SERS nanostructures with engineered active peptides against an immune checkpoint protein.

The immune checkpoint programmed death ligand 1 (PD-L1) protein is expressed by tumor cells and it suppresses the killer activity of CD8+ T-lymphocyte cells binding to the programmed death 1 (PD-1) protein of these immune cells. Binding to either PD-L1 or PD1 is used for avoiding the inactivation of CD8+ T-lymphocyte cells. We report, for the first time, Au plasmonic nanostructures with surface-enhanced Raman scattering (SERS) properties (SERS nanostructures) and functionalized with an engineered peptide (CLP002: Trp-His-Arg-Ser-Tyr-Tyr-Thr-Trp-Asn-Leu-Asn-Thr), which targets PD-L1. Molecular dynamics calculations are used to describe the interaction of the targeting peptide with PD-L1 in the region where the interaction with PD-1 occurs, showing also the poor targeting activity of a peptide with the same amino acids, but a scrambled sequence. The results are confirmed experimentally since a very good targeting activity is observed against the MDA-MB-231 breast adenocarcinoma cancer cell line, which overexpresses PD-L1. A good activity is observed, in particular, for SERS nanostructures where the CLP002-engineered peptide is linked to the nanostructure surface with a short charged amino acid sequence and a long PEG chain. The results show that the functionalized SERS nanostructures show very good targeting of the immune checkpoint PD-L1.

Differences and similarities in the conceptualization of COVID-19 and other diseases in the first Italian lockdown.

Several studies have highlighted the flexible character of our conceptual system. However, less is known about the construction of meaning and the impact of novel concepts on the structuring of our conceptual space. We addressed these questions by collecting free listing data from Italian participants on a newly-and yet nowadays critical-introduced concept, i.e., COVID-19, during the first Italian lockdown. We also collected data for other five illness-related concepts. Our results show that COVID-19's representation is mostly couched in the emotional sphere, predominantly evoking fear-linked to both possible health-related concerns and social-emotional ones. In contrast with initial public debates we found that participants did not assimilate COVID-19 neither completely to severe illnesses (e.g., tumor) nor completely to mild illnesses (e.g., flu). Moreover, we also found that COVID-19 has shaped conceptual relations of other concepts in the illness domain, making certain features and associations more salient (e.g., flu-fear; disease-mask). Overall, our results show for the first time how a novel, real concept molds existing conceptual relations, testifying the malleability of our conceptual system.

Molecular Sponge: pH-Driven Reversible Squeezing of Stimuli-Sensitive Peptide Monolayers.

The cyclic change of structure, thickness, and density, with pH switching from acidic (pH = 3) to basic (pH = 11) condition, has been revealed for chemisorbed monolayers of the peptide Lipo-Aib-Lys-Leu-Aib-Lys-Lys-Leu-Aib-Lys-Ile-Lol, a trichogin GA IV-analogue carrying Lys residues instead of Gly ones at positions 2, 5, 6, and 9, while a homologous peptide not containing Lys residues does not show any response to pH changes. Experimental and theoretical results, obtained by means of quartz crystal microbalance with dissipation monitoring, surface plasmon resonance, nanoplasmonic sensing technique, Fourier transform infrared-reflection attenuated spectroscopy and dynamic force spectroscopy, and molecular dynamics simulations provide detailed information on the overall monolayer structure changes with pH, including the analysis of the intra- and interchain peptide dynamics, the structure of the peptide layer/water/solid interface, as well as the position and role of solvation and nonsolvation water. The observed stimuli-responsive behavior of L1 peptide monolayers is accounted in terms of the occurrence of a pH-induced wetting/dewetting process, due to the pH-induced switching of the hydrophilic character of charged lysine groups to hydrophobic one of the same uncharged groups, along the peptide chain. This behavior in turn promotes the collective change of the aggregation state of the peptide chains. The present results may pave the way to critically reexamine the mechanism of stimuli-responsive systems.

Understanding the good and poor cell targeting activity of gold nanostructures functionalized with molecular units for the epidermal growth factor receptor.

Nanostructures can strongly interact with cells or other biological structures; furthermore when they are functionalized with targeting units, they are of great interest for a variety of applications in the biotechnology field like those for efficient imaging, diagnosis and therapy and in particular for cancer theranostics. Obtaining targeting with good specificity and sensitivity is a key necessity, which, however, is affected by the complexity of the interactions between the nanostructures and the biological components. In this work we report the study of specificity and sensitivity of gold nanoparticles functionalized with the peptide GE11 for the targeting of the epidermal growth factor receptor, expressed on many cells and, in particular, on many types of cancer cells. We show how a combination of spectroscopic measurements and molecular dynamics simulations allows the comprehension of the targeting activity of peptides linked to the surface of gold nanostructures and how the targeting is tuned by the presence of polyethylene glycol chains.

Innovative chemical gels meet enzymes: A smart combination for cleaning paper artworks.

HYPOTHESIS: Due to their highly retentive properties, innovative recently developed, semi-interpenetrated hydrogels made up of poly(vinyl pyrrolidone) (PVP) chains embedded in a poly(2-hydroxyethyl methacrylate) (p(HEMA)) network should be efficiently used as cleaning material for fragile and degraded paper artworks. In restoration practice, indeed the wet cleaning of these artworks is usually performed by immersion of paper in water, a procedure which may lead to several drawbacks, including paper fibers swelling and dissolution of water-soluble original components. EXPERIMENTS: This class of gels were yet presented in literature, but their interactions with paper materials and ability to be spiked with active enzymes (as cleaning agents), have not been analyzed. To establish the suitability of these hydrogels as paper cleaning materials, first, a rheological and microstructural characterization of the gels was performed. Moreover, diffusion of macromolecules inside gels was studied using fluorescence microscopy, to check if these innovative hydrogels can be used as carriers for hydrolytic enzymes. Indeed, pastes and glues are usually found in old paper artworks, and their removal is a very delicate operation that requires a selective action, which is granted by specific hydrolytic enzymes. At the same time, spectroscopic analyses on paper samples under investigation before and after cleaning treatment has been performed, thus assessing the capabilty of these gels as cleaning materials. FINDINGS: With the aim of demonstrating the versatility of these hydrogels, several case studies, i.e., the removal of grime and water-soluble cellulose degradation byproducts, the removal of animal glue and the removal of starch paste from real samples, are presented. Results obtained with these gels have been compared to those obtained by using another gel used for paper artworks cleaning, i.e., Gellan gel.

Self-assembly of a model peptide incorporating a hexa-histidine sequence attached to an oligo-alanine sequence, and binding to gold NTA/nickel nanoparticles.

Amyloid fibrils are formed by a model surfactant-like peptide (Ala)10-(His)6 containing a hexa-histidine tag. This peptide undergoes a remarkable two-step self-assembly process with two distinct critical aggregation concentrations (cac's), probed by fluorescence techniques. A micromolar range cac is ascribed to the formation of prefibrillar structures, whereas a millimolar range cac is associated with the formation of well-defined but more compact fibrils. We examine the labeling of these model tagged amyloid fibrils using Ni-NTA functionalized gold nanoparticles (Nanogold). Successful labeling is demonstrated via electron microscopy imaging. The specificity of tagging does not disrupt the ß-sheet structure of the peptide fibrils. Binding of fibrils and Nanogold is found to influence the circular dichroism associated with the gold nanoparticle plasmon absorption band. These results highlight a new approach to the fabrication of functionalized amyloid fibrils and the creation of peptide/nanoparticle hybrid materials.

Photophysical properties of 1,3,5-tris(2-naphthyl)benzene and related less-arylated compounds: experimental and theoretical investigations.

Recently, a growing interest has concerned compounds characterized by high chemical and photophysical stability and high quantum yield for their possible technological applications. 1,3,5-Tris(2-naphthyl)benzene (N3B), 1,3-bis(2-naphthyl)benzene (N2B), and 2-naphthyl-benzene (N1B) are promising compounds, but they needed a detailed photophysical characterization. In this context, theoretical and experimental investigations have been carried out. Steady-state and decay time fluorescence measurements indicate that the second naphthyl group, added in the meta position of N1B, perturbs the electronic levels, whereas the further naphthyl addition, leading to N3B, does not promote changes in all of the observed properties. The investigated compounds show a biexponential fluorescence decay that has been attributed to a rearrangement involving the exited states S(1) and S(2). The minimum structure corresponding to the S(1) and S(2) states has been obtained at the configuration interaction with single excitations (CIS) level of theory. For the ground-state geometry, a conformational analysis at the Hartree-Fock level has also been carried out. We have evaluated the energy gaps between electronic levels by using Zerner's intermediate neglect of differential overlap (ZINDO) method. The species involved in the fluorescence have been experimentally characterized, and the decay-associated spectra have been obtained.

Effect of peptide lipidation on membrane perturbing activity: a comparative study on two trichogin analogues.

The effect of lipidation on the membrane perturbing activity of peptaibol antibiotics was investigated by performing a comparative study on two synthetic analogues of the natural peptide trichogin GA IV. Both analogues were labeled with a hydrophobic fluorescent probe, but one of them lacked the N-terminal n-octanoyl chain, present in the natural peptide. Spectroscopic studies show that the fatty acyl chain produces two opposite effects: it increases the affinity of the monomeric peptide for the membrane phase, but, at the same time, it favors peptide aggregation in water, thus inhibiting membrane binding by reducing the effective monomer concentration. In the membrane phase the two analogues exhibit the same aggregation and orientation behavior, indicating that the n-octanoyl chain plays no specific role in determining their orientation or membrane perturbing activity. Indeed, the dependence of peptide-induced membrane leakage on total peptide concentration is basically the same for the two analogues, because the aforementioned opposite effects, caused by peptide lipidation, tend to balance. These findings make questionable the use of lipidation as a general method for increasing the peptide membrane-perturbing activity, as its validity seems to be restricted to parent compounds of limited overall hydrophobicity.

Inclusion of a photosensitizer in liposomes formed by DMPC/gemini surfactant: correlation between physicochemical and biological features of the complexes.

Mixed cationic liposomes composed by different ratios of dimyristoyl-sn-glycero-phosphatidylcoline (DMPC) and a cationic gemini surfactant have been studied by various physicochemical tools as vehicles for m-tetrahydroxyphenylchlorin (m-THPC), a photosensitizer used in photodynamic therapy. Entrapment and location of m-THPC within the lipid double layer have been evaluated by different techniques and the new formulations have been tested on a stabilized cell line from a human colon tumor, COLO206. A correlation between the physicochemical features of formulations and their efficiency as photosensitizers vector was found.

Mechanism of membrane activity of the antibiotic trichogin GA IV: a two-state transition controlled by peptide concentration.

Synthetic fluorescent analogs of the natural lipopeptide trichogin GA IV were used to investigate the peptide position and orientation in model membranes. A translocation assay based on Forster energy transfer indicates that trichogin is associated to both the outer and inner leaflet of the membrane, even at low concentration, when it is not active. Fluorescence quenching measurements, performed by using water soluble quenchers and quenchers positioned in the membrane at different depths, indicate that at low membrane-bound peptide/lipid ratios trichogin lies close to the region of polar headgroups. By increasing peptide concentration until membrane leakage takes place, a cooperative transition occurs and a significant fraction of the peptide becomes deeply buried into the bilayer. Remarkably, this change in peptide position is strictly coupled with peptide aggregation. Therefore, the mechanism of trichogin action can be envisaged as based on a two-state transition controlled by peptide concentration. One state is the monomeric, surface bound and inactive peptide, and the other state is a buried, aggregated form, which is responsible for membrane leakage and bioactivity.

Aggregation and water-membrane partition as major determinants of the activity of the antibiotic peptide trichogin GA IV.

Water-membrane partition and aggregation behavior are fundamental aspects of the biological activity of antibiotic peptides, natural compounds causing the death of pathogenic organisms by perturbing the permeability of their membranes. A synthetic fluorescent analog of the natural lipopeptaibol trichogin GA IV was used to study its interaction with model membranes. Time-resolved fluorescence data show that in water, an equilibrium between monomers and small aggregates is present, the two species having different affinity for membranes. Therefore, association curves are strongly dependent on peptide concentration. A similar heterogeneity is present in the membrane phase, which strongly suggests the occurrence of a monomer-aggregate equilibrium in this case, too. The relative population of each species was determined and a strong correlation between the concentration of membrane-bound aggregates and membrane leakage was found, thereby suggesting that liposome perturbation is due to peptide aggregates only. Light-scattering measurements demonstrate that leakage is not due to liposome micellization. Moreover, experiments with markers of different sizes show that molecules with a diameter of approximately 4 nm are released only to a minor extent. Overall, these results suggest that, within the concentration range explored, pore formation by peptide aggregates is the most likely mechanism of action for trichogin in membranes.