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The COVID-19 (Corona Virus Disease 19) pandemic, caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), is causing enormous difficulties in the world’s economies and there is uncertainty as to whether the current prophylactic measures will offer adequate protection globally after the appearance of virus variants that like that indicated as Omicron emerged in the presence of global vaccine-based immunization. While several studies are available describing the main differences in the spike protein of Omicron compared to the other variants previously emerged, there was no structural insights into the 3CL-protease (3CLpro) associated to the new variant. Herein, we performed a computational study based on genomic data and amino acid sequences available in the most updated COVID-19-related databases that allowed us to build upin silicothe 3D structure of Omicron 3CLpro. Moreover, by molecular dynamics simulation we demonstrated that currently available drugs acting as inhibitors of the SARS-CoV-2 main protease could be less effective in the case of Omicron variant due to the different chemical interactions in the binding site occurred after the recent amino acid mutations. Ultimately, our study highlights the need of exploitingin silicoand in vitro methods to discover novel 3CLproinhibitors starting from the computationally based structure we presented herein, and more in general to direct the major efforts to targeting the most conserved 3CLproregions that appeared unchanged in the context of the Omicron variant.

Cardio-cerebrovascular (CCV) disease contributes significantly to the global burden of disease, with dramatic consequences in terms of mortality and general health. Mitigate CCV risk factors is the key to reduce individual and population risk of CCV events. Evidence-based medicine and epidemiological investigations of risk factors are essential to optimize actions. To contribute to the knowledge of the burden of risk factors in determining CCV events in the individual patient and in the community. Clinical data and risk factors were collected through a longitudinal survey (1999) as part of a larger epidemiology and cardiovascular prevention project, namely the “VIP (Valle dell'Irno Prevention) Project”. We assessed the incidence of major cardiovascular events (MACE) and for each risk factor we calculated: prevalence, absolute risk, odds ratio (OR), additional risk (AR) = risk of exposed to the risk factor − risk of non-exposed, population attributable risk (PAR) = additional risk * prevalence, population attributable risk fraction (PAF) = PAR/total incidence of the disease. Comparing the MACE group with the non-MACE group, a statistically significant difference was found for the following: glomerular filtration rate (GFR), glucose and systolic blood pressure (SBP), BMI, diastolic blood pressure (DBP), cholesterol, triglycerides, creatinine and uric acid. GFR, glucose and SBP showed the highest OR. Age, creatinine, glycemia, SBP and uric acid were independent predictor of MACE. When calculating the PAF, the CCV risk factors with the greatest impact on MACE were: SBP (29.6%), triglyceridemia (19.4%) and metabolic syndrome (18.3%). The burden of risk factors on MACE changes substantially according to whether it is calculated in the single patient or in the population. It is crucial for physicians to take these differences into account when applying their own intervention to reduce CCV events.

Clays are used in various cosmetic formulations, such as sunscreens, toothpastes, deodorants, creams, hair cosmetics, makeups, nail polish, facial masks, and shampoos, among others, to improve the organoleptic and physicochemical characteristics, to increase the stability, or to facilitate elaboration. Together with their technological functionalities, clays are cosmetologically active ingredients with cleaning, anti-aging, anti-wrinkling, and sun-care functionalities. Talc, kaolinite, mica, and some smectites are the clay minerals used most frequently in cosmetic products, but several other phyllosilicates as well as modified and synthetic clays are also used. Sometimes, clays are useful in the design of cosmetics just because they are made of rigid, small, and anisometric particles. Kaolinite and mica are made of hard prismatic particles which are lightly abrasive over the skin, teeth, or hair. Electric charges in smectites result in ion-exchange capacities useful in the loading of active cosmetics but also adsorbing and cleaning waste substances. Intermediate net negative charges of smectites result in layer expansion in polar media and specific rheological properties that are very useful in cosmetic formulations. The absence of charged particles in talc and kaolin make them flow easily resulting in lubricant effects. Protection against radiation from the sun by clay particles and decorative effects complete the possibilities of clays in cosmetics. The nomenclature for clays used as ingredients in cosmetics follows historical use and the names of commercial products, rather than following strict compositional principles. In this sense, an effort was made here to correlate the names of the minerals that make up each of the clay-based cosmetic ingredients., The work has been supported by funding from the Spanish group CTS-946 and project P18-RT-3786.

Wound healing, especially of chronic wounds, is still an unmet therapeutic area since assessment and management are extremely complicated. Although many efforts have been made to treat wounds, all strategies have achieved limited results for chronic wounds. Stem cell-based therapy is considered a promising approach for complex wounds such as those occurring in diabetics. Mesenchymal stem cell transplantation significantly improves wound closure, angiogenesis and wound healing. However, cell therapy is complex, expensive and time-consuming. Recent studies have shown that stem cell-derived exosomes can be an exciting approach to treat wounds. Exosomes derived from mesenchymal stem cells can induce benefit in almost all stages of wound healing, including control of immune responses, inhibition of inflammation, promoting cell proliferation and angiogenesis, while reducing scar formation during the wound healing process. This review aimed at offering an updated overview of the use of exosomes in biological applications, such as wound healing, and addresses not only current applications but also new directions for this next-generation approach in wound healing.

The first aim of the present study is the development of a new ruthenium(III) complex, belonging to NAMI-A class, with a potentially high cytotoxic ability. The presence of a fully protected sugar moiety as ruthenium ligand should increase the complex ability to cross cellular membranes. Furthermore, it sets this molecule in the area of biocompatible agents as tumor drug. The second, more relevant, purpose is to verify the ruthenium complexes activity after loading into liposomes. We reported the characterization and in vitro biological assays of pyridine derivatives of ruthenium complexes loaded into Egg l -α-phosphatidylcholine cholesterol/DSPE-PEG liposomes. Dynamic light scattering estimates that the sizes of all obtained liposomes are in the 100 nm range. This value is suitable for in vivo use. The loading ability and release kinetic allowed selecting the best ratio between the lipid fraction and metal to be tested in cellular experiments. The growth inhibitory effects of both liposomal and free complex in PC-3 prostate cancer cell lines demonstrate a high cytotoxic ability of the liposome entrapped ruthenium (III) complex suggesting additional role further the antimetastatic function.

Four 4-nitrophenyl-functionalized benzofuran (BF1, BF2) and benzodifuran (BDF1, BDF2) compounds were synthesized by a convenient route based on the Craven reaction. All the compounds underwent a detailed chemical physical characterization to evaluate their structural, thermal and optical properties. An investigation on the therapeutic potential of the reported compounds was performed by analyzing their antiproliferative activity on prostatic tumour cells (PC-3). In both classes of compounds, anticancer potential is in direct correlation with the lipophilicity. From our study it emerged that antiproliferative activity was higher for benzofuran derivatives as compared to benzodifuran systems. Moreover, we report a mechanistic study relative to the most promising molecule, i.e. the apolar benzofuran BF1, that relates the antiproliferative properties found in our investigation to its ability to bind telomeric DNA (proven by CD and fluorescence techniques on tel(22) G4 DNA), and highlights its unexpected impact on cell cycle progression. (C) 2018 Elsevier B.V. All rights reserved.

The lipid phosphatase Ship2 binds the EphA2 receptor through a heterotypic Sam-Sam (Sterile alpha motif) interaction. Inhibitors of the Ship2-Sam/EphA2-Sam complex hold a certain potential as novel anticancer agents. The previously reported “KRI3” peptide binds Ship2-Sam working as a weak antagonist of the EphA2-Sam/Ship2-Sam interaction. Herein, the design and functional evaluation of KRI3 analogues, both linear and cyclic, are described. A multidisciplinary study was conducted through computational docking techniques, and conformational analyses by CD and NMR spectroscopies. The ability of new peptides to bind Ship2-Sam was analysed by NMR, MST and SPR assays. Studies on linear KRI3 analogues pointed out that aromatic interactions through tyrosines are important for the association with Ship2-Sam whereas, an increase of the net positive charge of the sequence or peptide cyclization through a disulfide bridge can favour unspecific interactions without a substantial improvement of the binding affinity to Ship2-Sam. Interestingly, preliminary cell-based assays demonstrated KRI3 cellular uptake even without the conjugation to a cell penetrating sequence with a main cytosolic localization. This work highlights important features of the KRI3 peptide that can be further exploited to design analogues able to hamper Sam-Sam interactions driven by electrostatic contacts.

In previous studies we demonstrated that radiofrequency (RF) electromagnetic fields (EMF) is able to reduce DNA damage induced by a subsequent treatment with genotoxic agents, resembling the adaptive response, a phenomenon well known in radiobiology. In this study we report on the capability of the culture medium from SH-SY5Y neuroblastoma cells exposed to 1950 MHz to elicit, in recipient non-exposed cells, a reduction of menadione-induced DNA damage (P < 0.05; comet assay), indicating the capability of non-ionizing radiation to elicit a bystander effect. A comparable reduction was also detected in cultures directly exposed to the same EMF conditions (P < 0.05), confirming the adaptive response. In the same exposure conditions, we also evidenced an increase of heat shock protein 70 (hsp70) in culture medium of cells exposed to RF with respect to sham exposed ones (P < 0.05; western blot analysis), while no differences were detected in the intracellular content of hsp70. On the whole, our results evidence a protective effect of RF against menadione-induced DNA damage in directly and non-directly exposed cells, and suggest hsp70 pathway to be investigated as one of the potential candidate underpinning the interaction between RF exposure and biological systems.

Herein, we described the synthesis of two l-phenylalanines α-derivatized with a terminal alkyne moiety whose structures differed by phenyl ring halogen substitution (two o-Cl in 1 vs. one p-Br in 2) and investigated their effect on biological macromolecules and living cells. We explored their interaction with quadruplex DNA (G4 DNA), using tel26 and c-myc as models, and bovine serum albumin (BSA). By CD spectroscopy, we found that 1 caused minor tel26 secondary structure changes, leading also to a slight thermal stabilization of this hybrid antiparallel/parallel G4 structure, while the c-myc parallel topology remained essentially unchanged upon 1 binding. Other CD evidences showed the ability of 1 to bind BSA, while molecular docking studies suggested that the same molecule could be housed into the hydrophobic cavity between sub-domains IIA, IIB, and IIIA of the protein. Furthermore, preliminary aggregation studies, based on concentration-dependent spectroscopic experiments, suggested the ability of 1 to aggregate forming noncovalent polymeric systems in aqueous solution. Differently from 1, the bromine-modified compound was able to bind Cu(II) ion, likely with the formation of a CuL2 complex, as found by UV spectroscopy. Finally, cell tests excluded any cytotoxic effect of both compounds toward normal cells, but showed slight antiproliferative effects of 2 on PC3 cancerous cells at 24 h, and of 1 on both T98G and MDA-MB-231 cancer cells at 48 h.

Herein, we present a spectroscopic (CD and UV) and SEM study of a phenylalanine derivative carrying a terminal alkyne moiety and indicated by us CF3IIIPhe, with particular attention to its interaction with Cu(II) cation and some biological macromolecules, as well as a preliminary evaluation of its effect on cancerous cells. CD spectroscopy evidenced the ability of CF3IIIPhe to interact with tel26 and c-myc, two quadruplex DNA (G4 DNA) models explored in this study. Other CD and UV studies revealed the ability of the unnatural amino acid to form aggregates in aqueous solution, to bind Cu(II) cation, and to interact with bovine serum albumin (BSA). Cellular studies demonstrated CF3IIIPhe antiproliferative activity on PC3 cells. Its ability to bind telomeric DNA was verified with tel26 by CD investigation and SEM analysis, that revealed a noteworthy change in DNA morphology (mainly based on nanosphere structures) by CF3IIIPhe, confirming its G4-DNA binding ability already evidenced by spectroscopy.

The lipid phosphatase Ship2 represents a drug discovery target for the treatment of different diseases, including cancer. Its C-terminal sterile alpha motif domain (Ship2-Sam) associates with the Sam domain from the EphA2 receptor (EphA2-Sam). This interaction is expected to mainly induce pro-oncogenic effects in cells therefore, inhibition of the Ship2-Sam/EphA2-Sam complex may represent an innovative route to discover anti-cancer therapeutics. In the present work, we designed and analyzed several peptide sequences encompassing the interaction interface of EphA2-Sam for Ship2-Sam. Peptide conformational analyses and interaction assays with Ship2-Sam conducted through diverse techniques (CD, NMR, SPR and MST), identified a positively charged penta-amino acid native motif in EphA2-Sam, that once repeated three times in tandem, binds Ship2-Sam. NMR experiments show that the peptide targets the negatively charged binding site of Ship2-Sam for EphA2-Sam. Preliminary in vitro cell-based assays indicate that -at 50 µM concentration- it induces necrosis of PC-3 prostate cancer cells with more cytotoxic effect on cancer cells than on normal dermal fibroblasts. This work represents a pioneering study that opens further opportunities for the development of inhibitors of the Ship2-Sam/EphA2-Sam complex for therapeutic applications.

Here we describe the synthesis, chromatographic purification, MS and NMR characterization of a new lactosyl-derivative, i.e. a lactosyl thiophenyl-substituted triazolyl-thione l-alanine (Lac-L-TTA). This amino acid–sugar conjugate was prepared by solution synthesis in analogy to the natural fructosyl-amino acids. Furthermore, we investigated the inhibition of PC-3 prostate cancer cell colony formation by this lactose derivative in comparison with the less polar fructose-based derivative, Fru-L-TTA. This let us to compare the properties of the artificial derivative, object of the present work, with the monosaccharide-based counterpart and to obtain a preliminary information on the influence of polarity on such biological activity. A significantly higher anticancer effect of Lac-L-TTA with respect to the fructose analogue emerged from our study suggesting that the anti-metastatic potential of fructosyl-amino acids can be enhanced by increasing the polarity of the compounds, for example by introducing disaccharide moieties in place of fructose.

The success of platinum complexes as chemiotherapeutic drugs pushed the scientific community to design new transition metal based complexes. to fight cancer and overcome the severe side effects and the insurgence of platinum resistance [1]..Among these compound ruthenium (III) complexes have been demonstrated very powerful against metastatic tumour but very poor is their action as cytotoxic agent.[2] The first aim of the present study was the development of a new ruthenium(III) complex, belonging to NAMI-A class, bearing to pyridine ligand a fully protected sugar moiety (RuPyTri). The presence of carbohydrate in coordination sphere should increase the complex ability to cross cellular membranes and sets this molecule in the area of biocompatible agents as tumour drug. The stability of the complex was studied and compared to RuPy complex in solution at different pH following UV-VIS spectra. The second, more relevant, purpose was to verify the ruthenium complexes activity after loading into liposomes. Liposomes can induce different mechanisms of delivery, increase the ability to cross cell membrane and stabilize the complexes reducing the water exchange. The lipid composition Egg L-α-phosphatidylcholine cholesterol / DSPE-PEG liposomes was selected to reproduce analog supramolecular aggregates in clinical use to vehicle doxorubicin (Doxil). Ruthenium complexes were loaded into liposomes using the passive equilibration loading method estimating the entrapped metal amount by ICP-MS. Full drug containing liposomes were structurally characterized by dynamic light scattering (DLS). Measurements confirm that sizes of all liposomes are in the 100 nm range, suitable for in vivo use. Loading ability and release kinetic allowed to select the best ratio between the lipid fraction and the metal to be tested in cellular experiments. Stability and drug release properties of ruthenium containing liposomes were confirmed in buffer. The growth inhibitory effects of both liposomal and free complex in PC3 prostate cancer cell lines validate a high cytotoxic ability of the liposome entrapped ruthenium (III) complexes.

Nodal is a potent embryonic morphogen belonging to the TGF-beta superfamily. Typically, it also binds to the ALK4/ActRIIB receptor complex in the presence of the co-receptor Cripto-1. Nodal expression is physiologically restricted to embryonic tissues and human embryonic stem cells, is absent in normal cells but re-emerges in several human cancers, including melanoma, breast, and colon cancer. Our aim was to obtain mAbs able to recognize Nodal on a major CBR (Cripto-Binding-Region) site and to block the Cripto-1-mediated signalling. To achieve this, antibodies were raised against hNodal(44-67) and mAbs generated by the hybridoma technology. We have selected one mAb, named 3D1, which strongly associates with full-length rhNodal (KD 1.4 nM) and recognizes the endogenous protein in a panel of human melanoma cell lines by western blot and FACS analyses. 3D1 inhibits the Nodal-Cripto-1 binding and blocks Smad2/3 phosphorylation. Data suggest that inhibition of the Nodal-Cripto-1 axis is a valid therapeutic approach against melanoma and 3D1 is a promising and interesting agent for blocking Nodal-Cripto mediated tumor development. These findings increase the interest for Nodal as both a diagnostic and prognostic marker and as a potential new target for therapeutic intervention.

A new dual-ligand liposomal doxorubicin delivery system, which couples targeting to enhanced cellular uptake and may lead to a more efficient drug delivery system, is here designed and synthetized. Liposomes based on the composition 1,2-dioleoyl-sn-glycero-3-phosphocholine/1,2-distearoyl-sn-glycero-3-phosphoethanolamine-Peg2000-R8/(C18)2-L5-SS-CCK8 (87/8/5 mol/mol/mol) were prepared and loaded with doxorubicin. Presence of the two peptides on the external surface is demonstrated by fluorescence resonance energy transfer assay. The combination of the R8 cell-penetrating peptide and of the CCK8 targeting peptide (homing peptide) on the liposome surface is obtained by combining pre-modification and post-modification methods. In the dual-ligand system, the CCK8 peptide is anchored to the liposome surface by using a disulfide bond. This chemical function is inserted in order to promote the selective cleavage of the homing peptide under the reductive conditions expected in proximity of the tumor site, thus allowing targeting and internalization of the liposomal drug. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.

A series of neutral and cationic gold(I) complexes bearing a glucopyranoside-incorporated N-heterocyclic carbene (NHC) ligand are synthetized and structurally characterized. Different secondary ligands (chlorido, phosphane, or sugar–NHC) are employed to tune the properties of the complexes. The antiproliferative effects of the compounds are evaluated against PC-3 prostate cancer cells and a panel of human tumor cell lines. The activities of the phosphane complexes are comparable to that observed for cisplatin. The combined results provide further insights into the biological behavior of NHC–gold complexes.

Five novel peptide amphiphiles (PAs), with common formula (C18)2-PEGx-CCK8 in which the CCK8 peptide and the (C18)2-hydrophobic moiety are spaced by polyethylene linkers of different length (PEG moieties with molecular weights of 700, 1000, 1500, 2000, and 3000 Daltons) are described. They act as potential target-selective nanocarriers towards tumor cells overexpressing cholecistokynin receptors. PAs self-assemble in supramolecular aggregates, with hydrodynamic radius ranging between 63 and 104 nm, as indicated by DLS measurements. Fluorescence studies suggested that, irrespective from the PEG length, the tryptophan residue located at the center of the CCK8 sequence is completely surrounded by water molecules at high mobility. This result indicates a potential capability of all formulated nanovectors to recognize the overexpressed CCK-2 receptors. CD data suggest that CCK8 peptide, in most of PAs in their aggregate form, adopts a conformation allowing the interaction with the receptor. Anyway, biological data obtained by flow cytometry analysis indicate that the five PAs have a different binding ability towards the CCK-2 receptors, with higher binding properties shown by PA containing PEG with MW of 2000 Dalton. Therefore, PEG2000 can be considered as the best spacer in the formulation of nanovectors based on CCK8 peptide amphiphiles.

Targeting regulatory RNA regions to interfere with the biosynthesis of a protein is an intriguing alternative to targeting a protein itself. Regulatory regions are often unique in sequence and/or structure and, thus, ideally suited for specific recognition with a low risk of undesired side effects. Targeting regulatory RNA elements, however, is complicated by their complex three-dimensional structure, which poses kinetic and thermodynamic constraints to the recognition by a complementary oligonucleotide. Oligonucleotide mimics, which shift the thermodynamic equilibrium towards complex formation and yield stable complexes with a target RNA, can overcome this problem. Peptide nucleic acids (PNA) represent such a promising class of molecules. PNA are very stable, non-ionic compounds and they are not sensitive to enzymatic degradation. Yet, PNA form specific base pairs with a target sequence. We have designed, synthesised and characterised PNA able to enter infected cells and to bind specifically to a control region of the genomic RNA of coxsackievirus B3 (CVB3), which is an important human pathogen. The results obtained by studying the interaction of such PNA with their RNA target, the entrance into the cell and the viral inhibition are herein presented. Copyright © 2005 European Peptide Society and John Wiley & Sons, Ltd.

Pure sterically stabilized micelles (SSM) of DSPE-PEG2000, and sterically stabilized mixed micelles (SSMM) containing PC or DOPC phospholipids (5, 10 or 20% mol/mol with respect to DSPE-PEG2000) are developed as delivery systems for the gold based cytotoxic drug Au(III)-dithiocarbamato complex AuL12. In particular, SSMM containing 5% of PC at 5 mM of lipid concentration encapsulates 61.0 ?g of AuL12 with a DL% of 1.13. The gold complex remains stable up to 72 h when incorporated in the aggregate, as indicated by UV-vis measurements. Incorporation in micelle composition of a low amount of the peptide derivative MonY-BN-AA1, containing a bombesin peptide analogue does not influence structural parameters of the micelles (diameter around 20 nm) neither the AuL12 loading parameters. Target selective properties of the peptide containing full aggregate on PC-3 cells overexpressing the GRP/bombesin receptors are observed by in vitro cytotoxic studies: a decrease of cell viability, ~50%, is obtained in cells treated with AuL12-targeted micelles at 10 ?M drug concentration for 48 h with respect to untargeted micelles. © 2014 Elsevier B.V.

Here, we report the synthesis, purification, ESI MS and NMR characterization, as well as the SEM analysis of a fructosyl thiophenyl-substituted triazolyl-thione l-alanine (denominated Fru-l-TTA). This novel fructosyl derivative was obtained by solution synthesis using the Amadori reaction, in analogy to other natural fructosyl-amino acids, and fully characterized. In particular, we report an accurate NMR/MS/SEM characterization of Fru-l-TTA alongside some biological properties, and investigated to compare the properties of the artificial derivative of this work with the natural counterparts. In particular, Fru-l-TTA shares with natural fructosyl-amino acids the possibility to inhibit the colony formation of prostate cancer cells and additionally decreases their migration.

Five novel peptide amphiphiles, PA-[700] ÷ PA-[3000], with common formula (C18)2-PEGx-CCK8 are described as potential target selective nanocarriers towards tumor cells overexpressing cholecistokynin receptors. DLS measurements indicate that PA-[700] ÷ PA-[2000] self-assemble in in supramolecular aggregates with a hydrodynamic radius ranged between 63 and 83 nm, while PA-[3000] shows a RH of 104 nm with an increase of ≈ 30% with respect to the other peptide derivatives. Fluorescence studies suggested that, irrespective from the PEG length on the PA, the tryptophan residue located at the center of the CCK8 sequence, is completely surrounded from water molecules at high mobility. This result indicates a potential capability of all formulated nanovectors to recognize the over-expressed CCK-2 receptors. Also CD data suggest that CCK8 peptide in all PAs in their aggregate form, with the exception of PA-[700], adopt a conformation allowing the interaction with the receptor. Anyway biological data obtained by flow cytometry analysis indicate that the five PAs with different PEG spacers have a different behavior in the binding ability of PA towards to the CCK2-R, with higher binding properties shown by PA-[2000]. Finally, proteolitic cleavage experiments indicate that in PA-[2000] supramolecular aggregates the CCK8 peptide present the Tyr-Met and Trp-Met cleavable amide bonds available for the enzymatic process, while in PA-[3000] the longer PEG moiety masks the active site in CCK8, thus preventing both enzymatic cleavage of the Tyr-Met bond and receptor interaction. Based on these results, PEG2000 can be considered as the best spacer for CCK8 peptide amphiphiles.

Vascular Endothelial Growth Factor mimetic peptides have interesting applications in therapeutic angiogenesis. Recently, we described the proangiogenic properties of a 15 mer peptide designed on the N-terminal helix 17-25 of VEGF. The peptide was stabilized introducing well known peptide chemical tools among which N- and C-terminal capping sequence. Here, we show that the C-terminal sequence does not affect the structural and biological properties of the full-length peptide. In fact, a C-terminal truncated analog peptide resulted in a well folded and stable helix retaining the ability to bind to VEGF receptors. This study will allow to develop smaller peptidomimetic analogs able to modulate the VEGF-dependent angiogenesis. © 2012 Elsevier Inc.

New liposomal aggregates, prepared by combining together, in a 90:10 molar ratio, two amphiphilic monomers, one containing two hydrocarbon chains in the hydrophobic region and the anionic DOTA chelating agent as hydrophilic moiety, and the other containing the same hydrophobic moiety and the CCK8 peptide, are described. The liposomal aggregates because of the presence of the specific moiety, constituted by the CCK8 peptide, which selectively recognizes CCK receptors on tumor cells are used as drug carriers with the aim to deliver into tumor cells the appropriate antitumor drug. The drug loading content and the releasing properties of the liposomal aggregates are studied by the use of the cytotoxic doxorubicin as drug model. The doxorubicin loading content determination reveals that above 95% of the total drug was uptaken with a corresponding drug/lipid w/w ratio of 0.134. The cellular uptake of the targeted liposomal doxorubicin with respect to the self-assembled, nonspecific, liposomal doxorubicin is evaluated using flow cytometry assays. The doxorubicin cell content for two types of cell systems, namely, A431 and HuVEC cells, for peptide derivatized liposomes was 70- and 8-fold higher, respectively, than for nontargeted liposomes, indicating that the bioactive CCK8 peptide is able to enhance the doxorubicin uptake into the carcinoma cells in vitro. The cytotoxicity effect of liposomal doxorubicin on A431 cells has been assessed by MTT assays: in presence of drug amounts ranged between 250 and 1000 ng/ml, incubation with peptide derivatized liposomes showed significantly lower cell survival compared with nontargeted liposomes.

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor. Its biological activity is mediated by the binding to the extracellular domain of two tyrosine kinase transmembrane receptors: VEGFR1 and VEGFR2. Deletion studies showed that VEGF binding site resides in the first three domains of VEGFR1 and in domains 2 and 3 of VEGFR2. In particular, the second extracellular domain of VEGFR1 (VEGFR1(D2)) contains most of the VEGF binding requirements. Here, we report an efficient expression protocol and the molecular characterization by spectroscopic techniques of VEGFR1(D2). The protein was expressed in E. coli and refolded from inclusion bodies. The recombinant protein assumes the correct fold as assessed by a combination of biochemical and functional assays as well as by NMR characterization. Furthermore, the recombinant VEGFR1(D2) was analyzed by circular dichroism and fluorescence spectroscopy. The protein obtained by this procedure is suitable for the structural characterization of the complexes with receptor binders and to be used in interaction/screening studies. 2010 Wiley Periodicals, Inc.

FcvarepsilonRIalpha found on the surface of mast cells and basophiles mediates allergic diseases, anaphylaxis and asthma through binding of IgE. Disrupting this interaction with anti-IgE mAbs has proven an efficient approach to control these diseases. The crystallographic structure of the complex formed between the IgE-Fc and FcvarepsilonRIalpha extracellular domain has shown that recognition is mediated by residues in the second Ig-like domain of the receptor (D2) and in the loop connecting the D1 and D2 domains. In an attempt to obtain specific IgE antagonists, we have designed and prepared a polypeptide named IgE-Trap that partially reproduces the IgE receptor-binding sites and binds with micromolar affinity to soluble IgE. The polypeptide contains loops C'-E [residues 129-134] and F-G [residues 151-161] from the D2 domain joined by a linker, and loop B-C [residues 110-113]. Peptide binding to IgE has been assessed by SPR analyses and the data fit with a biphasic model of interaction, in agreement with the two-site mechanism reported for the native receptor. The polypeptide binds to immobilized IgE in a dose-dependent manner with a K(D) estimated to be around 6muM, while it does not recognize IgG nor IgA. Polypeptide sub-domains involved in IgE binding have also been defined, showing that loop C'-E connected to loop B-C, but also the isolated loop B-C alone suffice to bind immunoglobulins E with high selectively though with reduced affinity compared to IgE-Trap. ELISA and cytometric assays on RBL2H3 cells demonstrate that the interacting peptides are able to displace the binding of IgE to receptor, confirming affinity and specificity of these ligands and suggesting a potential application as modulators of disorders associated with inappropriate IgE production.

Haptoglobin (Hpt) binds apolipoprotein A-I (ApoA-I), and impairs its stimulation of lecithin:cholesterol acyltransferase (LCAT). LCAT plays a major role in reverse cholesterol transport (RCT). Apolipoprotein E (ApoE), like ApoA-I, promotes different steps of RCT, including LCAT stimulation. ApoE contains amino acid sequences that are homologous with the ApoA-I region bound by Hpt and are involved in the interaction with LCAT. Therefore, Hpt was expected to also bind ApoE, and inhibit the ApoE stimulatory effect on LCAT. Western blotting and ELISA experiments demonstrated that the Hpt β-subunit binds ApoE. The affinity of Hpt for ApoE was higher than that for ApoA-I. High ratios of Hpt with either apolipoprotein, such as those associated with the acute phase of inflammation, inhibited, in vitro, the stimulatory effect of ApoE on the cholesterol esterification activity of LCAT. Hpt also impaired human hepatoblastoma-derived cell uptake of [3H]cholesterol from proteoliposomes containing ApoE or ApoA-I. We suggest that the interaction between Hpt and ApoE represents a mechanism by which inflammation affects atherosclerosis progression. Hpt might influence ApoE function in processes other than RCT. Structured digital abstract • MINT-7258778: Hpt beta chain (uniprotkb:P00738) binds (MI:0407) to APOE (uniprotkb:P02649) by filter binding (MI:0049) • MINT-7258829, MINT-7258868: Hpt (uniprotkb:P00738) binds (MI:0407) to APOA1 (uniprotkb:P02647) by competition binding (MI:0405) • MINT-7258848, MINT-7258819, MINT-7258877: APOE (uniprotkb:P02649) binds (MI:0407) to Hpt (uniprotkb:P00738) by competition binding (MI:0405) • MINT-7258791: Hpt (uniprotkb:P00738) binds (MI:0407) to APOE (uniprotkb:P02649) by pull down (MI:0096) • MINT-7258760: Hpt (uniprotkb:P00738) physically interacts (MI:0915) with APOE (uniprotkb:P02649) by pull down (MI:0096) • MINT-7258811: Hpt (uniprotkb:P00738) binds (MI:0407) to APOA1 (uniprotkb:P02647) by enzyme linked immunosorbent assay (MI:0411) • MINT-7258801: Hpt (uniprotkb:P00738) binds (MI:0407) to APOE (uniprotkb:P02649) by enzyme linked immunosorbent assay (MI:0411)

In this study, the induction of apoptosis after exposure to 900 MHz radiofrequency radiation (GSM signal) was investigated by assessing caspase 3 activation in exponentially growing Jurkat cells and in quiescent and proliferating human peripheral blood lymphocytes (PBLs). The exposure was carried out at an average specific absorption rate of 1.35 W/kg in a dual wire patch cell exposure system where the temperature of cell cultures was accurately controlled. After 1 h exposure to the radiofrequency field, a slight but statistically significant increase in caspase 3 activity, measured 6 h after exposure, was observed in Jurkat cells (32.4%) and in proliferating human PBLs (22%). In contrast, no effect was detected in quiescent human PBLs. In the same experimental conditions, apoptosis was also evaluated in Jurkat cells by Western blot analysis and in both cell types by flow cytometry. To evaluate late effects due to caspase 3 activity, flow cytometry was also employed to assess apoptosis and viability 24 h after radiofrequency-radiation exposure in both cell types. Neither the former nor the latter was affected. Since in recent years it has been reported that caspases are also involved in processes other than apoptosis, additional cell cycle studies were carried out on proliferating T cells exposed to radiofrequency radiation; however, we found no differences between sham-exposed and exposed cultures. Further studies are warranted to investigate the biological significance of our findings of a dose-response increase in caspase 3 activity after exposure to radiofrequency radiation.

The sea star Oreaster reticulatus contains, along with the known ophidiacerebrosides C–E, nine new glycosphingolipids named oreacerebrosides A–I. Their structures were elucidated by a combination of spectroscopic methods and chemical degradation. All compounds contain a 4,8,10-triunsaturated sphingoid base. Oreacerebrosides A–C have a β-glucopyranoside as the sugar residue, as found in ophidiacerebrosides C–E and in all known compounds of this type; in contrast, oreacerebrosides D–I are the first examples of β-galactosylceramides containing this unusual sphingoid base. Four representative glycosphingolipids were tested for cytotoxic activity on rat glioma C6 cells and were shown to be mildly cytotoxic; the glucosylceramides were more active than the galactosylceramides. In addition, oreacerebroside I, but not ophidiacerebroside E, was shown to exert proangiogenic activity and was able to increase VEGF-induced human endothelial cell proliferation.(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2007)

A new dual-ligand liposomal doxorubicin delivery system, which couples targeting to enhanced cellular uptake and may lead to a more efficient drug delivery system, is here designed and synthetized. Liposomes based on the composition 1,2-dioleoyl-sn-glycero-3-phosphocholine/1,2-distearoyl-sn-glycero-3-phosphoethanolamine-Peg2000-R8/(C18)2-L5-SS-CCK8 (87/8/5 mol/mol/mol) were prepared and loaded with doxorubicin. Presence of the two peptides on the external surface is demonstrated by fluorescence resonance energy transfer assay. The combination of the R8 cell-penetrating peptide and of the CCK8 targeting peptide (homing peptide) on the liposome surface is obtained by combining pre-modification and post-modification methods. In the dual-ligand system, the CCK8 peptide is anchored to the liposome surface by using a disulfide bond. This chemical function is inserted in order to promote the selective cleavage of the homing peptide under the reductive conditions expected in proximity of the tumor site, thus allowing targeting and internalization of the liposomal drug.

Previous studies reported that aged red wine, but not novel red wine or white wine protects human red blood cells from oxidative damage induced in vitro by H(2)O(2.) Here, we demonstrate that the beneficial properties of aged red wine are due, at least in part, to the presence of anthocyanins. We firstly measured the "antioxidant power" of an Italian red wine (Taurasi, Avellino) and that of its anthocyanin fractions by using Ferric Reducing Antioxidant Power Assay. Subsequently, we demonstrate that fractions containing anthocyanins lower ROS (reactive oxygen species) and methemoglobin production in human erythrocytes treated with H(2)O(2.) Finally, we reported that the protective effects of anthocyanins were also confirmed in an experimental model in which RBCs were deprived of catalase activity by treatment with 4 mM sodium azide. The results obtained clearly demonstrate that red wine anthocyanins protect human RBCs from oxidative stress.

Increased consumption of fruits and vegetables can represent an easy strategy to significantly reduce the incidence of cancer. From this observation, derived mostly from epidemiological data, the new field of chemoprevention has emerged in the primary and secondary prevention of cancer. Chemoprevention is defined as the use of natural or synthetic compounds able to stop, reverse, or delay the process of tumorigenesis in its early stages. A large number of phytochemicals are potentially capable of simultaneously inhibiting and modulating several key factors regulating cell proliferation in cancer cells. Quercetin is a flavonoid possessing potential chemopreventive properties. It is a functionally pleiotropic molecule, possessing multiple intracellular targets, affecting different cell signaling processes usually altered in cancer cells, with limited toxicity on normal cells. Simultaneously targeting multiple pathways may help to kill malignant cells and slow down the onset of drug resistance. Among the different substrates triggered by quercetin, we have reviewed the ability of the molecule to inhibit protein kinases involved in deregulated cell growth in cancer cells.

Five novel peptide amphiphiles (PAs), with common formula (C18)2-PEGx-CCK8 in which the CCK8 peptide and the (C18)2-hydrophobic moiety are spaced by polyethylene linkers of different length (PEG moieties with molecular weights of 700, 1000, 1500, 2000, and 3000 Daltons) are described. They act as potential target-selective nanocarriers towards tumor cells overexpressing cholecistokynin receptors. PAs self-assemble in supramolecular aggregates, with hydrodynamic radius ranging between 63 and 104 nm, as indicated by DLS measurements. Fluorescence studies suggested that, irrespective from the PEG length, the tryptophan residue located at the center of the CCK8 sequence is completely surrounded by water molecules at high mobility. This result indicates a potential capability of all formulated nanovectors to recognize the overexpressed CCK-2 receptors. CD data suggest that CCK8 peptide, in most of PAs in their aggregate form, adopts a conformation allowing the interaction with the receptor. Anyway, biological data obtained by flow cytometry analysis indicate that the five PAs have a different binding ability towards the CCK-2 receptors, with higher binding properties shown by PA containing PEG with MW of 2000 Dalton. Therefore, PEG2000 can be considered as the best spacer in the formulation of nanovectors based on CCK8 peptide amphiphiles.

Increased consumption of fruits and vegetables can represent an easy strategy to significantly reduce the incidence of cancer. From this observation, derived mostly from epidemiological data, the new field of chemoprevention has emerged in the primary and secondary prevention of cancer. Chemoprevention is defined as the use of natural or synthetic compounds able to stop, reverse, or delay the process of tumorigenesis in its early stages. A large number of phytochemicals are potentially capable of simultaneously inhibiting and modulating several key factors regulating cell proliferation in cancer cells. Quercetin is a flavonoid possessing potential chemopreventive properties. It is a functionally pleiotropic molecule, possessing multiple intracellular targets, affecting different cell signaling processes usually altered in cancer cells, with limited toxicity on normal cells. Simultaneously targeting multiple pathways may help to kill malignant cells and slow down the onset of drug resistance. Among the different substrates triggered by quercetin, we have reviewed the ability of the molecule to inhibit protein kinases involved in deregulated cell growth in cancer cells.

Until recently, the supposed preventive effects of red wine against cardiovascular diseases, the so-called "French Paradox", has been associated to its antioxidant properties. The interest in the anticancer capacity of polyphenols present in red wine strongly increased consequently to the enormous number of studies on resveratrol. In this study, using lyophilized red wine, we present evidence that its anticancer effect in a cellular model is mediated by apoptotic and autophagic cell death. Using a human osteosarcoma cell line, U2Os, we found that the lyophilized red wine was cytotoxic in a dose-dependent manner with a maximum effect in the range of 100-200 mu g/ml equivalents of gallic acid. A mixed phenotype of types I/II cell death was evidenced by means of specific assays following treatment of U2Os with lyophilized red wine, e.g., autophagy and apoptosis. We found that cell death induced by lyophilized red wine proceeded through a mechanism independent from its anti-oxidant activity and involving the inhibition of PI3K/Akt kinase signaling. Considering the relative low concentration of each single bioactive compound in lyophilized red wine, our study suggests the activation of synergistic mechanism able to inhibit growth in malignant cells.

The sulphonium compound S -adenosylmethionine (AdoMet) plays a central role in many metabolic reactions of cellular metabolism, acting both as a propylamine donor in the biosynthesis of polyamines as well as a methyl donor in the transmethylation reactions. Moreover, AdoMet is a key intermediate of the transsulphuration pathway by which methionine is converted into cysteine, a precursor of glutathione. The aim of this study was to investigate the methionine and AdoMet metabolism in bovine lenses cultured in the presence of labelled methionine, upon treatment with H 2 O 2 , as the experimental model for studying the molecular mechanisms responsible for the onset of senile cataract. The results reveal that one of the earliest changes following an oxidative stress is a severe impairment of protein synthesis. As far as the synthesis of AdoMet is concerned, a small but significant decrease in the conversion of labelled methionine into AdoMet occurs in treated lenses compared to the controls. In order to verify if the decreased AdoMet synthesis would lead in turn to alterations of methyl transfer reactions, we examined changes in the levels of various macromolecular methylations, such as protein methyl esterification and phospholipid methylation. The data clearly indicate that both the synthesis of AdoMet and the methyl transfer reactions could be significantly affected in eye lens upon an oxidative stress, suggesting that these alterations could be one of the biochemical events related to the ethiology of senile cataract. Finally, the question of whether or not H 2 O 2 -induced alterations of methionine and AdoMet metabolism could, in turn, affect some closely related metabolism, such as glutathione-associated reactions, is also discussed.

The proteasome is a multienzymatic complex that controls the half-life of the majority of intracellular proteins, including those involved in apoptosis and cell-cycle progression. Recently, proteasome inhibition has been shown to be an effective anticancer strategy, although its downregulation is often accompanied by severe undesired side effects. We previously reported that the inhibition of acylpeptide hydrolase (APEH) by the peptide SsCEI 4 can significantly affect the proteasome activity in A375 melanoma or Caco-2 adenocarcinoma cell lines, thus shedding new light on therapeutic strategies based on downstream regulation of proteasome functions. In this work, we investigated the functional correlation between APEH and proteasome in a panel of cancer cell lines, and evaluated the cell proliferation upon SsCEI 4-treatments. Results revealed that SsCEI 4 triggered a proliferative arrest specifically in osteosarcoma U2OS cells via downregulation of the APEH-proteasome system, with the accumulation of the typical hallmarks of proteasome: NF-kappaB, p21(Waf1), and polyubiquitinylated proteins. We found that the SsCEI 4 anti-proliferative effect involved a senescence-like growth arrest without noticeable cytotoxicity. These findings represent an important step toward understanding the mechanism(s) underlying the APEH-mediated downregulation of proteasome in order to design new molecules able to efficiently regulate the proteasome system for alternative therapeutic strategies.

We have recently reported on a class of IgE-binding peptides designed based on the crystallographic structure of the high affinity FceRI. Peptides contain receptor key residues located within the two distinct binding sites for IgE and selectively bind IgE with an affinity ranging between 6 and 60 µM. We have here designed and characterized a new molecule containing the receptor loops C′–E and B–C and an optimized linker for joining them made of a Lys side chain and a β-Ala. This new peptide shows an increased affinity (around 30 times) compared to the parent loop C′–E + B–C previously described, while retaining the same two-site mechanism of binding and the same selectivity. It also blocks the binding of IgE to the cell-anchored receptor and efficiently prevents histamine release from mast cells. These properties make the peptide a useful scaffold for the development of new anti-allergic drugs. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.