The role of water in hemoglobin function and stability

Effects of electric charges on hydrophobic forces. II.

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 …

Multi-Functional Nanogels for Tumor Targeting and Redox-Sensitive Drug and siRNA Delivery

(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 gl…

Physical Origin and Biological Significance of Solvent Induced Forces

The notion of solvent-induced forces (SIFs), as distinct from solute-solute forces acting through the solvent, is illustrated in terms of: i) the microscopic space-and time-resolved view provided by Molecular Dynamics Simulations; ii) the standard statistical-mechanical formulation, and iii) the inherent structures of water. It is shown that the origin of SIFs is in the non-additivity of the effects of solute-perturbation on the H-bond network in the solvent. This nonadditivity does not require non-additivity of water-water and solute-water interaction potentials. Two experimental studies, illustrating different aspects of SIFs are discussed in detail. One is the case of Human Adult Hemoglo…

K+ and Na+ effects on the gelation properties of κ-Carrageenan

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.

Mesoscopic gels at low agarose concentration: perturbation effects of ethanol

Aqueous agarose solutions at low concentrations (0.5 g/liter) were temperature quenched below the spinodal line to form mutually disconnected mesoscopic gels. In the presence of 6% ethanol, these solutions, obtained by quenching at the same temperature depth as in pure water, appear much more fluid, as determined by probe diffusion experiments. We show by static and dynamic light scattering that this can be explained by the solvent-mediated effects of ethanol, leading to a globular shape of mesoscopic agarose gels, rather than to an extended rodlike structure observed in pure water. Our findings show the significant effects of solvent perturbations on particle condensation and, therefore, m…

Recovery from Food Waste-Biscuit Doughs Enriched with Pomegranate Peel Powder as a Model of Fortified Aliment

This research was funded by the "IEV Programme de Cooperation Italie-Tunisie 2014-2020, Re-lancer une nouvelle economie (Re-Ne)". EU project code C-5-3.1-39.

Physico-chemical and mechanical characterization of in-situ forming xyloglucan gels incorporating a growth factor to promote cartilage reconstruction

Abstract 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 fo…

Curcumin-like compounds designed to modify amyloid beta peptide aggregation patterns

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 t…

Poly(vinyl alcohol)/κ-Carrageenan-based hydrogels enriched with the adhesive mussel protein Pvfp5β as 3D cell culture scaffold for tissue engineering applications

Many marine organisms such as sandcastle worms, barnacles and mussels, produce natural adhesives to attach to wet surfaces in aqueous tidal environments. In mussels, the adhesion is possible through the secretion of a protein-based water-resistant glue, composed of a mixture of proteins called mussel adhesive proteins (MAPs) or mussel foot proteins (mfps), that allow anchoring to almost any kind of surface in wet conditions [1]. The proteins confined to adhesive plaques are mfp-2, -3, -4, -5, and -6. All these proteins contain an atypically high concentration of the catecholic amino acid 3,4- dihydroxy-l-phenylalanine (DOPA), obtained by the post-translational enzymatic hydroxylation of tyr…

Amyloid β-peptide insertion in liposomes containing GM1-cholesterol domains.

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, bot…

Self-assembly of biopolymeric structures below the threshold of random cross-link percolation

Self-assembly of extended structures via cross-linking of individual biomolecules often occurs in solutions at concentrations well below the estimated threshold for random cross-link percolation. This requires solute-solute correlations. Here we study bovine serum albumin. Its unfolding causes the appearance of an instability region of the sol, not observed for native bovine serum albumin. As a consequence, spinodal demixing of the sol is observed. The thermodynamic phase transition corresponding to this demixing is the determinative symmetry-breaking step allowing the subsequent occurrence of (correlated) cross-linking and its progress up to the topological phase transition of gelation. Th…

(DIS)Assembly and Structural Stability of mtHsp60 and its Precursor NaÏve Form

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, (m…

Minimalism in radiation synthesis of biomedical functional nanogels.

A scalable, single-step, synthetic approach for the manufacture of biocompatible, functionalized micro- and nanogels is presented. In particular, poly(N-vinyl pyrrolidone)-grafted-(aminopropyl)methacrylamide microgels and nanogels were generated through e-beam irradiation of PVP aqueous solutions in the presence of a primary amino-group-carrying monomer. Particles with different hydrodynamic diameters and surface charge densities were obtained at the variance of the irradiation conditions. Chemical structure was investigated by different spectroscopic techniques. Fluorescent variants were generated through fluorescein isothiocyanate attachment to the primary amino groups grafted to PVP, to …

Recombinant mussel protein Pvfp5β enhances cell adhesion of poly(vinyl alcohol)/k-carrageenan hydrogel scaffolds

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 protein Pvfp-5β: A potential tissue bioadhesive

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 establis…

Human Hsp60 with Its Mitochondrial Import Signal Occurs in Solution as Heptamers and Tetradecamers Remarkably Stable over a Wide Range of Concentrations

It has been established that Hsp60 can accumulate in the cytosol in various pathological conditions, including cancer and chronic inflammatory diseases. Part or all of the cytosolic Hsp60 could be naive, namely, bear the mitochondrial import signal (MIS), but neither the structure nor the in solution oligomeric organization of this cytosolic molecule has still been elucidated. Here we present a detailed study of the structure and self-organization of naive cytosolic Hsp60 in solution. Results were obtained by different biophysical methods (light and X ray scattering, single molecule spectroscopy and hydrodynamics) that all together allowed us to assay a wide range of concentrations of Hsp60…

Structure and biological evaluation of amino-functionalized PVP nanogels for fast cellular internalization

Abstract Aminopropyl methacrylamide chloride-graft-poly(N-vinyl pyrrolidone) nanogels (NGs) were designed to exploit the favorable properties of poly(N-vinyl pyrrolidone) (PVP), such as its high affinity to water and complexation ability of ions, molecules and macromolecules, with the availability of primary amino groups for bioconjugation reactions. A thorough structural characterization of the nanoscalar networks was performed via 1 H NMR and solid state 13 C NMR spectroscopies, while solid state NMR relaxation time measurements completed the NGs description in terms of polymer network density. Information on the hydrodynamic size and surface charge densities were sought via dynamic light…

Mesoscopic gel at low agarose concentration in water: a dynamic light scattering study

Previous work in our laboratory has shown that at very low agarose concentration in water gelation still occurs within mutually disconnected, high concentration regions generated by spinodal demixing. The freely diffusing particles obtained in these conditions are studied in the present work by depolarized dynamic light scattering and probe diffusion experiments. These particles are found to behave as large (in fact, mesoscopic) polymer fibers entangled in a continuously rearranged mesh with scaling parameters typical of partially flexible, neutral chains. The present results allow specifying the notion of mesoscopic gelation. They also reveal that the same symmetry-breaking mechanism that …

Multiple interactions between molecular and supramolecular ordering

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 s…

Influence of gamma-irradiation on thermally-induced mesoscopic gelation of degalactosylated xyloglucans

Thermoresponsive degalactosylated xyloglucans have been already proposed as in situ gelling scaffolds for tissue engineering, due to their reversible macroscopic thermal gelation at body temperature and biodegradability. The highly branched, hydroxyl group-rich molecular structure renders xyloglucans interesting raw materials also in the form of micro/nanoparticles for application as nanoscalar drug delivery devices in cosmetic and pharmaceutical formulations. Owing to their natural source, xyloglucans show high average molecular weight, broad molecular weight distribution and poor water solubility, as large and compact aggregates usually form via inter-molecular hydrogen bonding. Co-60 gam…

Quantitative analysis of the impact of a human pathogenic mutation on the CCT5 chaperonin subunit using a proxy archaeal ortholog

The human chaperonin complex is a ~ 1 MDa nanomachine composed of two octameric rings formed from eight similar but non-identical subunits called CCT. Here, we are elucidating the mechanism of a heritable CCT5 subunit mutation that causes profound neuropathy in humans. In previous work, we introduced an equivalent mutation in an archaeal chaperonin that assembles into two octameric rings like in humans but in which all subunits are identical. We reported that the hexadecamer formed by the mutant subunit is unstable with impaired chaperoning functions. This study quantifies the loss of structural stability in the hexadecamer due to the pathogenic mutation, using differential scanning calorim…

Effects of solvent perturbation on gelation driven by spinodal demixing

We study effects of solvent perturbation on kinetic competition between spinodal demixing and gelation in agarose solutions at a concentration of 5 g/l. Two different cosolutes (tert-butyl alcohol and trimethyl amine N-oxide) known for altering in opposite way solvent-mediated interactions are chosen. By rheometry, static and dynamic light scattering experiments, we show that the cosolute presence shifts the boundary of the instability region of solution leaving unaffected temperature and polymer concentration values required for percolation. Results suggest that an appropriate choice of quenching temperature and solvent allows controlling the gelation time and the gel structural properties.

Relation between structural and release properties in a polysaccharide gel system.

Abstract The potential utility of κ-carrageenan gels for preparing drug release devices is here shown. Structural properties of κ-carrageenan gels prepared with different salt composition and containing Ketoprofen sodium salt, as model drug, have been evaluated with static light scattering and rheological measurements. These properties have been correlated with release profiles in vitro at pH 5.5. Release properties from gelled matrices have been compared with those obtained by two commercial products containing the same drug. Results show that: i) in this system it is possible to easily control the gel texture by using different cationic concentration; ii) the kinetics of drug release by κ…

Recombinant mussel protein Pvfp5β enhances cell adhesion of poly(vinyl alcohol)/k-carrageenan hydrogel scaffolds

Polymeric hydrogels are increasingly considered as scaffolds for tissue engineering due to their extraordinary resemblance with the extracellular matrix (ECM) of many tissues. As cell adhesion is a key factor in regulating important cell functions, hydrogel scaffolds are often functionalized or loaded with a variety of bioactive molecules that can promote adhesion. Interesting biomimetic approaches exploit the properties of mussel-inspired recombinant adhesive proteins. In this work, we prepared hydrogel scaffolds with a 50%w mixture of k-carrageenan (kC) and polyvinyl alcohol (PVA), by a two-step physical gelation process, and we coated them with Perna viridis foot protein-5 beta (Pvfp5 be…

Micro- and mesoscopic process interactions in protein coagulation

It has recently been recognized that pathological protein coagulation is responsible for lethal pathologies as diverse as amyloidosis, Alzheimer and TSE. Understanding the coagulation mechanisms is therefore stirring great interest. In previous studies we have shown that on profoundly different systems coagulation is the result of a strong interaction between two processes on different length scales (mesoscopic and microscopic). Here we report experiments on bovine serum albumin (BSA) showing that the overall mechanism is the result of at least 3 distinct and strongly intertwined processes, on both length scales: molecular conformational changes, solution demixing and intermolecular crossli…

Spinodal demixing, percolation and gelation of biostructural polymers

We present a variety of new experiments which concern the self-assembly of a polymeric network from homogeneous solutions of Agarose, a representative biostructural polysaccharide used for previous studies at our laboratories. They allow deriving a semi-quantitative phase diagram in the T, C plane. The diagram includes both the spinodal and gelation lines. Below a value of about 2% w/v, concentration is not sufficient for direct gelation; however, quenching of the sol from high temperatures to below the spinodal line initiates the spinodal demixing. The latter generates two sets of regions having respectively, higher- and lower-than-average polymer concentrations. In the higher-concentratio…

Spontaneous symmetry-breaking pathways: time-resolved study of agarose gelation

Abstract Extensive time-resolved studies of self-assembly of agarose gels, performed with the use of a variety of techniques allowed identification of the initial break of symmetry and the actual path leading to self-assembly at concentrations well below the random percolation threshold. The overall process is seen to occur through the following sequence: (i) break of symmetry in the sol, causing the spontaneous generation of mesoscopic polymer-rich and solvent-rich regions; (ii) percolation, or nearly percolation [see (iv) below], of polymer-rich regions through the sample, still in the sol state; (iii) start of polymer cross-linking within polymer-rich regions; (iv) progress of cross-link…

Collective properties of hydration: long range and specificity of hydrophobic interactions

We report results of molecular dynamics (MD) simulations of composite model solutes in explicit molecular water solvent, eliciting novel aspects of the recently demonstrated, strong many-body character of hydration. Our solutes consist of identical apolar (hydrophobic) elements in fixed configurations. Results show that the many-body character of PMF is sufficiently strong to cause 1) a remarkable extension of the range of hydrophobic interactions between pairs of solute elements, up to distances large enough to rule out pairwise interactions of any type, and 2) a SIF that drives one of the hydrophobic solute elements toward the solvent rather than away from it. These findings complement re…

Solvent-induced free energy landscape and solute-solvent dynamic coupling in a multielement solute

AbstractMolecular dynamics simulations using a simple multielement model solute with internal degrees of freedom and accounting for solvent-induced interactions to all orders in explicit water are reported. The potential energy landscape of the solute is flat in vacuo. However, the sole untruncated solvent-induced interactions between apolar (hydrophobic) and charged elements generate a rich landscape of potential of mean force exhibiting typical features of protein landscapes. Despite the simplicity of our solute, the depth of minima in this landscape is not far in size from free energies that stabilize protein conformations. Dynamical coupling between configurational switching of the syst…

High-energy radiation processing, a smart approach to obtain PVP-graft-AA nanogels

Abstract 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 irradiate…

Gelation of degalactosylated xyloglucan from nano to macroscopic scale

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 in…

The molecular anatomy of human Hsp60 and its effects on Amyloid-β peptide

Heat Shock Protein 60 (HSP60) is ubiquitous and highly conserved, being present in eukaryotes and prokaryotes, including pathogens. This chaperonin is typically considered a mitochondrial protein but it is also found in other intracellular sites, extracellularly and in circulation. HSP60 is an indispensable component of the Chaperoning System and plays a key role in protein quality control, preventing off-pathway folding events and refolding misfolded proteins. This makes HSP60 a putative therapeutic agent for neurodegenerative diseases associated with aggregation of misfolded proteins, for example, Alzheimer’s Disease. We produced and purified recombinant human HSP60 and investigated the e…

Large-scale radiation manufacturing of hierarchically assembled nanogels

Nanogels (NGs), or small particles formed by physically or chemically crosslinked polymer networks, represent a niche in the development of "smart" nanoparticles for drug delivery and diagnostics. They offer unique advantages over other systems, including a large and flexible surface for multivalent bio-conjugation; an internal 3D aqueous environment for incorporation and protection of (bio)molecular drugs; the possibility to entrap active metal or mineral cores for imaging or phototherapeutic purposes; stimuli-responsiveness to achieve temporal and/or site control of the release function and biocompatibility. The availability of inexpensive and robust preparation methodologies is at the ba…

Synaptosomes: new vesicles for neuronal mitochondrial transplantation

Abstract Background Mitochondrial dysfunction is a critical factor in the onset and progression of neurodegenerative diseases. Recently, mitochondrial transplantation has been advised as an innovative and attractive strategy to transfer and replace damaged mitochondria. Here we propose, for the first time, to use rat brain extracted synaptosomes, a subcellular fraction of isolated synaptic terminal that contains mitochondria, as mitochondrial delivery systems. Results Synaptosome preparation was validated by the presence of Synaptophysin and PSD95. Synaptosomes were characterized in terms of dimension, zeta potential, polydispersity index and number of particles/ml. Nile Red or CTX-FITCH la…

From Small Peptides to Large Proteins against Alzheimer'sDisease.

Alzheimer’s disease (AD) is the most common neurodegenerative disorder in the elderly. The two cardinal neuropathological hallmarks of AD are the senile plaques, which are extracellular deposits mainly constituted by beta-amyloids, and neurofibrillary tangles formed by abnormally phosphorylated Tau (p-Tau) located in the cytoplasm of neurons. Although the research has made relevant progress in the management of the disease, the treatment is still lacking. Only symptomatic medications exist for the disease, and, in the meantime, laboratories worldwide are investigating disease-modifying treatments for AD. In the present review, results centered on the use of peptides of different sizes invol…

Irreversible gelation of thermally unfolded proteins:structural and mechanical properties of lysozyme aggregates

The formation of protein aggregates is important in many fields of life science and technology. The morphological and mechanical properties of protein solutions depend upon the molecular conformation and thermodynamic and environmental conditions. Non-native or unfolded proteins may be kinetically trapped into irreversible aggregates and undergo precipitation or gelation. Here, we study the thermal aggregation of lysozyme in neutral solutions. We characterise the irreversible unfolding of lysozyme by differential scanning calorimetry. The structural properties of aggregates and their mechanisms of formation with the eventual gelation are studied at high temperature by spectroscopic, rheolog…

Temperature-induced self-assembly of degalactosylated xyloglucan at low concentration

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 …

Novel hydrogels based on a polyasparthydrazide. Synthesis and characterization

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 therm…

Hsp60, amateur chaperone in amyloid-beta fibrillogenesis

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), t…

Physics and biophysics of solvent induced forces: hydrophobic interactions and context-dependent hydration

Solvent induced forces (SIFs) among solutes derive from solvent structural modification due to solutes, and consequent thermodynamic drive towards minimization of related free energy costs. The role of SIFs in biomolecular conformation and function is appreciated by observing that typical SIF values fall within the 20–200 pN interval, and that proteins are stable by only a few kcal mol–1 (1 kcal mol–1 corresponds to 70 pN A). Here we study SIFs, in systems of increasing complexity, using Molecular Dynamics (MD) simulations which give time- and space-resolved details on the biologically significant scale of single protein residues and sidechains. Of particular biological relevance among our …

Interacting processes in protein coagulation

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 intermedia…

Moringa oleifera Leaf Powder as Functional Additive in Cookies to Protect SH-SY5Y Cells

The aim of this work is the evaluation of the addition of Moringa leaf powder (MLP) in cookies in terms of antioxidant properties, dough processability and sensorial properties of the cookies. The total content of biophenols and flavonoids in MLP was detected and the identification of the bioactive molecules was performed by HPLC-ESI-TOF-MS measurements, before and after oven treatment at 180 ◦C for 20 min. After a preliminary evaluation of the MLP water soluble fraction (MLPsf) cytotoxicity, its protective effect against an oxidative injury induced in the SH-SY5Y cells was assessed. Data evidence that the bioactive molecules present in MLPsf are effective in preventing ROS production and i…

Chaperonin of Group I: Oligomeric spectrum and biochemical and biological implications

Chaperonins play various physiological roles and can also be pathogenic. Elucidation of their structure, e.g., oligomeric status and post-translational modifications (PTM), is necessary to understand their functions and mechanisms of action in health and disease. Group I chaperonins form tetradecamers with two stacked heptameric rings. The tetradecamer is considered the typical functional complex for folding of client polypeptides. However, other forms such as the monomer and oligomers with smaller number of subunits than the classical tetradecamer, also occur in cells. The properties and functions of the monomer and oligomers, and their roles in chaperonin-associated diseases are still inc…

Oligomeric State and Holding Activity of Hsp60

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 differentia…

Entrapment of A Beta 1-40 peptide in unstructured aggregates

Recognizing the complexity of the fibrillogenesis process provides a solid ground for the development of therapeutic strategies aimed at preventing or inhibiting protein-protein aggregation. Under this perspective, it is meaningful to identify the possible aggregation pathways and their relative products. We found that Aβ-peptide dissolved in a pH 7.4 solution at small peptide concentration and low ionic strength forms globular aggregates without typical amyloid β-conformation. ThT binding kinetics was used to monitor aggregate formation. Circular dichroism spectroscopy, AFM imaging, static and dynamic light scattering were used for structural and morphological characterization of the aggre…

Structure and Stability of Hsp60 and Groel in Solution

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, …

Immunomorphological Pattern of Molecular Chaperones in Normal and Pathological Thyroid Tissues and Circulating Exosomes: Potential Use in Clinics

The thyroid is a major component of the endocrine system and its pathology can cause serious diseases, e.g., papillary carcinoma (PC). However, the carcinogenic mechanisms are poorly understood and clinical useful biomarkers are scarce. Therefore, we determined if there are quantitative patterns of molecular chaperones in the tumor tissue and circulating exosomes that may be useful in diagnosis and provide clues on their participation in carcinogenesis. Hsp27, Hsp60, Hsp70, and Hsp90 were quantified by immunohistochemistry in PC, benign goiter (BG), and normal peritumoral tissue (PT). The same chaperones were assessed in plasma exosomes from PC and BG patients before and after ablative surg…

Corrigendum to “Kinetics of Different Processes in Human Insulin Amyloid Formation” [J. Mol. Biol. 366/1 (2007) 258-274]

Mauro Manno⁎, Emanuela Fabiola Craparo, Alessandro Podesta, Donatella Bulone, Rita Carrotta, Vincenzo Martorana, Guido Tiana and Pier Luigi San Biagio Institute of Biophysics at Palermo Italian National Research Council, via U. La Malfa 153, I-90146 Palermo, Italy Dipartimento di Chimica e Tecnologie Farmaceutiche Universita di Palermo via Archirafi 32 I-90123 Palermo, Italy Department of Physics and CIMAINA, University of Milano, via Celoria 16, I-20133 Milano, Italy Department of Physics, University of Milano and INFN, via Celoria 16, I-20133 Milano, Italy

Structure of e-beam sculptured poly(N-vinylpyrrolidone) networks across different length-scales, from macro to nano

Abstract 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 t…

Kinetics of Insulin Aggregation: Disentanglement of Amyloid Fibrillation from Large-Size Cluster Formation

Kinetics of human insulin aggregation has been studied at pH 1.6 and 60 degrees C, when amyloid fibrils are formed. We developed a novel approach based on the analysis of scattered light intensity distribution, which allows distinguishing between small and large size aggregates. By this method, we observed an exponential growth of fibrillar aggregates implying a heterogeneous aggregation mechanism. Also, the apparent lag time observed, correlated with the major increase of thioflavin T fluorescence, has been assigned to the onset of large size cluster formation.

Oligonucleotides-decorated-poly(N-vinyl pyrrolidone) nanogels for gene delivery

Pulsed electron-beam irradiation of a semi-dilute poly(N-vinyl pyrrolidone) (PVP) aqueous solution in the presence of acrylic acid has led to a carboxyl functionalized nanogel system. Nanoparticles hydrodynamic size and surface charge density, in water and as a function of pH, were investigated by dynamic light scattering and laser doppler velocimetry, respectively. Nanogels (NGs) were proved not to be cytotoxic at the cellular level. Indeed, they rapidly bypass the cellular membrane to accumulate in specific cell portions of the cytoplasm, in the perinuclear area. The availability of pendant carboxyl groups on the crosslinked PVP NGs core prompted us to attempt their decoration with a sing…

Protofibril formation of amyloid beta-protein at low pH via a non-cooperative elongation mechanism.

Deposition of the amyloid beta-protein (Abeta) in senile or diffuse plaques is a distinctive feature of Alzheimer's disease. The role of Abeta aggregates in the etiology of the disease is still controversial. The formation of linear aggregates, known as amyloid fibrils, has been proposed as the onset and the cause of pathological deposition. Yet, recent findings suggest that a more crucial role is played by prefibrillar oligomeric assemblies of Abeta that are highly toxic in the extracellular environment. In the present work, the mechanism of protofibril formation is studied at pH 3.1, starting from a solution of oligomeric precursors. By combining static light scattering and photon correla…

Temporal control of xyloglucan self-assembly by radiation-induced degradation

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 as…

Microgel regions in dilute agarose solutions: the notion of non-gelling concentration, and the role of spinodal demixing

Abstract Freely drifting microgel regions are found in aqueous solutions of agarose, a representative biostructural polysaccharide, at concentrations between 0.01% and 0.05% w/v when quenched from 100°C to lower temperature. The size of these domains depends on the quench temperature and agarose concentration. The results agree with recent findings on the role that fluctuations within or close to the instability region of solution have as the initial step towards the self-assembly of supramolecular structures, and throw a new light on the notion of the lowest solute concentration needed for gelation.

Enthalpic and entropic contributions of water molecules to the functional T → R transition of human hemoglobin in solution

Generalized solvent-mediated forces contribute to free energy at the functional T → R transition of human hemoglobin A (HbA). Their contribution is here sorted out quantitatively in both its enthalpic and entropic parts, along with the average number of water molecules involved. The latter (about 75 waters in average) must be considered together with HbA as one statistically defined functional unit for oxygen transport. Their configurations are expected to undergo frequent structural rearrangements. Lifetimes of statistically relevant configurations do not need to (although, of course, they may) exceed by more than a factor 5 the normal H-bond lifetimes of the pure solvent. Compared to the …

Temporal control of xyloglucan self-assembly into layered structures by radiation-induced degradation

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…

E-beam irradiation and UV photocrosslinking of microemulsion-laden poly(N-vinyl-2-pyrrolidone) hydrogels for ‘‘in situ’’ encapsulation of volatile hydrophobic compounds

Gelled microemulsions are the subject of considerable scientific and commercial interest. Many efforts are currently devoted to improving their toxicological profile and functioning as biocompatible diffusion barrier for the controlled delivery of hydrophobic compounds. In the present investigation, a non-ionic polymeric surfactant was chosen to generate an oil-in-water microemulsion of a model fragrance in the presence of poly(N-vinyl-2-pyrrolidone) (PVP). The microemulsion was then subjected to either electron-beam or UV-irradiation to induce free-radical crosslinking of PVP at low temperature and in the absence of crosslinking agents, catalysts and initiators. Irradiation conditions with…

Additional file 1 of Synaptosomes: new vesicles for neuronal mitochondrial transplantation

Additional file 1: Figure S1. A) Morphological analysis of LAN5 cells incubated with different doses (5-10 and 20 µl, which correspond to concentration of 2.5 × 107; 5.1 × 107; 10.2 × 107 particles/100µl respectively) of synaptosomes (Synap). B) Nuclear staining by fluorescence probe Hoechst 3341 of LAN5 cells incubated with different doses (5-10 and 20 µl) of synaptosomes (Synap).