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Effect of Total Apple Polyphenols Extract as Inhibitors of Amyloid Protein Aggregation By: Guarrasi, Valeria; Rappa, Cinzia Giacoma; Costa, Maria Assunta; et al. JOURNAL OF CLINICAL GASTROENTEROLOGY Volume: 52 Supplement: 1 Pages: S94-S94 Published: NOV-DEC 2018

A growing body of evidence shows that Insulin, Insulin Receptor (IR) and IR signaling are involved in brain cognitive functions and their dysfunction is implicated in Alzheimer’s disease (AD) degeneration. Thus, administration of insulin could be a strategy for AD treatment. For this aim we have designed, synthesized and characterized a nanogel system (NG) to deliver insulin to the brain, as a tool for the development of a new therapy for AD. A carboxyl-functionalized poly(N-vinyl pyrrolidone) nanogel system produced by ionizing radiation was chosen as substrate for the covalent attachment of insulin or fluorescent molecules relevant for its characterization. Biocompatibility of the naked carrier was demonstrated by absence of cytotoxicity, oxidative stress and mitochondrial dysfunction. Hemocompatibility was demonstrated by hemolysis, coagulation time, leukocyte proliferation and inflammatory response tests. By immonufluorescence measurements we demonstrated that insulin conjugated to the NG (NG-In) is preotected by protease degradation and is able to bind and activate insulin receptor bringing to trigger the insulin signalling via AKT activation. Moreover, to provide consistent evidence on the functionality of the conjugated insulin on the glucose levels, the effect of NG-In was tested in mice demonstrating that plasma glucose levels was reduced. Neuroprotection of NG-In against dysfunction induced by amyloid β, a peptide mainly involved in AD, was verified. Finally, the potential of NG-In to be efficiently transported across the Blood Brain Barrier was demonstrated by using an in vitro system. All together these results indicated that the synthesized NG-In was a suitable vehicle system for insulin delivery in biomedicine and a very promising tool to develop new therapies for neurodegenerative diseases. The research eas supported by MIUR, FLAGSHIP PROJECT NanoMAX.

I nanogeli sono nanoparticelle polimeriche reticolate idrofile. La struttura polimerica del nanogelo permette la coniugazione di molecole che possano esplicare azioni terapeutiche, diagnostiche e di indirizzamento attivo su siti bersaglio. Le caratteristiche di flessibilità e conformabilità del nanogelo, e delle sue maglie, ne favoriscono l’attraversamento delle barriere biologiche, l’interazione di eventuali ligandi esposti sulla superfice con i recettori target, un’azione protettiva nei confronti del principio attivo incorporato. L’irraggiamento con radiazioni ionizzanti di soluzioni acquose di polimeri è una metodologia sintetica interamente “water-based”, che garantisce rese elevate e che può conferire sterilità al prodotto. [1] Lo studio presentato illustra la possibilità di produrre nanogeli a base di polivinilpirrolidone con le caratteristiche desiderate in termini di dimensioni e con gruppi funzionali disponibili per reazioni di coniugazione con l’insulina. L’obiettivo è quello di realizzare un nanovettore in grado di veicolare l’insulina al cervello attraverso la mucosa nasale. Studi recenti hanno dimostrato che l’insulina è in grado di ridurre la tossicità del peptide beta amiloide (A, coinvolto nella patogenesi della malattia di Alzheimer (MA), e di esplicare una funzione neuroprotettiva. [2, 3] Sono state studiate le caratteristiche dimensionali del nanosistema, la biocompatibilità e la funzione di protezione dalla tossicità indotta da Anei confronti di cellule di neuroblastoma.

Nanogels are nanoscalar polymeric networks, characterized by a dynamic internal structure and a flexible adaptive shape. When they are used as drug carriers, their flexibility and shape changing ability can facilitate the bypass of biological barriers, ensure protection of the payload and enable interaction of any attached ligand with its receptors. Poly(N-vinyl pyrrolidone)-co-acrylic acid nanogels (NGs) have been produced by ebeam irradiation of diluted aqueous solutions of a water-soluble polymer in the presence of a small amount of acrylic acid. In particular, industrial accelerators and the typical set-ups and doses applied for sterilization have been used. [1, 2] Particle size, molecular weight and functionalization can be controlled by a proper selection of polymer concentration and irradiation parameters, such as dose-rate and total dose imparted [3]. In recent years, a growing body of evidence has linked insulin resistance and insulin action to Alzheimer’s Disease (AD), a condition also referred to as Type 3 Diabetes (T3D) [4,5]. Recently, it has been demonstrated that insulin is capable of reducing toxicity induced by A oligomers, the protein mainly involved in AD onset, by inhibition of the intrinsic apoptotic pathway [6]. Moreover, activation of insulin signaling provides a neuroprotective mechanism to counteract oxidative stress, mitochondrial damage and neurodegeneration triggered by A oligomers in neuroblastoma cells [7]. In the present work NGs have been conjugated to insulin (NG-In) to defend the hormone from enzymatic degradation, facilitate its crossing through a blood brain barrier model and protect the LAN5 cells from the damages induced by A oligomers addiction.

Hsp60 is, typically, a mitochondrial protein, but it also occurs in the cytosol, vesicles, and plasma membrane, and in the intercellular space and biological fluids, e.g., blood. Changes in the levels and distribution of Hsp60 are linked to several pathologies, including cancer and chronic inflammatory and autoimmune disorders. What is the histopathological pattern of Hsp60 in the thyroid of Hashimoto’s patients? Are there indications of a pathogenic role of Hsp60 that may make Hashimoto’s thyroiditis a chaperonopathy? Experiments reported here provide information regarding those questions. We found by various immunomorphological techniques increased levels of Hsp60 in the thyroid from HT patients, localized to thyrocytes of small and degenerated follicles and to oncocytes (Hurtle cells). Immunofluorescence showed the chaperonin both inside the cells and also in the plasma membrane, especially in oncocytes. We also found that Hsp60 levels in the blood of HT patients were increased compared to controls and correlated with those of autoantibodies against two distinctive thyroidal proteins, thyroglobulin (TG) and thyroid peroxidase (TPO) (r=0.379, p=0.0103; r=0.484, p=0.0008; respectively). Molecular analysis of these two proteins in comparison with Hsp60 demonstrated various regions of high structural similarity shared by them, which could very well be immunologically crossreactive epitopes. Thus, it is likely that the three proteins potentiate each other as immunogens to elicit autoantibodies and, as antigens, to cause antigen-antibody reactions at those sites in which Hsp60 is exposed, for example the surface of oncocytes. This would lead to inflammation and oncocyte lysis with destruction of thyroidal tissue. The cytometric bead assay revealed that recombinant Hsp60 did not induce increment of cytokine production by peripheral blood mononuclear cells from HT patients. Consequently, we propose that Hsp60 is implicated in the pathogenesis of Hashimoto’s thyroiditis as autoantigen, via a participation of autoantibodies that also recognize TG and TPO, whereas participation of inflammatory cytokines induced by the chaperonin is unlikely. Supported by IEMEST (FC and AJLM)., Italian Journal of Anatomy and Embryology, Vol 118, No 2 (Supplement) 2013

Risulta comprovata nell’industria agroalimentare l’efficacia di oli essenziali di agrumi quale tecnologia per esaltare la sapidità di cibi e bevande. Ad oggi, il rinnovato interesse verso gli oli essenziali deriva dalle loro proprietà antibatteriche ed antifungine, che possono trovare ampia applicazione in sistemi hurdle-technology ai fini di prolungare la shelf-life di agroprodotti, specie in cibi minimally-processed e di IV gamma. Infatti, la necessità di ricercare nuove sostanze non di sintesi con proprietà antimicrobiche deriva dai cambiamenti legislativi in campo alimentare, dalla tendenza dei consumatori ad acquistare cibi processati naturalmente e dalla crescente resistenza dei patogeni alimentari ai comuni composti antimicrobici usati nel comparto alimentare. Gli EOs sono sostanze “generalmente riconosciute come sicure” (“generally recognized as safe”, GRAS) dalla U.S. FDA, per cui il loro potenziale utilizzo negli alimenti soddisfa non solo i requisiti legali, le esigenze dell’industria di trasformazione ma anche la richiesta dei consumatori. L’applicazione di tali biotecnologie, però, non può prescindere dalla caratterizzazione preliminare delle proprietà chimiche ed antimicrobiche degli oli essenziali. A tal fine, sono stati estratti gli oli essenziali (EOs) presenti nelle bucce di agrumi appartenenti a tre specie differenti del genere Citrus. Gli EOs sono stati caratterizzati mediante Gas-Cromatografia accoppiata a Spettrometria di Massa (GC-MS) ed identificati per confronto con libreria NIST, nel caso in cui mostravano una similarità maggiore del 90%. Per valutare l’attività antimicrobica, gli EOs sono stati provati in-vitro tramite un saggio di diffusione in pozzetto. A tal fine, gli EOs estratti sono stati testati contro 47 ceppi fungini alterativi e/o patogeni, precedentemente isolati da matrici alimentari. Gli EOs di limone hanno dimostrato possedere in vitro un’attività inibente della crescita fungina nettamente maggiore confrontata con gli oli estratti da mandarino ed arancia. La differente composizione chimica, evidenziata tramite GC/MS, è responsabile della diversa attività antifungina. In particolare, la presenza di idrocarburi monoterpenici ossigenati e sequiterpeni negli EOs con maggiore attività inibente (EO L1 e EO L2), suggerisce che siano proprio queste classi di composti a possedere le proprietà antifungine evidenziate nei test microbiologici.

Chemical (soluble solids content, titratable acidity, pH), morphological (weight, longitudinal and transversal diameter, thickness of the flesh, number of seeds, peel colour), and sensorial characteristics of four loquat cultivars (‘Algerie’, ‘Claudia’, ‘Nespolone di Trabia’ and ‘Sanfilippara’) from mature, organically-grown trees in Sicily, were analysed. The determinations were carried out by traditional instrumental techniques, by a panel test and by an electronic olfactory system. A panel of 10 trained judges was used to determine intensity of some attributes in the sensory profile of the different cultivars, while the electronic olfactory system was used to identify possible differences in the aromatic “fingerprint”. The main differences in the analytical evaluation among the four cultivars were the higher acidity and the lower fraction of soluble solids of ‘Sanfilippara’ and ‘Nespolone di Trabia’; while the highest value of soluble solids was observed in ‘Claudia’. These chemical characteristics were in agreement with the assessment of judges. In an overall sensory evaluation, ‘Claudia’ scored highest with attributes such as sweetness and juiciness, while ‘Sanfilippara’ had the most characteristic loquat odor and ‘Nespolone di Trabia’ the most grass odour. The responses of electronic olfactory system sensors were analyzed with a Principal Component Analysis (PCA). The plot of the two main components of the PCA analysis, PC1 and PC2, showed discrimination among the aromatic pattern of the four cultivars. The results show that the perception of volatile compounds by the human nose is important to evaluate the quality of loquat fruits, but, in this research, it is unable to discriminate the cultivars as well as the electronic nose.

The interest towards the application of essential oils (EOs) is increasing due to the antimicrobial potential to be used as “natural” alternative to the chemical preservation of foods [1]. EOs are aromatic oily liquids produced as secondary metabolites in various plants exerting different biological properties, such as antibacterial, antiviral, antimycotic and insecticidal. EOs are complex mixtures of lipophilic substances and the chemical composition, thereby the biological activities, is influenced by the raw plant material (genotype, part of the plant, harvest time, geographical, ecological conditions and cultural techniques) and extraction method [2].

In the recent years a growing body of evidence links insulin resistance and insulin action to neurodegenerative diseases, especially Alzheimer’s disease (AD). The importance of insulin in ageing as well as its role in cognition and other aspects of normal brain functions are well established. The hippocampus and cerebral cortex-distributed insulin and insulin receptor (IR) have been shown to be involved in brain cognitive functions. Conversely, deterioration of IR signaling is involved in agingrelated brain degeneration such as in AD and cognitive impairment in type 2 diabetes patients. Insulin administration, while maintaining euglycemia, improves memory in both healthy adults and Alzheimer’s disease patients. In the present review, some common links between AD and type 2 diabetes are presented. Furthermore, several biochemical aspects existing in both pathologies are highlighted.

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 aggregates. They appear stable or at least metastable with respect to fiber growth, therefore appearing as an incidental product in the pathway of fibrillogenesis

Background: alpha(s1)-Casein is one of the four types of caseins, the largest protein component of bovine milk. The lack of a compact folded conformation and the capability to form micelles suggest a relationship of alpha(s1)-casein with the class of the intrinsically disordered (or natively unfolded) proteins. These proteins are known to exert a stabilizing activity on biomolecules through specific interaction with hydrophobic surfaces. In the present work we focused on the effect of alpha(s1)-casein on the fibrillogenesis of 1-40 beta-amyloid peptide, involved in Alzheimer's disease. Methods: The aggregation kinetics of beta-peptide in presence and absence of alpha(s1)-casein was followed under shear at 37 degrees C by recording the Thioflavine fluorescence, usually taken as an indicator of fibers formation. Measurements of Static and Dynamic Light Scattering, Circular Dichroism, and AFM imaging were done to reveal the details of alpha(s1)-casein-A beta(1-40) interaction. Results and discussions: alpha(s1)-Casein addition sizably increases the lag-time of the nucleation phase and slows down the entire fibrillization process. alpha(s1)-Casein sequesters the amyloid peptide on its surface thus exerting a chaperone-like activity by means a colloidal inhibition mechanism. General significance: Insights on the working mechanism of natural chaperones in preventing or controlling the amyloid aggregation.

Fibril deposit formation of ainyloid P-protreiri (AP) iii the braiii is a liallmark oi Alzlieiiiicr disease (AD). Fibril: forination is triggered by 1nolecu1a.rc o~lformationacl hangeii and protein-protein interactions iiivolvirig partially unfo1de:i regioris of different AP peptide moleciiles. lncreasing cvi(.erice suggests that toxicity is linlced to diffusible AP oligoiners, which liave bccri foiind in soluble brain extracts of AI) patients, ratl-ier thail to the insoluble fibres. New thcrapcl tical approach, based 011 searchiiig molecules capable of regulating the peptide aggregation, is currently developiilg.

Recent evidences suggest that insulin delivery to the brain can be an important pharmacological therapy for some neurodegenerative pathologies, including Alzheimer disease (AD). Due to the presence of the Blood Brain Barrier, a suitable carrier and an appropriate route of administration are required to increase the efficacy and safety of the treatment. Here, poly(N-vinyl pyrrolidone)-based nanogels (NG), synthetized by e-beam irradiation, alone and with covalently attached insulin (NG-In) were characterized for biocompatibility and brain delivery features in a mouse model. Preliminarily, the biodistribution of the "empty" nanocarrier after intraperitoneal (i.p.) injection was investigated by using a fluorescent-labeled NG. By fluorescence spectroscopy, SEM and dynamic light scattering analyses we established that urine clearance occurs in 24 h. Histological liver and kidneys inspections indicated that no morphological alterations of tissues occurred and no immunological response was activated after NG injection. Furthermore, after administration of the insulin-conjugated nanogels (NG-In) through the intranasal route (i.n.) no alteration or immunogenic response of the nasal mucosa was observed, suggesting that the formulation is well tolerated in mouse. Moreover, an enhancement of NG-In delivery to the different brain areas and of its biological activity, measured as Akt activation levels, with reference to free insulin administration was demonstrated. Taken together, these results indicate that the synthesized NG-In enhances brain insulin delivery upon i.n. administration and strongly encourage its further evaluation as therapeutic agent against some neurodegenerative diseases.

Human Hsp60 chaperonin and its bacterial homolog GroEL, in association with the corresponding co-chaperonins Hsp10 and GroES, constitute important chaperone systems promoting the proper folding of several mitochondrial proteins. Hsp60 is also currently described as a ubiquitous molecule with multiple roles both in health conditions and in several diseases. Naïve Hsp60 bearing the mitochondrial import signal has been recently demonstrated to present different oligomeric organizations with respect to GroEL, suggesting new possible physiological functions. Here we present a combined investigation with circular dichroism and small-angle X-ray scattering of structure, self-organization, and stability of naïve Hsp60 in solution in comparison with bacterial GroEL. Experiments have been performed in different concentrations of guanidine hydrochloride, monitoring the dissociation of tetradecamers into heptamers and monomers, until unfolding. GroEL is proved to be more stable with respect to Hsp60, and the unfolding free energy as well as its dependence on denaturant concentration is obtained. © 2015 Elsevier B.V.

The present work is focused on the study of the characteristics of the fruits of four main lemon (Citrus limon L. Burm.) varieties cultivated in Sicily: 'Femminello Comune', 'Monachello', 'Zagara Bianca', and 'Femminello Santa Teresa'. Physical and chemical properties as well as aroma compounds were analysed as quality discrimination factors. Also the effect of the storage conditions was verified. Standard experimental techniques were used to determine: weight, longitudinal and transverse diameters, pH, titratable acidity, total soluble solids, juice percentage and total polyphenols. The volatile component was analysed by i) a gas chromatographer with a mass spectrometer detector and ii) an electronic olfactory system ("e-nose") equipped with an array of six MOS sensors. Most of the physical and chemical parameters analysed allowed a statistically significant discrimination among the factors cultivar and storage time. The aromatic pattern was similar for all the cultivars. Differences were observed after the storage at 18° C, particularly for the cultivar 'Monachello'.

Injectable polymer scaffolds are particularly attractive for guided tissue growth and drug/cell delivery with minimally invasive intervention. In the present work, "all-polymeric" gelling systems based on pectins and water-soluble maltose-conjugated chitosans (CM) have been developed. Maltose-conjugated chitosan has been synthesized at three different molar ratios, as evaluated by FITR analysis and fluorimetric titration. A thorough rheological characterization of the blends and their parent solutions has been performed. Macroscopic gelation has been achieved by mixing the high esterification degree pectins with CM at higher maltose grafted to chitosan contents. Gels form in a few minutes and reach their full strength in less than two hours. These features encourage their further development as scaffold for tissue engineering. (C) 2014 Elsevier Ltd. All rights reserved.

The role Hsp60 might play in various inflammatory and autoimmune diseases is under investigation, but little information exists pertaining to Hashimoto’s thyroiditis (HT). With the aim to fill this gap, in the present work, we directed our attention to Hsp60 participation in HT pathogenesis. We found Hsp60 levels increased in the blood of HT patients compared to controls. The chaperonin was immunolocalized in thyroid tissue specimens from patients with HT, both in thyrocytes and oncocytes (Hurthle cells) with higher levels compared to controls (goiter). In oncocytes, we found Hsp60 not only in the cytoplasm but also on the plasma membrane, as shown by double immunofluorescence performed on fine needle aspiration cytology. By bioinformatics, we found regions in the Hsp60 molecule with remarkable structural similarity with the thyroglobulin (TG) and thyroid peroxidase (TPO) molecules, which supports the notion that autoantibodies against TG and TPO are likely to recognize Hsp60 on the plasma membrane of oncocytes. This was also supported by data obtained by ELISA, showing that anti-TG and anti-TPO antibodies cross-react with human recombinant Hsp60. Antibody-antigen (Hsp60) reaction on the cell surface could very well mediate thyroid cell damage and destruction, perpetuating inflammation. Experiments with recombinant Hsp60 did not show stimulation of cytokine production by peripheral blood mononuclear cells from HT patients. All together, these results led us to hypothesize that Hsp60 may be an active player in HT pathogenesis via an antibody-mediated immune mechanism.

In the present study the rheological properties of hydrogels obtained through UV irradiation of an aqueous solution of methacrylated inulin (INUMA) alone or in the presence of four different crosslinkers, have been recorded as a function of irradiation time, at two different wavelengths (320 and 365 nm) and fixed frequency and amplitude by oscillatory experiments. Each hydrogel has been analyzed by measurements of the frequency-dependency of the elastic modulus, G ′, in the linear viscoelastic region. The amount of polymeric chains giving an elastic response has been correlated to the chemical nature of the crosslinker used together with INUMA. Finally the release properties of the hydrogel exhibiting the highest elastic modulus have been tested by using 5-Fluorouracil, as a model drug, and two different loading procedures. The rheological characterization allowed us to determine the most effective condition (appropriate crosslinker, wavelength and irradiation time) to obtain a strong hydrogel that was able to give a modified drug release in simulated gastrointestinal fluids.

Chemical (soluble solids content, titratable acidity, pH), morphological (weight, longitudinal and transversal diameter, thickness of the flesh, number of seeds, peel colour), and sensorial characteristics of four loquat cultivars (‘Algerie’, ‘Claudia’, ‘Nespolone di Trabia’ and ‘Sanfilippara’) from mature, organically-grown trees in Sicily, were analysed. The determinations were carried out by traditional instrumental techniques, by a panel test and by an electronic olfactory system. A panel of 10 trained judges was used to determine intensity of some attributes in the sensory profile of the different cultivars, while the electronic olfactory system was used to identify possible differences in the aromatic “fingerprint”. The main differences in the analytical evaluation among the four cultivars were the higher acidity and the lower fraction of soluble solids of ‘Sanfilippara’ and ‘Nespolone di Trabia’; while the highest value of soluble solids was observed in ‘Claudia’. These chemical characteristics were in agreement with the assessment of judges. In an overall sensory evaluation, ‘Claudia’ scored highest with attributes such as sweetness and juiciness, while ‘Sanfilippara’ had the most characteristic loquat odor and ‘Nespolone di Trabia’ the most grass odour. The responses of electronic olfactory system sensors were analyzed with a Principal Component Analysis (PCA). The plot of the two main components of the PCA analysis, PC1 and PC2, showed discrimination among the aromatic pattern of the four cultivars. The results show that the perception of volatile compounds by the human nose is important to evaluate the quality of loquat fruits, but, in this research, it is unable to discriminate the cultivars as well as the electronic nose.

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, rheological and scattering techniques. The experiments show that irreversible micron-sized aggregates are organised into larger clusters according to a classical mechanism of diffusion and coagulation, which leads to a percolative transition at high concentrations. At a smaller length scale, optical and atomic force microscopy images reveal the existence of compact aggregates, which are the origin of the aggregation irreversibility.

Protein cold-gelation has recently received particular attention for its relevance in bio and food technology. In this work, we report a study on bovine serum albumin cold-gelation induced by copper or zinc ions. Metal-induced cold-gelation of proteins requires two steps: during the first one, the heat treatment causes protein partial unfolding and aggregation; then, after cooling the solution to room temperature, gels are formed upon the addition of metal ions. The thermally induced behaviour has been mainly investigated through different techniques: Fourier transform infrared (FTIR) spectroscopy, circular dichroism, dynamic light scattering (DLS) and rheology. Data have shown that the aggregation process is mainly due to protein conformational changes--alpha-helices into beta-aggregates-forming small aggregated structures with a mean diameter of about 20 nm a few minutes after heating. After metal ion addition, the viscoelastic properties of the gels have been investigated by rheological measurements. The behaviour of the elastic and viscous moduli as a function of time is discussed in terms of ion concentration and type. Our results show that: (1) the elastic behaviour depends on ion concentration and (2) at a given ion concentration, gels obtained in the presence of zinc exhibit an elastic value larger than that observed in the Cu(2+) case. Data suggest that cold-gelation is the result of different mechanisms: the ion-mediated protein-protein interaction and the bridging effect due to the presence of divalent ions in solution.