Search

EAK16 (AEAEAKAKAEAEAKAK) and seven alternating polar/hydrophobic oligopeptides (derived from EAK16 by primary structure modifications) were tested for their resistance to free radical attack, in view of their possible use as biomimetic coatings of biomedical devices; in fact, oxidative radical stress, mainly due to hydroxyl radicals ((OH)-O-center dot), is a common physiological condition occurring under acute or chronic inflammatory response. (OH)-O-center dot radicals, mimicking an endogenous radical stress, were generated by gamma-radiolysis of water solutions. IR and Raman spectroscopies were used to investigate the damages induced by free radicals on the oligopeptides, in particular on their folding and inter-chain interactions. Some oligopeptides, i.e. EAK16 (pept1), pept3 (obtained by KO substitution), pept5 (AAbu substitution), pept7 (insertion at the N-terminus of the RGD sequence), and pept8 (RGD insertion and scrambling of the sequence) were not severely affected by the treatment and retained their prevalent structure (i.e. -helix for pept8 and -sheet for the others). The other peptides, i.e. pept2 (obtained by ED substitution), pept4 (ED and KO substitutions) and pept6 (AY substitution), showed significant conformational changes (i.e. increase in the -helix content) upon irradiation. In pept2 and pept4, modifications in hydrophobic and ionic interactions were observed; the latter were mainly ascribed to the decarboxylation of acidic amino acids. In pept6, the main target of the (OH)-O-center dot attack was Tyr, as deduced by the strong variations observed in Tyr bands both in IR and Raman spectra. This study may contribute to gain insight into the different biological performances of the EAK16-derived peptides and to identify the most adequate materials usable for bone regeneration.

Self-assembling peptides are a category of peptides which undergo spontaneous assembling into ordered nanostructures. These designed peptides have attracted huge interest in the field of nanotechnology for its potential application in areas such as biomedical nanotechnology, cell culturing, molecular electronics, and more. In the emerging field of tissue engineering, the development of synthetic materials promoting cell growth has led to the study of regularly alternating polar/non-polar amphiphilic oligopeptides, such as EAK16 (AEAEAKAK)2, also called LEGO peptides, which have been considered particularly promising. Self-assembling LEGO peptides have a preferential ?-sheet structure, are resistant to proteolytic cleavage, and are able to form an insoluble macroscopic membrane under physiological conditions. Their ability to create such stable structures derive from the hydrophobic interactions between the aliphatic groups of non-ionic residues and the complementary ionic bonds between acidic and basic amino acids. This stability can be enhanced by the pH regulation and the presence of monovalent ions. This chapter will be focused on some approaches useful for elucidating the influence of the sequence modifications and the interactions with a surface on the self-assembly capability of differently synthesised peptides. Eight different oligopeptides (from 16 to 19 residues), derived from EAK16, have been analysed by IR and Raman spectroscopies that are particularly useful for obtaining qualitative and quantitative information on the secondary structure of these peptides. Several modifications in the primary structure of peptides have been considered, namely acidic and/or basic substitutions (Glu -> Asp; Lys -> Orn), changes in the length of the aliphatic side chain of the spacer residues (Ala -> Abu or Ala -> Tyr), the addition of RGD (known to promote osteoblast adhesion), or scrambling of the sequence. As these peptides are widely used as biocompatible coatings of metallic implants, the peptide folding after adsorption on different surfaces, in particular on titanium oxide, will be also discussed. Finally, it will be shown that not all the oligopeptides examined can self-assemble into a homogeneous multilayer on metallic surfaces; however, most of the peptides take a prevailing ?-sheet structure which guarantees the best peptide-surface interaction. In particular, the interactions of polar, ionic and aromatic residues with surfaces will be discussed more in detail.

The design of composite scaffolds with slow degradation kinetics imposes the assessment of the time-course of degradation to predict the long-term in vitro behavior. In this work, the effect of hydroxyapatite (HA) particles on the hydrolytic degradation of poly e-caprolactone composite scaffold was investigated. The study of accelerated degradation mechanisms in alkaline medium enabled analysing comparable degradation profiles at different times. The accurate qualitative and quantitative study of morphology by scanning electron microscopy supported by image analysis demonstrated only a negligible effect on the structural porosity, to be ascribed to the addition of micrometric HA as a filler. Moreover, by comparing the Raman spectra with thermal analysis thermogravimetry and differential scanning calorimetry) the role of HA on the composite degradation mechanism was defined, by separately quantifying the contribution of HA particles in the bulk and on the surface, on the bone formation as a function of modifications induced in the pore morphology, as well as physical and chemical properties of the polymer matrix. Indeed, HA particles alter the poly e-caprolactone crystallinity inducing a ‘‘shielding’’ effect of the polymer matrix. Meanwhile, the slight reduction of pore size as a function of the increasing HA content and the improvement of the effective hydrophilicity of the scaffolds also influence the degradation by faster mechanisms. Finally, it has been proven that the presence of HA enhances the scaffold bioactivity and human osteoblast cell response, remarking the active role of bioactive signals on the promotion of the surface mineralization and, as a consequence, on the cell–material interaction

The employment of composite scaffolds with a well-organized architecture and multi-scale porosity certainly represents a valuable approach for achieving a tissue engineered construct to reproduce the middle and long-term behaviour of hierarchically complex tissues such as spongy bone. In this paper, fibre-reinforced composites scaffold for bone tissue engineering applications is described. These are composed of poly-L-lactide acid (PLLA) fibres embedded in a porous poly(3-caprolactone) matrix, and were obtained by synergistic use of phase inversion/particulate leaching technique and filament winding technology. Porosity degree as high as 79.7% was achieved, the bimodal pore size distribution showing peaks at ca 10 and 200 mm diameter, respectively, accounting for 53.7% and 46.3% of the total porosity. In vitro degradation was carried out in PBS and SBF without significant degradation of the scaffold after 35 days, while in NaOH solution, a linear increase of weight lost was observed with preferential degradation of PLLA component. Subsequently, marrow stromal cells (MSC) and human osteoblasts (HOB) reached a plateau at 3 weeks, while at 5 weeks the number of cells was almost the same. Human marrow stromal cell and trabecular osteoblasts rapidly proliferate on the scaffold up to 3 weeks, promoting an oriented migration of bone cells along the fibre arrangement. Moreover, the role of seeded HOB and MSC on composite degradation mechanism was assessed by demonstrating a more relevant contribution to PLLA degradation of MSC when compared to HOB. The novel PCL/PLLA composite scaffolds thus showed promise whenever tuneable porosity, controlled degradability and guided cell–material interaction are simultaneously requested.

The addition of Mn in bioceramic formulation is gaining interest in the field of bone implants. Mn activates human osteoblast (h-osteoblast) integrins, enhancing cell proliferation with a dose-dependent effect, whereas Mn-enriched glasses induce inhibition of Gram-negative or Gram-positive bacteria and fungi. In an effort to further optimize Mn-containing scaffolds’ beneficial interaction with h-osteoblasts, a selective and specific covalent functionalization with a bioactive peptide was carried out. The anchoring of a peptide, mapped on the BMP-2 wrist epitope, to the scaffold was performed by a reaction between an aldehyde group of the peptide and the aminic groups of silanized Mn-containing bioceramic. SEM-EDX, FT-IR, and Raman studies confirmed the presence of the peptide grafted onto the scaffold. In in vitro assays, a significant improvement in h-osteoblast proliferation, gene expression, and calcium salt deposition after 7 days was detected in the functionalized Mn-containing bioceramic compared to the controls.

In the last decades, silk fibroin and wool keratin have been considered functional materials for biomedical applications. In this study, fabrics containing silk fibers from Bombyx mori and Tussah silk fibers from Antheraea pernyi, as well as wool keratin fabrics, were grafted with phosmer CL and phosmer M (commercial names, i.e., methacrylate monomers containing phosphate groups in the molecular side chain) with different weight gains. Both phosmers were recently proposed as flame retarding agents, and their chemical composition suggested a possible application in bone tissue engineering. IR and Raman spectroscopy were used to disclose the possible structural changes induced by grafting and identify the most reactive amino acids towards the phosmers. The same techniques were used to investigate the nucleation of a calcium phosphate phase on the surface of the samples (i.e., bioactivity) after ageing in simulated body fluid (SBF). The phosmers were found to polymerize onto the biopolymers efficiently, and tyrosine and serine underwent phosphorylation (monitored through the strengthening of the Raman band at 1600 cm−1 and the weakening of the Raman band at 1400 cm−1, respectively). In grafted wool keratin, cysteic acid and other oxidation products of disulphide bridges were detected together with sulphated residues. Only slight conformational changes were observed upon grafting, generally towards an enrichment in ordered domains, suggesting that the amorphous regions were more prone to react (and, sometimes, degrade). All samples were shown to be bioactive, with a weight gain of up to 8%. The most bioactive samples contained the highest phosmers amounts, i.e., the highest amounts of phosphate nucleating sites. The sulphate/sulphonate groups present in grafted wool samples appeared to increase bioactivity, as shown by the five-fold increase of the IR phosphate band at 1040 cm−1.

Cyanobacteria are oxygenic phototrophs that have an essential role in soil N2 fixation, fertility, and water retention. Cyanobacteria are also natural sources of bioactive metabolites beneficial to improve plant vigor and potentially active against fungal plant pathogens. Therefore, we studied the antifungal activity of water extract (WE) and phycobiliproteins (PBPs) from Anabaena minutissima strain BEA 0300B against the fungal plant pathogen Botrytis cinerea on tomato fruits and in vitro. The water extract and PBPs were characterized by using FT-IR and FT-Raman spectroscopies. Both water extract (5 mg/mL) and PBPs (ranged from 0.3 to 4.8 mg/mL) reduced disease incidence and disease severity on tomato fruits and mycelium growth and colony forming units in vitro. For mycelium growth, a linear PBP dose-response was found. Tomato fruits were also characterized by FT-IR and FT-Raman spectroscopies in order to evaluate structural modifications induced by pathogen and PBP treatment. PBPs preserved cutin and pectin structures by pathogen challenge. In conclusion, A. minutissima can be considered a potential tool for future large-scale experiments for plant disease control.

Cadophora luteo-olivacea represents a critical problem for kiwifruit in the post-harvest phase, mainly for its little note epidemiology. The study presented some results about the possibility of preserving kiwifruit from skin pitting symptoms using alternative methods to fungicides. By in vitro assays, antagonist mechanisms of action against pathogen isolates were tested. Trichoderma harzianum (Th1) showed the highest inhibitory activity against C. luteo-olivacea isolates by volatile, non-volatile, and by dual culture assay, displaying an inhibition respectively by 90%, 70.6%, and 78.8%, and with respect to Aureobasidium pullulans (L1 and L8) by 23.3% and 25.8%, 50% and 34.7%, and 22.5% and 23.6%, respectively. Further, the sensitivity on CFU and mycelial growth of C. luteo-olivacea isolates to fludioxonil, and CaCl2 was tested, displaying interesting EC50 values (0.36 and 0.92 g L−1, 22.5 g L−1, respectively). The effect of Brassica nigra defatted meal was tested as biofumigation assays and through FT-IR (Fourier-Transform Infrared) spectroscopy. The above-mentioned treatments were applied in vivo to evaluate their efficacy on kiwifruits. Our data demonstrated that alternative solutions could be considered to control postharvest pathogens such as C. luteo-olivacea.

20 pags., 5 figs., 3 tabs. This article belongs to the Special Issue Peptides and Proteins as Innovative Biomaterials, The surface-enhanced Raman scattering (SERS) spectra of three amphiphilic oligopeptides derived from EAK16 (AEAEAKAK) were examined to study systematic amino acid substitution effects on the corresponding interaction with Ag colloidal nanoparticles. Such self-assembling molecular systems, known as “molecular Lego”, are of particular interest for their uses in tissue engineering and as biomimetic coatings for medical devices because they can form insoluble macroscopic membranes under physiological conditions. Spectra were collected for both native and gamma-irradiated samples. Quantum mechanical data on two of the examined oligopeptides were also obtained to clarify the assignment of the prominent significative bands observed in the spectra. In general, the peptide–nanoparticles interaction occurs through the COO groups, with the amide bond and the aliphatic chain close to the colloid surface. After gamma irradiation, mimicking a free oxidative radical attack, the SERS spectra of the biomaterials show that COO groups still provide the main peptide–nanoparticle interactions. However, the spatial arrangement of the peptides is different, exhibiting a systematic decrease in the distance between aliphatic chains and colloid nanoparticles.

Investment casting is a leading technique to manufacture very reliable components. The design and the optimization of this process are usually performed by simulation software and/or by means of simplified models based on heat transfer coefficients obtained by solving the inverse heat conduction problem (IHCP). Both methods require experimental tests, that involve temperature measurements, aiming at validating the investment casting simulations or evaluating the heat transfer coefficients in the IHCP. Typically, all these temperature measurements are performed with thermocouples that are characterized by several limits, such as heat transfer alteration, approximate location of the measurement points, and spatial-limited evaluation of the heat transfer coefficients. In order to overcome these limits, the infrared thermography is proposed as an alternative measurement technique. The infrared thermography allows to obtain complete and spatial-accurate information about the surface temperature of an investigated body which, in particular, is a ceramic shell mould in the present work. Since the infrared thermography is a 2D technique, the purpose of this work is to provide a procedure to experimentally reconstruct the 3D temperature surface distribution of this ceramic shell mould in a real industrial environment with all its space and time constraints.

Five alternating polar/non-polar peptides derived from the self-assembling peptide EAK-16 (AEAEAKAK)2 were examined in comparison with the EAK-16 parent form (pept1). The peptides were studied for their possible use as biomimetic materials due to their auto-assembling properties and to the presence, in two of them, of the RGD sequence, an active modulator of cell adhesion. The use of SERS allows the detection of peptides at very low concentrations (10-5-10-6 M), a feature of extreme interest to check their presence in the aqueous environment surrounding a metal implant and to study the effects of systematic amino acid substitution along the peptide chain on the corresponding interaction with the Ag colloidal nanoparticles. Quantum-mechanical data on two of the examined peptides were carried out and were very useful for clarifying the bands assignment debated in the literature. The results indicate that, in general, the peptide-nanoparticle interaction takes place through the carboxylate groups. The SERS spectrum displays an enhancement of the bands attributed to carboxylate vibrations, indicating that these groups directly interact with the nanoparticles. The most prominent band of carboxylate groups appears at 1393 cm-1 (symmetrical COO- stretching), intensified and red shifted of about 10 cm-1, as compared to the FT-Raman spectrum. This effect can be attributed to the proximity of the COO- group to the surface and to a charge transfer mechanism. Moreover, other bands can be attributed to COO- vibrations: 909 cm-1 (C-COO- stretching), 655 cm-1 (COO- bending) and 563 cm-1 (COO- wagging). The theoretical calculations pointed out that the last two bands are mixed with amide motions. As regards the other examined peptides, the spacer substitution in the sequence is a factor able to affect the peptide-Ag particles interaction: the increase in the hydrophobic chain length (Ala substituted by 2-aminobutanoic acid) favors the interaction by NH3+ groups, although the charge transfer interaction with the COO- ions is still the main interaction with the colloid. In fact, the presence of many SERS bands attributable to the NH3+ moieties of the Lys amino acid side chains, indicates the existence of interactions between the NH3+ groups and the nanoparticles, mediated by the Cl- anions present in the colloidal solution. The substitution of Ala with an aromatic Tyr residue strongly affects the interaction mechanism: the Tyr residue lies in a position close to perpendicular to the silver surface, partly as tyrosinate ion.

Ibuprofen (IbuH), 2-(4-isobutylphenyl) propionic acid, is a well-known nonsteroidal anti-inflammatory drug and is a promising antiatherosclerotic agent. Since IbuH has demonstrated to affect cell membranes structure, the study of the interaction between the membrane components, like phospholipids, and the drug, is of paramount interest. Moreover, liposomes can be used as drug carriers. In this paper, the effect of increasing amounts of Ibuprofen at neutral and acidic pH on the behaviour of dimyristoylphosphatidylcholine (DMPC) liposomes was investigated by means of Raman and differential scanning calorimetry (DSC) techniques. The results showed that pH influenced noticeably the liposome structure. A simple ‘solution-like’ model can explain the system when small or high IbuH or IbuNa (IbuH sodium salt forming at neutral and basic pH) amounts are present (IbuH/DMPC molar ratio ≤1/8.3 or ≥1/2.2 or IbuNa/DMPC molar ratio ≤1/17.5 or ≥1/4), whereas at intermediate amounts, two DSC peaks appeared: these systems could be described as a mixture of ‘phase II’ domains inserted within a structure of smaller and further ramified ‘phase I’ domains. Both DSC and Raman data suggested IbuH can penetrate within the apolar bilayer at pH 3, while at pH 7, the setting up of polar interactions of different strength between the carboxylic groups of IbuNa and the choline head of the DMPC, hamper a further penetration of the drug. Similar results were obtained both inserting directly IbuH molecules into liposomes as well as acidifying IbuNa water solutions, suggesting a possible use of liposomes as drug carriers when polarity is pH dependent.

BACKGROUND: Metal ions are implicated in protein aggregation processes of several neurodegenerative pathologies, where the protein deposition occurs, and in the biotechnology field like the food technology where many processes in food manufacturing are based on thermal treatments. OBJECTIVE: The influence of Cu2+ or Zn2+ ions on the thermal aggregation process of Bovine beta-lactoglobulin (BLG) and Bovine Serum Albumin (BSA), two protein models, was studied with the aim of delineating the role of these ions in the protein aggregation kinetics and to clarify the related molecular mechanisms. METHODS: The protein structure changes were monitored by Raman spectroscopy, whereas the aggregate growth was followed by Dynamic Light Scattering measurements. RESULTS: Both metal ions are able to favour the BLG aggregation, whereas only Zn2+ ions have a promoter effect on the thermal aggregation of BSA. The reason of this different behaviour is that the BLG aggregation evolution is manly affected by the redistribution of charges, whereas that of BSA by the metal coordination binding which depends on metal. CONCLUSIONS: Raman spectroscopy, combined with dynamic light scattering experiments, was very useful in identifying the role played by Cu2+ and Zn2+ on the aggregation pathways of BLG and BSA. The results provide evidence for the role of histidine residues both in the redistribution of charges and in the two modes of metal binding that take place in BLG- and BSA-containing systems, respectively.

Tissue Engineering is a multidisciplinary field aimed at the creation of biological substitutes that restore and maintain the biological function of a damaged tissue. The key of the success of these biomedical devices lies into surface interactions with living tissues, therefore a common strategy is to create biomimetic surfaces that helps cells to colonize the biomaterial, leading to tissue healing. Vibrational spectroscopy, in particular Raman, apart from being mainly surface and non-destructive technique, is extremely sensitive to changes in structure and molecular interactions, thus its use in investigating biomimetic devices is increasing. In particular, we have recently used vibrational spectroscopies to investigate different biomimetic materials and to test some of their proprieties: - self-assembling peptides adsorbed on titanium surfaces for bone implants [1], analyzed before and after attack from free radicals (obtained by gamma-radiolysis and mimicking inflammation processes) [2], with the aim to evaluate their capability in resisting to oxidative stress; - composite (ceramic-polymer, polymer-polymer) bioresorbable biomaterials [3, 4]; - hydroxyapatite nanomaterials functionalized with proteins to increase biocompatibility [5-7]. [1] M. Di Foggia, P. Taddei, A. Torreggiani, M. Dettin, A. Tinti, J. Raman Spectrosc. 42 (2011) 276-285. [2] M. Di Foggia, A. Torreggiani, P. Taddei, M. Dettin, A. Tinti, J. Raman Spectrosc. 44 (2013) 1446-1450. [3] V. Guarino, F. Causa, P. Taddei, M. Di Foggia, G. Ciapetti, D. Martini, C. Fagnano, N. Baldini, L. Ambrosio, Biomaterials 29 (2008) 3662-3670. [4] V. Guarino, P. Taddei, M. Di Foggia, C. Fagnano, G. Ciapetti, L. Ambrosio, Tissue Eng. A 15 (2009) 3655-3668. [5] M. Iafisco, B. Palazzo, G. Falini, M. Di Foggia, S. Bonora, S. Nicolis, L. Casella, N. Roveri, Langmuir 24 (2008) 4924-4930. [6] M. Iafisco, M. Di Foggia, S. Bonora, M. Prat, N. Roveri, Dalton Trans. 40 (2011) 820-827. [7] M. Iafisco, E. Varoni, M. Di Foggia, S. Pietronave, M. Fini, N. Roveri, L. Rimondini, M. Prat, Colloids Surf.s B 90 (2012) 1-7.

Molecular basis of the role played by Cu 2 + and Zn 2 + ions during the thermal aggregation processes of beta-lactoglobulin (BLG) was studied by using a joint application of different techniques. In particular, Raman spectroscopy was very useful in identifying the different effects caused by the two metals at molecular level (i.e. changes in His protonation state, disulfides bridge conformation, and micro-environment of aromatic residues), evidencing the primary importance of the protein charge distribution during the aggregation process. Both metal ions are able to act on this factor and favor the protein aggregation, but Zn 2 + is able to alter the natural conformational state of BLG, causing a slight unfolding, whereas Cu 2 + ions play a role only during the thermal treatment. Thus, Zn 2 + ions favor the formation of bigger aggregates and branched fibril-like structures, whereas for Cu 2 + ions a greater number of cross-beta structures during thermal incubation and finally, fibrillar structures. The aggregation process occurs in two phases, as suggested by the measurements on the time evolution of the BLG aggregates: the first one is characterized by a partial unfolding of the protein and aggregate growth, forming oligomers and protofibrils, whereas the second one is characterized by further supramolecular assembly, leading to the formation of fibrils.

The investment casting process and its key parameters are described in order to give a quite detailed knowledge of the main indicators of this production method, for its nature prone to have high costs of rework or scrap. Initially, a schematic flow of the main processes is provided; afterwards the main processes are discussed in more detailed manner with their manufacturing methods and product specifications. These concepts are indispensable to introduce the discussion on the critical parameters for investment casting process and some proposal to implement a few new methods for quality assurance of the processes and products. Finally a description of the economic impact of the critical manufacturing operations related to some specific case studies and possible goals have been identified.

A serious problem in the production of gas turbine blades is the detection of residual ceramic cores inside the cooling passages; in fact, the presence of even small ceramic pieces affects turbine performance and may cause difficulties in successive manufacturing. Therefore, it is important to have a non-destructive technique that must be capable of detecting tiny ceramic fragments in a fast and easy way. In this perspective, the suitability of infrared thermography was investigated within cooperation between the University of Naples and the Europea Microfusioni Aerospaziali S.p.A. (EMA). Several blades of three different types were inspected revealing that in many cases infrared thermography can discover small ceramic fragments which were missed by X-ray inspection. In addition, infrared thermography allows gaining of information about other types of anomalies (e.g., surface defects) during the same testing step (by eventually changing the test parameters) and then saving time and money. The obtained results look promising in view of introducing infrared thermography among industrial instrumentation as an alternative to, or integrated with, the most currently utilized non-destructive techniques.