous for their application in drug delivery. This detailed insight on peptide-membrane interactions is important for understanding the mechanism of peptide-based excipients and the influence of peptide sequence modifications.

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 cellular uptake and in the following processes are still unclear. Here, we use fluorescence microscopy methods to analyze the interactions between the multifunctional peptide Transportan 10 (TP10) and the giant plasma membrane vesicles (GPMVs) derived from cancer cells. This aims to highlight the molecular mechanisms underlying functional interactions which bring its translocation across the membrane or cytotoxic mechanisms leading to membrane collapse and disruption. The Fluorescence Lifetime Imaging Microscopy (FLIM) method coupled with the phasor approach analysis proved to be the winning choice for following highly dynamic spatially heterogeneous events in real-time and highlighting aspects of such complex phenomena. Thanks to the presented approach, we were able to identify and monitor TP10 translocation into the lumen, internalization, and membrane-induced modifications depending on the peptide concentration regime.

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 properties of these aggregates can be readily tuned by selecting the pH of the solution during production. In particular, the presence of ß-structures typical of amyloids as well as an environment characterized by a lower dielectric constant seem to enhance metal binding affinity revealing that hydrophobicity and water accessibility of the material are key features affecting the adsorption efficiency. Presented results provide new knowledge on how raw plant proteins can be valorised for the production of new biomaterials. This may offer extraordinary opportunities towards the design and production of new tailorable biosorbents which can also be exploited for several cycles of purification with minimal reduction in performance. SYNOPSIS: Innovative, sustainable plant-protein biomaterials with tunable properties are presented as green solution for water purification from lead(II) and the structure-function relationship is discussed.

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. METHODS: By means of quantitative fluorescence imaging and complementary spectroscopic methods, we were able to characterise α-casein association state and the course of events induced by pH changes, which regulate the interaction of this molecule with membranes. RESULTS: The study of these complex dynamic events revealed that the initial conformation of the protein critically regulates the fate of α-casein, size and structure of the newly formed aggregates and their effect on membrane structures. Disassembly of micelles due to modification in electrostatic interactions results in increased membrane structure rigidity which accompanies the formation of protein lipid flower-like co-aggregates with protein molecules localised in the external part. GENERAL SIGNIFICANCE: These results may contribute to the comprehension of how the initial state of a protein establishes the course of events that occur upon changes in the molecular environment. These events which may occur in cells may be essential to functional, pathological or therapeutical properties specifically associated to casein proteins.

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- and minimally invasive strategies mainly for topical application.

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

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 readability. In this work, we demonstrate that the fluorescein-Au3+complex provides a rapid, selective, and sensitive tool for the quantification of creatinine concentrations in ranges typical of sweat and urine. UV-visible absorption, diffuse reflectance spectroscopy, steady state and time resolved fluorescence spectroscopy were used to shed light on the molecular mechanisms involved in the changes of optical properties, which underlie the multiplexed sensor analytical reply. Interestingly, sensing can be performed in solution or on solid nylon support accessing different physiological concentrations from micromolar to millimolar range. As a proof-of-concept, the nylon-based platform was used to demonstrate its effectiveness in creatinine detection on a solid and flexible substrate, showing its analytical colorimetric properties as an easy and disposable creatinine point-of-care test.

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

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-5ß, one of the three byssal plaque proteins secreted by the Asian green mussel Perna viridis, and the component responsible for initiating interactions with the substrate. We demonstrate that Pvfp-5ß has a stably folded structure in agreement with the presence in the sequence of two EGF motifs. The structure is highly rigid except for a few residues affected by slow local motions in the µs-ms time scale, and differs from the model calculated by artificial intelligence methods for the relative orientation of the EGF modules, which is something where computational methods still underperform. We also show that Pvfp-5ß is able to coacervate even with no DOPA modification, giving thus insights both for understanding the adhesion mechanism of adhesive mussel proteins, and developing of biomaterials.

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

Amyloid 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 simulations were employed to investigate the initial interaction between the surfactant and insulin. Addition of polysorbate 80 slowed down, but did not prevent, aggregation of insulin. Amyloid spherulites formed under all conditions, with a higher content of intermolecular beta-sheets in the presence of the surfactant above its critical micelle concentration. In addition, a denser packing was observed, leading to a more stable aggregate. Molecular dynamics simulations suggested a tendency for insulin to form dimers in the presence of the surfactant, indicating a change in protein-protein interactions. It is thus shown that surfactants not only alter aggregation kinetics, but also affect physicochemical properties of any aggregates formed.

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 on the adsorption ability and affinity has been evaluated revealing the complexity of adsorption mechanisms which are the result of the balance of specific interactions with functional groups in protein structure and not specific ones common to all polypeptide chains, and possibly related to amyloid state and to modification of particulates hydration layer.

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 native protein conformation is destabilized and as a consequence, the simultaneous occurrence of conformational changes and protein aggregation is observed in both conditions. The effects of the extracellular addition of native protein, oligomers and mature fibrils were tested on LAN5 neuroblastoma cells by MTS assay. Results showed the toxicity of the first two species while a negligible effect was detected for amyloid fibrils. Both native and oligomeric aggregates were found to be able to activate apoptosis exclusively by extrinsic pathway through caspase 8 activation. Those results suggest that cytotoxicity mechanisms arise from specific membrane interactions with reactive conformations of destabilized molecules occurring during the amyloidal aggregation pathway. Those conformations, populated when native or preformed oligomers are incubated, are unavailable to bind cell membrane proteins. This happens because they are recruited in the mature fibrillar structure which-as a consequence-turns out to be non-toxic.

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 interleukin-6 and thus contribute to the establishment of a pro-inflammatory, Th17 permissive environment in AITL. We further hypothesized that the AITL clone itself could be responsible for the preferential accumulation of MCs at sites of infiltration through the synthesis of CXCL-13 and its interaction with the CXCR3 and CXCR5 receptors expressed on MCs. Consistent with this hypothesis, we observed MCs efficiently migrating in response to CXCL-13. On these bases, we conclude that MCs have a role in molding the immunological microenvironment of AITL toward the maintenance of pro-inflammatory conditions prone to Th17 generation and autoimmunity.