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Quatsome nanovesicles, formed through the self-assembly of cholesterol (CHOL) and cetyltrimethylammonium bromide (CTAB) in water, have shown long-term stability in terms of size and morphology, while at the same time exhibiting high CHOL-CTAB intermolecular binding energies. We hypothesize that CHOL/CTAB quatsomes are indeed thermodynamically stable nanovesicles, and investigate the mechanism underlying their formation., This work was supported by funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 712949 (TECNIOspring PLUS) and from the Agency for Business Competitiveness of the Government of Catalonia. The production of quatsomes and part of their characterization has been performed by the ICTS “NANBIOSIS”, more specifically by the Biomaterial Processing and Nanostructuring Unit (U6), Unit of the CIBER in Bioengineering, Biomaterials & Nanomedicine (CIBER-BBN) located at the Institute of Materials Science of Barcelona (ICMAB-CSIC). ICMAB-CSIC acknowledges support from the MINECO through the Severo Ochoa Programme for Centres of Excellence in R&D (SEV-2015-0496 and CEX2019-000917-S). Authors acknowledge financial support from the Spanish Ministry of Science and Innovation through grants “MOL4BIO” (PID2019-105622RB-I00), “SimBioSoft” (PID2021-124297NB-C33) and the FUNFUTURE-FIP-2020 Severo Ochoa project, from Generalitat de Catalunya through grant 2017-SGR-918, from CSIC through grant 2019AEP133, and from the European Commission through the H2020 PHOENIX project (contract no. 953110). We acknowledge the support of the Israel scienceIsrael science Foundation, grant 1117/2016, and thank Dr. Inbal Ionita for her professional assistance in the cryo-TEM analysis. We thank Jannik Nedergaard Pedersen and Beatrice Plazzotta for help with the SAXS measurements. The simulations reported here were performed using the Cori Supercomputing facility of the National Energy Research Scientific Computing Center (NERSC), a U.S. Department of Energy Office of Science User Facility operated under Contract No. DE-AC02-05CH11231., With funding from the Spanish government through the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2019-000917-S).

Similar to its bacterial homolog GroEL, Hsp60 in oligomeric conformation is known to work as a folding machine, with the assistance of co-chaperonin Hsp10 and ATP. However, recent results have evidenced that Hsp60 can stabilize aggregation-prone molecules in the absence of Hsp10 and ATP by a different, “holding-like” mechanism. Here, we investigated the relationship between the oligomeric conformation of Hsp60 and its ability to inhibit fibrillization of the Ab40 peptide. The monomeric or tetradecameric form of the protein was isolated, and its effect on beta-amyloid aggregation was separately tested. The structural stability of the two forms of Hsp60 was also investigated using differential scanning calorimetry (DSC), light scattering, and circular dichroism. The results showed that the protein in monomeric form is less stable, but more effective against amyloid fibrillization. This greater functionality is attributed to the disordered nature of the domains involved in subunit contacts.

Polymeric hydrogels are increasingly being considered as a scaffold for tissue engineering because they show similarity to the extracellular matrix (ECM) of many tissues. To control various cellular processes, hydrogels are often functionalized or loaded with various bioactive molecules such as: specific ligands for adhesion receptors, growth factors, hormones, enzymes, and other natural or synthetic regulators of cellular behavior [1]. Cell adhesion is essential for cell communication and regulation of the cell cycle and is therefore of vital importance in tissue engineering. Biomimetic approaches have been investigated to facilitate cell-scaffold adhesion interactions. In particular, the recombinant mussel Perna viridis foot protein 5β (Pvfp5β) was developed. Mussel adhesion is made possible by the secretion of a waterproof protein-based adhesive composed of a mixture of proteins called mussel adhesive protein (MAP) or mussel foot protein (mfps). This allows it to be fixed to almost any type of surface when wet [2]. In this work, a two-step physical gelling process is used to create hydrogel scaffolds with a 50 wt% mixture of k-carrageenan (kC) and polyvinyl alcohol (PVA), which are coated with Pvfp5β. The mechanical and morphological properties of hydrogels were investigated both after conditioning in a typical cell culture medium and after coating with Pvfp5β. The protein was strongly adsorbed on the surface of the hydrogel and, as shown by confocal analysis, was able to interact primarily with the kC component of the scaffold and penetrate into it to some depth. NIH-3T3 mouse embryonic fibroblasts were seeded in hydrogel and cultured for up to 2 weeks. The role of Pvfp5β in promoting cell adhesion, diffusion and colonization of scaffolds was verified. References [1] H. W. Ooi, S. Hafeez, C. A. van Blitterswijk, L. Moroni, and M. B. Baker, Mater. Horiz., 2017, 4 (6), 1020–40. [2] Y. He, C. Sun, F. Jiang, B. Yang, J. Li, C. Zhong, L. Zheng, H. Ding, Soft Matter, 2018, 14, 7145–7154

Fractal analysis can be properly applied to complex structures, like physical and chemical networks formed by particles or polymers, when they exhibit self-similarity over an extended range of length scales and, hence, can be profitably used not only for their morphological characterization but also for individuating possible relationships between morphology and mechanisms of aggregation and crosslinking, as well as between morphology and physical properties. Several experimental methods are available to determine the fractal dimension of gel networks, including various scattering techniques and microscopies, permeability measurements and rheology. The present study regards the self-assembly kinetics of High Methoxyl Pectin (HMP) solutions with different pectin and sucrose concentrations investigated by rheological measurements to highlight the effects of pectin and sucrose concentrations on the gel point and to evaluate the degree of compactness of the incipient gel networks through an interpretation of the viscoelastic response at the sol-gel transition.

During their lifecycle, many marine organisms rely on natural adhesives to attach to wet surfaces for movement and self-defence in aqueous tidal environments. Adhesive proteins from mussels are biocompatible and elicit only minimal immune responses in humans. Therefore these proteins have received increased attention for their potential applications in medicine, biomaterials and biotechnology. The Asian green mussel Perna viridis secretes several byssal plaque proteins, molecules that help anchor the mussel to surfaces. Among these proteins, protein-5β (Pvfp-5β) initiates interactions with the substrate, displacing interfacial water molecules before binding to the surface. Here, we established the first recombinant expression in Escherichia coli of Pvfp-5β. We characterized recombinant Pvfp-5β, finding that despite displaying a CD spectrum consistent with features of a random coil, the protein is correctly folded as indicated by MS and NMR analyses. Pvfp-5β folds as a β-sheet–rich protein as expected for an epidermal growth factor (EGF)-like module. We examined the effects of Pvfp-5β on cell viability and adhesion capacity in NIH-3T3 and HeLa cell lines, revealing that Pvfp-5β has no cytotoxic effects at the protein concentrations used and provides good cell-adhesion strength on both glass and plastic plates. Our findings suggest that the adhesive properties of recombinant Pvfp-5β make it an efficient surface coating material, potentially suitable for biomedical applications including regeneration of damaged tissues.

Optimization of microwave-assisted extraction (MAE) of water-soluble pectin (WSP) from Opuntia ficus indica cladodes was performed using Response Surface Methodology. The effect of extraction time (X-1), microwave power (X-2), pH (X-3) and solid-to-liquid ratio (X-4) on the extraction yield was examined. The optimum conditions of MAE were as follows: X-1 = 2.15 min; X-2 = 517 W; X-3 = 2.26 and X-4 = 2g/30.6 mL. The maximum obtained yield of pectin extraction was 12.57%. Total carbohydrate content of WSP is about 95.5% including 34.4% of Galacturonic acid. Pectin-related proteins represent only the 0.66% of WSP mass. HPSEC and light scattering analyses reveal that WSP is mostly constituted of high molecular pectin and FTIR measurements show that the microwave treatment does not alter the chemical structure of WSP, in which Galacturonic acid content and yield are 34.4% and 4.33%, respectively. Overall, application of MAE can give rise to high quality pectin. (C) 2016 Elsevier Ltd. All rights reserved.

BACKGROUND: Molecular chaperones are a very special class of proteins that play essential roles in many cellular processes like folding, targeting and transport of proteins. Moreover, recent evidence indicates that chaperones can act as potentially strong suppressor agents in Alzheimer's disease (AD). Indeed, in vitro experiments demonstrate that several chaperones are able to significantly slow down or suppress aggregation of Aβ peptide and in vivo studies reveal that treatment with specific chaperones or their overexpression can ameliorate some distinct pathological signs characterizing AD. METHODS: Here we investigate using a biophysical approach (fluorescence, circular dichroism (CD), transmission electron (TEM) and atomic force (AFM) microscopy, size exclusion chromatography (SEC)) the effect of the human chaperonin Hsp60 on Aβ fibrillogenesis. RESULTS: We found that Hsp60 powerfully inhibits Aβ amyloid aggregation, by closing molecular pathways leading to peptide fibrillogenesis. CONCLUSIONS: We observe that Hsp60 inhibits Aβ aggregation through a more complex mechanism than a simple folding chaperone action. The action is specifically directed toward the early oligomeric species behaving as aggregation seeds for on-pathway amyloid fibrillogenesis. GENERAL SIGNIFICANCE: Understanding the specificity of the molecular interactions of Hsp60 with amyloid Aβ peptide allowed us to emphasize the important aspects to be taken into consideration when considering the recent promising therapeutic strategies for neurodegeneration. Copyright © 2016 Elsevier B.V. All rights reserved.

Partially degalactosylated xyloglucan from tamarind seeds (Deg-XG) is a very appealing biopolymer for the production of in situ gelling systems at physiological temperature. In this work, we observe that the morphology of hydrogels evolves towards high degrees of structural organization with time, yielding to dense stacks of thin membranes within 24 h of incubation at 37 °C. We also explore the possibility offered by gamma irradiation of controlling the time scale of this phenomenon, the final morphology and mechanical properties of the system. Structural and molecular modifications of Deg-XG with dose are investigated by FTIR, dynamic light scattering (DLS) and rotational viscosimetry. The impact on gelation ability and gel strength is studied by rheological analysis. The morphology evolution is investigated by SEM analysis, and absence of cytotoxicity verified by MTS assay and optical microscopy of neuroblastoma cells.

Chaperonopathies are diseases in which abnormal chaperones play an etiopathogenic role. A chaperone is mutated or otherwise abnormal (e.g., modified by an aberrant posttranslational modification) in structure/function. To understand the pathogenic mechanisms of chaperonopathies, it is necessary to elucidate the impact of the pathogenic mutation or posttranslational modification on the chaperone molecule's properties and functions. This impact is usually subtle because if it were more than subtle the overall effect on the cell and organism would be catastrophic, lethal. This is because most chaperones are essential for life and, if damaged in structure/function too strongly, there would be death of the cell/organism, and no phenotype, i.e., there would be no patients with chaperonopathies. Consequently, diagnostic procedures and analysis of defects of the abnormal chaperones require a multipronged method for assessing the chaperone molecule from various angles. Here, we present such a method that includes assessing the intrinsic properties and the chaperoning functions of chaperone molecules.

Chaperonopathies are diseases in which abnormal chaperones play an etiopathogenic role. A chaperone is mutated or otherwise abnormal (e.g., modified by an aberrant posttranslational modification) in structure/ function. To understand the pathogenic mechanisms of chaperonopathies, it is necessary to elucidate the impact of the pathogenic mutation or posttranslational modification on the chaperone molecule's properties and functions. This impact is usually subtle because if it were more than subtle the overall effect on the cell and organism would be catastrophic, lethal. This is because most chaperones are essential for life and, if damaged in structure/function too strongly, there would be death of the cell/organism, and no phenotype, i.e., there would be no patients with chaperonopathies. Consequently, diagnostic procedures and analysis of defects of the abnormal chaperones require a multipronged method for assessing the chaperone molecule from various angles. Here, we present such a method that includes assessing the intrinsic properties and the chaperoning functions of chaperone molecules.

This work aims to fill the gap in the present knowledge about the structure of pectin from Opuntia ficus-indica. The water-soluble pectin (WSP) fraction, extracted with the Microwave Assisted Extraction (MAE), was further deproteinated (dWSP) and analyzed through several biophysical and biochemical techniques. HPSEC, light scattering and FTIR data showed that dWSP is low methylated high molecular weight pectin. The biochemical structure of dWSP, after methanolysis, silylation, carboxyl reduction showed that dWSP belongs to rhamnogalacturonan I class. Then, dWSP was heat-modified (HM) to obtain small-molecular weight deproteinated fraction (HM-dWSP). Both species, dWSP and HM-dWSP, were tested in LAN5 and NIH 3T3 model cells to study their biological effect. Results indicated that both dWSP and HM-dWSP exerted cytotoxic activity affecting selectively LAN5 cancer cells, without any effect on NIH 3T3 normal cells.

Several natural and synthetic polysaccharides are able to form, under appropriate conditions, supramolecular structures, typically physical hydrogels, and, together with their biocompatibility, this explains their wide use in food, pharmaceutical and biomedical sectors. In the case of high methoxyl pectins (HMP) the gel formation is promoted by the presence of cosolutes (sugars or polyols) and low pH. The present investigation mainly regards the structuring kinetics of aqueous HMP solutions at add pH (3.1) with the same pectin concentration (0.2% w/w) and different sucrose concentrations (from 56 to 65% w/w). Preliminary viscosity tests were performed to individuate the threshold of the sol region. A sequence of consecutive frequency sweeps was applied to each sample immediately after its preparation. The time evolution of the linear viscoelastic behavior is described by the sigmoidal profiles of both moduli at each applied frequency and more thoroughly defined through the change of the mechanical spectrum, i.e. the variation of the parameters of the generalized Maxwell model or the Friedrich-Braun model which are both suitable to provide a satisfactory data fitting. In particular, the equilibrium modulus G(e) offers a significant description of the gelation kinetics and its sucrose dependence.

International audience; Curcumin is a natural polyphenol able to bind the amyloid beta peptide, which is related to Alzheimer's disease, and modify its self-assembly pathway. This paper focuses on a multi-disciplinary study that starts from the design of curcumin-like compounds with the key chemical features required for inhibiting amyloid beta aggregation, and reports the effects of these compounds on the in vitro aggregation of amyloid beta peptides. Chemoinformatic screening was performed through the calculation of molecular descriptors that were able to highlight the drug-like profile, followed by docking studies with an amyloid beta peptide fibril. The computational design underlined two different scaffolds that were easily synthesized in good yields. In vitro experiments, ranging from fluorescence spectroscopy and confocal microscopy up to small angle X-ray scattering, provided evidence that the synthesized compounds are able to modify the aggregation pattern of amyloid beta peptides both in the secondary structures, and in terms of the overall structure dimensions. The cytotoxic potential of the synthesized compounds was finally tested in vitro with a model neuronal cell line (LAN5). The overall view of this study suggests new concepts and potential difficulties in the design of novel drugs against diverse amyloidoses, including Alzheimer's disease

alpha-Casein is known to inhibit the aggregation of several proteins, including the amyloid beta-peptide, by mechanisms that are not yet completely clear. We studied its effects on insulin, a system extensively used to investigate the properties of amyloids, many of which are common to all proteins and peptides. In particular, as for other proteins, insulin aggregation is affected by secondary nucleation pathways. We found that alpha-casein strongly delays insulin amyloid formation, even at extremely low doses, when the aggregation process is characterized by secondary nucleation. At difference, it has a vanishing inhibitory effect on the initial oligomer formation, which is observed at high concentration and does not involve any secondary nucleation pathway. These results indicate that an efficient inhibition of amyloid formation can be achieved by chaperone-like systems, by sequestering the early aggregates, before they can trigger the exponential proliferation brought about by secondary nucleation mechanisms.

Poly(N-vinylpyrrolidone)-grafted-acrylic acid biocompatible nanogels (NGs) were prepared using an exiting industrial-type electron accelerator and setups, starting from semi-dilute aqueous solutions of a commercial PVP and the acrylic acid monomer. As a result, NGs with tunable size and structure can be obtained quantitatively. Sterility was also imparted at the integrated dose absorbed. The chemical structure of the NGs produced was confirmed through Fourier Transformer Infrared Spectroscopy (FT-IR). The molecular and physico-chemical properties of NGs, such as the hydrodynamic dimensions and surface charge densities, for various polymer and monomer concentrations in the irradiated solutions, are discussed here.

The development of growth factors is very promising in the field of tissue regeneration but specifically designed formulations have to be developed in order to enable such new biological entities (NBEs). In particular, the range of therapeutic concentrations is usually very low compared to other active proteins and the confinement in the target site can be of crucial importance. In-situ forming scaffolds are very promising solutions for minimally invasive intervention in cartilage reconstruction and targeting of NBEs. In this work injectable, in-situ forming gels of a temperature responsive partially degalactosylated xyloglucan (Deg-XG) incorporating the growth factor FGF-18 are formulated and characterized. In particular, injectability and shear viscosity at room temperature, time-to-gel at body temperature, morphology and mechanical properties of gels are investigated. The highly hydrophobic growth factor is favorably incorporated and retained by the gel. Gels undergo a slow erosion process when immersed in PBS at 37 °C that opens up their porous structure. The prolonged hydrothermal treatment leads to structural rearrangements towards tougher networks with increased dynamic shear modulus. Preliminary biological evaluations confirm absence of cytotoxicity and the ability of these scaffolds to host cells and promote their proliferation.

Study of macromolecular structure and dynamics of networks formed by pulsed electron-beam irradiation of poly(N-vinyl pyrrolidone) (PVP) aqueous solutions, at relatively low energy per pulse and across different concentration regimes, provides the basis for the understanding of a new generation of functional nanostructures. Networks are the result of the follow–up reactions initiated by a continuous series of electron pulse-generated hydroxyl radicals, which may have a different fate at the variance of polymer concentration. Different spectroscopic techniques, FT-IR, 13 C { 1 H} CP-MAS NMR and Raman, applied to characterize the formed networks, describe a profound modification of the chemical structure when network size is approaching the nanoscale. Static light scattering measurements provide further information on the average weight molecular weight modification of PVP when forming nanogel particles. From the simultaneous control of network size and modification of chemical functionality stems also an intrinsic fluorescence of these nanogels never observed before. Altogether the obtained radiation-sculptured nanogels exhibit interesting multifunctionality that, coupled with the already proven biocompatibility, can be exploited in the biomedical field.

(1) Background: A new family of nanosystems able to discern between normal and tumor cells and to release a therapeutic agent in controlled way were synthetized by e-beam irradiation. This technique permits to obtain biocompatible, sterile, carboxyl-functionalized polyvinylpyrrolidone (PVP-co-acrylic acid) nanogels (NGs); (2) Methods: Here, we performed a targeting strategy based on the recognition of over-expressed proteins on tumor cells, like the folate receptor. The selective targeting was demonstrated by co-culture studies and flow cytometry analysis, using folate conjugated NGs. Moreover, nanoparticles were conjugated to a chemotherapeutic drug or to a pro-apoptotic siRNA through a glutathione sensitive spacer, in order to obtain a controlled release mechanism, specific for cancer cells. The drug efficiency was tested on tumor and healthy cells by flow cytometric analysis, confocal and epifluorescence microscopy and cytotoxicity assay; the siRNA effect was investigated by RNAi experiment; (3) Results: The data obtained showed that the use of NGs permits a faster cargo release in cancer cells, in response to high cytosolic glutathione level, also improving their efficacy; (4) Conclusion: The possibility of releasing biological molecules in a controlled way and to recognize a specific tumor target allows overcoming the typical limits of the classic cancer therapy.

The inhibition of the aggregation in protein solutions is currently a subject of great interest in many research fields, from the study of protein-misfolding related diseases to pharmaceutics, biotechnology, and food science. α(s1)-Casein, one of the four types of caseins, which are the largest protein component of bovine milk, has been found to hinder the aggregation process of several proteins, including the amyloid β-peptide, involved in Alzheimer's disease. To shed light into the mechanisms by which casein exerts this chaperon-like protective action, we studied its effect on the different steps of the aggregation process of concanavalin A, by means of both static and dynamic light scattering, thioflavin T and ANS fluorescence, circular dichroism, and atomic force microscopy. Our results show that casein has a poor effect on the first step of the process leading to the formation of amyloid-like structures. On the contrary, it has a marked effect on the second step of the process, ascribable to clusters condensation and compaction, up to the formation of very large aggregates. Such an effect requires a molar ratio of casein larger than that necessary to inhibit the fibrillogenesis of the amyloid β-peptide, thus, suggesting a different mechanism of interaction of casein, depending on both conformational properties and relative size of the aggregating molecules.

In this work we describe the chemical and physical changes of Sicilian extra virgin olive oil (EVOO) heated at 90° in a conventional oven for one month. The effects of the thermal treatment along days on volatile compounds and vis- cosity were monitored using electronic nose, gas chromatography and rheology. Data obtained by these techniques were combined in order to create a data matrix for the analysis, and the unsupervised method Principal Component Analysis (PCA) was used. A synergistic effect was obtained by combining all data, thus allowing discriminating oil samples ac- cording to their oxidative status.

Pectin gels at different polymer concentrations were used as matrices for the encapsulation of a volatile flavour (limonene). This provides a useful model system for studying the influence of the matrix viscoelastic properties on the flavour release towards the gel headspace. The electronic nose technique and principal component analysis (PCA), a multivariate data analysis, were used for detecting changes in the fingerprint of the released vapour as a function of pectin concentration. Samples with different composition were also studied by rheological measurements in order to discriminate the effects of polymer concentration on the gelation kinetics from those due to the addition of limonene and the detergent used to dissolve it.The combined approach, rheometry-electronic nose, allows obtaining a direct semi-quantitative correlation between the expected decrease of flavour release intensity and the increasing solid like character of the matrix due to trapping effect. In fact, the comparison of the viscoelastic properties for matrixes with and without the flavour, together with direct observation by optical microscopy, suggests that the release modulation is mainly due to interaction of the gelling pectin with the microemulsion of detergent and flavour. Fil: Monge, Maria Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina Fil: Negri, Ricardo Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química, Física de los Materiales, Medioambiente y Energía. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química, Física de los Materiales, Medioambiente y Energía; Argentina Fil: Giacomazza, Daniela. Consiglio Nazionale delle Ricerche; Italia Fil: Bulone, Donatella. Consiglio Nazionale delle Ricerche; Italia

Molecular chaperones are a class of proteins able to prevent non-specific aggregation of mitochondrial proteins and to promote their proper folding. Among them, human Hsp60 is currently considered as a ubiquitous molecule with multiple roles both in maintaining health conditions and as a trigger of several diseases. Of particular interest is its role in neurodegenerative disorders since it is able to inhibit the formation of amyloid fibrils.Hsp60 structure was considered, until recent years, analogue to the one of its bacterial homolog GroEL, one of the most investigated chaperones, whose crystallographic structure is a homo-tetradecamer, made up of two seven member rings. On the contrary, it has been recently demonstrated by some of us that Hsp60 shows an equilibrium of tetradecamers and heptamers in solution.We present the results of combined investigations including complementary approaches such as molecular dynamics (MD), circular dichroism (CD) and small-angle X-ray scattering (SAXS) concerning structure, self-organization, and stability of naive Hsp60 in solution in comparison with bacterial GroEL. We analyzed the structural differences between the two proteins in solution, evidencing their different oligomeric equilibria, too. Experiments at increasing concentrations of guanidinium hydrochloride provided further information on the stability of the oligomeric species, on the dissociation of tetradecamers into heptamers and monomers, until the unfolding. The unfolding free energy was obtained, proving that GroEL is more stable in respect to Hsp60.

Curcumin is a natural polyphenol able to bind the amyloid beta peptide, which is related to Alzheimer's disease, and modify its self-assembly pathway. This paper focuses on a multi-disciplinary study that starts from the design of curcumin-like compounds with the key chemical features required for inhibiting amyloid beta aggregation, and reports the effects of these compounds on the in vitro aggregation of amyloid beta peptides. Chemoinformatic screening was performed through the calculation of molecular descriptors that were able to highlight the drug-like profile, followed by docking studies with an amyloid beta peptide fibril. The computational design underlined two different scaffolds that were easily synthesized in good yields. In vitro experiments, ranging from fluorescence spectroscopy and confocal microscopy up to small angle X-ray scattering, provided evidence that the synthesized compounds are able to modify the aggregation pattern of amyloid beta peptides both in the secondary structures, and in terms of the overall structure dimensions. The cytotoxic potential of the synthesized compounds was finally tested in vitro with a model neuronal cell line (LAN5). The overall view of this study suggests new concepts and potential difficulties in the design of novel drugs against diverse amyloidoses, including Alzheimer's disease.

Several biopolymers are widely employed in food, pharmaceutical and biomedical sectors by virtue of their ability to generate supramolecular structures, typically physical hydrogels. In the case of high methoxyl pectins (HMP) the gel formation is promoted by the presence of cosolutes (sugars or polyols) and low pH. The present investigation regards the structuring kinetics of aqueous HMP solutions having different polymer concentration and equal sucrose content at 20°C. A sequence of consecutive frequency sweep was applied to each sample immediately after its preparation. The time evolution of the linear viscoelastic behavior is described by the sigmoidal profiles of both moduli at each applied frequency and more thoroughly defined through the change of the mechanical spectrum, i.e. the variation of the parameters of the generalized Maxwell model or the Friedrich-Braun model which are both suitable to provide a satisfactory data fitting.

Xyloglucan is a natural polysaccharide present as storage material in seeds and structural materials in the primary cells walls of higher plants. Xyloglucan from tamarind seed is composed of B(1,4)-D-glucan backbone, partially substituted by a-(1,6)-linked xylose units, some of which are B-D-galactosylated at O-2. In its native form the polymer is water-soluble and forms gel in the presence of moderate amounts of alcohols. When a given fraction of galactosyl residues are removed, the polymer acquires the ability to form gels in aqueous solution at physiological temperatures, a property of great interest for biomedical/pharmaceutical applications. Gelation on increasing temperature can be ascribed to hydrophobic interactions that drive the system into a region of thermodynamic instability. In the highly concentrated regions generated by demixing, the dangling segments of polymeric chains "condensate" into additional intra- and inter molecular bonds forming a more compact structure. The drammatic viscosity increase prevents phase separation to occur and leads to gel formation. Gel morphology and properties are controlled by the relative rate of condensation and gelation, and they are both affected by the molecular weight distribution of the biopolymer. Gamma irradiation of the solid polymer in the form of a dry powder has been carried out to tailor the molecular weight of the polymer without significant alteration of its chemical structure. As a result, temperature induced gelation leads to gels with vaious degrees of structural order, from disordered porous networks for the non-irradiated system to a fairly ordered stack of thin membranes for the system irradiated at 20 kGy. Gamma irradiation of the polymer as a solid materials in the presence of different gaseous atmospheres and in aqueous solution have been carried out in the attempt to elucidate the radiation chemistry of xyloglucan.

Partially degalactosylated xyloglucans are able to form thermo-reversible gels in aqueous solution at physiological temperature. This property makes them very promising materials for tissue engineering. Moreover, due to a highly branched, hydroxyl group-rich molecular structure, xyloglucans are particularly interesting for manufacturing of micro/nanoparticles to be used as nano-scalar delivery devices of active ingredients in cosmetic and pharmaceutical formulations. Here we present results on the temperature-induced self-assembly of degalactosylated xyloglucan at varying polymer concentration from dilute to semi-dilute regime. Our aim is to investigate the gelation mechanism for gaining information valuable in tailoring structural and mechanical properties of either nanogel particles or macrogel depots. We used static and dynamic light scattering, rheology, and scanning electron microscopy to follow the gelation kinetics and characterize the system in its final state. Results obtained at low polymer concentration show that, on increasing temperature, the polymer chains and pre-existing clusters form larger structures having higher density and homogeneity over the length scale of a few nanometers. Despite the system polydispersity, signatures of concomitant coil-globule and LCST transition can be envisaged. Both demixing and coil-to globule transition are induced by the worsening of the solvent quality with increasing temperature. At increasing polymer concentration, gelation on macroscopic scale occurs quickly, but continuous adjustments are observed over times comparable to those required for nanogel formation. The microstructure of the gel evolves from a disordered arrangement of partially folded sheets, forming large and heterogeneous cavities, to a layered microstructure with regularly stacked sheets connected by filaments and transverse walls. The morphological evolution of the gel suggests the formation of several crosslinking lines suturing the polymeric membranes formed by ribbon-like structures self-assembly.

Neuronal membrane damage is related to the early impairments appearing in Alzheimer's disease due to the interaction of the amyloid β-peptide (Aβ) with the phospholipid bilayer. In particular, the ganglioside GM1, present with cholesterol in lipid rafts, seems to be able to initiate Aβ aggregation on membrane. We studied the thermodynamic and structural effects of the presence of GM1 on the interaction between Aβ and liposomes, a good membrane model system. Isothermal Titration Calorimetry highlighted the importance of the presence of GM1 in recruiting monomeric Aβ toward the lipid bilayer. Light and Small Angle X-ray Scattering revealed a different pattern for GM1 containing liposomes, both before and after interaction with Aβ. The results suggest that the interaction with GM1 brings to insertion of Aβ in the bilayer, producing a structural perturbation down to the internal layers of the liposome, as demonstrated by the obtained electron density profiles.

The effects of K(+), Na(+) ions and their mixture on the conformational transition and macroscopic gel properties of kappa-Carrageenan system have been studied using different experimental techniques. The macroscopic gelation properties of kappa-Carrageenan were found to be dependent upon cosolute type. Indeed, a more ordered and strong gel was obtained in the presence of K(+) with respect to Na(+) ions. The gel properties obtained using mixtures of two cosolutes are shown to depend on the [K(+)]/[Na(+)] ratio.

Experimental determination of stability conditions or instability boundaries in protein solution is a powerful tool for testing interaction models and for use in seeking an understanding of the mechanisms leading to protein aggregation. In a recent work on lysozyme solutions at different NaCl concentrations, we have determined the instability boundaries (spinodal line) of a liquid–liquid phase separation, metastable with respect to crystallization. Here, we review previous results, pointing out the scaling critical behaviour of isothermal compressibility with respect to spinodal temperatures, and discuss interaction models and the addition of an entropic term. The measured slowing down of concentration fluctuation dynamics is clearly related to the divergence of the fluctuation correlation length on approaching each point of the spinodal line. This observed off-critical slowing down calls for a crucial role for enhanced fluctuations in the crystallization process.

Many soft materials, such as gels or glasses, exhibit both a fast and a very slow relaxation behavior, often related to thermally activated processes restoring ergodicity. Pusey and Van Megen (Physica A 157 (1989) 705), have elaborated a theory that allows the usage of standard light scattering techniques to treat systems that are dynamically arrested, or non-ergodic, over the experimental time-scale. This theory concerning the distribution of intensity scattered by non-ergodic media is here extended, by taking into account second order temporal coherence of scattered radiation. The time-integrated intensity distribution function so obtained allows to distinguish between fast and slow contributions when the two time scales are not (or not yet) completely separated. Thus, by simple and quick static light scattering measurements one can follow an ergodic-to-non-ergodic transition. We present an experiment on gelation kinetics of sucrose-pectin systems, which illustrates the quality of the method, and show how the gel network is formed out of a homogeneous solution. © 2004 Elsevier B.V. All rights reserved.

Heat shock protein 60kDa is a molecular chaperone (GroEL human homolog) that assists protein folding in mitochondria (mtHsp60). It is synthesized in the cell cytoplasm as a higher molecular weight precursor form (p-mtHsp60) containing a N-terminal targeting sequence, that is cleaved after import into the mitochondrial matrix [1, 2].It has been established, and demonstrated by various techniques, Hsp60 can accumulate in the cytosol, in various pathological conditions (i.e., cancer and chronic inflammatory diseases). The cytosolical Hsp60 accumulation mechanism may occur with or without mitochondrial release concomitantly, so that in the cytosol the two types of 60 kDa chaperonin proteins, (mtHsp60 and its precursor naive form, p-mtHsp60) could coexist [3]. It has been recently observed that in a wide range of concentration, the precursor naive Hsp60 is able to assemble in both heptamers and tetradecamers [4].Key questions still unanswered pertain to the differences in structure-function features that might exist between the well-studied prokaryotic GroEL and the largely unexplored eukaryotic Hsp60 proteins. Moreover, studies on human Hsp60 structure and oligomeric state in vitro could help to validate its role in physiological or pathological cases. In order to pursue this goal, we investigated the (dis)assembly and thermal stability of mtHsp60, p-mtHsp60 and GroEL in vitro, by means of Differential Scanning Calorimetry (DSC) and Isothermal Titration Calorimetry (ITC). Complementar Circular Dicroism (CD) measurements were done to follow the change in the secondary structure due to unfolding.[1] Ellis RJ, 2007, Adv Exp Med Biol., 594: 1-13.[2] Jonathan D, 2000, JHC., 48(1): 45-56.[3] D. Chandra, 2007, J. Biol. Chem., 282: 31289-31301.[4] Vilasi S. et al, 2014, Plos One., 9(5): e97657.

Xyloglucan is a natural polysaccharide having a cellulose-like backbone and hydroxyl groups-rich side-chains. In its native form the polymer is water-soluble and forms gel only in presence of selected co-solutes. When a given fraction of galactosyl residues are removed by enzymatic reaction, the polymer acquires the ability to form a gel in aqueous solution at physiological temperatures, a property of great interest for biomedical/pharmaceutical applications. This work presents data on the effect of a temperature increase on degalactosylated xyloglucan dispersed in water at concentration low enough not to run into macroscopic gelation. Results obtained over a wide interval of length scales show that, on increasing temperature, individual polymer chains and pre-existing clusters self-assemble into larger structures. The process implies a structural rearrangement over a few nanometers scale and an increase of dynamics homogeneity. The relation of these findings to coil-globule transition and phase separation is discussed.

This work concerns the aggregation properties of (Gly-Val-Gly-Val-Pro)(251) rec, a polypentapeptide reflecting a highly conserved repetitive unit of the bioelastomer, elastin. On raising the temperature of aqueous solutions above 25 degrees C, this polypeptide was already known to undergo concurrent conformational changes (hydrophobic folding), phase separation, and self-assembly with formation of aggregated three-stranded filaments composed of dynamic polypeptide helices, called beta-spirals. Aggregates obtained from the solution can be shaped into bands that acquire entropic elastic properties upon gamma-irradiation and can perform a variety of energy conversions. Previous studies have shown that aggregation is prompted by the (diverging) critical fluctuations of concentration occurring in the solution, in vicinity of its spinodal line. Here, we present combined circular dicroism (CD) and light scattering experiments, and independent fittings of experimental data to the theoretical spinodal and binodal (coexistence) lines. Results show the following logical and causal sequence of processes: (a) Smooth and progressive conformational changes promoted by concentration fluctuations occurring as temperature is raised "pull down" (in the temperature scale) the instability region of the solution. (b) This further promotes critical fluctuations. (c) The related locally high concentration prompts a further substantial conformational change ending in triple-helix formation and coacervation. (d) This intertwining of processes, covering different length scales (from that of individual peptides to the mesoscopic one of demixed regions), is related to the fact that solvent-induced interactions play a strong role over the entire scale span. These results concur with other recent ones in pointing out that process interactions over many length-scales probably reflect a frequent if not ubiquitous pattern in protein aggregation. This may be highly relevant to the desirable deep understanding of such phenomenon, whose interests cover many fields.

We study by molecular-dynamics simulations the effect of electric charges of either sign on hydrophobic interactions and on the dynamics of hydration water, using explicit water and very simplified solutes. Results show that the presence of a charged solute can disrupt the "hydrophobic contact bond" between two apolar solutes nearby, by forcing them towards a different configuration. As a consequence of different structural changes of the solvent caused by charges of opposite sign, the effect is markedly charge-sign-dependent. Analogous weaker effects appear to be induced by the presence of one additional apolar element. The dynamics of hydration water around each solute is also seen to be strongly influenced by the presence of other (charged or uncharged) nearby solutes. Comparison between our results on hydration water dynamics around charged solutes and available experimental data allows sorting out the effects of solute charge sign and size. Our results also offer a plain interpretation of the equivalence of the effects on water structure due to solute ions and to high pressures. These results reflect at a basic paradigmatic level the immensely more complex cases of well-known phenomena such as salting-in and salting-out, and of protein conformational changes caused, e.g., by the arrival of a charged or of an apolar group (phosphorilation or methylation). As it will be discussed, they help in the direction of Delbruck's desirable "progress towards a radical physical explanation" for this class of phenomena.

α,β-polyasparthydrazide (PAHy), a synthetic water-soluble biocompatible polymer, was chemically crosslinked with ethyleneglycol diglycidylether (EGDGE), in order to obtain water swellable microparticies. These were characterized by means of FT-IR spectrophotometry and by means of particle size distribution analysis. The mean pore size of the prepared gels as various crosslinking ratios and the fractal dimensions were determined by light scattering measurements. Swelling measurements gave evidence of the high affinity of PAHy-EGDGE microparticles towards aqueous media at different pH values. The physical state of the prepared networks was evaluated by means of X-rays diffractometry and thermal analysis. A peculiar effect of the degree of cross-linking on the glass transition temperature (T g ) value was shown. In vitro chemical and enzymatic stability studies suggested that the prepared samples do not undergo any degradation alter treatment at pH 1. 7.4 and 10 as well as after incubation with enzymes such as peptin and α-chymotrypsin.

Molecular dynamics and potential of mean force (PMF) calculations are performed on an aqueous sucrose solution of an ion pair to investigate how ionic interactions are modified by the presence of the sugar. The presence of sucrose is shown to deepen the PMF minimum for Na–Cl ion pair, with an overall behavior compatible with continuum electrostatics. The comparison of PMF and hydration of the Na–Cl ion pair in pure water and in concentrated aqueous sucrose solution clearly reveals the preferential hydration of ions. The relevance of these results to biological systems is discussed.

A strong interest is currently focused on protein self-association and deposit. This usually involves conformational changes of the entire protein or of a fragment. It can occur even at low concentrations and is responsible for pathologies such as systemic amyloidosis, Alzheimer's and Prion diseases, and other neurodegenerative pathologies. Readily available proteins, exhibiting at low concentration self-association properties related to conformational changes, offer very convenient model systems capable of providing insight into this class of problems. Here we report experiments on bovine serum albumin, showing that the process of conformational change of this protein towards an intermediate form required for coagulation occurs simultaneously and interacts with two more processes: mesoscopic demixing of the solution and protein cross-linking. This pathway of three interacting processes allows coagulation even at very low concentrations, and it has been recently observed also in the case of a nonpeptidic polymer. It could therefore be a fairly common feature in polymer coagulation/gelation. Proteins 1999;37:116-120.

Molecular chaperones play essential and several roles in many cellular processes, including protein folding, targeting, transport, and are essential in fighting the consequences of protein misfolding and aggregation or enhancing disaggregation of toxic aggregates by clearance mechanisms. In particular, recent studies have attributed to some chaperones the capability to directly interact with amyloid aggregation-prone species of peptide involved in Alzheimer Disease in solution and/or suppress the associated toxicity. Among them are the principal heat-shock proteins (HSPs), the small HSPs, but also molecules the exhibit marked chaperon like activity, like milk caseins or BRICHOS domains. There are several mechanisms by which chaperones exert their protective action, many of which involve protein disordered regions. However, in many cases, the scarcity of structural data has impeded an understanding of the recognition and antiaggregation mechanisms, that result crucially important when considering how to screen for and characterize potential inhibitors of amyloidosis. Here we show how a human chaperonin, Hsp60, and intrinsically disorder colloids with marked chaperon-like activity, the milk caseins, can specifically influence Aβ40 fibrillogenesis. Inhibition studies are correlated with investigation on structural, self organization and stability properties of the chaperones under study performed by a battery of biophysical methods and aimed to understand what are molecular determinants responsible for their inhibitory action.

We report studies of the interplay among processes of molecular conformational changes, spinodal demixing of the solution, and molecular crosslinking involved in the physical gelation of a biopolysaccharide-water system. Multiple interactions and kinetic competition among these processes were studied under largely different absolute and relative values of their individual rates by appropriate choices of the quenching temperature at constant polymer concentration. Quenching temperature strongly affects the rate of growth but not the final value of the fractal dimension of the gel. Kinetic competition plays a central role in determining the final conformation of individual molecules and the structure and properties of the final gel. This behavior highlights the frustrated nature of the system, and the need of bringing kinetics sharply into focus in gelation theories. General aspects of the present findings and, specifically, the interplay of molecular conformation changes, solution demixing, and molecular crosslinking extend the relevance of these studies to the fast growing field of amyloid condensation and Prion diseases.