New self-assembled nano-particles were developed by chemical conjugation of natural fatty acids to the backbone of hyaluronan (HA). The chemical structure and the self-association behavior of them were studied by FT-IR, NMR, fluorescence and dynamic light scattering. The HA derivatives form stable spherical shape aggregates, as assessed by transmission electron microscopy. This journal is

The critical role of integrins in tumor progressionand metastasis has stimulated intense efforts to identifypharmacological agents that can modulate integrin function.In recent years, avb3 and avb5 integrin antagonists weredemonstrated to be effective in blocking tumor progression.RGDechi-hCit, a chimeric peptide containing a cyclic RGDmotif linked to an echistatin C-terminal fragment, is able torecognize selectively avb3 integrin both in vitro and in vivo.High-resolution molecular details of the selective avb3 recognitionof the peptide are certainly required, nonethelessRGDechi-hCit internalization limited the use of classical incell NMR experiments. To overcome such limitations, weused WM266 isolated cellular membranes to accomplisha detailed NMR interaction study that, combined with a computationalanalysis, provides significant structural insightsinto avb3 molecular recognition by RGDechi-hCit. Remarkably,on the basis of the identified molecular determinants,we design a RGDechi-hCit mutant that is selective for avb5integrin.

Acylpeptide hydrolase (APEH), one of the four members of the prolyl oligopeptidase class, catalyses the removal of N-acylated amino acids from acetylated peptides and it has been postulated to play a key role in protein degradation machinery. Disruption of protein turnover has been established as an effective strategy to down-regulate the ubiquitin-proteasome system (UPS) and as a promising approach in anticancer therapy.Here, we illustrate a new pathway modulating UPS and proteasome activity through inhibition of APEH. To find novel molecules able to down-regulate APEH activity, we screened a set of synthetic peptides, reproducing the reactive-site loop of a known archaeal inhibitor of APEH (SsCEI), and the conjugated linoleic acid (CLA) isomers. A 12-mer SsCEI peptide and the trans10-cis12 isomer of CLA, were identified as specific APEH inhibitors and their effects on cell-based assays were paralleled by a dose-dependent reduction of proteasome activity and the activation of the pro-apoptotic caspase cascade. Moreover, cell treatment with the individual compounds increased the cytoplasm levels of several classic hallmarks of proteasome inhibition, such as NFkappaB, p21, and misfolded or polyubiquitinylated proteins, and additive effects were observed in cells exposed to a combination of both inhibitors without any cytotoxicity. Remarkably, transfection of human bronchial epithelial cells with APEH siRNA, promoted a marked accumulation of a mutant of the cystic fibrosis transmembrane conductance regulator (CFTR), herein used as a model of misfolded protein typically degraded by UPS. Finally, molecular modeling studies, to gain insights into the APEH inhibition by the trans10-cis12 CLA isomer, were performed.Our study supports a previously unrecognized role of APEH as a negative effector of proteasome activity by an unknown mechanism and opens new perspectives for the development of strategies aimed at modulation of cancer progression.

Competitive peptides displacing AKAP121 from mitochondria in the tissue and in the cells were used to investigate the role of AKAP121 in mitochondrial function, reactive oxygen species (ROS) generation, and cell survival. Displacement of AKAP121 from mitochondria by synthetic peptides triggers the death program in cardiomyocytes. Under pathological conditions in vivo, in a rat model of cardiac hypertrophy induced by ascending aorta banding, the levels of AKAP121 are significantly down-regulated. Disappearance of AKAP121 is associated with mitochondrial dysfunction, high oxidative stress, and apoptosis. In vivo delocalization of AKAP121 by competitive peptides replicates some of the molecular signatures induced by pressure overload: mitochondrial dysfunction, increased mitochondrial ROS, and apoptosis.

A set of nine columbite samples (Fe,Mn)(Nb,Ta)(2)O-6, preserved in the Mineralogical Museum, Naples University Federico II (Italy) and originating from several world-wide pegmatitic occurrences, was studied by electron-probe microanalysis and single crystal X-ray diffraction. All the samples are columbite-(Fe), except for two samples corresponding to columbite-(Mn). For seven samples Ta/(Nb+Ta) is <0.05 and for two samples <0.2. The (Ti+Sn)(4+) content of all samples is <0.12 pfu. Structure refinements showed that five samples displayed strong (Fe,Mn)(2+) vs. (Nb,Ta)(5+) disorder at the two octahedral sites A and B, whereas four samples were highly ordered. Two samples could be interpreted as "pseudo-ixiolite". The degree of cation order at A and B was calculated from cell dimensions and scattering behaviour at A and B. Both methods are in good agreement. The octahedral coordination of (Nb, Ta)5+ in columbite is characterized by out-of-center distortion, thus the average distortion at the B site was probed by calculating the difference between the longest and shortest B-O distance. For a Nb-rich and (Ti,Sn)-poor data set, the min-max distortional parameter shows very good linear correlation with the order parameter calculated from site occupancy.

In malignant melanoma (MM), one of the most aggressive cancers, changes in integrin expression and intracellular control of integrin function are involved in the conversion from a stationary to a migratory and invasive phenotype, key step toward the progression of thistumor. Overexpression of alphav beta 3 integrin is linked to a more metastatic phenotype. Therefore, development of anti-?alphav beta 3 agents able to counteract the progression of melanoma would be helpful for the disease treatment. A new highly selective ligand of alphav beta 3, referred to as RGDechi-hCit, containing a cyclic RGD motif with two echistatin moieties, has been demonstrated to have anti-angiogenic properties against endothelial cells in animal models of angiogenesis. Aim of this study was to evaluate the in vitro effects of the RGDechi-hCit peptide on MM cell lines. C Cytofluorimetric analysis allowed the characterization of cell surface expression of alphav beta 3 integrin on seven MM cell lines. Cell proliferation, adhesion, and migration assays were carried out on these MM cells in the presence of the?alphav beta 3-antagonist RGDechi-hCit. Proliferation was not significantly inhibited by RGDechi-hCit but, striking morphological changes were detected in MM cell lines highly expressing alphav beta 3, suggesting a specific role of this integrin in adhesion and migration. Assays on fibronectin-coated plates showed indeed a significant RGDechi-hCit dose-dependent inhibitory effect on both adhesion (p = 0.024) and migration (p < 0.001).Our data demonstrate anti-adhesion and anti-migration, but not anti-proliferative, activities of RGDechi-hCit against MM cells.

Understanding the mechanism of action of antimicrobial peptides is pivotal to the design of new and more active peptides. In the last few years it has become clear that the behavior of antimicrobial peptides on membrane model systems does not always translate to cells; therefore the need to develop methods aimed at capturing details of the interactions of peptides with bacterial cells is compelling. In this work we analyzed binding of two peptides, namely temporin B and TB_KKG6A, to Escherichia coli cells and to Escherichia coli LPS. Temporin B is a natural peptide active against Gram positive bacteria but inactive against Gram negative bacteria, TB_KKG6A is an analogue of temporin B showing activity against both Gram positive and Gram negative bacteria. We found that binding to cells occurs only for the active peptide TB_KKG6A; stoichiometry and affinity constant of this peptide toward Escherichia coli cells were determined.

Herein, we report the synthesis and biological characterization of the new peptide psi RGDechi as the first step toward novel targeted theranostics in melanoma. This pseudopeptide is designed from our previously reported RGDechi peptide, known to bind selectively alpha(v)beta(3) integrin, and differs for a modified amide bond at the main protease cleavage site. This chemical modification drastically reduces the enzymatic degradation in serum, compared to its parental peptide, resulting in an overall magnification of the biological activity on a highly expressing alpha(v)beta(3) human metastatic melanoma cell line. Selective inhibition of cell adhesion, wound healing, and invasion are demonstrated; near infrared fluorescent t psi RGDechi derivative is able to detect alpha(v)beta(3) integrin in human melanoma xenografts in a selective fashion. More, molecular docking studies confirm that psi RGDechi recognizes the receptor similarly to RGDechi. All these findings pave the way for the future employment of this novel peptide as promising targeting probe and therapeutic agent in melanoma disease.

An efficient and rapid procedure for synthesizing S-linked glycopeptides is reported. The approach uses activated molecular sieves as a base to promote the selective S-alkylation of readily prepared cysteine-containing peptides, upon reaction of appropriate glycosyl halides. Considering the very mild conditions employed, the chemoselective linkage of the electrophilic sugar with a peptide sulfhydryl group occurred in satisfactory yield, allowing the incorporation of mono and disaccharide moieties. The sugar-peptide conjugates obtained from ?-d-glycosyl derivatives adopt a ?-S-configuration, indicating the high stereoselectivity of the substitution reaction.

The growing interest in peptide nucleic acid (PNA) oligomers has led to the development of a very wide variety of PNA derivatives. Among others, the introduction of charged chiral groups on a PNA oligomer has proven effective in improving DNA binding ability, complexation direction and cellular uptake. In particular, the introduction of three adjacent chiral monomers based on d-Lys in the middle of the PNA sequence ( d-Lys-PNA) has produced noteworthy results in modulating the directionality of the binding with the DNA complementary strand and in mismatch detection. Here, through a molecular dynamics approach, a comparative study has been carried out to investigate the structural properties that drive the interaction of the chiral d-Lys-PNA and the corresponding achiral PNA system with DNA as well as RNA complementary strands, starting from the crystal structure of d-Lys-PNA in complex with DNA. The results obtained complement experimental data and indicate that the binding with the RNA molecule, compared to DNA, is differently affected by the addition of three d-Lys groups on the PNA backbone, suggesting that this modification could be taken into account for the development of new PNA-based molecules able to discriminate between DNA and RNA. © 2014 Elsevier Masson SAS.

The crystal chemistry of wavellite from Zbirov (Czech Republic), ideally Al-3(PO4)(2)(OH,F)(3)center dot 5H(2)O, was addressed by means of a multi-methodological approach based on electron microprobe analysis (EMPA) using wave-dispersive spectroscopy, single-crystal X-ray diffraction, powder and single-crystal infrared spectroscopy and Raman spectroscopy. The EMPA data showed the presence of significant F replacing OH in the sample studied. The structure was solved in the Pcmn orthorhombic space group, with the following unit-cell constants: a = 9.6422(7), b = 17.4146(15), c = 7.0094(2) angstrom, V = 1176.98(10) angstrom(3). Phosphorus atoms display tetrahedral (PO4) coordination, while Al cations display octahedral coordination. The mineral framework can be viewed as the repetition of cationic arrays made up of AlO6 polyhedra, bridged by PO4 groups and further joined by O-H center dot center dot center dot O hydrogen bonds. The single-crystal unpolarized Fourier transform infrared (FTIR) spectrum shows combination bands indicating the presence of both OH and H2O in the structure. Both FTIR and Raman spectra show a broad absorption extending from 3600 to 2800 cm(-1) resulting from the overlapping of several components due to the water molecules and the OH group. The frequencies observed are comparable to those expected on the basis of the Libowitzky relationship for the range of D-H center dot center dot center dot A bond systems in the structure.

The structural characterization of Al-2(PO4)(OH)(3) augelite, a natural orthophosphate coming from Rapid Creek, Yukon, Canada, was carried out by means of a multi-methodological approach based on WDS-EMPA chemical analysis, single crystal X-ray diffraction, and infrared spectroscopy. The structure was solved in the C2/m monoclinic space group, with the following unit cell constants: a = 13.0740(10), b = 7.9690(7), c = 5.0910(7) angstrom, beta = 112.282(5), V = 490.81(9) angstrom(3). Phosphorus atoms display tetrahedral (PO4) coordination, while aluminum cations display octahedral and unusual five-fold coordination. The framework of the mineral can be viewed as the repetition of cationic arrays made up by AlO6/AlO5 polyhedra, bridged by PO4 groups, and further joined by O-H..O hydrogen bonds: such interactions were also correlated to IR spectroscopy analysis, by means of Libowitzky formula applied on OH stretching vibrations.

Synthetic models of receptors that have relevant biological roles are valuable tools for studying receptors itself and the corresponding ligands. Their properties can be validated at first by their capacity to fold in solution under native-like conditions and to assume conformations structurally and functionally equivalent to those in the native receptor. In this context, a new strategy to prepare the two-fragments synthetic receptor model HER2-DIVMP, an independent structural and functional motif of HER2, has been developed and the folding properties have been investigated. The strategy is based on a one-step cysteine co-oxidation procedure in slightly alkaline aqueous buffers, whereby the two separate peptide chains are allowed to self-assemble in solution. Under these conditions, the two chains spontaneously form the expected heterodimer with the correct pattern of disulfide bridges. To gain insights on the folding mechanism, we investigated the folding of two scrambled variants of the constituent peptide chains.

We report the application of the arsenical complex FlAsH-EDT2 for the identification of oligonucleotide sequences.  We designed PNA sequences conjugated to either a tetracysteine motif and to split tetracysteine sequences, that are recognized by FlAsH.  The effect of conjugation of the PNA to the tetracysteine peptide and RNA hybridization on the fluorescence of the arsenical complex has been investigated.  The reconstitution of the tetracysteine motif, starting from 15-mer PNAs conjugated to split tetracysteine sequences and hybridized to a complementary oligonucleotide was also explored.bject]

A protocol to run CD experiments in the presence of Gram positive bacterial cells was set up. The secondary structure of the antimicrobial peptides temporin L and TB_KKG6A with S. epidermidis cells was determined. CD experiments were also reported for temporin L in the presence of Gram negative bacterium E. coli cells.

The interaction between Herceptin (Fab)-derived dimeric peptide and HER2-DIVMP, a synthetic peptide mimicking the subdomain IV of the receptor HER2, was monitored by fluorescence spectroscopy method. The obtained results confirmed that the adopted mimetic receptor fragment represents a reliable model system to monitor the ability of selected ligands to bind the receptor target, avoiding the involvement of cells or cellular extracts in a preliminary binding assay. Moreover, HER2-DIVMP has the characteristics of being easily integrated in rapid, efficient and inexpensive screening method for optimizing small molecules as targeting ligands for the receptor-mediated anticancer therapy.

An environmentally sustainable and energy-efficient synthetic process has been developed to prepare hyaluronan-based nano-sized material. It consists in a microwave-promoted acylation of the hydroxyl function of the polysaccharide with natural fatty acids, performed under solvent-free conditions. The efficient interaction of the solid reagents with the MW radiation accounts for the obtained high yielded products. The self-assembly process of the obtained compounds very fast occurred in an aqueous medium under MW-radiation, thus allowing the development of a green protocol for the nano-particles preparation.

HER2 transmembrane receptor is an important target in immunotherapy treatment of breast and gastroesophageal cancer. Molecular imaging of HER2 expression may provide essential prognostic and predictive information concerning disseminated cancer and aid in selection of an optimal therapy. Radiolabeled low molecular weight peptide ligands are particularly attractive as probes for molecular imaging, since they reach and bind to the target and clear from non-target organs and blood stream faster than bulky antibodies. In this study, we evaluated a potential HER2-imaging probe, an A9 nonapeptide, derived from the trastuzumab-Fab portion. Its cellular uptake was investigated by mass spectrometry analysis of the cytoplasmic cellular extracts. Moreover, based on in-silico modeling, DTPA chelator was conjugated to N-terminus of A9. In-111-labeled A9 demonstrated nanomolar affinity to HER2-expressing BT474 cells and favorable biodistribution profile in NMRI mice. This study suggests that the peptide A9 represents a good lead candidate for development of molecular probe, to be used for imaging purposes and for the delivery of cytotoxic agents.

Integrins are heterodimeric receptors that play a critical role in cell-cell and cell-matrix adhesion processes. Among them, ?V?3 integrin, that recognizes the aminoacidic RGD triad, is reported to be involved in angiogenesis, tissue repair and tumor growth. We have recently synthesized a new and selective ligand of ?V?3 receptor, referred to as RGDechiHCit, that contains a cyclic RGD motif and two echistatin moieties.MethodsThe aim of this study is to evaluate in vitro and in vivo the effects of RGDechiHCit. Therefore, we assessed its properties in cellular (endothelial cells [EC], and vascular smooth muscle cells [VSMC]) and animal models (Wistar Kyoto rats and c57Bl/6 mice) of angiogenesis.ResultsIn EC, but not VSMC, RGDechiHCit inhibits intracellular mitogenic signaling and cell proliferation. Furthermore, RGDechiHCit blocks the ability of EC to form tubes on Matrigel. In vivo, wound healing is delayed in presence of RGDechiHCit. Similarly, Matrigel plugs demonstrate an antiangiogenic effect of RGDechiHCit.ConclusionsOur data indicate the importance of RGDechiHCit in the selective inhibition of endothelial ?V?3 integrin in vitro and in vivo. Such inhibition opens new fields of investigation on the mechanisms of angiogenesis, offering clinical implications for treatment of pathophysiological conditions such as cancer, proliferative retinopathy and inflammatory disease.

Fluorescence titrations allowed us to study the interaction process between Herceptin (Fab)-derived peptides and a synthetic peptide mimicking a subdomain IV of the receptor HER2 (HER2-DIVMP). For some of the investigated peptide/HER2-DIVMP complexes a nanomolar dissociation constant was found. The performed interaction studies were completely immune from interferences of other receptor domains not covered by the design, thus decreasing the possibilities of selecting potential ligands able to bind other subtypes of HER2 receptor family. Our results demonstrate that the adopted receptor fragment approach represents an efficient methodology for selecting new molecules as lead structures specific for the receptor target. For these reasons the optimized compounds could be employed as delivery agents for the receptor-mediated anticancer therapy. © 2012 Elsevier Masson SAS. All rights reserved.

The ability of PNA to interact with DNA double stranded has been recently investigated. In a decoy approach these interactions are of great importance as may lead to inhibition of interactions of DNA sequences to specific transcription factors and may be employed as a strategy for the inhibition of gene transcription alternative to the antisense strategy (targeting transcription factors mRNAs) and the transcription factor decoy approach (targeting transcription factors). We explored the ability of PNA and PNAs with modified monomers to bind to DNA and to interfere in the formation of DNA/transcription factor complex. We report a procedure for the synthesis of Fmoc-gamma-hydroxymetyl PNA, the synthesis and CD analysis of PNA oligomers containing the modified monomer in different positions and EMSA assays to test the: (a) binding to double stranded DNA and (b) inhibition of DNA-protein interactions.

New eco-friendly materials have been developed via esterification of several natural fatty acids by pectin alcoholic functions. The quaternization of the obtained pectin-fatty acid conjugates was performed as subsequent synthetic step. By using microwave heating and solvent-free conditions, the conjugation procedure of fatty acid chains and quaternary ammonium groups to the natural polysaccharide was clean, fast and efficient. In other words, it can be considered a low environmental impact process.

HER2 receptor, for its involvement in tumorigenesis, has been largely studied as topic in cancer research. In particular, the employment of trastuzumab (Herceptin), a humanized anti-HER2 antibody, showed several clinical benefits in the therapy against the breast cancer. Moreover, for its accessible extracellular domain, this receptor is considered an ideal target to deliver anticancer drugs for the receptormediated anticancer therapy. By now, monoclonal antibody and its fragments, affibody, and some peptides have been employed as targeting agents in order to deliver various drugs to HER2 positive tumor cells. In particular, the ability to perform a fast and reliable screening of a large number of peptide molecules would make possible the selection of highly specific compounds to the receptor target. In this regard, the availability of preparing a simplified synthetic model which is a good mimetic of the receptor target and can be used in a reliable screening method of ligands would be of a strategic importance for the development of selective HER2-targeting peptide molecules. Herein, we illustrate the importance of HER2-targeted anticancer therapies. We also report on a synthetic and effective mimetic of the receptor, which revealed to be a useful tool for the selection of specific HER2 ligands.

Protein disulfide oxidoreductases (PDOs) are ubiquitous redox enzymes that catalyse dithiol-disulfide exchange reactions. PDOs have been well studied in bacteria and eukarya, and they have been described in a number of thermophilic and hyperthermophilic species, where they play a critical role in the structural stabilization of intracellular proteins. In this study, the effect of high hydrostatic pressure on the structural properties of PDO from the hyperthermophilic archaeon Pyrococcus furiosus (PfPDO) was analysed in order to gain insights on the possible mechanisms used to endure extreme environmental conditions. The protein is highly thermostable and the data indicate that PfPDO is highly piezostable as well, and that different areas of the protein have a different local compressibility and resistance to high hydrostatic pressure. In particular, the results show that alpha-helices are more sensitive to pressure up to 5 kbar, whilst within 5-9 kbar the loss of beta-sheets is more pronounced than the loss of alpha-helices. Examination of the PfPDO structure and calculations of the solvent accessible surface areas for each amino acid indicate that 42% of the PfPDO residues are buried and that the protein contains four small internal hydrophobic cavities. These findings are discussed in terms of important factors contributing to the high piezostability and thermostability of the enzyme.

Hollow structures containing pockets and pores formed by oligopeptides and proteins are involved in numerous biological processes. Except for a small number of hollows associated with secondary structures, most voids in nature are associated with tertiary and quaternary structures of proteins. One of the most important aspect of natural hollow structures is the exquisite complementarity between their sizes and functions and those of the corresponding guest molecules, process, and reactions. With their complementarity, natural cavities and pores provide microenviroments that lead to a specific binding, catalysis, transportation, and other functions. Since the discovery of crown ether, many macrocycles have been created as host for various guests. The majority of synthetic macrocycles and their acyclic analogs have flexible backbones and thus collapsible cavities. Looking to the example by nature pore-, or cavity-containing secondary structures, work described in this paper stemmed from the development of synthetic peptides containing any cyclic residues insert in well-defined positions of the main chain. This cyclic unit immobilize the curvature into the corresponding backbones, leads an enforced helical (or ring with cyclic peptides) conformation. As a result, a variety of reliably folded, modifiable scaffold can now be constructed. The well-defined crescent helical conformation contain non collassable internal cavities having multiple, introverted amide bonds. Changing the backbone curvature by tuning the cyclic unit (geometry and/or position) leads to crescents ring dimension or helical with cavities of tunable sizes. We synthesized a series of molecules inserting cyclic units in well-defined position obtaining the designed, natural-like hollow structure. The computational and spectroscopic integrated study of these models allowed us to identify different new structures that will be discussed.[1] Fenude E.., Roggio A.M SardiniaChem2006 Giornata di Studio Dedicata alla Chimica Organica delle Molecole Biologicamente Attive, Cagliari 31 Maggio 2006 [2] E. Fenude, S. Dedola, M. Fais VII Convegno "Complex Systems: structure, properties, reactivity and dynamics" Alghero, 2005[3] Navarro, E.; Fenude, E.; Celda, B. Biopolymers, 73, 2004,229-241 [4] Saviano, M.; .Zaccaria, L.; Lombardi,A.; Pedone,C.; Di Blasio,B.; Sun, X.; Lorenzi,G.P.;J.of Inclusion Phenomena and Molecular Recognition in Chemistry, 1994, 18, p.27-36

Aggregation of amyloid ? (A?) peptides into fibrils has been implicated in the pathogenesis of Alzheimer's disease (AD). As a result, in recent years, substantial efforts have been expended in the study of the mechanism of aggregation of the A? peptide as well as of its inhibition by potential drug molecules. In this context, we have built a model of the A?(17-42) deca-oligomer using the solid-state NMR (ssNMR) structure of the A?(17-42) penta-oligomer as a reference. Both the penta- and deca-oligomer systems have been studied by all-atom molecular dynamics (MD) simulations and used as target systems for the investigation of the mechanism of action of a trehalose-derived A? aggregation inhibitor. In the deca-oligomer all the main structural features of the putative fibrillar state are retained. Moreover, the simulations reveal a remarkable gain in stability as the oligomer grows. MD studies of the inhibitor in complex with the penta- and deca-oligomers indicate a significant destabilization of the structure beyond the hampering of the addition of successive A? peptides at the ends of the fibril due to the presence of the inhibitor molecule. Our work provides an easy and effective approach which could be useful for the in silico development of potential drug molecules acting at different stages of the progression of A?-related diseases. © 2013 The Royal Society of Chemistry.

In malignant melanoma (MM), overexpression of alfavbeta3 integrin is linked to a more metastatic phenotype. Development of anti-alfavbeta3 agents able to counteract melanoma progression would be helpful for disease treatment. A new selective ligand of alfavbeta3, RGDechi-hCit, has anti-angiogenic properties against endothelial cells in angiogenesis animal models. Aim of this study was to evaluate the in vitro effects of the RGDechi-hCit peptide on MM cell lines. Material and Methods: Cytofluorimetric analysis characterized cell surface expression of alfavbeta3 integrin on seven MM cell lines. Cell proliferation, adhesion, and migration assays were carried out using the alfavbeta3-antagonist RGDechi-hCit. Results: Proliferation was not significantly inhibited by RGDechi-hCit, though striking morphological changes were detected in MM cell lines highly expressing alfavbeta3. Conversely, assays on fibronectin-coated plates showed a significant RGDechi-hCit dose-dependent inhibitory effect on both adhesion and migration. Conclusions: Data demonstrate anti-adhesion and anti-migration, but not anti-proliferative, activities of RGDechi-hCit against MM cells.

The delivery of peptide nucleic acids (PNAs) to cells is a very challenging task. We report here that a liposomal formulation composed of egg PC/cholesterol/DSPE-PEG2000 can be loaded, according to different encapsulation techniques, with PNA or fluorescent PNA oligomers. PNA loaded liposomes efficiently and quickly promote the uptake of a PNA targeting the microRNA miR-210 in human erythroleukemic K562 cells. By using this innovative delivery system for PNA, down-regulation of miR-210 is achieved at a low PNA concentration.

An attractive strategy to contrast the Alzheimer disease (AD) is represented by the development of ?-sheet breaker peptides (BSB). ?-sheet breakers constitute a class of compounds which have shown a good efficacy in preventing the A? fibrillogenesis; however, their mechanism of action has not been precisely understood. In this context, we have studied the structural basis underlying the inhibitory effect of A?(1-42) fibrillogenesis explicated by two promising trehalose-conjugated BSB peptides using an all-atom molecular dynamics (MD) approach. Our simulations suggest that the binding on the two protofibril ends occurs through different binding modes. In particular, binding on the odd edge (chain A) is guided by a well defined hydrophobic cleft, which is common to both ligands. Moreover, targeting chain A entails a significant structure destabilization leading to a partial loss of ? structure and is an energetically favoured process. A significant contribution of the trehalose moiety to the stability of the complexes emerged from our results. The energetically favoured hydrophobic cleft detected on chain A could represent a good starting point for the design of new molecules with improved anti-aggregating features. © 2013 The Royal Society of Chemistry.

The cyclic pentapeptide c(RGDfK) is a high affinity ligand of alphaVbeta3 integrin. It was an analog of Cilengitide (EMD 121974) developed to be employed as tracer for cancer diagnosis and therapy by functionalisation of its Lys side-chain. Solution-phase and solid-phase synthetic approaches were previously reported. In the attempt to improve solid-phase synthesis of the cyclopeptide circumventing cyclodimerisation reactions, a systematic study of the synthetic conditions was performed, evaluating and optimising parameters directly involved in the ring closure step. The three-dimensional orthogonal solid-phase strategy developed in this study yields the desired c(RGDfK) peptide with no cyclodimerisation by-products. The protocols described allow the modification of the peptide directly on the solid support in order to obtain novel derivatives for biomedical applications.

Negli ultimi venticinque anni il metodo della diffrazione da polveri è stato applicato con successo crescente per risolvere strutture cristalline (composti organici, inorganici, metallorganici). Nonostante ciò, la soluzione strutturale da polveri è ancora oggi una sfida perché i problemi del metodo, dovuti principalmente all'interpretazione dei dati sperimentali, devono essere ancora risolti.

Peptide Nucleic Acids (PNAs) still represent a growing research area thanks to their potential applications in many fields of science from chemistry and biology to medicine. In these years, structural investigations by means of either experimental or computational techniques have proved to be very useful for the understanding of the structural organization and the binding properties of PNA. In this context, we here report an all-atoms Molecular Dynamics (MD) study of a PNA-PNA duplex and a PNA-DNA hetero-duplex with the well known GROMACS simulation package, by using new force field parameters properly derived for PNA molecules. The good agreement of our results with the crystallographic and NMR data, available for both the systems under investigation, confirms the validity of our approach. Moreover, our simulations reveal new interesting features related to the conformational-dynamic behavior of the studied systems, thus demonstrating the ability of MD simulations to gain insights into the dynamic properties of biologically relevant systems. This force field parametrization represents a good starting point for the implementation of a computational platform, based on the GROMACS package, useful for the rational design of modified PNA molecules with improved conformational features for selective binding toward DNA or RNA. This journal is © 2014 the Owner Societies.

Nm23-H1 is one of the most interesting candidate genes for a relevant role in Neuroblastoma pathogenesis. H-Prune is the most characterized Nm23-H1 binding partner, and its overexpression has been shown in different human cancers. Our study focuses on the role of the Nm23-H1/h-Prune protein complex in Neuroblastoma. Using NMR spectroscopy, we performed a conformational analysis of the h-Prune C-terminal to identify the amino acids involved in the interaction with Nm23-H1. We developed a competitive permeable peptide (CPP) to impair the formation of the Nm23-H1/h-Prune complex and demonstrated that CPP causes impairment of cell motility, substantial impairment of tumor growth and metastases formation. Meta-analysis performed on three Neuroblastoma cohorts showed Nm23-H1 as the gene highly associated to Neuroblastoma aggressiveness. We also identified two other proteins (PTPRA and TRIM22) with expression levels significantly affected by CPP. These data suggest a new avenue for potential clinical application of CPP in Neuroblastoma treatment.

Here, silica-coated PbS quantum dots (QDs) with photoluminescence emission properties in the near-infrared (NIR) region are proposed as potential effective single particle optical nanoprobes for future in vivo imaging of tumors.. The dispersibility in aqueous medium of hydrophobic PbS QDs was accomplished by growing a silica shell on their surface by exploiting a base assisted water-in-oil microemulsion method. The silica-coated PbS QDs were then conjugated with a specifically designed cyclic arginine-glycine-aspartic acid (cRGD) peptide that is able to specifically recognize alpha nu beta 3 integrins, which are overexpressed in angiogenic tumor-induced vasculatures and on some solid tumors, to achieve tumor-specific targeting. The cRGD peptide PbS silica-coated QDs were systematically characterized, at each step of their preparation, by means of complementary optical and structural techniques, demonstrating appropriate colloidal stability and the maintenance of their optical futures in aqueous solutions. The cellular uptake of cRGD peptide functionalized luminescent nanostructures in human melanoma cells, where overexpression of alpha nu beta 3 was observed, was assessed by means of confocal microscopy analysis and cytometric study. The selectivity of the cRGD peptide PbS silica-coated QDs for the alpha nu beta 3 integrin was established, consequently highlighting the significant potential of the developed NIR emitting nanostructures as optically traceable nanoprobes for future alpha nu beta 3 integrin receptor in vivo targeting in the NIR region.

Odin is a protein belonging to the ANKS family, and has two tandem Sam domains. The first, Odin-Sam1, binds to the Sam domain of the EphA2 receptor (EphA2-Sam); this interaction could be crucial for the regulation of receptor endocytosis and might have an impact on cancer. Odin-Sam1 associates with EphA2-Sam by adopting a "mid-loop/end-helix" model. In this study three peptide sequences, encompassing the mid-loop interacting portion of Odin-Sam1 and its C-terminal ?5 helix, were designed. Their conformational properties were analyzed by CD and NMR. In addition, their abilities to interact with EphA2-Sam were investigated by SPR studies. The peptides adopt a predominantly disordered state in aqueous buffer, but a higher helical content is evident in the presence of the cosolvent trifluoroethanol. Dissociation constants towards EphA2-Sam were in the high micromolar range. The structural findings suggest further routes for the design of potential anti-cancer therapeutics as inhibitors of EphA2-Sam heterotypic interactions.

Specific inhibition of the copper-containing peptidylglycine alpha-hydroxylating monooxygenase (PHM), which catalyzes the post-translational modification of peptides involved in carcinogenesis and tumor progression, constitutes a new approach for combating cancer. We carried out a structure-activity study of new compounds derived from a well-known PHM substrate analogue, the olefinic compound 4-phenyl-3-butenoic acid (PBA). We designed, synthesized, and tested various PBA derivatives both in vitro and in silico. We show that it is possible to increase PBA affinity for PHM by appropriate functionalization of its aromatic nucleus. Compound 2d, for example, bears a meta-benzyloxy substituent, and exhibits better inhibition features (K(i) = 3.9 mu m, k(inact)/K(i) = 427 M(-1) s(-1)) than the parent PBA (K(i) = 19 mu m, k(inact)/K(i) = 82 M(-1) s(-1)). Docking calculations also suggest two different binding modes for PBA derivatives; these results will aid in the development of further PHM inhibitors with improved features.

alpha v beta 3 integrin is an important tumor marker widely expressed on the surface of cancer cells. Recently, we reported some biological features of RGDechi-hCit, an alpha v beta 3 selective peptide antagonist. In the present work, we mainly investigated the pro-apoptotic activity of the molecule and its ability to penetrate the membrane of WM266 cells, human malignant melanoma cells expressing high levels of alpha v beta 3 integrin. For the first time we demonstrated the pro-apoptotic effect and the ability of RGDechi-hCit to enter into cell overexpressing alpha v beta 3 integrin mainly by clathrin- and caveolin-mediated endocytosis. Furthermore, we deepened and confirmed the selectivity, anti-adhesion, and anti-proliferative features of the peptide. Altogether these experiments give insight into the biological behavior of RGDechi-hCit and have important implications for the employment of the peptide as a new selective carrier to deliver drugs into the cell and as a therapeutic and diagnostic tool for metastatic melanoma. Moreover, since the peptide shows a pro-apoptotic effect, a great perspective could be the development of a new class of selective systems containing RGDechi-hCit and pro-apoptotic molecules or other therapeutic agents to attain a synergic action.

The study of molecules that self-assemble through noncovalent interactions is one of the most attractive topics in supramolecular chemistry. The use of short peptides or modified nucleotides as building blocks for the aggregates is particularly intriguing because these are very easy to synthesize; moreover, subtle changes in the chemical structure of such building blocks may drastically affect the properties of the aggregates. The ability of peptide nucleic acids (PNA) to aggregate has been very little explored, despite its practical applications. In this work we investigated the self-assembling properties of a PNA dimer, conjugated at the N-terminus to a fluorenylmethoxycarbonyl group. This PNA dimer forms nano-aggregates at low concentration in CHCl3/CH3OH mixtures. The aggregates retain very interesting fluorescent properties (high quantum yield in the visible region with lifetimes on the nanosecond scale), which make them promising materials for applications in optoelectronics.

This work is focused on the crystal-chemical characterization of the cationic and anionic components of sodalite-group minerals (SGM) occurring in various igneous-to-metamorphic rocks and ejecta from the alkaline-potassic Somma-Vesuvius volcano (southern Italy). A combination of different analytical techniques, i.e., XRD, SEM, EPMA, SIMS, FTIR, and mu-FTIR-FPA imaging, were used. Minor and trace volatile elements (H, C, and F) have been quantified for the first time (to the authors' knowledge) by SIMS, and in situ ion probe data were integrated with information derived from FTIR spectroscopy. The studied samples correspond to sodalite sensu stricto, nosean, and hauyne. SIMS measurements for H, F, and C (quantified as H2O, F, and CO2 wt.%, respectively) show contents 0.02-5.0 wt.% H2O, 0.01-0.14 wt.% F, and 0.08-2.95 wt.% CO2. Within the single crystals, the F and, to a lesser extent, CO2 concentrations are homogeneous, whereas, in terms of H2O, they range from relatively homogenous to strongly zoned. Single-crystal FTIR spectra of SGM can be grouped into sodalites and noseans/hauynes, according to the occurrence of the (CO2)-C-12 absorption at 2340 cm(-1). The absorption due to H2O or OH groups occurs as a very broad band extending from 3700 to 3000 cm(-1). FTIR data show the presence of CO32- in all samples. The collected data allow us to fully characterize the SGM from Somma-Vesuvius, and also provide some constraints regarding their genesis. These data can contribute to a more detailed understanding of the crystal chemistry of SGM, their geological environments of formation, and possibly of the technological characteristics of their synthetic analogues.

The present work deals with the synthesis and characterization of a novel nucleoamino acid derivative based on a l-tyrosine moiety to which a thymine nucleobase was anchored by means of an amide bond to the N-alpha group. This derivative, denominated by us TyrT, belongs to the family of the nucleobase-amino acid conjugates that show a wide range of biological activities, frequently associated with their ability to interact with nucleic acids. In this respect, the interaction of TyrT with poly(A), a proposed RNA target for anticancer strategies, was studied by circular dichroism (CD) which suggested its ability to bind this RNA. Moreover, the modification of the morphology of a sample of TyrT in the presence of poly(A) was visualised by scanning electron microscopy (SEM) which was in agreement with the evidence that the thyminyl l-tyrosine interacts with poly(A). Finally, computational analyses have been performed to hypothesize the binding mode from a structural point of view, suggesting that the binding is mainly kept via hydrophobic contacts, reproducing a stacking-like interaction between the thymine ring of TyrT and the two successive adenine rings of a poly(A) model.

Streamlined access to S-glycosylated Fmocaminoacids was developed. The process provides diverseglycosylated modified amino acids in high yield and stereoselectivitytaking advantage of the in situ generation of aglycosylthiolate obtained from carbohydrate acetates in a fewsteps. Mild basic conditions make the conjugation reactioncompatible with Fmoc-iodo-amino acids. To validate thestrategy the glycosylated building blocks were used for SPPSand the unprecedented incorporation of a long thiooligosaccharideto the peptide chain was demonstrated.

The structural investigation of the ligand/target interactions represents a challenging task in the field of drug discovery or lead compound optimization. In the present study, a computational approach allowed the identification of the binding site of A9 peptide, within a synthetic model of HER2 receptor (HER2-DIVMP). To this aim, molecular docking calculations and molecular dynamics simulations were employed, taking into account experimental data obtained by fluorescence studies. The computational model was further validated by performing fluorescence binding studies between the ligand A9 and HER2-DIVMP mutants, prepared by replacing key amino acid residues. A new binding pocket of HER2-DIVMP was identified, which could be fruitfully exploited for future lead-optimization studies.

Ribosome biogenesis is closely linked to the cell growth and proliferation. Dysregulation of this process causes several diseases collectively known as ribosomopathies. One of them is the Shwachman-Diamond Syndrome, and the SBDS protein mutated in this disease participates with EFL1 in the cytoplasmic maturation of the 60S subunit. Recently, we have shown that the interaction of EFL1 with SBDS resulted in a decrease of the Michaelis-Menten constant (KM) for GTP and thus SBDS acts as a GEF for EFL1 (1). Subsequent studies demonstrated that SBDS greatly debilitates the interaction of EFL1 with GDP without altering that for GTP. The interaction of EFL1 alone or in complex with SBDS to guanine nucleotides is followed by a conformational rearrangement. Understanding the molecular strategy used by SBDS to disrupt the binding of EFL1 for GDP and the associated conformational changes will be key to understand their mode of action and alterations occurring in the disease. The structure of the GTPase EFL1 is not known and its crystallization has been unsuccessful at least in our hands. In this study, we aim to show the conformational changes resulting from the interactions between EFL1 and its binding partners, the SBDS protein and the guanine nucleotides using SAXS technique (2, 3). SAXS will provide structural information of the proteins and their conformational changes (4). For the SAXS data analysis we have built models of EFL1 using by EF-2 as homology template and of SBDS using the crystal structures of the archaea orthologues.

The ECA celebrates in 2012 its 15th anniversary, but coordinated European crystallographic activities date back to 1973, i.e., the first ECM in Bordeaux. Indeed, the IUCr was founded in 1946 (perhaps one of the few cases where worldwide coordination started at the very beginning of a science) to allow the development of crystallography through the exchange of notions and expertises. The rapid development and the fundamental role of crystallography in many different branches of science, as well as the awareness that Europe has a common cultural (and hopefully soon political) house became increasingly evident at all the ECMs, which regularly alternated with the IUCr congresses. There were also opportunities for educational targets as testified by many satellite meetings and workshops, aimed to point out the frontiers of crystallography. At the beginning, the background concepts and techniques in crystallography were entrusted to the national academic institutions. However, following the giant developments of the methodology, especially through efficient and easy-to-handle software packages, crystallography is no longer perceived as a science deserving academic courses. Hence, the strictly crystallographic community has the duty to preserve the knowledge of fundamental crystallography, which only allows critical usage of the procedures, attainment of well established targets and further progresses in the methodologies. The Italian community has a long tradition in the organization of national schools of crystallography, which in the last years have also developed undeniable international character in terms of both teachers and participants. One year ago, the AIC proposed to validate this trend and launched the idea of annual European schools devoted to the principles and applications of crystallography. All the countries, adhering to the ECA, shall alternate in the task of planning and organizing an educational event under the control and sponsorship of the ECA itself. The goal is not only that of increasing the number of crystallography users but also forming conscious scientists. Attendants with different curricula will eventually learn how to exploit the crystallographic tools in a well designed strategy, also aimed to the interpretation of the results to reach specific goals. Participation of teachers and students from different countries will favor contacts and collaborations, which will act as potential seeds for joint European research projects. Each school shall illustrate different aspects of the discipline, at least within the time scale of a three year PhD curriculum, so that the multi-disciplinary character of crystallography will fully emerge. The relevance of each ECS proposal shall be validated by the ECA council by examining and comparing the bids prepared by national or associated communities. High educational contents, opportune infra-structures and fund raising strategies must be illustrated to support a truly adequate internation

Liposomal formulations composed of phospholipids with different unsaturation degrees, head groups and at different cholesterol content have been tested for the encapsulation of Peptide Nucleic Acid (PNA) oligomers. The best loading capability (177 ?g, ER% = 87.2) was obtained for pure liposomes of phosphatidylglycerol (DOPG) with negatively charged head group. The insertion of a 10-20% of cholesterol in DOPG based liposomes provides a slight decrease (~160 ?g) of the PNA loading. On the other hand, the cholesterol addition (20-30%) slows down the PNA's release (~27%) in fetal bovine serum from the liposomal formulation. Based on the encapsulation and the release properties, PEGylated DOPG liposomes with a percentage of cholesterol of 10-20% are the optimal formulation for the loading of PNA-a210.

The inhibition of amyloid formation is a promisingtherapeutic approach for the treatment of neurodegenerativediseases. Peptide-based inhibitors, which have widely beeninvestigated, are generally derived from original amyloid sequences.Most interesting, trehalose, a non-reducing disaccharide of ?-glucose, is effective in preventing the aggregation of numerousproteins. We have determined that the development of hybridcompounds may provide new molecules with improved propertiesthat might sinergically increase the potency of their single moieties.In this work, the ability of the C-terminal trehalose conjugated Ac-LPFFD-Th derivative to slow down the A? aggregation process wasinvestigated by means of different biophysical techniques, includingTh-T fluorescence, DLS, ESI-MS and NMR. Moreover, wedemonstrate that Ac-LPFFD-Th modifies the aggregation features ofA? and protects neurons from A? oligomers' toxic insult.

The invention relates to a functionalized silk fibroin security marker comprising one, two, three or more different security taggants selected from the group consisting of metallic particles, preferably metallic nanoparticles; magnetic particles, preferably magnetic nanoparticles; and peptide sequences, use of the inventive security marker within the substrate and/or on the surface of a security document, a functionalized security document comprising the inventive security marker as well as processes for manufg. and authenticating the inventive functionalized silk fibroin security marker and inventive security document.