Mini-review: combinatorial approaches for the design of novel coating systems

Combinatorial and high throughput experimental methods are being applied to the design and development of novel polymers and coatings used in a number of application areas. Methods have been developed for polymer synthesis and screening and for the development of polymer thin film and coating libraries and the screening of these libraries for key properties such as surface energy and modulus. Combinatorial and high throughput methods enable the efficient exploration of a large number of compositional variables over a wide range. In the development of coatings for use in the marine environment, the key challenge is in the development of screening methods that can predict good performance. A number of assays are under development that will permit the rapid screening of the interaction of coatings with representative marine organisms.     

Thermoplastic,rubber-like marine antifouling coatings with micro-structures via mechanical embossing

Abstract

New processing routes and materials for non-biocidal, antifouling (AF) coatings with an improved performance are currently much sought after for a range of marine applications. Here, the processing, physical properties and marine AF performance of a fluorinated coating based on a thermoplastic (non-crosslinked) fluorinated polymer are reported. It was found that the addition of lubricating oil and hydrodynamic drag reducing microstructures improved the AF properties substantially, i.e. the settlement of a marine biofilm, containing mixed microalgae including diatoms, was reduced to low levels. More importantly, the remaining fouling was removed from the coatings at low hydrodynamic shear rates and promising AF properties were obtained. Moreover, additional potential benefits were revealed originating from the thermoplastic nature of the coating material which might result in significant cost reductions.     

Minireview: Challenges of High Throughput Screening Against Cell Surface Receptors

Abstract

Excessive or inappropriate activation of cell surface receptors can mediate the development of disease. Receptors, therefore, are a focus for drug discovery activities. Empirical screening is important in the search for novel compounds acting at receptors. Technical developments and the application of molecular biology have facilitated access to receptors of interest and have provided efficient screening methods capable of very high throughput. Reliability in high throughput screening requires the use of appropriate methodology, good screen design and effective validation and quality control processes. Validation should aim to establish that the basic experimental design is sound. In developing software to handle high throughput screening data, a fundamental requirement is to provide performance monitoring and error trapping facilities. Additional requirements are automatic data capture from instruments, on-line data reduction and analysis and transfer of results to central databases. As data volumes increase through effective high throughput screening, conventional interrogation methods become less appropriate and are being augmented by newer computing techniques referred to as knowledge mapping or database mining. Targeting cell surface receptors has been very successful as an approach to drug discovery. If the challenges of high throughput empirical screening are addressed effectively, cell surface receptors will provide new opportunities for improved therapy in the coming years.     

Combinatorial chemistry in the agrosciences

Combinatorial chemistry and high throughput screening have had a profound effect upon the way in which agrochemical companies conduct their lead discovery research. The article reviews recent applications of combinatorial synthesis in the lead discovery process for new fungicides, herbicides and insecticides. The role and importance of bioavailability guidelines, natural products, privileged structures, virtual screening and X-ray crystallographic protein structures on the design of solid- and solution-phase compound libraries is discussed and illustrated.     

A comparison between different fouling-release elastomer coatings containing surface-active polymers

Surface-active polymers derived from styrene monomers containing siloxane (S), fluoroalkyl (F) and/or ethoxylated (E) side chains were blended with an elastomer matrix, either poly(dimethyl siloxane) (PDMS) or poly(styrene-b-(ethylene-co-butylene)-b-styrene) (SEBS), and spray-coated on top of PDMS or SEBS preformed films. By contact angle and X-ray photoelectron spectroscopy measurements, it was found that the surface-active polymer preferentially populated the outermost layers of the coating, despite its low content in the blend. However, the self-segregation process and the response to the external environment strongly depended on both the chemistry of the polymer and the type of matrix used for the blend. Additionally, mechanical testing showed that the elastic modulus of SEBS-based coatings was one order of magnitude higher than that of the corresponding PDMS-based coatings. The coatings were subjected to laboratory bioassays with the marine alga Ulva linza. PDMS-based coatings had superior fouling-release properties compared to the SEBS-based coatings.     

Toxicity Screening of a Combinatorial Library: Correlation of Cytotoxicity and Gene Induction to Compound Structure

Combinatorial chemistry has increased the number of compounds available for efficacy and safety assessment by several orders of magnitude and has made high throughput assays essential. To test whether higher throughput toxicity assays could be of utility in screening compounds in early development, a selected set of combinatorial chemistry compounds was screened for induction of 70-Kd heat shock protein (HSP70) and 45-Kd growth arrest and DNA damage protein (GADD45) mRNA levels as well as cytotoxicity, in HepG2 cells, using a 96-well microtiter plate format. Both assays, the branched DNA (Quantigene) assay for mRNA levels and MTT for cytotoxicity, were robust enough to be incorporated into a screening format using a single replicate and a single concentration of compound. Significantly, a structure/toxicity correlation was established with this set of compounds with cytotoxicity and gene induction patterns linked to compound structure. Therefore, this type of early screening may be useful in identifying toxic substituents, enabling the design of libraries with less potential for toxicity. While structure/toxicity correlations were observed, no relationship was observed between GADD45 gene induction and mutagenesis as measured by the Ames bacterial reverse mutation assay.     

USE OF FLUORESCENCE POLARIZATION DETECTION FOR THE MEASUREMENT OF FLUOPEPTIDE™ BINDING TO G PROTEIN-COUPLED RECEPTORS

ABSTRACT

G protein-coupled receptors (GPCRs) represent the single largest molecular target of therapeutic drugs currently on the market, and are also the most common target in high throughput screening assays designed to identify potential new drug candidates. A large percentage of these assays are now formatted as radioligand binding assays. Fluorescence polarization ligand binding assays can offer a non-rad alternative to radioligand binding assays. In addition, fluorescence polarization assays are a homogenous format that is easy to automate for high throughput screening. We have developed a series of peptide ligands labeled with the fluorescent dye BODIPY® TMR whose binding to GPCRs can be detected using fluorescence polarization methodology. BODIPY® TMR has advantages over the more commonly used fluorescein dye in high throughput screening (HTS) assays due to the fact that its excitation and emission spectra are red-shifted approximately 50 nm relative to fluorescein. Assays based on BODIPY® TMR ligands are therefore less susceptible to interference from tissue auto-fluorescence in the assay matrix, or the effects of colored or fluorescent compounds in the screening libraries. A series of BODIPY® TMR labeled peptides have been prepared that bind to a range of GPCRs including melanin concentrating hormone, bradykinin, and melanocortin receptors. Conditions have been optimized in order to utilize a comparable amount of receptor membrane preparation as is used in a radioligand binding assay. The assays are formatted in 384-well microplates with a standard volume of 40 µL. We have compared the assays across the different fluorescence polarization (FP) readers available to determine the parameters for each instrument necessary to achieve the required precision.     

Microfluidic synthesis of composite cross‐gradient materials for investigating cell–biomaterial interactions

Combinatorial material synthesis is a powerful approach for creating composite material libraries for the high‐throughput screening of cell–material interactions. Although current combinatorial screening platforms have been tremendously successful in identifying target (termed “hit”) materials from composite material libraries, new material synthesis approaches are needed to further optimize the concentrations and blending ratios of the component materials. Here we employed a microfluidic platform to rapidly synthesize composite materials containing cross‐gradients of gelatin and chitosan for investigating cell–biomaterial interactions. The microfluidic synthesis of the cross‐gradient was optimized experimentally and theoretically to produce quantitatively controllable variations in the concentrations and blending ratios of the two components. The anisotropic chemical compositions of the gelatin/chitosan cross‐gradients were characterized by Fourier transform infrared spectrometry and X‐ray photoelectron spectrometry. The three‐dimensional (3D) porous gelatin/chitosan cross‐gradient materials were shown to regulate the cellular morphology and proliferation of smooth muscle cells (SMCs) in a gradient‐dependent manner. We envision that our microfluidic cross‐gradient platform may accelerate the material development processes involved in a wide range of biomedical applications. Biotechnol. Bioeng. 2011; 108:175–185. © 2010 Wiley Periodicals, Inc.     

Variation in adhesion strength of Balanus eburneus,crassostrea virginica and hydroides dianthus to fouling‐release coatings

This study compared the shear adhesion strength of barnacles, oysters and tubeworms on eight RTV 11‐based silicone fouling‐release coatings containing different silicone oil additives. It was found that adhesion strength differed among species and coating types. In most cases, oysters and tubeworms had higher adhesion strengths than barnacles. Barnacle adhesion strength was reduced on all coatings containing oil additives; however, this was not generally true for oysters and tubeworms. The difference in the adhesion strength among the three organisms tested in this study emphasizes the importance of understanding the fundamental interaction between marine invertebrate adhesives and the substratum.     

随机突变文库构建与筛选研究进展

定向进化是一个循环过程,在构建多样化基因序列和筛选功能基因变体之间交替进行.该技术目前已被广泛应用于DNA序列、基因功能和蛋白质结构的优化和分析.定向进化包括随机基因文库的生成、基因在合适宿主中的表达和突变文库的筛选.构建基因文库的关键是库容量和突变多样性,而筛选变体的关键是高灵敏度和高通量.文中讨论了定向进化技术的最...     

Diatom community structure on commercially available ship hull coatings

Diatoms are primary colonizers of both antifouling and fouling-release ship hull coatings. There are few published studies which report on diatom community development on modern ship hull coatings. This study reports diatom communities on eight commercial marine ship hull coatings exposed at three static immersion sites along the east coast of Florida, viz. Daytona, Sebastian, and Miami. The coatings tested were three ablative copper systems (Ameron ABC-3, International BRA-640, and Hempel Olympic 76600), two copper-free biocidal systems (E-Paint SN-1, Sherwin Williams HMF), and three fouling-release (FR) systems (International Intersleek 700, International Intersleek 900, and Hempel Hempasil). One hundred and twenty-seven species comprising 44 genera were identified, including some of the more commonly known foulers, viz. Achnanthes, Amphora, Cocconeis, Entomoneis, Licmophora, Melosira, Navicula, Nitzschia, Synedra, and Toxarium. A significant difference was seen among sites, with the more estuarine site, Sebastian, having lower overall diatom abundance and higher diversity than Daytona and Miami. Copper coatings were primarily fouled by Amphora delicatissima and Entomoneis pseudoduplex. Copper-free coatings were fouled by Cyclophora tenuis, A. delicatissima, Achnanthes manifera, and Amphora bigibba. FR surfaces were typified by C. tenuis, and several species of Amphora. The presence of C. tenuis is new to the biofouling literature, but as new coatings are developed, this diatom may be one of many that prove to be problematic for static immersion. Results show coatings can be significantly influenced by geographical area, highlighting the need to test ship hull coatings in locations similar to where they will be utilized.     

Interplay of metagenomics and in vitro compartmentalization

In recent years, the application of approaches for harvesting DNA from the environment, the so‐called, ‘metagenomic approaches’ has proven to be highly successful for the identification, isolation and generation of novel enzymes. Functional screening for the desired catalytic activity is one of the key steps in mining metagenomic libraries, as it does not rely on sequence homology. In this mini‐review, we survey high‐throughput screening tools, originally developed for directed evolution experiments, which can be readily adapted for the screening of large libraries. In particular, we focus on the use of in vitro compartmentalization (IVC) approaches to address potential advantages and problems the merger of culture‐independent and IVC techniques might bring on the mining of enzyme activities in microbial communities.     

Investigation of the antimicrobial activity of soy peptides by developing a high throughput drug screening assay

BackgroundAntimicrobial resistance is a great concern in the medical community, as well as food industry. Soy peptides were tested against bacterial biofilms for their antimicrobial activity. A high throughput drug screening assay was developed using microfluidic technology, RAMAN spectroscopy, and optical microscopy for rapid screening of antimicrobials and rapid identification of pathogens.MethodsSynthesized PGTAVFK and IKAFKEATKVDKVVVLWTA soy peptides were tested against Pseudomonas aeruginosa and Listeria monocytogenes using a microdilution assay. Microfluidic technology in combination with Surface Enhanced RAMAN Spectroscopy (SERS) and optical microscopy was used for rapid screening of soy peptides, pathogen identification, and to visualize the impact of selected peptides.ResultsThe PGTAVFK peptide did not significantly affect P. aeruginosa, although it had an inhibitory effect on L. monocytogenes above a concentration of 625 µM. IKAFKEATKVDKVVVLWTA was effective against both P. aeruginosa and L. monocytogenes above a concentration of 37.2 µM. High throughput drug screening assays were able to reduce the screening and bacterial detection time to 4 h. SERS spectra was used to distinguish the two bacterial species.ConclusionsPGTAVFK and IKAFKEATKVDKVVVLWTA soy peptides showed antimicrobial activity against P. aeruginosa and L. monocytogenes. Development of high throughput assays could streamline the drug screening and bacterial detection process.General significanceThe results of this study show that the antimicrobial properties, biocompatibility, and biodegradability of soy peptides could possibly make them an alternative to the ineffective antimicrobials and antibiotics currently used in the food and medical fields. High throughput drug screening assays could help hasten pre-clinical trials in the medical field.     

Antibacterial activity of resin based coatings containing marine microbial extracts

Bioactive agents produced by marine surface‐associated bacteria have been incorporated into experimental antifouling coatings. It was found that coating formulation can affect the action spectra of bacterial extracts involved. Little work has been done on the effect of permeability on the leaching of bioactive compounds from antifouling coatings. The formulations with 1.5% and 1.0% alginate produced significantly larger zones (p < 0.05) than with 0.5% alginate. Test formulations continued to leach active compounds after continuous immersion in seawater for 120 h. The bacterial strains used to produce the active compounds were identified by biochemical tests and/or the analysis of the DNA from a 500 bp section of the gene coding for the 16S rRNA subunit. Strains used were tentatively identified as Planococcus citreus, seven Bacillus strains (including three Bacillus pumilus and three Bacillus lautus strains), Cytophaga fucicola, Cytophaga uliginosa and a Pseudoalteromonas spp.     

Amphiphilic hydrolyzable polydimethylsiloxane-b-poly(ethyleneglycol methacrylate-co-trialkylsilyl methacrylate) block copolymers for marine coatings. II. Antifouling laboratory tests and field trials

Abstract

Poly(dimethylsiloxane) (PDMS) elastomer coatings containing an amphiphilic hydrolyzable diblock copolymer additive were prepared and their potential as marine antifouling and antiadhesion materials was tested. The block copolymer additive consisted of a PDMS first block and a random poly(trialkylsilyl methacrylate (TRSiMA, R?=?butyl, isopropyl)-co-poly(ethyleneglycol) methacrylate (PEGMA) copolymer second block. PDMS-b-TRSiMA block copolymer additives without PEGMA units were also used as additives. The amphiphilic character of the coating surface was assessed in water using the captive air bubble technique for measurements of static and dynamic contact angles. The attachment of macro- and microorganisms on the coatings was evaluated by field tests and by performing adhesion tests to the barnacle Amphibalanus amphitrite and the green alga Ulva rigida. All the additive-based PDMS coatings showed better antiadhesion properties to A. amphitrite larvae than to U. rigida spores. Field tests provided meaningful information on the antifouling and fouling release activity of coatings over an immersion period of 23?months.     

Combining a bio-based polymer and a natural antifoulant into an eco-friendly antifouling coating

Abstract

Biodegradable polymers are promising binders and carriers for natural antifoulants. In the present study, an antifouling (AF) coating was developed by adding a non-toxic AF compound (butenolide) to a bio-based and biodegradable poly(lactic acid)-based polyurethane. Mass loss measurement showed that the polymer degraded in seawater at a rate of 0.013?mg cm^?2?day^?1. Measurements showed that butenolide was released from the coatings into seawater over a period of at least three months. Both the concentration of butenolide in the coatings and the ambient temperature determined the release rate of butenolide. The results further demonstrate that incorporating rosin into the coatings increase the self-renewal rate of the polymer and facilitated the long-term release of butenolide from the coating. The results show that poly(lactic acid)-based polyurethane is a suitable polymer for butenolide-based AF coatings.     

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.