Factors influencing initial bacterial adhesion to antifouling surfaces studied by single-cell force spectroscopy.

Biofilm formation, a major concern for healthcare systems, is initiated when bacteria adhere to surfaces. Escherichia coli adhesion is mediated by appendages, including type-1 fimbriae and curli amyloid fibers. Antifouling surfaces prevent the adhesion of bacteria to combat biofilm formation. Here, we used single-cell force-spectroscopy to study the interaction between E. coli and glass or two antifouling surfaces: the tripeptide DOPA-Phe(4F)-Phe(4F)-OMe and poly(ethylene glycol) polymer-brush. Our results indicate that both antifoulants significantly deter E. coli initial adhesion. By using two mutant strains expressing no type-1 fimbriae or curli amyloids, we studied the adhesion mechanism. Our results suggest that the bacteria adhere to different antifoulants via separate mechanisms. Finally, we show that some bacteria adhere much better than others, illustrating how the variability of bacterial cultures affects biofilm formation. Our results emphasize how additional study at the single-cell level can enhance our understanding of bacterial adhesion, thus leading to novel antifouling technologies.

Self-Assembly of a Dipeptide with a Reduced Amount of Copper into Antifungal and Antibacterial Particles.

With the growing concern over the environmental impact and health risks associated with conventional pesticides, there is a great need for developing safer and more sustainable alternatives. This study demonstrates the self-assembly of antimicrobial and antifungal spherical particles by a dipeptide utilizing a reduced amount of copper salt compared to the commonly employed formulation. The particles can be sprayed on a surface and form an antimicrobial coating. The effectiveness of the coating against the bacteria Pectobacterium brasiliense, a common pathogen affecting potato crops, was demonstrated, as the coating reduced the bacterial load by 7.3 log. Moreover, a comprehensive field trial was conducted, where the formulation was applied to potato seeds. Remarkably, it exhibited good efficacy against three prevalent potato pathogens (P. brasiliense, Pythium spp., and Spongospora subterranea) while demonstrating no phytotoxic effects on the potatoes. These findings highlight the tremendous potential of this formulation as a nonphytotoxic alternative to replace hazardous pesticides currently available in the market.

Enzyme-Driven, Switchable Catalysis Based on Dynamic Self-Assembly of Peptides.

Covalent regulatory systems of enzymes are widely used to modulate biological enzyme activities. Inspired by the regulation of reactive-site phosphorylation in organisms, we developed peptide-based catecholase mimetics with switchable catalytic activity and high selectivity through the co-assembly of nanofibers comprising peptides and copper ions (Cu2+ ). Through careful design and modification of the peptide backbone structure based on the change in the free energy of the system, we identified the peptide with the most effective reversible catalytic activity. Kinase/phosphatase switches were used to control the reversible transition of nanofiber formation and depolymerization, as well as to modulate the active-site microenvironment. Notably, the self-assembly and disassembly processes of nanofibers were simulated using coarse-grained molecular dynamics. Furthermore, theoretical calculations confirmed the coordination of the peptide and Cu2+ , forming a zipper-like four-ligand structure at the catalytically active center of the nanofibers. Additionally, we conducted a comprehensive analysis of the catalytic mechanism. This study opens novel avenues for designing biomimetic enzymes with ordered structures and dynamic catalytic activities.

Antimicrobial dichloroisocyanurate-salts for controlled release of chlorine.

Sodium dichloroisocyanurate (Na-DCC), a disinfectant known for rapid decomposition in water, loses its effectiveness with complete release of free available chlorine (FAC) in under an hour. To overcome this, a series of chlorine rich transition metal complexes/tetrabutylammonium (TBA) salts of DCC, including 2Na[Cu(DCC)4], 2Na[Fe(DCC)4], 2Na[Co(DCC)4]·6H2O, 2Na[Ni(DCC)4]·6H2O, and TBA[DCC]·4H2O have been developed for extended chlorine release studies. The DCC-salts are synthesized based on the metathesis reaction process and are characterized using IR, NMR, CHN analyses, TGA,DSC, and Lovi bond colorimeter. The DCC-salts displayed poor water solubility and low decomposition chlorine release profile compared to Na-DCC. The water solubility of DCC-salts was reduced by a factor of 5.37 to 2500 compared to Na-DCC. The decomposition release of FAC from DCC-salts has been studied over time in comparison to Na-DCC in distilled water using a Lovi-bond colorimeter. DCC-salts displayed controlled FAC release profiles that varied from 1-13 days depending on the type of metal/TBA unit in them, whereas the parent Na-DCC displayed complete FAC release in about 0.91 h. For a proof of concept, the controlled release of metal from one of the DCC-metal complex salts, i.e., copper from the Cu-DCC is also investigated with a function of time in distilled water at RT. The 100% release of copper from Cu-DCC was identified over a period of 10 days. In addition, the applicability of DCC-salts as excellent antiviral agents against the bacteriophage T4 and antibacterial agents against Erwinia, Pseudomonas aeruginosa PA014 (Gram-negative), and Staphylococcus epidermidis (Gram-positive) compared to Na-DCC has been demonstrated.

Engineered Cross-Linked Silane with Urea Polymer Thin Durable Coatings onto Polymeric Films for Controlled Antiviral Release of Activated Chlorine and Essential Oils.

In March 2020, the World Health Organization announced a pandemic attributed to SARS-CoV-2, a novel beta-coronavirus, which spread widely from China. As a result, the need for antiviral surfaces has increased significantly. Here, the preparation and characterization of new antiviral coatings on polycarbonate (PC) for controlled release of activated chlorine (Cl+) and thymol separately and combined are described. Thin coatings were prepared by polymerization of 1-[3-(trimethoxysilyl)propyl] urea (TMSPU) in ethanol/water basic solution by modified Stöber polymerization, followed by spreading the formed dispersion onto surface-oxidized PC film using a Mayer rod with appropriate thickness. Activated Cl-releasing coating was prepared by chlorination of the PC/SiO2-urea film with NaOCl through the urea amide groups to form a Cl-amine derivatized coating. Thymol releasing coating was prepared by linking thymol to TMSPU or its polymer via hydrogen bonds between thymol hydroxyl and urea amide groups. The activity towards T4 bacteriophage and canine coronavirus (CCV) was measured. PC/SiO2-urea-thymol enhanced bacteriophage persistence, while PC/SiO2-urea-Cl reduced its amount by 84%. Temperature-dependent release is presented. Surprisingly, the combination of thymol and chlorine had an improved antiviral activity, reducing the amount of both viruses by four orders of magnitude, indicating synergistic activity. For CCV, coating with only thymol was inactive, while SiO2-urea-Cl reduced it below a detectable level.

Enzymatic polymerization of enantiomeric L-3,4-dihydroxyphenylalanine into films with enhanced rigidity and stability.

L-3,4-dihydroxyphenylalanine is an important molecule in the adhesion of mussels, and as an oxidative precursor of natural melanin, it plays an important role in living system. Here, we investigate the effect of the molecular chirality of 3,4-dihydroxyphenylalanine on the properties of the self-assembled films by tyrosinase-induced oxidative polymerization. The kinetics and morphology of pure enantiomers are completely altered upon their co-assembly, allowing the fabrication of layer-to-layer stacked nanostructures and films with improved structural and thermal stability. The different molecular arrangements and self-assembly mechanisms of the L+D-racemic mixtures, whose oxidation products have increased binding energy, resulting in stronger intermolecular forces, which significantly increases the elastic modulus. This study provides a simple pathway for the fabrication of biomimetic polymeric materials with enhanced physicochemical properties by controlling the chirality of monomers.

In Situ Measurements of Cell Mechanical Properties Using Force Spectroscopy.

Mechanobiology focuses on how physical forces and the mechanical properties of cells and whole tissues affect their function. The mechanical properties of cells are of particular interest to developmental biology and stem cell differentiation, lymphocyte activation and phagocytic action in phagocytes, and development of malignant tumors and metastases. These properties can be measured on whole tissue and cell culture. Advances in instrument sensitivity and design, as well as improved techniques and scientific know-how achieved over the past few decades, allow researchers to study the mechanical properties of single cells and even at the subcellular level. Particularly, nanoindentation measurements using atomic force microscopy (AFM) mechanically probes single cells and even allows mapping of these traits. This chapter discusses these measurements from the experimental design to the analysis.

Peptide-capped Au and Ag nanoparticles: Detection of heavy metals and photochemical core/shell formation.

We present a short peptide of only six amino acids that can be used in ambient conditions to simultaneously reduce either Au3+ or Ag+ ions, forming nanoparticles, and function as a stabilizing capping agent. At acidic pH, Hg2+ ions oxidize the silver nanoparticles and Fe2+ ions promote the aggregation of the gold nanoparticles. At alkaline conditions, Mn2+ ions induce the aggregation of the silver nanoparticles. Through the absorbance changes of these processes, these peptide-capped nanoparticles demonstrated a fast, selective, and sensitive pH-dependent detection system. The limit of detection of Hg2+, Mn2+, and Fe2+ was 319 nм, 184 nм, and 320 nм, respectively. Furthermore, the formed gold nanoparticles were successfully enveloped by a silver shell in a peptide-mediated photoreduction process. These bimetallic Au@Ag core/shell nanoparticles were characterized using UV-vis spectroscopy, high-resolution scanning transmission electron microscopy (HR-STEM), and energy dispersive X-ray spectroscopy (EDS). While prior studies used peptides as ligands for nanoparticles, the versatile abilities of the novel peptide presented in this study display the promising potential of using peptides for nanoparticles synthesis. This is because a single peptide can be used in a single-step one-pot synthesis to prepare and stabilize AuNPs, AgNPs, and Au@Ag core/shell nanoparticles, while also allowing to selectively probe different metal ions.

Fabrication of antimicrobial polymeric films by compression molding of peptide assemblies and polyethylene.

This paper presents compression molding of peptide assemblies with low-density polyethylene (LDPE) for the robust production of antimicrobial polymeric films. These films show a significant reduction of colony-forming units and plaque-forming units. Moreover, they significantly inhibited the growth of three different fungi. These innovative active polymeric films can potentially be applied for medical device wrapping, food packaging, and agriculture applications.

Platform for Active Vaccine Formulation Using a Two-Mode Enhancement Mechanism of Epitope Presentation by Pickering Emulsion.

The efficiency of epitope-based vaccination (subunit vaccines) is tightly correlated with heterogeneity and the high density of epitope presentation, which maximizes the potential antigenic determinants. Here, we developed a two-mode platform for intensifying the epitope presentation of subunit vaccines. The two-mode epitope presentation enhancement includes a covalent attachment of high concentrations of SARS-CoV-2-S1 peptide epitope to the surface of virus-like-particles (VLPs) and the subsequent assembly of VLP/epitope conjugates on the oil droplet surface at an oil/water interface of an emulsion as Pickering stabilizers. The resultant emulsions were stable for weeks in ambient conditions, and our platform was challenged using the epitope of the SARS-CoV-2-S1 peptide that served as a model epitope in this study. In vivo assays showed that the αSARS-CoV-2-S1 immunoglobulin G (IgG) titers of the studied mouse antisera, developed against the SARS-CoV-2-S1 peptide under different epitope preparation conditions, showed an order of magnitude higher IgG titers in the studied VLP-based emulsions than epitopes dissolved in water and epitopes administered with an adjuvant, thereby confirming the efficacy of the formulation. This VLP-based Pickering emulsion platform is a fully synthetic approach that can be readily applied for vaccine development to a wide range of pathogens.

Urinary Stent Development and Evaluation Models: In Vitro, Ex Vivo and In Vivo-A European Network of Multidisciplinary Research to Improve Urinary Stents (ENIUS) Initiative.

Background: When trying to modify urinary stents, certain pre-clinical steps have to be followed before clinical evaluation in humans. Usually, the process starts as an in silico assessment. The urinary tract is a highly complex, dynamic and variable environment, which makes a computer simulation closely reflecting physiological conditions extremely challenging. Therefore, the pre-clinical evaluation needs to go through further steps of in vitro, ex vivo and in vivo assessments. Methods and materials: Within the European Network of Multidisciplinary Research to Improve Urinary Stents (ENIUS), the authors summarized and evaluated stent assessment models in silico, in vitro, ex vivo and in vivo. The topic and relevant sub-topics were researched in a systematic literature search in Embase, Scope, Web of Science and PubMed. Clinicaltrials.gov was consulted for ongoing trials. Articles were selected systematically according to guidelines with non-relevant, non-complete, and non-English or Spanish language articles excluded. Results: In the first part of this paper, we critically evaluate in vitro stent assessment models used over the last five decades, outlining briefly their strengths and weaknesses. In the second part, we provide a step-by-step guide on what to consider when setting up an ex vivo model for stent evaluation on the example of a biodegradable stent. Lastly, the third part lists and discusses the pros and cons of available animal models for urinary stent evaluation, this being the final step before human trials. Conclusions: We hope that this overview can provide a practical guide and a critical discussion of the experimental pre-clinical evaluation steps needed, which will help interested readers in choosing the right methodology from the start of a stent evaluation process once an in silico assessment has been completed. Only a transparent multidisciplinary approach using the correct methodology will lead to a successful clinical implementation of any new or modified stent.

Understanding the Adhesion Mechanism of Hydroxyapatite-Binding Peptide.

Understanding the interactions between the protein collagen and hydroxyapatite is of high importance for understanding biomineralization and bone formation. Here, we undertook a reductionist approach and studied the interactions between a short peptide and hydroxyapatite. The peptide was selected from a phage-display library for its high affinity to hydroxyapatite. To study its interactions with hydroxyapatite, we performed an alanine scan to determine the contribution of each residue. The interactions of the different peptide derivatives were studied using a quartz crystal microbalance with dissipation monitoring and with single-molecule force spectroscopy by atomic force microscopy. Our results suggest that the peptide binds via electrostatic interactions between cationic moieties of the peptide and the negatively charged groups on the crystal surface. Furthermore, our findings show that cationic residues have a crucial role in binding. Using molecular dynamics simulations, we show that the peptide structure is a contributing factor to the adhesion mechanism. These results suggest that even small conformational changes can have a significant effect on peptide adhesion. We suggest that a bent structure of the peptide allows it to strongly bind hydroxyapatite. The results presented in this study improve our understanding of peptide adhesion to hydroxyapatite. On top of physical interactions between the peptide and the surface, peptide structure contributes to adhesion. Unveiling these processes contributes to our understanding of more complex biological systems. Furthermore, it may help in the design of de novo peptides to be used as functional groups for modifying the surface of hydroxyapatite.

A Novel Copper-Binding Peptide That Self-Assembles Into a Transparent Antibacterial and Antiviral Coating.

The health, economy, and quality of life all over the world are greatly affected by bacterial infections and viral outbreaks. Bacterial cells and viruses, such as influenza, can spread through contaminated surfaces and fomites. Therefore, a possible way to fight these pathogens is to utilize antibacterial and antiviral coatings, which reduce their numbers on contaminated surfaces. Here, we present a novel short peptide that can self-assemble, adhere to various surfaces, and bind different metal ions such as copper, which provides the surface with antibacterial and antiviral properties. For these functions, the peptide incorporates the amino acid 3,4-dihydroxyphenylalanine (DOPA), which provides the peptide with adhesive capabilities; a diphenylalanine motif that induces the self-assembly of the peptide; the metal-binding hexahistidine sequence. Our results demonstrate that the coating, which releases monovalent cuprous ions and hydrogen peroxide, provides the surfaces with significant antibacterial and antiviral properties. Additionally, the coating remains transparent, which is favorable for many objects and especially for display screens.

Antiviral Activity of Peptide-Based Assemblies.

The COVID-19 pandemic highlighted the importance of developing surfaces and coatings with antiviral activity. Here, we present, for the first time, peptide-based assemblies that can kill viruses. The minimal inhibitory concentration (MIC) of the assemblies is in the range tens of micrograms per milliliter. This value is 2 orders of magnitude smaller than the MIC of metal nanoparticles. When applied on a surface, by drop casting, the peptide spherical assemblies adhere to the surface and form an antiviral coating against both RNA- and DNA-based viruses including coronavirus. Our results show that the coating reduced the number of T4 bacteriophages (DNA-based virus) by 3 log, compared with an untreated surface and 6 log, when compared with a stock solution. Importantly, we showed that this coating completely inactivated canine coronavirus (RNA-based virus). This peptide-based coating can be useful wherever sterile surfaces are needed to reduce the risk of viral transmission.

Antiviral Polymers Based on N-Halamine Polyurea.

N-halamines are a commonly applied class of antimicrobial agents used for a variety of applications relating to human health. Here, we present the modulation of the common polymers polyurea and polyguanidine with the N-halamine technology. The N-H bonds in either polymer were converted to N-Cl or N-Br bonds capable of releasing Cl+ or Br+ cations to aqueous media as antiviral agents. Controlled release of the oxidizing agents was monitored for a period of 4 weeks. Antiviral activity was evaluated against the T4 bacteriophage as well as against the highly stable plant virus belonging to the Tobamovirus genus, tomato brown rugose fruit virus. The incorporation of the N-halamine technology on commonly used polymers has effectively introduced antiviral functionality for a wide variety of potential applications.

Interactions of Microorganisms with Lipid Langmuir Layers.

Preventing microbial contamination of aquatic environments is crucial for the proper supply of drinking water. Hence, understanding the interactions that govern bacterial and virus adsorption to surfaces is crucial to prevent infection transmittance. Here, we describe a new approach for studying the organization and interactions of various microorganisms, namely, Escherichia coli (E. coli) bacteria, E. coli-specific bacteriophage T4, and plant cucumber green mottle mosaic viruses (CGMMV), at the air/water interface using the Langmuir-Blodgett (LB) technique. CGMMV were found as applicable candidates for further studying their interactions with Langmuir lipid monolayers. The zwitterionic, positively, and negatively charged LB lipid monolayers with adsorbed viruses were deposited onto solid supports and characterized by atomic force microscopy. Using polymerase chain reaction, we indicated that the adsorption of CGMMV onto the LB monolayer is a result of electrostatic interactions. These insights are useful in engineering membrane filters that prevent biofouling for efficient purification systems.

Non-covalently embedded oxytocin in alkanethiol monolayer as Zn2+ selective biosensor.

Peptides are commonly used as biosensors for analytes such as metal ions as they have natural binding preferences. In our previous peptide-based impedimetric metal ion biosensors, a monolayer of the peptide was anchored covalently to the electrode. Binding of metal ions resulted in a conformational change of the oxytocin peptide in the monolayer, which was measured using electrochemical impedance spectroscopy. Here, we demonstrate that sensing can be achieved also when the oxytocin is non-covalently integrated into an alkanethiol host monolayer. We show that ion-binding cause morphological changes to the dense host layer, which translates into enhanced impedimetric signals compared to direct covalent assembly strategies. This biosensor proved selective and sensitive for Zn2+ ions in the range of nano- to micro-molar concentrations. This strategy offers an approach to utilize peptide flexibility in monitoring their response to the environment while embedded in a hydrophobic monolayer.

Structural preferences of an anti-fouling peptide: From single chain to small molecular assemblies.

The structural features of a tripeptide constituted by two different non-coded amino acids, 3,4-dihydroxy-L-phenylalanine (L-DOPA) and 4-fluoro-Phenylalanine, (Phe(4F)), have been investigated by means of classical mechanics simulations. This tripeptide had been characterised as an antifouling agent with great adhesion capabilities. In this work, its conformational preferences have been described in two different environments (gas phase and water solution), at three different pHs and with different degrees of terminal capping. At the same time, the structural dynamics of small aggregates of the tripeptide have been investigated and their ability to stabilise ß-sheet based assemblies has been studied. The reported results describe the complexity of the tripeptide conformational preferences due to both the amphiphilic nature of its side chains, and the effect of the ionisation state resulting from the solution conditions. The investigations performed with small tripeptide assemblies in water solution reproduced the previously reported structural features, such as the polymorphism of its aggregates as a function of the pH. At edge pH values, the electrostatic screening imposed by the ions present in the solution facilitates the aggregation of the tripeptide chains, while at neutral pH and low concentrations of ionised species, the polar groups and the hydrogen bond capable groups impose their strength and lead to the disaggregation of the peptide clusters by favouring the solvation of individual chains rather than stabilising the aggregated states.

Nucleobase morpholino ß amino acids as molecular chimeras for the preparation of photoluminescent materials from ribonucleosides.

Bioinspired smart materials represent a tremendously growing research field and the obtainment of new building blocks is at the molecular basis of this technology progress. In this work, colloidal materials have been prepared in few steps starting from ribonucleosides. Nucleobase morpholino ß-amino acids are the chimera key intermediates allowing Phe-Phe dipeptides' functionalization with adenine and thymine. The obtained compounds self-aggregate showing enhanced photoluminescent features, such as deep blue fluorescence and phosphorescence emissions.

Combining chemistry and topography to fight biofilm formation: Fabrication of micropatterned surfaces with a peptide-based coating.

This paper describes the fabrication of antifouling surfaces by the combination of topography and peptide chemistry. The topography of the surface mimics the skin of the shark that can resist biofouling by having a certain microtopography. A peptide-based coating that resists fouling self-assembles on these surfaces. In biofilm formation assays, performed under static conditions, the resulting combination (micropattern with peptide coating) has superior antifouling properties against the Gram-negative and Gram-positive strains tested (Escherichia coli and Staphylococcus epidermidis, respectively) when compared to both micropatterned and peptide-coated surfaces. The same behavior was observed in dynamic assays performed in a parallel plate flow chamber (PPFC) setup, where E. coli could not attach to the micropatterned surface coated with peptide during the 30 min of initial adhesion. These assays, mimicking physiological shear stress conditions, suggest that the peptide-coated surface with micropatterned topography may be promising in reducing adhesion and subsequent biofilm formation in biomedical devices such as urinary catheters and stents, and cardiovascular, dental and orthopedic implants.