Surface-immobilised antimicrobial peptoids

Surface modification techniques that create surfaces capable of killing adherent bacteria are promising solutions to infections associated with implantable medical devices. Antimicrobial (AM) peptoid oligomers (ampetoids) that were designed to mimic helical AM peptides were synthesised with a peptoid spacer chain to allow mobility and an adhesive peptide moiety for easy and robust immobilisation onto substrata. TiO₂ substrata were modified with the ampetoids and subsequently backfilled with an antifouling (AF) polypeptoid polymer in order to create polymer surface coatings composed of both AM (active) and AF (passive) peptoid functionalities. Confocal microscopy images showed that the membranes of adherent E. coli cells were damaged after 2-h exposure to the modified substrata, suggesting that ampetoids retain AM properties even when immobilised on substrata.     

Self-decontaminating photocatalytic zinc oxide nanorod coatings for prevention of marine microfouling: a mesocosm study

The antifouling (AF) properties of zinc oxide (ZnO) nanorod coated glass substrata were investigated in an out-door mesocosm experiment under natural sunlight (14:10 light: dark photoperiod) over a period of five days. The total bacterial density (a six-fold reduction) and viability (a three-fold reduction) was significantly reduced by nanocoatings in the presence of sunlight. In the absence of sunlight, coated and control substrata were colonized equally by bacteria. MiSeq Illumina sequencing of 16S rRNA genes revealed distinct bacterial communities on the nanocoated and control substrata in the presence and absence of light. Diatom communities also varied on nanocoated substrata in the presence and the absence of light. The observed AF activity of the ZnO nanocoatings is attributed to the formation of reactive oxygen species (ROS) through photocatalysis in the presence of sunlight. These nanocoatings are a significant step towards the production of an environmentally friendly AF coating that utilizes a sustainable supply of sunlight.     

Pioneer marine biofilms on artificial surfaces including antifouling coatings immersed in two contrasting French Mediterranean coast sites

Marine biofilm communities that developed on artificial substrata were investigated using molecular and microscopic approaches. Polystyrene, Teflon® and four antifouling (AF) paints were immersed for 2 weeks at two contrasting sites near Toulon on the French Mediterranean coast (Toulon military harbour and the natural protected area of Porquerolles Island). Biofilms comprising bacteria and diatoms were detected on all the coatings. The population structure as well as the densities of the microorganisms differed in terms of both sites and coatings. Lower fouling densities were observed at Porquerolles Island compared to Toulon harbour. All bacterial communities (analysed by PCR-DGGE) showed related structure, controlled both by the sites and the type of substrata. Pioneer microalgal communities were dominated by the same two diatom species, viz. Licmophora gracilis and Cylindrotheca closterium, at both sites, irrespective of the substrata involved. However, the density of diatoms followed the same trend at both sites with a significant effect of all the AF coatings compared to Teflon and polystyrene.     

Pioneer marine biofilms on artificial surfaces including antifouling coatings immersed in two contrasting French Mediterranean coast sites

Marine biofilm communities that developed on artificial substrata were investigated using molecular and microscopic approaches. Polystyrene, Teflon? and four antifouling (AF) paints were immersed for 2 weeks at two contrasting sites near Toulon on the French Mediterranean coast (Toulon military harbour and the natural protected area of Porquerolles Island). Biofilms comprising bacteria and diatoms were detected on all the coatings. The population structure as well as the densities of the microorganisms differed in terms of both sites and coatings. Lower fouling densities were observed at Porquerolles Island compared to Toulon harbour. All bacterial communities (analysed by PCR-DGGE) showed related structure, controlled both by the sites and the type of substrata. Pioneer microalgal communities were dominated by the same two diatom species, viz. Licmophora gracilis and Cylindrotheca closterium, at both sites, irrespective of the substrata involved. However, the density of diatoms followed the same trend at both sites with a significant effect of all the AF coatings compared to Teflon and polystyrene.     

Peptoid origins

Peptoid oligomers were initially developed as part of a larger basic research effort to accelerate the drug-discovery process in the biotech/biopharma industry. Their ease of synthesis, stability, and structural similarity to polypeptides made them ideal candidates for the combinatorial discovery of novel peptidomimetic drug candidates. Diverse libraries of short peptoid oligomers provided one of the first demonstrations in the mid-1990s that high-affinity ligands to pharmaceutically relevant receptors could be discovered from combinatorial libraries of synthetic compounds. The solid-phase submonomer method of peptoid synthesis was so efficient and general that it soon became possible to explore the properties of longer polypeptoid chains in a variety of areas beyond drug discovery (e.g., diagnostics, drug delivery, and materials science). Exploration into protein-mimetic materials soon followed, with the fundamental goal of folding a non-natural sequence-specific heteropolymer into defined secondary or tertiary structures. This effort first yielded the peptoid helix and much later the peptoid sheet, both of which are secondary-structure mimetics that are close relatives to their natural counterparts. These crucial discoveries have brought us closer to building proteinlike structure and function from a non-natural polymer and have provided great insight into the rules governing polymer and protein folding. The accessibility of peptoid synthesis to chemists and nonchemists alike, along with a lack of information-rich non-natural polymers available to study, has led to a rapid growth in the field of peptoid science by many new investigators. This work provides an overview of the initial discovery and early developments in the peptoid field.     

Polysulfone and polyacrylate-based zwitterionic coatings for the prevention and easy removal of marine biofouling

A series of polysulfone and polyacrylate-based zwitterionic coatings were prepared on epoxy-primed aluminum substrata and characterized for their antifouling (AF) and fouling-release (FR) properties towards marine bacteria, microalgae and barnacles. The zwitterionic polymer coatings provided minimal resistance against bacterial biofilm retention and microalgal cell attachment, but facilitated good removal of attached microbial biomass by exposure to water-jet apparatus generated hydrodynamic shearing forces. Increasing the ion content of the coatings improved the AF properties, but required a stronger adhesive bond to the epoxy-primed aluminum substratum to prevent coating swelling and dissolution. Grafted poly(sulfobetaine) (gpSBMA), the most promising zwitterionic coating identified from microfouling evaluations, enabled the removal of four out of five barnacles reattached to its surface without incurring damage to their baseplates. This significant result indicated that gpSBMA relied predominately on its surface chemistry for its FR properties since it was very thin (~1–2 µm) relative to commercial coating standards (>200 µm).     

Physical and chemical characterization of the polysaccharide capsule of the marine bacterium,Hyphomonas strain MHS-3

Hyphomonas MHS-3 is a biphasic, marine bacterium that synthesizes an exopolysaccharide (EPS) capsule, which has a role in attaching the adherent, prosthecate developmental stages to solid substrata. To correlate structure with function, we characterized this integral EPS. It has a relatively homogeneous molecular weight of approximately 60000 daltons, is acidic, and putatively contains large concentrations ofN-acetylgalactosamine (GalNAc). The theoretical identity of the anionic component of the polymer, and the similarities betweenHyphomonas MHS-3 EPS and other adhesive marine/aquatic bacterial EPS are discussed.     

A VEGFR2 Antagonist and Other Peptoids Evade Immune Recognition

We have recently reported a peptoid (N-alkyl-oligoglycine) molecule that binds to the Vascular Endothelial Growth Factor Receptor 2 (VEGFR2) with high affinity and specificity. Moreover, this peptoid is capable of inhibiting VEGFR2 function in vivo (Udugamasooriya et al. J Am Chem Soc 130:5744–5745, 2008) and thus is a lead compound for anti-angiogenic agents. Moreover, the assay developed to identify this VEGFR2 inhibitor is likely to be a general route to peptoid antagonists or agonists of integral membrane receptors. Therefore, it is important to determine whether the VEGFR2-targeted peptoid, and indeed peptoids in general, are inherently immunogenic since an anti-peptoid immune response would significantly complicate their development as therapeutic candidates. In this study, the VEGFR2-targeted peptoid as well as other peptoids of varying lengths were injected into mice along with an immunostimulatory agent. We demonstrate that no significant anti-peptoid immune response is induced. It is further shown that this is not a trivial result of the lack of immunogenicity of a particular peptoid sequence, since conjugation of the peptoids to carrier proteins such as KLH prior to injection induces a robust anti-peptoid immune response. We conclude that free peptoid molecules are not immunogenic, probably due to a lack of T cell epitopes and that peptoid-based therapeutics are therefore not likely to be hindered by anti-peptoid antibody production in most cases.     

Fecundity of Hoplopleura scapteromydis (Phthiraptera: Anoplura, Hoplopleuridae), ectoparasite of Scapteromys aquaticus (Rodentia: Muridae) in Rio de La Plata marshlands, Argentina.

Density and age structure of lice collected from captured Scapteromys aquaticus rodents were studied to estimate the fecundity of Hoplopleura scapteromydis. The number of eggs with a visible embryo inside (E), nymphs (N), adult males (AM), and adult females (AF) were recorded for each rodent. For the ith rodent, the fecundity of H. scapteromydis, F(i), was estimated as F(i) = [[E(i) + N(i)]/2]/AF(i)/T, where T represents the period of preimaginal development (unknown and arbitrarily considered as T = 1), and the sex ratio of the preimaginal stages was supposed to be 1:1. In order to look for density-dependent effects, F(i) was plotted against AM(i), this being an independent estimation of infrapopulation density. The number of rodents suitable for AF and AM calculations was 38 (57% of the parasitized animals). Almost 95% had a low-to-moderate louse burden (1 < AM < 30) and were captured every season, whereas only 3% had a heavy (61 < AM < 70, captured in winter) or very heavy (AM > 80, captured in summer) louse burden. The extreme values of F were 0.63 and 18 (1.3 and 36 if both sexes were considered). High-to-moderate F-values (F > 5) were estimated in only 5 rodents that exhibited low louse density, whereas low F-values (F < 5) were found at all louse densities. Notwithstanding the tendency toward an inverse relationship between fecundity and infrapopulation density, the correlation was not significantly different from 0.     

Catabolism of 1,5-anhydro-D-fructose in Sinorhizobium morelense S-30.7.5: discovery, characterization, and overexpression of a new 1,5-anhydro-D-fructose reductase and its application in sugar analysis and rare sugar synthesis

The bacterium Sinorhizobium morelense S-30.7.5 was isolated by a microbial screening using the sugar 1,5-anhydro-D-fructose (AF) as the sole carbon source. This strain metabolized AF by a novel pathway involving its reduction to 1,5-anhydro-D-mannitol (AM) and the further conversion of AM to D-mannose by C-1 oxygenation. Growth studies showed that the AF metabolizing capability is not confined to S. morelense S-30.7.5 but is a more common feature among the Rhizobiaceae. The AF reducing enzyme was purified and characterized as a new NADPH-dependent monomeric reductase (AFR, EC 1.1.1.-) of 35.1 kDa. It catalyzed the stereoselective reduction of AF to AM and also the conversion of a number of 2-keto aldoses (osones) to the corresponding manno-configurated aldoses. In contrast, common aldoses and ketoses, as well as nonsugar aldehydes and ketones, were not reduced. A database search using the N-terminal AFR sequence retrieved a putative 35-kDa oxidoreductase encoded by the open reading frame Smc04400 localized on the chromosome of Sinorhizobium meliloti 1021. Based on sequence information for this locus, the afr gene was cloned from S. morelense S-30.7.5 and overexpressed in Escherichia coli. In addition to the oxidoreductase of S. meliloti 1021, AFR showed high sequence similarities to putative oxidoreductases of Mesorhizobium loti, Brucella suis, and B. melitensis but not to any oxidoreductase with known functions. AFR could be assigned to the GFO/IDH/MocA family on the basis of highly conserved common structural features. His6-tagged AFR was used to demonstrate the utility of this enzyme for AF analysis and synthesis of AM, as well as related derivatives.     

The effects of potential larval supply, settlement and post-settlement processes on the distribution of two species of filter-feeding caddisflies

1. Hydropsychid caddisfly larvae are often abundant in fast flow habitats and on highly textured substrata. Such patterns are usually inferred to result from habitat preference, but the roles of larval supply to the benthos and post‐settlement events are rarely examined for stream populations. We describe a manipulative field experiment that examined larval supply, habitat preference and post‐settlement events simultaneously for two co‐occurring species of hydropsychids. 2. Ten artificial channels were constructed in a natural riffle to create fast and slow flow habitats and within each channel we placed six artificial substrata whose surfaces had been modified to create three different texture treatments. We measured discharge through channels and monitored hydropsychid colonisation of substrata every 3–4 days. Half of the substrata had all hydropsychids and nets removed every 3–4 days (short‐term colonisation) whereas the remaining half were counted but nets and hydropsychids left attached (long‐term colonisation). Short‐term counts of nets and individuals were summed to allow comparison with long‐term colonisation substrata. 3. Smicrophylax sp. AV2 larvae were more abundant in fast flow channels than slow flow channels, but these differences were proportional to discharge through each of the channels, suggesting that supply of settlers can explain this pattern. Smicrophylax larvae were least abundant on smooth substrata, which suggests that this species selects habitats based on surface texture. Alternatively, Asmicridea sp. AV1 larvae were only found in fast flow channels and this most likely reflects an active habitat choice by this species, but there was no significant difference between different texture treatments. 4. There were no differences between summed, short‐ and long‐term counts of recruits for either species, but there were more nets than larvae, especially in slow flow channels, by the end of the experiment, suggesting that larval mortality or re‐dispersal after settlement could be considerable. 5. Our results indicate that supply, habitat selection at settlement and post‐settlement processes all contributed variously to the distribution of hydropsychid caddisfly larvae, but that each species was affected differentially by these factors. Larval supply and post‐settlement processes are rarely examined by stream researchers and our results demonstrate these factors deserve much more consideration. Calculating accurate larval supply rates to sites is challenging, but we suggest that such detailed information is necessary if we are to sort out what sets limits to distributions and the underlying population structure of stream invertebrate populations.     

Application of capillary electrophoresis to the separation of structurally diverse N-(substituted)-glycine–peptoid combinatorial mixtures

The capillary electrophoresis (CE)-based separation of five N-(substituted)-glycine (NSG)–peptoid mixtures with a wide range of physical and chemical properties was studied. A CE separation, initially developed using a single representative peptoid mixture, with a backround electrolyte (BGE) modified by the addition of both methyl-β-cyclodextrin and heptane sulfonic acid was found to provide good separations of most of the combinatorial mixtures investigated. For those mixtures not separated well by this procedure, the use of SDS micelles in conjunction with methyl-β-cyclodextrin resulted in dramatic improvements in the separation. While no single set of separation conditions proved sufficient for all of the NSG–peptoid combinatorial mixtures, the two methods were able to provide separation sufficient for characterization of a set of mixtures with a wide range of physical and chemical properties. The efficiency of the CE-based separation of the combinatorial mixtures studied was compared to a reversed-phase liquid chromatographic method using gradient elution.     

Amphiphilic triblock copolymers with PEGylated hydrocarbon structures as environmentally friendly marine antifouling and fouling-release coatings

The ideal marine antifouling (AF)/fouling-release (FR) coating should be non-toxic, while effectively either resisting the attachment of marine organisms (AF) or significantly reducing their strength of attachment (FR). Many recent studies have shown that amphiphilic polymeric materials provide a promising solution to producing such coatings due to their surface dual functionality. In this work, poly(ethylene glycol) (PEG) of different molecular weights (M_w?=?350, 550) was coupled to a saturated difunctional alkyl alcohol to generate amphiphilic surfactants (PEG-hydrocarbon-OH). The resulting macromolecules were then used as side chains to covalently modify a pre-synthesized PS₈?_K-b-P(E/B)₂₅?_K-b-PI₁₀?_K (SEBI or K3) triblock copolymer, and the final polymers were applied to glass substrata through an established multilayer surface coating technique to prepare fouling resistant coatings. The coated surfaces were characterized with AFM, XPS and NEXAFS, and evaluated in laboratory assays with two important fouling algae, Ulva linza (a green macroalga) and Navicula incerta, a biofilm-forming diatom. The results suggest that these polymer-coated surfaces undergo surface reconstruction upon changing the contact medium (polymer/air vs polymer/water), due to the preferential interfacial aggregation of the PEG segment on the surface in water. The amphiphilic polymer-coated surfaces showed promising results as both AF and FR coatings. The sample with longer PEG chain lengths (M_w?=?550?g?mol^?1) exhibited excellent properties against both algae, highlighting the importance of the chemical structures on ultimate biological performance. Besides reporting synthesis and characterization of this new type of amphiphilic surface material, this work also provides insight into the nature of PEG/hydrocarbon amphiphilic coatings, and this understanding may help in the design of future generations of fluorine-free, environmentally friendly AF/FR polymeric coatings.     

A peptoid antagonist of VEGF receptor 2 recognizes a 'hotspot' in the extracellular domain distinct from the hormone-binding site

Antagonists of VEGF-mediated angiogenesis are of great interest clinically for the treatment of solid tumors and certain forms of macular degeneration. We recently described a novel peptoid antagonist of VEGF Receptor 2 (VEGFR2) that binds to the extracellular domain of the receptor and inhibits VEGF-mediated autophosphorylation and subsequent downstream signaling. Given the structural similarities between peptides and peptoids, an obvious model for the mode of action of the peptoid is that it competes with VEGF for binding to VEGFR2. However, we present evidence here that this is not the case and that VEGF and the peptoid antagonist recognize non-overlapping surfaces located within the first three immunoglobulin-like subdomains of the receptor. These data argue that the peptoid inhibits receptor-mediated autophosphorylation by a novel allosteric mechanism that may prevent the receptor from acquiring the conformation necessary to propagate downstream signals.     

Peptoid conformational free energy landscapes from implicit-solvent molecular simulations in AMBER

To test the accuracy of existing AMBER force field models in predicting peptoid conformation and dynamics, we simulated a set of model peptoid molecules recently examined by Butterfoss et al. (JACS 2009, 131, 16798-16807) using QM methods as well as three peptoid sequences with experimentally determined structures. We found that AMBER force fields, when used with a Generalized Born/Surface Area (GBSA) implicit solvation model, could accurately reproduce the peptoid torsional landscape as well as the major conformers of known peptoid structures. Enhanced sampling by replica exchange molecular dynamics (REMD) using temperatures from 300 to 800 K was used to sample over cis-trans isomerization barriers. Compared to (Nrch)5 and cyclo-octasarcosyl, the free energy of N-(2-nitro-3-hydroxyl phenyl)glycine-N-(phenyl)glycine has the most "foldable" free energy landscape, due to deep trans-amide minima dictated by N-aryl sidechains. For peptoids with (S)-N (1-phenylethyl) (Nspe) side chains, we observe a discrepancy in backbone dihedral propensities between molecular simulations and QM calculations, which may be due to force field effects or the inability to capture n --> n* interactions. For these residues, an empirical phi-angle biasing potential can "rescue" the backbone propensities seen in QM. This approach can serve as a general strategy for addressing force fields without resorting to a complete reparameterization. Overall, this study demonstrates the utility of implicit-solvent REMD simulations for efficient sampling to predict peptoid conformational landscapes, providing a potential tool for first-principles design of sequences with specific folding properties.     

Growth rate control of adherent bacterial populations.

We report a novel in vitro method which, through application of appropriate nutrient limitations, enables growth rate control of adherent bacterial populations. Exponentially growing cells are collected by pressure filtration onto cellulose acetate membranes. Following inversion into the bases of modified fermentors, membranes and bacteria are perfused with fresh medium. Newly formed and loosely attached cells are eluted with spent medium. Steady-state conditions (dependent upon the medium flow rate) at which the adherent bacterial biomass is constant and proportional to the limiting nutrient concentrations are rapidly achieved, and within limits, the growth rate is proportional to the medium flow rate. Scanning electron microscopic studies showed that such populations consist of individual cells embedded within an extracellular polymer matrix.     

Growth rate control of adherent bacterial populations

We report a novel in vitro method which, through application of appropriate nutrient limitations, enables growth rate control of adherent bacterial populations. Exponentially growing cells are collected by pressure filtration onto cellulose acetate membranes. Following inversion into the bases of modified fermentors, membranes and bacteria are perfused with fresh medium. Newly formed and loosely attached cells are eluted with spent medium. Steady-state conditions (dependent upon the medium flow rate) at which the adherent bacterial biomass is constant and proportional to the limiting nutrient concentrations are rapidly achieved, and within limits, the growth rate is proportional to the medium flow rate. Scanning electron microscopic studies showed that such populations consist of individual cells embedded within an extracellular polymer matrix.     

Use of the environmentally sensitive fluorophore 4-N,N-dimethylamino-1,8-naphthalimide to study peptoid helix structures

Peptoids, oligomers of N-substituted glycine, have been valuable targets for study and diverse application as peptidomimetics and as nanomaterials. Their conformational heterogeneity has made the study of peptoid structures using high-resolution analyses challenging, limiting our understanding of the physiochemical features that mediate peptoid folding. Here, we introduce a new method for the study of peptoid structure that relies on the environmentally sensitive fluorescence properties of 4-N,N-dimethylamino-1,8-naphthalimide (4-DMN). We have prepared a 4-DMN-functionalized primary amine that is compatible with the traditional submonomer peptoid synthesis methods and incorporated it sequence-specifically into 11 of 13 new peptoids. When included as a peptoid side chain modification, the fluorescence emission intensity of 4-DMN correlates with predictions of the fluorophore's local polarity within a putative structure. 4-DMN fluorescence is maximized when the fluorophore is placed in the middle of the hydrophobic face of an amphiphilic helical peptoid. When the fluorophore is placed near the peptoid terminus or on a polar face of an amphiphilic sequence, 4-DMN fluorescence is diminished. Disruption of the peptoid secondary structure or amphiphilicity also modulates 4-DMN fluorescence. The peptoids' helical secondary structures are moderately disrupted by inclusion of a 4-DMN-modified side chain as evaluated by changes in the peptoids' CD spectral features. This new method for peptoid structure evaluation should be a valuable complement to existing peptoid structural analysis tools.     

Incorporation of chemoselective functionalities into peptoids via solid-phase submonomer synthesis

A simple route to the introduction of a number of chemoselective functional groups into peptoids (oligo(N-substituted glycines)) by an extension of the standard solid-phase submonomer method is reported. The following groups were introduced: aminooxyacetamide, N-(carbamoylmethyl)acetohydrazide, mercaptoacetamide, 2-pyridinesulfenylmercaptoacetamide, and aldehyde-terminated peptoids. The method uses commercially available reagents, is fully compatible with standard peptoid submonomer synthesis conditions, is easily automated, and generates the desired functionalized peptoid in high yield and purity. Peptoids with suitable pairs of chemoselective ligation groups were joined in high yield.