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.

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.

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.

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.

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.

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.

Amphiphilic Peptide with Dual Functionality Resists Biofouling.

Biofouling, the accumulation of organisms on surfaces, can lead to several undesirable phenomena, including hospital-acquired infections, blockage of water purification systems, and food contamination. The solution to the problem should be nontoxic and environmentally friendly, so that it could be applied on different surfaces and could come into contact with food, water, or human tissues. Peptides can provide such a solution, since they are biocompatible and biodegradable materials that can resist biofouling, either by preventing the attachment of organisms to the surface (antifouling) or by killing the bacteria (antimicrobial activity). This paper presents an amphiphilic peptide with antifouling, antimicrobial, and adhesive properties. The peptide adheres to titanium surfaces and inhibits the adhesion of both Gram-negative and Gram-positive bacteria to surfaces. In addition, it reduces the growth of bacteria in solution. This peptide has both antifouling and antimicrobial properties, which could be useful in health care systems, food packaging, and other systems that suffer from biocontamination.

Fast and synchronized fluctuations of cortical actin negatively correlate with nucleoli liquid-liquid phase separation in T cells.

Liquid-liquid phase separation is an important mechanism by which eukaryotic cells functionally organize their intracellular content and has been related to cell malignancy and neurodegenerative diseases. These cells also undergo ATP-driven mechanical fluctuations, yet the effect of these fluctuations on the liquid-liquid phase separation remains poorly understood. Here, we employ high-resolution microscopy and atomic force microscopy of live Jurkat T cells to characterize the spectrum of their mechanical fluctuations, and to relate these fluctuations to the extent of nucleoli liquid-liquid phase separation (LLPS). We find distinct fluctuation of the cytoskeleton and of the cell diameter around 110 Hz, which depend on ATP and on myosin activity. Importantly, these fluctuations negatively correlate to nucleoli LLPS. According to a model of cell viscoelasticity, we propose that these fluctuations generate mechanical work that increases intracellular homogeneity by inhibiting LLPS. Thus, active mechanical fluctuations serve as an intracellular regulatory mechanism that could affect multiple pathophysiological conditions.

A peptide coating preventing the attachment of Porphyromonas gingivalis on the surfaces of dental implants.

OBJECTIVES: The aim of this study was to investigate whether a peptide-based coating can prevent the adhesion of Porphyromonas gingivalis, a key human pathogen associated with periodontitis and peri-implantitis. BACKGROUND: Nonsurgical and surgical interventions have been used for the treatment of peri-implantitis; however, the effectiveness of these approaches is usually unsatisfactory. The main reason is that dental plaque on the surface of the implant is difficult to remove due to its rough surface and thread design. Recently, a peptide-based coating for implant surfaces that can reject the adhesion of Escherichia coli and improve the attachment of host cells was developed. METHODS: A salivary pellicle was created on the surfaces of peptide-coated bare discs and verified with anti-human immunoglobulin G, A and M, and anti-fibrinogen. Early colonizers, Veillonella parvula and Streptococcus sobrinus, and the later colonizer, Porphyromonas gingivalis, were labelled with green and red fluorescent dyes, respectively, and seeded on the discs. Bacterial attachment was semi-quantified by fluorescence intensity. RESULTS: The salivary pellicle was evenly distributed on the discs, with or without the peptide coating, with an average thickness of 3.84 µm. A multi-species dental biofilm was created on the salivary pellicle. The peptide coating resulted in an approximate 25% reduction in the attachment of Veillonella parvula and Streptococcus sobrinus, and a 50% reduction in Porphyromonas gingivalis, when compared to control, uncoated implant discs. CONCLUSION: The novel peptide-based coating can inhibit the attachment of Porphyromonas gingivalis. It may have the potential to impede the development of peri-implantitis.

The effect of end-group substitution on surface self-assembly of peptides.

Biofouling, the undesirable accumulation of organisms onto surfaces, affects many areas including health, water, and energy. We previously designed a tripeptide that self-assembles into a coating that prevents biofouling. The peptide comprises three amino acids: DOPA, which allows its adhesion to the surface, and two fluorinated phenylalanine residues that direct its self-assembly into a coating and acquire it with antifouling properties. This short peptide has an ester group at its C-terminus. To examine the importance of this end group for the self-assembly and antifouling properties of the peptide, we synthesized and characterized tripeptides with different end groups (ester, amide, or carboxylic group). Our results indicate that different groups at the C-terminus of the peptide can lead to a change in the peptide assembly on the surface and its adsorption process. However, this change only affects the antifouling properties of the coating toward Gram-positive bacteria (Staphylococcus epidermidis), whereas Gram-negative bacteria (Escherichia coli) are not affected.

Rational Design of Amphiphilic Peptides and Its Effect on Antifouling Performance.

Biofouling, the unwanted adhesion of organisms to surfaces, has a negative impact on energy, food, water, and health resources. One possible strategy to fight biofouling is to modify the surface using a peptide-based coating that will change the surface properties. We reveal the importance of rational design and positioning of individual amino acids in an amphiphilic peptide sequence. By just manipulating the position of the amino acids within the peptide chain having the same chemical composition, we improved the antifouling performance of an amphiphilic peptide-based coating, Phe(4-F)-Lys-DOPA, by 30%. We have judiciously tailored the peptide configurations to achieve the best antifouling performance by (i) positioning the amino acid lysine adjacent to the DOPA moiety in the linear peptide chain for better adhesion, (ii) having a linear fluorinated N-terminal to improve the packing density of the film by straightening the peptide chain, and (iii) placing DOPA at the C-terminal. We have also compared the antifouling performances of amphiphilic, hydrophobic, hydrophilic, and alternately arranged peptides. Our results show a reduction of ∼80% in bacterial adhesion for an amphiphilic peptide-coated surface when compared to a bare titanium surface. This work provides important strategic design guidelines for future peptide-related materials that have effective antifouling properties.

The role of hydrophobic, aromatic and electrostatic interactions between amino acid residues and a titanium dioxide surface.

Understanding the nature of interactions between inorganic surfaces and biomolecules, such as amino acids and peptides, can enhance the development of new materials. Here, we present single molecule force spectroscopy (SMFS) measurements of the interactions between an atomic force microscopy (AFM) probe, modified with various amino acids, and a titanium dioxide surface. Specifically, we study the affinity of amino acids toward a titanium dioxide surface bearing hydrophobic (Leu), aromatic (Phe) and hydrophilic (Orn) residues. We find that aromatic interactions dominate over aliphatic in their affinity to the titanium dioxide surface. In addition, we show that by combining aromatic and hydrophilic moieties in a single amino acid (NH2-Phe), the adhesion of the latter to the surface increases. Furthermore, the affinity of positively charged amino acids to the titanium dioxide surface is higher than that of uncharged, and can be increased more, with elevating the pH of the buffer above the pKa of the basic residues. The kinetic and thermodynamic parameters imply that the dynamics of the surface-amino acid interface are mostly governed by hydrophobic interactions.

Self-assembly of an amphipathic ααß-tripeptide into cationic spherical particles for intracellular delivery.

The development of molecular carriers able to carry molecules directly into the cell is an area of intensive research. Cationic nanoparticles are effective delivery systems for several classes of molecules, such as anticancer agents, oligonucleotides and antibodies. Indeed, a cationic charge on the outer surface allows a rapid cellular uptake together with the possibility of carrying negatively charged molecules. In this work, we studied the self-assembly of an ultra-short ααß-tripeptide containing an l-Arg-l-Ala sequence and an unnatural fluorine substituted ß2,3-diaryl-amino acid. The presence of the unnatural ß2,3-diaryl-amino acid allowed us to obtain a protease stable sequence. Furthermore, an arginine guanidinium group triggered the formation of spherical assemblies that were able to load small molecules and enter cells. These spherical architectures, thus, represent interesting candidates for the delivery of exogenous entities directly into cells.

Peptide fibrils as monomer storage of the covalent HIV-1 integrase inhibitor.

We have recently reported the covalent inhibition of HIV-1 integrase by an N-terminal succinimide-modified lens epithelium-derived growth factor (361-370) peptide. We also showed that this peptide is proteolytically stable. Here, we show that this inhibitor is stored as fibrils that serve as a stock for the inhibitory monomers. The fibrils increase the local concentration of the peptide at the target protein. When the monomers bind integrase, the equilibrium between the fibrils and their monomers shifts towards the formation of peptide monomers. The combination of fibril formation and subsequent proteolytic stability of the peptide may bring to new strategy for developing therapeutic agents. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Review insights into the interactions of amino acids and peptides with inorganic materials using single molecule force spectroscopy.

Understanding the interactions between proteins and inorganic surfaces is important for the development of new biomaterials and implants as they interface with the immune response by proteins. In addition, the adsorption of proteins to inorganic surfaces leads to the formation of a conditioning layer that facilitates bacterial attachments and biofilm formation. As biofilm provides bacterial resistance to antibiotics, biofilm formation is an undesirable process that could be prevented by resisting protein interactions with the substrate. Moreover, the interaction between proteins and inorganic materials is the basis for the formation of composite materials in nature. Understanding the underlying forces that governs these interactions would lead to the design of new and unique composite materials in vitro. This review focuses on the insights gained using single-molecule force spectroscopy by AFM on these interactions. This tool provides molecular information, at the single molecule level, on the interaction between a molecule on the AFM tip and a substrate.

Elucidating the mechanism of interaction between peptides and inorganic surfaces.

Understanding the mechanism of interaction between peptides and inorganic materials is of high importance for the development of new composite materials. Here, we combined an experimental approach along with molecular simulations in order to gain insights into this binding process. Using single molecule force spectroscopy by atomic force microscopy and molecular simulations we studied the binding of a peptide towards an inorganic substrate. By performing alanine scan we examined the propensity of each amino acid in the peptide sequence to bind the substrate (mica). Our results indicate that this binding is not controlled by the specific sequence of the peptide, but rather by its conformational freedom in solution versus its freedom when it is in proximity to the substrate. When the conformational freedom of the peptide is identical in both environments, the peptide will not adhere to the substrate. However, when the conformational freedom is reduced, i.e., when the peptide is in close proximity to the substrate, binding will occur. These results shed light on the interaction between peptides and inorganic materials.

Optimization of liganded polyethylenimine polyethylene glycol vector for nucleic acid delivery.

The delivery of nucleic acids into cells is an attractive approach for cancer therapy. Polyethylenimine (PEI) is among the most efficient nonviral carriers. Recent studies have demonstrated that PEI can be conjugated to targeting ligands, such as epidermal growth factor (EGF) and transferrin (Schaffert et al., 2011; Abourbeh et al., 2012; Ogris et al., 1999). Herein we present a simplified protocol for producing homogeneous preparations of PEGylated linear PEI: LPEI-PEG2k. We generated two well-characterized copolymers, with ratios of LPEI to PEG of 1:1 and 1:3. These copolymers were further conjugated through disulfide bonds to a Her-2 targeting moiety, Her-2 affibody. This reaction yielded two triconjugates that target Her-2 overexpressing tumors. Polyplexes were formed by complexing plasmid DNA with the triconjugates. We characterized the biophysical properties of the conjugates, and found that the triconjugate 1:3 polyplex had lower ζ potential, larger particle size, and more heterogeneous shape than the triconjugate 1:1 polyplex. Triconjugate 1:1 and triconjugate 1:3 polyplexes were highly selective toward cells that overexpress Her-2 receptors, but triconjugate 1:1 polyplex was more efficient at gene delivery. Our studies show that the biophysical and biological properties of the conjugates can be profoundly affected by the ratio of LPEI:PEG2k:ligand. The procedure described here can be adapted to generate a variety of triconjugates, simply by changing the targeting moiety.

Self-assembly of azide containing dipeptides.

Functional structures and materials are formed spontaneously in nature through the process of self-assembly. Mimicking this process in vitro will lead to the formation of new substances that would impact many areas including energy production and storage, biomaterials and implants, and drug delivery. The considerable structural diversity of peptides makes them appealing building blocks for self-assembly in vitro. This paper describes the self-assembly of three aromatic dipeptides containing an azide moiety: H-Phe(4-azido)-Phe(4-azido)-OH, H-Phe(4-azido)-Phe-OH, and H-Phe-Phe(4-azido)-OH. The peptide H-Phe(4-azido)-Phe(4-azido)-OH self-assembled into porous spherical structures, whereas the peptides H-Phe(4-azido)-Phe-OH and H-Phe-Phe(4-azido)-OH did not form any ordered structures under the examined experimental conditions. The azido group of the peptide can serve as a photo cross-linking agent upon irradiation with UV light. To examine the effect of this group and its activity on the self-assembled structures, we irradiated the assemblies in solution for different time periods. Using electron microscopy, we determined that the porous spherical assemblies formed by the peptide H-Phe(4-azido)-Phe(4-azido)-OH underwent a structural change upon irradiation. In addition, using FT-IR, we detected the chemical change of the peptide azido group. Moreover, using indentation experiments with atomic force microscopy, we showed that the Young's modulus of the spherical assemblies increased after 20 min of irradiation with UV light. Overall, irradiating the solution of the peptide assemblies containing the azido group resulted in a change both in the morphology and mechanical properties of the peptide-based structures. These ordered assemblies or their peptide monomer building blocks can potentially be incorporated into other peptide assemblies to generate stiffer and more stable materials.

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.

Probing the interaction of individual amino acids with inorganic surfaces using atomic force spectroscopy.

This article describes single-molecule force spectroscopy measurements of the interaction between individual amino acid residues and inorganic surfaces in an aqueous solution. In each measurement, there is an amino acid residue, lysine, glutamate, phenylalanine, leucine, or glutamine, and each represents a class of amino acids (positively or negatively charged, aromatic, nonpolar, and polar). Force-distance curves measured the interaction of the individual amino acid bound to a silicon atomic force microscope (AFM) tip with a silcon substrate, cut from a single-crystal wafer, or mica. Using this method, we were able to measure low adhesion forces (below 300 pN) and could clearly determine the strength of interactions between the individual amino acid residues and the inorganic substrate. In addition, we observed how changes in the pH and ionic strength of the solution affected the adsorption of the residues to the substrates. Our results pinpoint the important role of hydrophobic interactions among the amino acids and the substrate, where hydrophobic phenylalanine exhibited the strongest adhesion to a silicon substrate. Additionally, electrostatic interactions also contributed to the adsorption of amino acid residues to inorganic substrates. A change in the pH or ionic strength values of the buffer altered the strength of interactions among the amino acids and the substrate. We concluded that the interplay between the hydrophobic forces and electrostatic interactions will determine the strength of adsorption among the amino acids and the surface. Overall, these results contribute to our understanding of the interaction at the organic-inorganic interface. These results may have implications for our perception of the specificity of peptide binding to inorganic surfaces. Consequently, it would possibly lead to a better design of composite materials and devices.