Publications

Histones possess significant antimicrobial potential, yet their activity against biofilms remains underexplored. Moreover, concerns regarding adverse effects limit their clinical implementation. We investigated the antibacterial efficacy of human recombi nant histone H1 subtypes against Pseudomonas aeruginosa PAO1, both planktonic and in biofilms. After the in vitro tests, toxicity and efficacy were assessed in a P. aeruginosa PAO1 infection model using Galleria mellonella larvae. Histones were also evaluated in combination with ciprofloxacin (Cpx) and gentamicin (Gm). Our results demonstrate antimicrobial activity of all three histones against P. aeruginosa PAO1, with H1.0 and H1.4 showing efficacy at lower concentrations. The bactericidal effect was associated with a mechanism of membrane disruption. In vitro studies using static and dynamic models showed that H1.4 had antibiofilm potential by reducing cell biomass. Neither H1.0 nor H1.4 showed toxicity in G. mellonella larvae, and both increased larvae survival when infected with P. aeruginosa PAO1. Although in vitro synergism was observed between ciprofloxacin and H1.0, no improvement over the antibiotic alone was noted in vivo. Differences in antibacterial and antibiofilm activity were attributed to sequence and structural variations among histone subtypes. Moreover, the efficacy of H1.0 and H1.4 was influenced by the presence and strength of the extracellular matrix. These findings suggest histones hold promise for combating acute and chronic infections caused by pathogens such as P. aeruginosa.

JTD Keywords: Antimicrobial peptide, Antimicrobial peptides, Binding, Biofilm, Galleria mellonella, Host-defense, Peptid, Protein, Proteins, Toxicity, Treatment

Mesenchymal stem cells (MSCs) interact with their surroundings via integrins, which link to the actin cytoskeleton and translate physical cues into biochemical signals through mechanotransduction. N-cadherins enable cell-cell communication and are also linked to the cytoskeleton. This crosstalk between integrins and cadherins modulates MSC mechanotransduction and fate. Here we show the role of this crosstalk in the mechanosensing of viscosity using supported lipid bilayers as substrates of varying viscosity. We functionalize these lipid bilayers with adhesion peptides for integrins (RGD) and N-cadherins (HAVDI), to demonstrate that integrins and cadherins compete for the actin cytoskeleton, leading to an altered MSC mechanosensing response. This response is characterised by a weaker integrin adhesion to the environment when cadherin ligation occurs. We model this competition via a modified molecular clutch model, which drives the integrin/cadherin crosstalk in response to surface viscosity, ultimately controlling MSC lineage commitment. The crosstalk between cell-cell and cell-matrix adhesions regulates stem cell fate. Here, the authors reveal a critical role for matrix viscosity in controlling this crosstalk, which they explain via a modified molecular clutch model.

JTD Keywords: Actin cytoskeleton, Adhesion, Animals, Arginyl-glycyl-aspartic acid, Cadherins, Cell adhesion, Cell communication, Fibronectin, Force transmission, Humans, Hydrogel, Integrins, Lipid bilayers, Matrix, Mechanotransduction, Mechanotransduction, cellular, Mesenchymal stem cells, Mobility, Oligopeptides, Osteogenic differentiation, Substrate stiffness, Vinculin, Viscosity

Hydrogels with cell adhesive moieties stand out as promising materials to enhance tissue healing and regeneration. Nonetheless, bacterial infections of the implants represent an unmet major concern. In the present work, we developed an alginate hydrogel modified with a multifunctional peptide containing the RGD cell adhesive motif in combination with an antibacterial peptide derived from the 1-11 region of lactoferrin (LF). The RGD-LF branched peptide was successfully anchored to the alginate backbone by carbodiimide chemistry, as demonstrated by 1H NMR and fluorescence measurements. The functionalized hydrogel presented desirable physicochemical properties (porosity, swelling and rheological behavior) to develop biomaterials for tissue engineering. The viability of mesenchymal stem cells (MSCs) on the peptide-functionalized hydrogels was excellent, with values higher than 85 % at day 1, and higher than 95 % after 14 days in culture. Moreover, the biological characterization demonstrated the ability of the hydrogels to significantly enhance ALP activity of MSCs as well as to decrease bacterial colonization of both Gram-positive and Gram-negative models. Such results prove the potential of the functionalized hydrogels as novel biomaterials for tissue engineering, simultaneously displaying cell adhesive activity and the capacity to prevent bacterial contamination, a dual bioactivity commonly not found for these types of hydrogels. In this work we report on the functionalization of an alginate hydrogel with a tailor-made multifunctional peptide containing the cell adhesive RGD motif and the LF1-11 antibacterial peptide. Such novel multifunctional biomaterial ensures the viability of human mesenchymal stem cells, enhances ALP activity and decreases bacterial infections of both Gram-positive and Gram-negative models. image

JTD Keywords: Alginate hydrogel, Alginates, Anti-bacterial agents, Antimicrobial peptid, Antimicrobial peptide, Antimicrobial peptides, Arginyl-glycyl-aspartic acid, Biocompatible materials, Biofunctionalization, Bone, Cell adhesion, Cell survival, Composite hydrogels, Cross-linking, Hlf1-11 peptide, Human lactoferrin, Humans, Hydrogels, Immobilization, Mesenchymal stem cells, Multifunctional peptide, Oligopeptides, Peptides, Physical-properties, Scaffolds, Surfac, Tissue engineering

Conventional osteogenic platforms utilize active growth factors to repair bone defects that are extensive in size, but they can adversely affect patient health. Here, an unconventional osteogenic platform is reported that functions by promoting capture of inactive osteogenic growth factor molecules to the site of cell growth for subsequent integrin-mediated activation, using a recombinant fragment of latent transforming growth factor beta-binding protein-1 (rLTBP1). It is shown that rLTBP1 binds to the growth-factor- and integrin-binding domains of fibronectin on poly(ethyl acrylate) surfaces, which immobilizes rLTBP1 and promotes the binding of latency associated peptide (LAP), within which inactive transforming growth factor beta 1 (TGF-beta 1) is bound. rLTBP1 facilitates the interaction of LAP with integrin beta 1 and the subsequent mechanically driven release of TGF-beta 1 to stimulate canonical TGF-beta 1 signaling, activating osteogenic marker expression in vitro and complete regeneration of a critical-sized bone defect in vivo. An osteogenic platform that functions by capturing inactive growth factor molecules is engineered to overcome conventional challenges associated with the use of active growth factors. The platform triggers capture of inactive transforming growth factor beta-1 for its subsequent integrin-mediated activation which activates osteogenic downstream signaling in vitro and fully repairs critical-sized bone defect in vivo. image

JTD Keywords: Animals, Bone, Bone defect, Bone regeneration, Cell proliferation, Cells, Chemical activation, Defects, Differentiation, Fibronectin, Fibronectins, Growth factor, Growth factors, Humans, Integrin beta1, Integrins, Latency associated peptides, Latent tgf-beta binding proteins, Ltbp1, Osteogenesis, Osteogenic, Protein binding, Recombinant proteins, Release, Repair, Signal transduction, Surface properties, Tgf-beta, Tgf-β1, Transforming growth factor beta1, Transforming growth factors

A new generation of fully bioresorbable metallic Zn-based alloys could be used for stenting applications; however, the initial surface degradation delays stent re-endothelialization. Thus, this work proposes a dual strategy to control the corrosion and accelerate the endothelialization of ZnMg and ZnAg biodegradable alloys. First, a stable polycaprolactone (PCL) coating is obtained and followed by its functionalization with either linear RGD (Arg-Gly-Asp) or REDV (Arg-Glu-Asp-Val) peptides or a dual peptide-based platform combining both sequences (RGD-REDV). Scratching tests showed neither delamination nor detachment of the polymeric coating. Potentiodynamic polarization (PDP) and electrochemical impedance spectroscopy (EIS) measurements confirmed the corrosion resistance after PCL coating by revealing lower current density and higher absolute impedance values. X-ray photoelectron spectroscopy (XPS) and fluorescent microscopy confirmed the correct peptide immobilization onto PCL coated Zn alloys. The functionalized samples exhibited enhanced human umbilical vein endothelial cells (HUVEC) adhesion. The higher number of adhered cells to the functionalized surfaces with the RGD-REDV platform demonstrates the synergistic effect of combining both RGD and REDV sequences. Higher corrosion resistance together with enhanced endothelialization indicates that the dual functionalization of Zn alloys with PCL and peptide-based RGD-REDV platform holds great potential to overcome the clinical limitations of current biodegradable metal stents.

JTD Keywords: Binary alloys, Biodegradable metals, Bioresorbable, Cardiovascular applications, Cell adhesive peptides, Corrosion, Corrosion resistance, Corrosion resistant alloys, Corrosion resistant coatings, Degradation, Dual peptide-based platform, Electrochemical corrosion, Electrochemical impedance spectroscopy, Endothelial cells, Endothelialization, Functionalization, Functionalizations, In-vitro, Magnesium alloys, Metallics, Mg alloy, Peptides, Polycaprolactone coating, Polymer-coatings, Rgd-functionalization, Silver alloys, Stents, Surface, X ray photoelectron spectroscopy, Zinc, Zinc alloys, Zn alloys, Zn-based alloys

Current strategies to identify ligands for brain delivery select candidates based on preferential binding to cell-membrane components (CMC) on brain endothelial cells (EC). However, such strategies generate ligands with inherent brain specificity limitations, as the CMC (e.g., the transferrin receptor TfR1) are also significantly expressed on peripheral EC. Therefore, novel strategies are required to identify molecules allowing increased specificity of therapy brain delivery. Here, we demonstrate that, while individual CMC are shared between brain EC and peripheral EC, their endocytic internalization rate is markedly different. Such differential endocytic rate may be harnessed to identify molecular tags for brain targeting based on their selective retention on the surface of brain EC, thereby generating 'artificial' targets specifically on the brain vasculature. By quantifying the retention of labelled proteins on the cell membrane, we measured the general endocytic rate of primary brain EC to be less than half that of primary peripheral (liver and lung) EC. In addition, through bio-panning of phage-displayed peptide libraries, we unbiasedly probed the endocytic rate of individual CMC of liver, lung and brain endothelial cells. We identified phage-displayed peptides which bind to CMC common to all three endothelia phenotypes, but which are preferentially endocytosed into peripheral EC, resulting in selective retention on the surface of brain EC. Furthermore, we demonstrate that the synthesized free-form peptides are capable of generating artificial cell-surface targets for the intracellular delivery of model proteins into brain EC with increasing specificity over time. The developed identification paradigm, therefore, demonstrates that the lower endocytic rate of individual CMC on brain EC can be harnessed to identify peptides capable of generating 'artificial' targets for the selective delivery of proteins into the brain vasculature. In addition, our approach identifies brain-targeting peptides which would have been overlooked by conventional identification strategies, thereby increasing the repertoire of candidates to achieve specific therapy brain delivery.© 2023. The Author(s).

JTD Keywords: brain endothelium, endocytic rates, ligand identification, nanoparticles, phage-display, Barrier, Brain, Brain endothelium, Brain targeting, Endocytic rates, Endothelial cells, Endothelium, Ligand identification, Peptides, Phage-display

Clathrin-mediated endocytosis (CME) is an essential cellular process, conserved among eukaryotes. Yeast constitutes a powerful genetic model to dissect the complex endocytic machinery, yet there is a lack of specific pharmacological agents to interfere with CME in these organisms. TL2 is a light-regulated peptide inhibitor targeting the AP2-β-adaptin/β-arrestin interaction and that can photocontrol CME with high spatiotemporal precision in mammalian cells. Here, we study endocytic protein dynamics by live-cell imaging of the fluorescently tagged coat-associated protein Sla1-GFP, demonstrating that TL2 retains its inhibitory activity in S. cerevisiae spheroplasts. This is despite the β-adaptin/β-arrestin interaction not being conserved in yeast. Our data indicate that the AP2 α-adaptin is the functional target of activated TL2. We identified as interacting partners for the α-appendage, the Eps15 and epsin homologues Ede1 and Ent1. This demonstrates that endocytic cargo loading and sensing can be executed by conserved molecular interfaces, regardless of the proteins involved.© 2023 The Author(s).

JTD Keywords: adapters, alpha-appendage, azobenzene, cross-linker, mechanism, peptides, proteins, receptor, trafficking, Actin polymerization, Biochemistry, Biological sciences, Cell biology, Molecular biology, Natural sciences

The functionalization of 3D-printed poly-l-lactic acid (PLLA) and poly(l-lactic-co-ε-caprolactone) (PLCL) bioresorbable stents has been successfully achieved with linear RGDS and YIGSR peptides, as well as a dual platform containing both motifs within a single biomolecule.

JTD Keywords: adsorbed fibrinogen, chemistry, endothelialization, immobilization, platelets, plla, selectivity, surface, titanium, Absorbable implants, Cell adhesion, Endothelial cells, In-vitro hemocompatibility, Peptides, Polymers, Printing, three-dimensional, Stents, Tyrosyl-isoleucyl-glycyl-seryl-arginine

Mimicking bone extracellular matrix (ECM) is paramount to develop novel biomaterials for bone tissue engineering. In this regard, the combination of integrin-binding ligands together with osteogenic peptides represents a powerful approach to recapitulate the healing microenvironment of bone. In the present work, we designed polyethylene glycol (PEG)-based hydrogels functionalized with cell instructive multifunctional biomimetic peptides (either with cyclic RGD-DWIVA or cyclic RGD-cyclic DWIVA) and cross-linked with matrix metalloproteinases (MMPs)-degradable sequences to enable dynamic enzymatic biodegradation and cell spreading and differentiation. The analysis of the intrinsic properties of the hydrogel revealed relevant mechanical properties, porosity, swelling and degradability to engineer hydrogels for bone tissue engineering. Moreover, the engineered hydrogels were able to promote human mesenchymal stem cells (MSCs) spreading and significantly improve their osteogenic differentiation. Thus, these novel hydrogels could be a promising candidate for applications in bone tissue engineering, such as acellular systems to be implanted and regenerate bone or in stem cells therapy.Copyright © 2023 Oliver-Cervelló, Martin-Gómez, Gonzalez-Garcia, Salmeron-Sanchez, Ginebra and Mas-Moruno.

JTD Keywords: biomaterials, cross-linking, dwiva, functionalization, hydrogel, integrin, kinetics, marrow stromal cells, matrices, multifunctionality, myogenic differentiation, osteogenic differentiation, regeneration, stem-cells, Biomimetic peptides, Dwiva, Functionalization, Hydrogel, Multifunctionality, Osteogenic differentiation, Poly(ethylene glycol) hydrogels

CRENKA [Cys-Arg-(NMe)Glu-Lys-Ala, where (NMe)Glu refers to N-methyl-Glu], an anti-cancer pentapeptide that induces prostate tumor necrosis and significant reduction in tumor growth, was engineered to increase the resistance to endogenous proteases of its parent peptide, CREKA (Cys-Arg-Glu-Lys-Ala). Considering their high tendency to aggregate, the self-assembly of CRENKA and CREKA into well-defined and ordered structures has been examined as a function of peptide concentration and pH. Spectroscopic studies and atomistic molecular dynamics simulations reveal significant differences between the secondary structures of CREKA and CRENKA. Thus, the restrictions imposed by the (NMe)Glu residue reduce the conformational variability of CRENKA with respect to CREKA, which significantly affects the formation of well-defined and ordered self-assembly morphologies. Aggregates with poorly defined morphology are obtained from solutions with low and moderate CREKA concentrations at pH 4, whereas well-defined dendritic microstructures with fractal geometry are obtained from CRENKA solutions with similar peptide concentrations at pH 4 and 7. The formation of dendritic structures is proposed to follow a two-step mechanism: (1) pseudo-spherical particles are pre-nucleated through a diffusion-limited aggregation process, pre-defining the dendritic geometry, and (2) such pre-nucleated structures coalesce by incorporating conformationally restrained CRENKA molecules from the solution to their surfaces, forming a continuous dendritic structure. Instead, no regular assembly is obtained from solutions with high peptide concentrations, as their dynamics is dominated by strong repulsive peptide-peptide electrostatic interactions, and from solutions at pH 10, in which the total peptide charge is zero. Overall, results demonstrate that dendritic structures are only obtained when the molecular charge of CRENKA, which is controlled through the pH, favors kinetics over thermodynamics during the self-assembly process.

JTD Keywords: aggregation, amphiphilic peptides, breast-cancer, cells, design, oxidative stress, resistance, strategy, Molecular-dynamics

Cell-material interactions are regulated by mimicking bone extracellular matrix on the surface of biomaterials. In this regard, reproducing the extracellular conditions that promote integrin and growth factor (GF) signaling is a major goal to trigger bone regeneration. Thus, the use of synthetic osteogenic domains derived from bone morphogenetic protein 2 (BMP-2) is gaining increasing attention, as this strategy is devoid of the clinical risks associated with this molecule. In this work, the wrist and knuckle epitopes of BMP-2 are screened to identify peptides with potential osteogenic properties. The most active sequences (the DWIVA motif and its cyclic version) are combined with the cell adhesive RGD peptide (linear and cyclic variants), to produce tailor-made biomimetic peptides presenting the bioactive cues in a chemically and geometrically defined manner. Such multifunctional peptides are next used to functionalize titanium surfaces. Biological characterization with mesenchymal stem cells demonstrates the ability of the biointerfaces to synergistically enhance cell adhesion and osteogenic differentiation. Furthermore, in vivo studies in rat calvarial defects prove the capacity of the biomimetic coatings to improve new bone formation and reduce fibrous tissue thickness. These results highlight the potential of mimicking integrin-GF signaling with synthetic peptides, without the need for exogenous GFs.© 2022 The Authors. Advanced Healthcare Materials published by Wiley-VCH GmbH.

JTD Keywords: adhesion formation, bmp-2, cell adhesions, in-vivo, integrin, mesenchymal stem-cells, morphogenetic protein-2, multifunctionality, osteoblastic differentiation, osteogenic differentiation, rgd-dwiva, rgd-peptides, titanium biofunctionalization, titanium surfaces, Animals, Biocompatible materials, Biomimetic peptides, Bone morphogenetic protein 2, Bone regeneration, Cell adhesions, Cell differentiation, Epitopes, Extracellular matrix, Integrins, Marrow stromal cells, Multifunctionality, Osteogenesis, Osteogenic differentiation, Peptides, Rats, Rgd-dwiva, Titanium, Titanium biofunctionalization

Peptide derivatives and, most specifically, their self-assembled supramolecular structures are being considered in the design of novel biofunctional materials. Although the self-assembly of triphenylalanine homopeptides has been found to be more versatile than that of homopeptides containing an even number of residues (i.e. diphe-nylalanine and tetraphenylalanine), only uncapped triphenylalanine (FFF) and a highly aromatic analog blocked at both the N-and C-termini with fluorenyl-containing groups (Fmoc-FFF-OFm), have been deeply studied before. In this work, we have examined the self-assembly of a triphenylalanine derivative bearing 9-fluorenylme-thyloxycarbonyl and benzyl ester end-capping groups at the N-and C-termini, respectively (Fmoc-FFF-OBzl). The antiparallel arrangement clearly dominates in beta-sheets formed by Fmoc-FFF-OBzl, whereas the parallel and antiparallel dispositions are almost isoenergetic in Fmoc-FFF-OFm beta-sheets and the parallel one is slightly favored for FFF. The effects of both the peptide concentration and the mediu m on the self-assembly process have been examined considering Fmoc-FFF-OBzl solutions in a wide variety of solvent:co-solvent mixtures. In addi-tion, Fmoc-FFF-OBzl supramolecular structures have been compared to those obtained for FFF and Fmoc-FFF-OFm under identical experimental conditions. The strength of pi-pi stacking interactions involving the end-capping groups plays a crucial role in the nucleation and growth of supramolecular structures, which de-termines the resulting morphology. Finally, the influence of a non-invasive external stimulus, ultrasounds, on the nucleation and growth of supramolecular structures has been examined. Overall, FFF-based peptides provide a wide range of supramolecular structures that can be of interest in the biotechnological field.

JTD Keywords: aromatic interactions, beta-sheet, hierarchical structures, phenylalanine homopeptides, supramolecular structures, Amino-acids, Aromatic interactions, Beta-sheet, Fmoc, Hierarchical struc tures, Hydrogels, Phenylalanine homopeptides, Solvent, Spectroscopy, Supramolecular structures, Triphenylalanine

The increasing resistance of bacteria to existing antibiotics constitutes a major public health threat globally. Most current antibiotic treatments are hindered by poor delivery to the infection site, leading to undesired off-target effects and drug resistance development and spread. Here, we describe micro- and nanomotors that effectively and autonomously deliver antibiotic payloads to the target area. The active motion and antimicrobial activity of the silica-based robots are driven by catalysis of the enzyme urease and antimicrobial peptides, respectively. These antimicrobial motors show micromolar bactericidal activity in vitro against different Gram-positive and Gram-negative pathogenic bacterial strains and act by rapidly depolarizing their membrane. Finally, they demonstrated autonomous anti-infective efficacy in vivo in a clinically relevant abscess infection mouse model. In summary, our motors combine navigation, catalytic conversion, and bactericidal capacity to deliver antimicrobial payloads to specific infection sites. This technology represents a much-needed tool to direct therapeutics to their target to help combat drug-resistant infections.

JTD Keywords: antibiotic-resistance, antimicrobial peptides, autonomous treatment, bacterial infection, delivery, ll-37, nanomotors, nanoparticles, peptide, self-propulsion, tissue, vitro, wasp venom, Antibiotic-resistance, Antimicrobial peptides, Autonomous treatment, Bacterial infection, Delivery, Ll-37, Mesoporous silica nanoparticles, Nanomotors, Nanoparticles, Peptide, Self-propulsion, Tissue, Vitro, Wasp venom

Endothelial coverage of an exposed cardiovascular stent surface leads to the occurrence of restenosis and late-stent thrombosis several months after implantation. To overcome this difficulty, modification of stent surfaces with topographical or biochemical features may be performed to increase endothelial cells’ (ECs) adhesion and/or migration. This work combines both strategies on cobalt-chromium (CoCr) alloy and studies the potential synergistic effect of linear patterned surfaces that are obtained by direct laser interference patterning (DLIP), coupled with the use of Arg-Gly-Asp (RGD) and Tyr-Ile-Gly-Ser-Arg (YIGSR) peptides. An extensive characterization of the modified surfaces was performed by using AFM, XPS, surface charge, electrochemical analysis and fluorescent methods. The biological response was studied in terms of EC adhesion, migration and proliferation assays. CoCr surfaces were successfully patterned with a periodicity of 10 µm and two different depths, D (≈79 and 762 nm). RGD and YIGSR were immobilized on the surfaces by CPTES silanization. Early EC adhesion was increased on the peptide-functionalized surfaces, especially for YIGSR compared to RGD. High-depth patterns generated 80% of ECs’ alignment within the topographical lines and enhanced EC migration. It is noteworthy that the combined use of the two strategies synergistically accelerated the ECs’ migration and proliferation, proving the potential of this strategy to enhance stent endothelialization.

JTD Keywords: adhesion, bare-metal, biofunctionalization, biomaterials, cell adhesive peptides, cobalt-chromium alloy, direct laser interference patterning (dlip), endothelial cell migration, functionalization, matrix, proliferation, selectivity, shear-stress, surfaces, Biofunctionalization, Cell adhesive peptides, Cobalt-chromium alloy, Direct laser interference patterning (dlip), Drug-eluting stents, Endothelial cell migration

Unlike the attachment of soft epithelial skin tissue to penetrating solid natural structures like fingernails and teeth, sealing around percutaneous/permucosal devices such as dental implants is hindered by inflammation and epidermal down growth. Here, we employed a dual keratinocyte-adhesive peptide and anti-inflammatory biomolecule coating on titanium to promote oral epithelial tissue attachment. For minimizing inflammation-triggered epidermal down growth, we coated pristine and oxygen plasma pre-treated polished titanium (pTi) with conjugated linoleic acid (CLA). Further, in order to aid in soft tissue attachment via the formation of hemidesmosomes, adhesive structures by oral keratinocytes, we coated the anionic linoleic acid (LA) adsorbed titanium with cationic cell adhesive peptides (CAP), LamLG3, a peptide derived from Laminin 332, the major extracellular matrix component of the basement membrane in skin tissue and Net1, derived from Netrin-1, a neural chemoattractant capable of epithelial cell attachment via alpha 6 beta 4 integrins. The dual CLA-CAP coatings on pTi were characterized by X-ray photoelectron spectroscopy and dynamic water contact angle measurements. The proliferation of human oral keratinocytes (TERT-2/OKF6) was accelerated on the peptide coated titanium while also promoting the expression of Col XVII and beta-4 integrin, two markers for hemidesmosomes. Simultaneously, CLA coating suppressed the production of inducible nitric oxide synthase (anti-iNOS); a pro-inflammatory M1 marker expressed in lipopolysaccharide (LPS) stimulated murine macrophages (RAW 264.7) and elevated expression of anti-CD206, associated to an anti-inflammatory M2 macrophage phenotype. Taken together, the dual keratinocyte-adhesive peptide and anti-inflammatory biomolecule coating on titanium can help reduce inflammation and promote permucosal/peri-implant soft tissue sealing.

JTD Keywords: Adhesives, Animal, Animals, Anti-inflammatories, Anti-inflammatory agents, Antiinflammatory agent, Biomolecules, Bone, Cell adhesion, Cell-adhesives, Coatings, Conjugated linoleic acid, Conjugated linoleic-acid, Contact angle, Hemidesmosome, Hemidesmosomes, Human, Humans, Hydroxyapatite, Inflammation, Integrins, Keratinocyte, Keratinocytes, Linoleic acid, Macrophages, Mice, Mouse, Nitric oxide, Oral implants, Pathology, Peptides, Skin tissue, Soft tissue, Supplementation, Surface properties, Surface property, Tissue, Titania, Titanium, X ray photoelectron spectroscopy

Polypeptide-based nanoparticles offer unique advantages from a nanomedicine perspective such as biocompatibility, biodegradability, and stimuli-responsive properties to (patho)physiological conditions. Conventionally, self-assembled polypeptide nanostructures are prepared by first synthesizing their constituent amphiphilic polypeptides followed by postpolymerization self-assembly. Herein, we describe the one-pot synthesis of oxidation-sensitive supramolecular micelles and vesicles. This was achieved by polymerization-induced self-assembly (PISA) of the N-carboxyanhydride (NCA) precursor of methionine using poly(ethylene oxide) as a stabilizing and hydrophilic block in dimethyl sulfoxide (DMSO). By adjusting the hydrophobic block length and concentration, we obtained a range of morphologies from spherical to wormlike micelles, to vesicles. Remarkably, the secondary structure of polypeptides greatly influenced the final morphology of the assemblies. Surprisingly, wormlike micellar morphologies were obtained for a wide range of methionine block lengths and solid contents, with spherical micelles restricted to very short hydrophobic lengths. Wormlike micelles further assembled into oxidation-sensitive, self-standing gels in the reaction pot. Both vesicles and wormlike micelles obtained using this method demonstrated to degrade under controlled oxidant conditions, which would expand their biomedical applications such as in sustained drug release or as cellular scaffolds in tissue engineering.

JTD Keywords: alpha-amino-acid, hydrogels, leuchs anhydrides, platform, polypeptides, transformation, triggered cargo release, Amino acids, Amphiphilics, Biocompatibility, Biodegradability, Block lengths, Controlled drug delivery, Dimethyl sulfoxide, Ethylene, Gels, Hydrophobicity, Medical nanotechnology, Methionine, Micelles, Morphology, One-pot synthesis, Organic solvents, Oxidation, Physiological condition, Polyethylene oxides, Post-polymerization, Ring-opening polymerization, Scaffolds (biology), Self assembly, Stimuli-responsive properties, Supramolecular chemistry, Supramolecular gels, Supramolecular micelles, Wormlike micelle

Contact-based antimicrobials, as antibiotic-free technologies that use non-specific interactions with bacterial cells to exert antimicrobial activity, are a prospective solution in fighting the global issue of bacterial resistance. A very simplified approach to their design considers the direct bonding of cationic guanidine-containing amino acids to the surface of nano-gold carriers. The structure enables antimicrobial activity due to a high density of cationic surface charges. This opens a set of novel questions that are important for their effective engineering, particularly regarding (i) chemistry and events that take place at the interface between NPs and cells, (ii) the direct influence of a charge (and its change) on interactions with bacterial and mammalian cells, and (iii) the stability of structures (and their antimicrobial activity) in the presence of enzymes, which are addressed in this paper. Because of the ability of amino acid-functionalized nano-gold to retain structural and functional activity, even after exposure to a range of physicochemical stimuli, they provide an excellent nanotechnological platform for designing highly effective contact-based antimicrobials and their applications.

JTD Keywords: agents, antibiotic-free technology, arginine, charged amino acids, contact-based antimicrobials, discovery, enzyme-resistant antimicrobials, functionalized gold, peptides, polymers, resistant, Antibiotic-free technology, Charged amino acids, Contact-based antimicrobials, Enzyme-resistant antimicrobials, Functionalized gold, Nanoparticles

Cell response to force regulates essential processes in health and disease. However, the fundamental mechanical variables that cells sense and respond to remain unclear. Here we show that the rate of force application (loading rate) drives mechanosensing, as predicted by a molecular clutch model. By applying dynamic force regimes to cells through substrate stretching, optical tweezers, and atomic force microscopy, we find that increasing loading rates trigger talin-dependent mechanosensing, leading to adhesion growth and reinforcement, and YAP nuclear localization. However, above a given threshold the actin cytoskeleton softens, decreasing loading rates and preventing reinforcement. By stretching rat lungs in vivo, we show that a similar phenomenon may occur. Our results show that cell sensing of external forces and of passive mechanical parameters (like tissue stiffness) can be understood through the same mechanisms, driven by the properties under force of the mechanosensing molecules involved. Cells sense mechanical forces from their environment, but the precise mechanical variable sensed by cells is unclear. Here, the authors show that cells can sense the rate of force application, known as the loading rate, with effects on YAP nuclear localization and cytoskeletal stiffness remodelling.

JTD Keywords: Actin cytoskeleton, Actin filament, Actin-filament, Adhesion, Animal, Animals, Atomic force microscopy, Breathing, Cell, Cell adhesion, Cell culture, Cell nucleus, Cells, cultured, Cytoplasm, Extracellular-matrix, Fibroblast, Fibroblasts, Fibronectin, Frequency, Gene knockdown, Gene knockdown techniques, Genetics, Germfree animal, Integrin, Intracellular signaling peptides and proteins, Knockout mouse, Lung, Male, Mechanotransduction, Mechanotransduction, cellular, Metabolism, Mice, Mice, knockout, Microscopy, atomic force, Mouse, Optical tweezers, Paxillin, Physiology, Primary cell culture, Pxn protein, mouse, Rat, Rats, Rats, sprague-dawley, Respiration, Signal peptide, Softening, Specific pathogen-free organisms, Sprague dawley rat, Stress, Substrate, Substrate rigidity, Talin, Talin protein, mouse, Tln2 protein, mouse, Traction, Transmission, Ultrastructure, Yap1 protein, rat

A dual approach employing peptidic biofunctionalization and laser micro-patterns on dental zirconia was explored, with the aim of providing a flexible tool to improve tissue integration of restorations. Direct laser interference patterning with a femtosecond Ti:Sapphire laser was employed, and two periodic grooved patterns were produced with a periodicity of 3 and 10 μm. A platform containing the cell-adhesive RGD and the osteogenic DWIVA peptides was used to functionalize the grooved surfaces. Topography and surface damage were characterized by confocal laser scanning (CLSM), scanning electron and scanning transmission electron microscopy techniques. The surface patterns exhibited a high homogeneity and subsurface damage was found in the form of nano-cracks and nano-pores, at the bottom of the valleys. Accelerated tests in water steam were carried out to assess hydrothermal degradation resistance, which slightly decreased after the laser treatment. Interestingly, the detrimental effects of the laser modification were reverted by a post-laser thermal treatment. The attachment of the molecule was verified trough fluorescence CLSM and X-ray photoelectron spectroscopy. Finally, the biological properties of the surfaces were studied in human mesenchymal stem cells. Cell adhesion, morphology, migration and differentiation were investigated. Cells on grooved surfaces displayed an elongated morphology and aligned along the patterns. On these surfaces, migration was greatly enhanced along the grooves, but also highly restricted in the perpendicular direction as compared to flat specimens. After biofunctionalization, cell number and cell area increased and well-developed cell cytoskeletons were observed. However, no effects on cell migration were found for the peptidic platform. Although some osteogenic potential was found in specimens grooved with a periodicity of 10 μm, the largest effects were observed from the biomolecule, which favored upregulation of several genes related to osteoblastic differentiation in all the surfaces.

JTD Keywords: alumina toughened zirconia, cell alignment, grain-size, implants, interference, laser patterning, osteogenic differentiation, osteointegration, peptides, surface functionalization, surface-topography, tissue, titanium surface, Laser patterning, Low-temperature degradation, Osteointegration, Peptides, Surface functionalization, Zirconia

© 2020 Wiley-VCH GmbH Recreating the healing microenvironment is essential to regulate cell–material interactions and ensure the integration of biomaterials. To repair bone, such bioactivity can be achieved by mimicking its extracellular matrix (ECM) and by stimulating integrin and growth factor (GF) signaling. However, current approaches relying on the use of GFs, such as bone morphogenetic protein 2 (BMP-2), entail clinical risks. Here, a biomimetic peptide integrating the RGD cell adhesive sequence and the osteogenic DWIVA motif derived from the wrist epitope of BMP-2 is presented. The approach offers the advantage of having a spatial control over the single binding of integrins and BMP receptors. Such multifunctional platform is designed to incorporate 3,4-dihydroxyphenylalanine to bind metallic oxides with high affinity in a one step process. Functionalization of glass substrates with the engineered peptide is characterized by physicochemical methods, proving a successful surface modification. The biomimetic interfaces significantly improve the adhesion of C2C12 cells, inhibit myotube formation, and activate the BMP-dependent signaling via p38. These effects are not observed on surfaces displaying only one bioactive motif, a mixture of both motifs or soluble DWIVA. These data prove the biological potential of recreating the ECM and engaging in integrin and GF crosstalk via molecular-based mimics.

JTD Keywords: binding, biomaterials, biomimetic peptides, bone, cell adhesion, cell differentiation, differentiation, dwiva, multifunctional coatings, osseointegration, osteoblasts, rgd, surface, surface functionalization, Biomimetic peptides, Biomimetics, Cell adhesion, Cell differentiation, Dwiva, Integrins, Intercellular signaling peptides and proteins, Matrix-bound bmp-2, Peptides, Rgd, Surface functionalization

© 2020 The Authors. Advanced Science published by Wiley-VCH GmbH The establishment of tumor microenvironment using biomimetic in vitro models that recapitulate key tumor hallmarks including the tumor supporting extracellular matrix (ECM) is in high demand for accelerating the discovery and preclinical validation of more effective anticancer therapeutics. To date, ECM-mimetic hydrogels have been widely explored for 3D in vitro disease modeling owing to their bioactive properties that can be further adapted to the biochemical and biophysical properties of native tumors. Gathering on this momentum, herein the current landscape of intrinsically bioactive protein and peptide hydrogels that have been employed for 3D tumor modeling are discussed. Initially, the importance of recreating such microenvironment and the main considerations for generating ECM-mimetic 3D hydrogel in vitro tumor models are showcased. A comprehensive discussion focusing protein, peptide, or hybrid ECM-mimetic platforms employed for modeling cancer cells/stroma cross-talk and for the preclinical evaluation of candidate anticancer therapies is also provided. Further development of tumor-tunable, proteinaceous or peptide 3D microtesting platforms with microenvironment-specific biophysical and biomolecular cues will contribute to better mimic the in vivo scenario, and improve the predictability of preclinical screening of generalized or personalized therapeutics.

JTD Keywords: 3d in vitro models, cancers, hydrogels, peptides, 3d in vitro models, Cancers, Hydrogels, Peptides, Proteins

© 2021 by the authors. Licensee MDPI, Basel, Switzerland. Achieving intracellular delivery of protein therapeutics within cells remains a significant challenge. Although custom formulations are available for some protein therapeutics, the development of non‐toxic delivery systems that can incorporate a variety of active protein cargo and maintain their stability, is a topic of great relevance. This study utilized ionic polyphosphazenes (PZ) that can assemble into supramolecular complexes through non‐covalent interactions with different types of protein cargo. We tested a PEGylated graft copolymer (PZ‐PEG) and a pyrrolidone containing linear derivative (PZ‐PYR) for their ability to intracellularly deliver FITC‐avidin, a model protein. In endothelial cells, PZ‐PYR/protein exhibited both faster internalization and higher uptake levels than PZ‐PEG/protein, while in cancer cells both polymers achieved similar uptake levels over time, although the internalization rate was slower for PZ‐PYR/protein. Uptake was mediated by endocytosis through multiple mechanisms, PZ‐PEG/avidin colocalized more profusely with endo-lysosomes, and PZ‐PYR/avidin achieved greater cytosolic delivery. Consequently, a PZ‐PYR-delivered anti‐F‐actin antibody was able to bind to cytosolic actin filaments without needing cell permeabilization. Similarly, a cell‐impermeable Bax‐BH3 peptide known to induce apoptosis, decreased cell viability when complexed with PZ‐PYR, demonstrating endo‐lysosomal escape. These biodegradable PZs were non‐toxic to cells and represent a promising platform for drug delivery of protein therapeutics.

JTD Keywords: cytosolic delivery, cytotoxicity, delivery of apoptotic peptides, endosomal escape, intracellular delivery of antibody, intracellular protein delivery, Cytosolic delivery, Cytotoxicity, Delivery of apoptotic peptides, Endosomal escape, Intracellular delivery of antibody, Intracellular protein delivery, Polyphosphazene polymers

Plaques of the amyloid beta (A beta) peptide are a pathological hallmark of Alzheimer's disease (AD), the most common form of dementia. Mutations in A beta also cause familial forms of AD (fAD). Here, we use deep mutational scanning to quantify the effects of >14,000 mutations on the aggregation of A beta. The resulting genetic landscape reveals mechanistic insights into fibril nucleation, including the importance of charge and gatekeeper residues in the disordered region outside of the amyloid core in preventing nucleation. Strikingly, unlike computational predictors and previous measurements, the empirical nucleation scores accurately identify all known dominant fAD mutations in A beta, genetically validating that the mechanism of nucleation in a cell-based assay is likely to be very similar to the mechanism that causes the human disease. These results provide the first comprehensive atlas of how mutations alter the formation of any amyloid fibril and a resource for the interpretation of genetic variation in A beta.

JTD Keywords: aggregation, kinetics, oligomers, onset, rates, state, Aggregation, Alzheimer disease, Alzheimer's, Amyloid, Amyloid beta-peptides, Computational biology, Deep mutagenesis, Dna mutational analysis, Genetics, Genomics, High-throughput nucleotide sequencing, Kinetics, Mutation, Nucleation, Oligomers, Onset, Plasmids, Precursor protein, Rates, S. cerevisiae, Saccharomyces cerevisiae, State, Systems biology

© The Royal Society of Chemistry 2021. A novel and versatile toolkit approach for the functionalization of biomaterials of different nature is described. This methodology is based on the solid-phase conjugation of specific anchoring units onto a resin-bound azido-functionalized peptide by using click chemistry. A synergistic multifunctional peptidic scaffold with cell adhesive properties was used as a model compound to showcase the versatility of this new approach. Titanium, gold and polylactic acid surfaces were biofunctionalized by this method, as validated by physicochemical surface characterization with XPS.In vitroassays using mesenchymal stem cells showed enhanced cell adhesion on the functionalized samples, proving the capacity of this strategy to efficiently bioactivate different types of biomaterials.

JTD Keywords: Biocompatible materials, Click chemistry, Peptides, Protein conformation

The targeted delivery of therapeutic compounds to the brain is arguably the most significant open problem in drug delivery today. Nanoparticles (NPs) based on peptides and designed using the emerging principles of molecular engineering show enormous promise in overcoming many of the barriers to brain delivery faced by NPs made of more traditional materials. However, shortcomings in our understanding of peptide self-assembly and blood–brain barrier (BBB) transport mechanisms pose significant obstacles to progress in this area. In this review, we discuss recent work in engineering peptide nanocarriers for the delivery of therapeutic compounds to the brain: from synthesis, to self-assembly, to in vivo studies, as well as discussing in detail the biological hurdles that a nanoparticle must overcome to reach the brain.

JTD Keywords: Alzheimer's disease, Blood-brain barrier, Drug delivery, Glioma, Parkinson's disease, Peptides, Self-assembly, Transcytosis

The introduction of 3D bioprinting to fabricate living constructs with tailored architecture has provided a new paradigm for biofabrication, with the potential to overcome several drawbacks of conventional scaffold-based tissue regeneration strategies. Hydrogel-based materials are suitable candidates regarding cell biocompatibility but often display poor mechanical properties. Self-assembling peptides are a promising source of biomaterials to be used as 3D scaffolds based on their similarity to extracellular matrices (structurally and mechanically). In this study, an advanced bioink for biofabrication is presented based on the optimization of a RAD16-I-based biomaterial. The strategy followed to build 3D predefined structures by 3D printing is based on an enhancement of bioink viscosity by adding methylcellulose (MC) to a RAD16-I solution. The resultant constructs display high shape fidelity and stability and embedded human mesenchymal stem cells present high viability after 7 days of culture. Moreover, cells are also able to differentiate to the adipogenic lineage, suggesting the suitability of this novel biomaterial for soft tissue engineering applications.

JTD Keywords: 3D bioprinting, Biofabrication, Bioinks, Self-assembling peptides, Tissue engineering

Strategies to inhibit initial bacterial adhesion are extremely important to prevent infection on biomaterial surfaces. However, the simultaneous attraction of desired eukaryotic cells remains a challenge for successful biomaterial-host tissue integration. Here we describe a method for the development of a trifunctional coating that repels contaminating bacteria, kills those that adhere, and promotes osteoblast adhesion. To this end, titanium surfaces were functionalized by electrodeposition of an antifouling polyethylene glycol (PEG) layer and subsequent binding of a peptidic platform with cell-adhesive and bactericidal properties. The physicochemical characterization of the samples via SEM, contact angle, FTIR and XPS analysis verified the successful binding of the PEG layer and the biomolecules, without altering the morphology and topography of the samples. PEG coatings inhibited protein adsorption and osteoblast-like (SaOS-2) attachment; however, the presence of cell adhesive domains rescued osteoblast adhesion, yielding higher values of cell attachment and spreading compared to controls (p < 0.05). Finally, the antibacterial potential of the coating was measured by live/dead assays and SEM using S. sanguinis as a model of early colonizer in oral biofilms. The presence of PEG layers significantly reduced bacterial attachment on the surfaces (p < 0.05). This antibacterial potential was further increased by the bactericidal peptide, yielding values of bacterial adhesion below 0.2% (p < 0.05). The balance between the risk of infection and the optimal osteointegration of a biomaterial is often described as “the race for the surface”, in which contaminating bacteria and host tissue cells compete to colonize the implant. In the present work, we have developed a multifunctional coating for a titanium surface that promotes the attachment and spreading of osteoblasts, while very efficiently inhibits bacterial colonization, thus holding promise for application in bone replacing applications.

JTD Keywords: Polyethylene glycol, Antibacterial, Osteointegration, Multifunctionality, Peptides, Titanium

The rational design of nanomedicines is a challenging task given the complex architectures required for the construction of nanosized carriers with embedded therapeutic properties and the complex interface of these materials with the biological environment. Herein, an unexpected charge-like attraction mechanism of self-assembly for star-shaped polyglutamates in nonsalty aqueous solutions is identified, which matches the ubiquitous “ordinary–extraordinary” phenomenon previously described by physicists. For the first time, a bottom-up methodology for the stabilization of these nanosized soft-assembled star-shaped polyglutamates is also described, enabling the translation of theoretical research into nanomaterials with applicability within the drug-delivery field. Covalent capture of these labile assemblies provides access to unprecedented architectures to be used as nanocarriers. The enhanced in vitro and in vivo properties of these novel nanoconstructs as drug-delivery systems highlight the potential of this approach for tumor-localized as well as lymphotropic delivery.

JTD Keywords: Charge-like, Drug delivery, Polymer therapeutics, Polypeptides, Self-assembly

The electronic spin filtering capability of a single chiral helical peptide is measured. A ferromagnetic electrode source is employed to inject spin-polarized electrons in an asymmetric single-molecule junction bridging an α-helical peptide sequence of known chirality. The conductance comparison between both isomers allows the direct determination of the polarization power of an individual chiral molecule.

JTD Keywords: Alpha-helical peptides, Chiral transport, Single-molecule wires, Spin-polarization power, Spin-polarized transmission

In dentistry and orthopedics, it is well accepted that implant fixation is a major goal. However, an emerging concern is bacterial infection. Infection of metallic implants can be catastrophic and significantly reduce patient quality of life. Accordingly, in this work, we focus on multifunctional coatings to simultaneously address and mitigate both these problems. We have developed a tailor-made peptide-based chemical platform that integrates the well-known RGD cell adhesive sequence and the lactoferrin-derived LF1-11 antimicrobial peptide. The platform was covalently grafted on titanium via silanization and the functionalization process characterized by contact angle, XPS, and QCM-D. The presence of the platform statistically improved the adhesion, proliferation and mineralization of osteoblast-like cells compared to control surfaces. At the same time, colonization by representative bacterial strains was significantly reduced on the surfaces. Furthermore, the biological potency of the multifunctional platform was verified in a co-culture in vitro model. Our findings demonstrate that this multifunctional approach can be useful to functionalize biomaterials to both improve cell integration and reduce the risk of bacterial infection.

JTD Keywords: Antimicrobial peptides, Cell adhesive peptides, Multifunctionality, Osseointegration, Surface functionalization

Biomimetic surface modification with peptides that have specific cell-binding moieties is a promising approach to improve endothelialization of metal-based stents. In this study, we functionalized CoCr surfaces with RGDS, REDV, YIGSR peptides and their combinations to promote endothelial cells (ECs) adhesion and proliferation. An extensive characterization of the functionalized surfaces was performed by XPS analysis, surface charge and quartz crystal microbalance with dissipation monitoring (QCM-D), which demonstrated the successful immobilization of the peptides to the surface. Cell studies demonstrated that the covalent functionalization of CoCr surfaces with an equimolar combination of RGDS and YIGSR represents the most powerful strategy to enhance the early stages of ECs adhesion and proliferation, indicating a positive synergistic effect between the two peptide motifs. Although these peptide sequences slightly increased smooth muscle cells (SMCs) adhesion, these values were ten times lower than those observed for ECs. The combination of RGDS with the REDV sequence did not show synergistic effects in promoting the adhesion or proliferation of ECs. The strategy presented in this study holds great potential to overcome clinical limitations of current metal stents by enhancing their capacity to support surface endothelialization.

JTD Keywords: Cell adhesive peptides, CoCr alloy, Endothelialization, HUVEC proliferation, SMCs adhesion, Surface functionalization

Surface modification stands out as a versatile technique to create instructive biomaterials that are able to actively direct stem cell fate. Chemical functionalization of titanium has been used in this work to stimulate the differentiation of human mesenchymal stem cells (hMSCs) into the osteoblastic lineage, by covalently anchoring a synthetic double-branched molecule (PTF) to the metal that allows a finely controlled presentation of peptidic motifs. In detail, the effect of the RGD adhesive peptide and its synergy motif PHSRN is studied, comparing a random distribution of the two peptides with the chemically-tailored disposition within the custom made synthetic platform, which mimics the interspacing between the motifs observed in fibronectin. Contact angle measurement and XPS analysis are used to prove the efficiency of functionalization. We demonstrate that, by rationally designing ligands, stem cell response can be efficiently guided towards the osteogenic phenotype: In vitro, PTF-functionalized surfaces support hMSCs adhesion, with higher cell area and formation of focal contacts, expression of the integrin receptor α5β1 and the osteogenic marker Runx2, and deposition a highly mineralized matrix, reaching values of mineralization comparable to fibronectin. Our strategy is also demonstrated to be efficient in promoting new bone growth in vivo in a rat calvarial defect. These results highlight the efficacy of chemical control over the presentation of bioactive peptides; such systems may be used to engineer bioactive surfaces with improved osseointegrative properties, or can be easily tuned to generate multi-functional coatings requiring a tailored disposition of the peptidic motifs. Statement of significance Organic coatings have been proposed as a solution to foster osseointegration of orthopedic implants. Among them, extracellular matrix-derived peptide motifs are an interesting biomimetic strategy to harness cell-surface interactions. Nonetheless, the combination of multiple peptide motifs in a controlled manner is essential to achieve receptor specificity and fully exploit the potentiality of synthetic peptides. Herein, we covalently graft to titanium a double branched molecule to guide stem cell fate in vitro and generate an osseoinductive titanium surface in vivo. Such synthetic ligand allows for the simultaneous presentation of two bioactive motifs, thus is ideal to test the effect of synergic sequences, such as RGD and PHSRN, and is a clear example of the versatility and feasibility of rationally designed biomolecules.

JTD Keywords: hMSCs, Integrin-binding peptides, Osseointegration, RGD-PHSRN, Titanium

Lung disease models are useful to study how cell engraftment, proliferation and differentiation are modulated in lung bioengineering. The aim of this work was to characterize the local stiffness of decellularized lungs in aged and fibrotic mice. Mice (2- and 24-month old; 14 of each) with lung fibrosis (N=20) and healthy controls (N=8) were euthanized after 11 days of intratracheal bleomycin (fibrosis) or saline (controls) infusion. The lungs were excised, decellularized by a conventional detergent-based (sodium-dodecyl sulfate) procedure and slices of the acellular lungs were prepared to measure the local stiffness by means of atomic force microscopy. The local stiffness of the different sites in acellular fibrotic lungs was very inhomogeneous within the lung and increased according to the degree of the structural fibrotic lesion. Local stiffness of the acellular lungs did not show statistically significant differences caused by age. The group of mice most affected by fibrosis exhibited local stiffness that were ~2-fold higher than in the control mice: from 27.2±1.64 to 64.8±7.1. kPa in the alveolar septa, from 56.6±4.6 to 99.9±11.7. kPa in the visceral pleura, from 41.1±8.0 to 105.2±13.6. kPa in the tunica adventitia, and from 79.3±7.2 to 146.6±28.8. kPa in the tunica intima. Since acellular lungs from mice with bleomycin-induced fibrosis present considerable micromechanical inhomogeneity, this model can be a useful tool to better investigate how different degrees of extracellular matrix lesion modulate cell fate in the process of organ bioengineering from decellularized lungs.

JTD Keywords: Ageing, Atomic force microscopy, Decellularization, Lung fibrosis, Tissue engineering, Atomic force microscopy, Biological organs, Peptides, Sodium dodecyl sulfate, Sodium sulfate, Tissue engineering, Ageing, Decellularization, Extracellular matrices, Healthy controls, Inhomogeneities, Lung fibrosis, Micro-mechanical, Statistically significant difference, Mammals, bleomycin, adventitia, animal experiment, animal model, article, atomic force microscopy, bleomycin-induced pulmonary fibrosis, cell fate, controlled study, extracellular matrix, female, intima, lung alveolus, lung fibrosis, lung mechanics, mechanical probe, microenvironment, mouse, nonhuman, pleura, priority journal, rigidity, tissue engineering

Control of membrane traffic: Photoswitchable inhibitors of protein-protein interactions were applied to photoregulate clathrin-mediated endocytosis (CME) in living cells. Traffic light (TL) peptides acting as "stop" and "go" signals for membrane traffic can be used to dissect the role of CME in receptor internalization and in cell growth, division, and differentiation.

JTD Keywords: Clathrin-mediated endocytosis, Optopharmacology, Peptides, Photoswitches, Protein-protein interactions

The self-assembly of peptides and proteins into amyloid fibrils of nanometric thickness and up to several micrometres in length, a phenomenon widely observed in biological systems, has recently aroused a growing interest in nanotechnology and nanomedicine. Here we have applied atomic force microscopy and single molecule force spectroscopy to study the amyloidogenesis of a peptide derived from human amylin and of its reverse sequence. The spontaneous formation of protofibrils and their orientation along well-defined directions on graphite and DMSO-coated graphite substrates make the studied peptides interesting candidates for nanotechnological applications. The measured binding forces between peptides correlate with the number of hydrogen bonds between individual peptides inside the fibril structure according to molecular dynamics simulations.

JTD Keywords: Amyloid fibril, Amyloidogenesis, Binding forces, Fibril structure, Graphite substrate, Molecular dynamics simulations, Nanometrics, Protofibrils, Single molecule force spectroscopy, Spontaneous formation, Atomic force microscopy, Atomic spectroscopy, Graphite, Hydrogen bonds, Medical nanotechnology, Molecular dynamics, Molecular physics, Self assembly, Thickness measurement, Peptides

Five peptide sequences corresponding to the E1 protein of GBV-C [NCCAPEDIGFCLEGGCLV (P7), APEDIGFCLEGGCLVALG (P8), FCLEGGCLVALGCTICTD (P10), QAGLAVRPGKSAAQLVGE (P18), and AQLVGELGSLYGPLSVSA (P22)] were synthesized because they were capable of interfering with the HIV-1 fusion peptide (HIV-1 FP)-vesicle interaction. In this work the interaction of these peptides with the HIV-1 FP, as well as with membrane models, was analyzed to corroborate their inhibition ability and to understand if the interaction with the fusion peptide takes place in solution or at the membrane level. Several studies were carried out on aggregation and membrane fusion, surface Plasmon resonance, and conformational analysis by circular dichroism. Moreover, in vitro toxicity assays, including cytotoxicity studies in 3T3 fibroblasts and hemolysis assays in human red blood cells, were performed to evaluate if these peptides could be potentially used in anti-HIV-1 therapy. Results show that P10 is not capable of inhibiting membrane fusion caused by HIV-1 and it aggregates liposomes and fuses membranes, thus we decided to discard it for futures studies. P18 and P22 do not inhibit membrane fusion, but they inhibit the ability of HIV-1 FP to form pores in bilayers, thus we have not discarded them yet. P7 and P8 were selected as the best candidates for future studies because they are capable of inhibiting membrane fusion and the interaction of HIV-1 FP with bilayers. Therefore, these peptides could be potentially used in future anti-HIV-1 research. Part of the gang: Liposomes are deposited on a surface plasmon resonance chip (see AFM image of the chip) to observe the interaction of peptides corresponding to the E1 envelop protein of the hepatitis G virus with membranes to show how they reduce the interaction of the HIV-1 fusion peptide.

JTD Keywords: HIV-1 fusion protein, Liposomes, Membranes, Peptides, Viruses

Two Cys-containing analogues of the anticancer drug Kahalalide F are synthesized and conjugated to 20 and 40 nm gold nanoparticles (GNPs). The resulting complexes are characterized by different analytical techniques to confirm the attachment of peptide to the GNPs. The self-assembly capacity of a peptide dramatically influences the final ratio number of molecules per nanoparticle, saturating the nanoparticle surface and prompting multilayered capping on the surface. In such way, the nanoparticle could act as a concentrator for the delivery of drugs, thereby increasing bioactivity. The GNP sizes and the conjugation have influence on the biological activities. Kahalalide F analogues conjugated with GNPs are located subcellularly at lysosome-like bodies, which may be related to the action mechanism of Kahalalide F. The results suggest that the selective delivery and activity of Kahalalide F analogues can be improved by conjugating the peptides to GNPs.

JTD Keywords: Electrical detection, Cellular uptake, Drug-delivery, Cancer-cells, Peptide, Size, Surface, Absorption, Scattering, Therapy