Investigation of N-Substituted Morpholine Structures in an Amphiphilic PDMS-Based Antifouling and Fouling-Release Coating.

Biofouling is a major disruptive process affecting the fuel efficiency and durability of maritime vessel coatings. Previous research has shown that amphiphilic coatings consisting of a siloxane backbone functionalized with hydrophilic moieties are effective marine antifouling and fouling-release materials. Poly(ethylene glycol) (PEG) has been the primary hydrophilic component used in such systems. Recently, the morpholine group has emerged as a promising compact alternative in antifouling membranes but is yet to be studied against marine foulants. In this work, the use of morpholine moieties to generate amphiphilicity in a poly(dimethylsiloxane) (PDMS)-based antifouling and fouling-release coating was explored. Two separate coating sets were investigated. The first set examined the incorporation of an N-substituted morpholine amine, and while these coatings showed promising fouling-release properties for Ulva linza, they had unusually high settlement of spores compared to controls. Based on those results, a second set of materials was synthesized using an N-substituted morpholine amide to probe the source of the high settlement and was found to significantly improve antifouling performance. Both coating sets included PEG controls with varying lengths to compare the viability of the morpholine structures as alternative hydrophilic groups. Surfaces were evaluated through a combination of bubble contact angle goniometry, profilometry, X-ray photoelectron spectroscopy (XPS), and marine bioassays against two soft fouling species, U. linza and Navicula incerta, known to have different adhesion characteristics.

Low Fouling Polysulfobetaines with Variable Hydrophobic Content.

Amphiphilic polymer coatings combining hydrophilic elements, in particular zwitterionic groups, and hydrophobic elements comprise a promising strategy to decrease biofouling. However, the influence of the content of the hydrophobic component in zwitterionic coatings on the interfacial molecular reorganization dynamics and the anti-fouling performance is not well understood. Therefore, coatings of amphiphilic copolymers of sulfobetaine methacrylate 3-[N-2'-(methacryloyloxy)ethyl-N,N-dimethyl]-ammonio propane-1-sulfonate (SPE) are prepared which contain increasing amounts of hydrophobic n-butyl methacrylate (BMA). Their fouling resistance is compared to that of their homopolymers PSPE and PBMA. The photo-crosslinked coatings form hydrogel films with a hydrophilic surface. Fouling by the proteins fibrinogen and lysozyme as well as by the diatom Navicula perminuta and the green algae Ulva linza is assessed in laboratory assays. While biofouling is strongly reduced by all zwitterionic coatings, the best fouling resistance is obtained for the amphiphilic copolymers. Also in preliminary field tests, the anti-fouling performance of the amphiphilic copolymer films is superior to that of both homopolymers. When the coatings are exposed to a marine environment, the reduced susceptibility to silt incorporation, in particular compared to the most hydrophilic polyzwitterion PSPE, likely contributes to the improved fouling resistance.

Amphiphilic Nitroxide-Bearing Siloxane-Based Block Copolymer Coatings for Enhanced Marine Fouling Release.

The buildup of organic matter and organisms on surfaces exposed to marine environments, known as biofouling, is a disruptive and costly process affecting maritime operations. Previous research has identified some of the surface characteristics particularly suited to the creation of antifouling and fouling-release surfaces, but there remains room for improvement against both macrofouling and microfouling organisms. Characterization of their adhesives has shown that many rely on oxidative chemistries. In this work, we explore the incorporation of the stable radical 2,2,6,6-tetramethylpipiderin-1-oxyl (TEMPO) as a component in an amphiphilic block copolymer system to act as an inhibitor for marine cements, disrupting adhesion of macrofouling organisms. Using polystyrene-b-poly(dimethylsiloxane-r-vinylmethysiloxane) block copolymers, pendent vinyl groups were functionalized with TEMPO and poly(ethylene glycol) to construct an amphiphilic material with redox active character. The antifouling and fouling-release performance of these materials was investigated through settlement and removal assays of three model fouling organisms and correlated to surface structure and chemistry. Surfaces showed significant antifouling character and fouling-release performance was increased substantially toward barnacles by the incorporation of stable radicals, indicating their potential for marine antifouling applications.

Degradable hyaluronic acid/chitosan polyelectrolyte multilayers with marine fouling-release properties.

Polysaccharide multilayers consisting of hyaluronic acid and chitosan were prepared by layer-by-layer assembly. To be used in seawater, the multilayers were crosslinked to a different degree using thermal or chemical methods. ATR-FTIR revealed different amide densities as a result of the crosslinking conditions. AFM showed that the crosslinking affected the roughness and swelling behavior of the coatings. The stability and degradability of the multilayers in aqueous environments were monitored with spectroscopic ellipsometry. The resistance of the coatings against non-specific protein adsorption was characterized by SPR spectroscopy. Settlement assays using Ulva linza zoospores and removal assays using the diatom Navicula incerta showed that the slowly degradable coatings were less prone to fouling than the strongly crosslinked ones. Thus, the coatings were a suitable model system to show that crosslinking the multilayers under mild conditions and equipping the coatings with controlled degradation rates enhances their antifouling and fouling-release properties against marine fouling organisms.

Effect of Dipole Orientation in Mixed, Charge-Equilibrated Self-assembled Monolayers on Protein Adsorption and Marine Biofouling.

While zwitterionic interfaces are known for their excellent low-fouling properties, the underlying molecular principles are still under debate. In particular, the role of the zwitterion orientation at the interface has been discussed recently. For elucidation of the effect of this parameter, self-assembled monolayers (SAMs) on gold were prepared from stoichiometric mixtures of oppositely charged alkyl thiols bearing either a quaternary ammonium or a carboxylate moiety. The alkyl chain length of the cationic component (11-mercaptoundecyl)-N,N,N-trimethylammonium, which controls the distance of the positively charged end group from the substrate's surface, was kept constant. In contrast, the anionic component and, correspondingly, the distance of the negatively charged carboxylate groups from the surface was varied by changing the alkyl chain length in the thiol molecules from 7 (8-mercaptooctanoic acid) to 11 (12-mercaptododecanoic acid) to 15 (16-mercaptohexadecanoic acid). In this way, the charge neutrality of the coating was maintained, but the charged groups exposed at the interface to water were varied, and thus, the orientation of the dipoles in the SAMs was altered. In model biofouling studies, protein adsorption, diatom accumulation, and the settlement of zoospores were all affected by the altered charge distribution. This demonstrates the importance of the dipole orientation in mixed-charged SAMs for their inertness to nonspecific protein adsorption and the accumulation of marine organisms. Overall, biofouling was lowest when both the anionic and the cationic groups were placed at the same distance from the substrate's surface.

Antifouling Properties of Dendritic Polyglycerols against Marine Macrofouling Organisms.

Dendritic polyglycerols (PGs) were synthesized and postmodified by grafting of poly(ethylene glycol) (PEG) and polypropylene glycol (PPG) diglycidyl ether groups, and their antifouling and fouling-release properties were tested. Coating characterization by spectroscopic ellipsometry, contact angle goniometry, attenuated total internal reflection-Fourier transform infrared spectroscopy (ATR-FTIR), and atomic force microscopy showed brushlike morphologies with a high degree of microscale roughness and the ability to absorb large amounts of water within seconds. PGs with three different thicknesses were tested in laboratory assays against settlement of larvae of the barnacle Balanus improvisus and against the settlement and removal of zoospores of the alga Ulva linza. Very low coating thicknesses, e.g., 11 nm, reduced the settlement of barnacles, under static conditions, to 2% compared with 55% for an octadecyltrichlorosilane reference surface. In contrast, zoospores of U. linza settled readily but the vast majority were removed by exposure to a shear force of 52 Pa. Both PEG and PPG modification increased the antifouling properties of the PG films, providing a direct comparison of the ultralow fouling properties of all three polymers. Both, the modified and the nonmodified PGs are promising components for incorporation into amphiphilic fouling-resistant coatings.

Layer-by-layer constructed hyaluronic acid/chitosan multilayers as antifouling and fouling-release coatings.

Polyelectrolyte multilayers (PEMs) consisting of hyaluronic acid (HA) and chitosan (Ch) are extensively studied for biomedical applications and suppress bacterial and protein attachment. Here, we prepared and tested HA/Ch PEMs as marine fouling-release coatings. PEMs were constructed by layer-by-layer assembly using spin coating. The multilayers were crosslinked for enhanced stability in the sea water environment by chemical and thermal treatment. Protein-repelling properties of the crosslinked multilayers were investigated by surface plasmon resonance spectroscopy. The marine antifouling and fouling-release properties were tested against the settlement of zoospores of the green alga Ulva linza and the subsequent development and removal of sporelings. With spin coating and thermal crosslinking, a thick yet homogeneous coating was obtained with antifouling properties against marine algal zoospores indicating the potential of these compounds for application in protective coatings.

Transcriptional characterisation of the Exaiptasia pallida pedal disc.

BACKGROUND: Biological adhesion (bioadhesion), enables organisms to attach to surfaces as well as to a range of other targets. Bioadhesion evolved numerous times independently and is ubiquitous throughout the kingdoms of life. To date, investigations have focussed on various taxa of animals, plants and bacteria, but the fundamental processes underlying bioadhesion and the degree of conservation in different biological systems remain poorly understood. This study had two aims: 1) To characterise tissue-specific gene regulation in the pedal disc of the model cnidarian Exaiptasia pallida, and 2) to elucidate putative genes involved in pedal disc adhesion. RESULTS: Five hundred and forty-seven genes were differentially expressed in the pedal disc compared to the rest of the animal. Four hundred and twenty-seven genes were significantly upregulated and 120 genes were significantly downregulated. Forty-one condensed gene ontology terms and 19 protein superfamily classifications were enriched in the pedal disc. Eight condensed gene ontology terms and 11 protein superfamily classifications were depleted. Enriched superfamilies were consistent with classifications identified previously as important for the bioadhesion of unrelated marine invertebrates. A host of genes involved in regulation of extracellular matrix generation and degradation were identified, as well as others related to development and immunity. Ab initio prediction identified 173 upregulated genes that putatively code for extracellularly secreted proteins. CONCLUSION: The analytical workflow facilitated identification of genes putatively involved in adhesion, immunity, defence and development of the E. pallida pedal disc. When defence, immunity and development-related genes were identified, those remaining corresponded most closely to formation of the extracellular matrix (ECM), implicating ECM in the adhesion of anemones to surfaces. This study therefore provides a valuable high-throughput resource for the bioadhesion community and lays a foundation for further targeted research to elucidate bioadhesion in the Cnidaria.

Common Variants in 6 Lipid-Related Genes Discovered by High-Resolution DNA Melting Analysis and Their Association with Plasma Lipids.

BACKGROUND: Total cholesterol was among the earliest identified risk factors for coronary heart disease (CHD). We sought to identify genetic variants in six genes associated with lipid metabolism and estimate their respective contribution to risk for CHD. METHODS: For 6 lipid-associated genes (LCAT, CETP, LIPC, LPL, SCARB1, and ApoF) we scanned exons, 5' and 3' untranslated regions, and donor and acceptor splice sites for variants using Hi-Res Melting® curve analysis (HRMCA) with confirmation by cycle sequencing. Healthy subjects were used for SNP discovery (n=64), haplotype determination/tagging SNP discovery (n=339), and lipid association testing (n=786). RESULTS: In 17,840 bases of interrogated sequence, 90 variant SNPs were identified; 19 (21.1%) previously unreported. Thirty-four variants (37.8%) were exonic(16 non-synonymous), 28 (31.1%) in intron-exon boundaries, and 28 (31.1%) in the 5' and 3' untranslated regions. Compared to cycle sequencing, HRMCA had sensitivity of 99.4% and specificity of 97.7%. Tagging SNPs (n=38) explained >90% of the variation in the 6 genes and identified linkage disequilibrium (LD) groups. Significant beneficial lipid profiles were observed for CETP LD group 2, LIPC LD groups 1 and 7, and SCARB1 LD groups 1, 3 and 4. Risk profiles worsened for CETP LD group 3, LPL LD group 4. CONCLUSIONS: These findings demonstrate the feasibility, sensitivity, and specificity of HRMCA for SNP discovery. Variants identified in these genes may be used to predict lipid-associated risk and reclassification of clinical CHD risk.

Joint effects of common genetic variants from multiple genes and pathways on the risk of premature coronary artery disease.

OBJECTIVE: The aim of this study is to discover common variants in 6 lipid metabolic genes and construct and validate a genetic risk score (GRS) based on the joint effects of genetic variants in multiple genes from lipid and other pathobiologic pathways. BACKGROUND: Explaining the genetic basis of coronary artery disease (CAD) is incomplete. Discovery and aggregation of genetic variants from multiple pathways may advance this objective. METHODS: Premature CAD cases (n = 1,947) and CAD-free controls (n = 1,036) were selected from our angiographic registry. In a discovery phase, single nucleotide polymorphisms (SNPs) at 56 loci from internal discovery and external reports were tested for associations with biomarkers and CAD: 28 promising SNPs were then tested jointly for CAD associations, and a GRS consisting of SNPs contributing independently was constructed and validated in a replication set of familial cases and population-based controls (n = 1,320). RESULTS: Five variants contributed jointly to CAD prediction in a multigenic GRS model: odds ratio 1.24 (95% CI 1.16-1.33) per risk allele, P = 8.2 x 10(-11), adjusted OR 2.03 (1.53-2.70), fourth versus first quartile. 5-SNP genetic risk score had minor impact on area under the receiver operating characteristic curve (P > .05) but resulted in substantial net reclassification improvement: 0.16 overall, 0.28 in intermediate-risk patients (both P < .0001). GRS(5) predicted familial CAD with similar magnitude in the validation set. CONCLUSIONS: The Intermountain Healthcare's Coronary Genetics study demonstrates the ability of a multigenic, multipathway GRS to improve discrimination of angiographic CAD. Genetic risk scores promise to increase understanding of the genetic basis of CAD and improve identification of individuals at increased CAD risk.

Multiple less common genetic variants explain the association of the cholesteryl ester transfer protein gene with coronary artery disease.

OBJECTIVES: The objective of this study was to identify associations of the cholesteryl ester transfer protein (CETP) gene with coronary artery disease (CAD) with tagging (t) single nucleotide polymorphisms (SNPs) chosen to optimally account for intra-genic variation. BACKGROUND: The CETP gene plays a critical role in lipoprotein metabolism, but the common and well-studied TaqIB variant is inconsistently predictive of CAD. METHODS: From a deoxyribonucleic acid bank of 10,020 individuals, nondiabetic nonsmoking patients (n = 4,811) with angiographically defined, clinically significant CAD (> or =70% stenosis) or normal coronaries were genotyped for 11 CETP tSNPs. Myocardial infarction (MI) and lipid levels were evaluated as secondary end points. RESULTS: Analysis of single tSNPs, corrected for multiple comparisons (p < 0.00485), identified allele +1086A to be associated with CAD (p = 0.0034). Suggestive allelic and significant genotypic associations were found for -631AA (odds ratio [OR] = 3.95, p = 0.004 vs. CC) and +2389GA (OR = 1.21, p = 0.003 vs. GG). Haplotype analysis by linkage disequilibrium (LD) group revealed a CAD association for LD group B (p = 0.0025 across T+1086A, C+878T, C+408T) and near significance for LD group A (p = 0.013 across C-631A, MspI, G+2389A). A weak protective trend for TaqIB was eliminated by adjustment for other tSNPs, and haplotype analyses suggested that TaqIB was simply a marker for other tSNPs or haplotypes. No tSNP or haplotype associations with MI were found. CONCLUSIONS: Multiple, less common SNPs and haplotype variants underlie CETP-related CAD risk, for which the common TaqIB variant is simply a poor marker. The occurrence of risk-related variants on separate haplotypes suggests genetic-risk complexity and allelic heterogeneity. (Database Registry of the Intermountain Heart Collaborative Study; http://clinicaltrials.gov/ct/show/NCT00406185?order=1; NCT00406185).

Genotypes of the cytochrome p450 isoform, CYP2C9, and the vitamin K epoxide reductase complex subunit 1 conjointly determine stable warfarin dose: a prospective study.

BACKGROUND: Warfarin has a narrow therapeutic range and wide inter-individual dosing requirements that may be related to functional variants of genes affecting warfarin metabolism (i.e., CYP2C9) and activity (i.e., vitamin K epoxide reductase complex subunit 1-VKORC1). We hypothesized that variants in these two genes explain a substantial proportion of variability in stable warfarin dose and could be used as a basis for improved dosing algorithms. METHODS: Consecutive consenting outpatients (n = 213) with stable INR (2-3) for >1 month were enrolled. Buccal DNA was extracted using a Qiagen mini-column and CYP2C9*2 and VKORC1 genotyping performed by the Taqman 3' nuclease assay. Sequencing for CYP2C9*3, genotyping was done using Big Dye v3.1 terminator chemistry Dose by genotype was assessed by linear regression. RESULTS: Weekly warfarin dose averaged 30.8 +/- 13.9 mg/week; average INR was 2.42 +/- 0.72. CYP2C9*2/*3 genotype distribution was: CC/AA (wild-type [WT]) = 71.4%, CT/AA = 18.3%, CC/AC = 9.4%, and CT/AC = 1%; VKORC1 genotypes were CC (WT) = 36.6%, CT = 50.7%, and TT = 12.7%. Warfarin doses (mg/week) varied by genotype: for CYP2C9, 33.3 mg/week for WT (CC/AA), 27.2 mg/week for CT/AA (P = 0.04 vs. WT), 23.0 mg/week for CC/AC (P = 0.003), and 6.0 mg/week for CT/AC (P < 0.001), representing dose reductions of 18-31% for single and 82% for double variant carriers; for VKORC1: 38.4 mg/week for WT (CC), 28.6 mg/week for CT (P < 0.001 vs. WT), 20.95 mg/week for TT (P < 0.001). In multiple linear regression, genotype was the dominant predictor of warfarin dose (P = 2.4 x 10(-15)); weak predictors were age, weight, and sex. Genotype-based modeling explained 33% of dose-variance, compared with 12% for clinical variables alone. CONCLUSION: In this large prospective study of warfarin genetic dose-determinants, carriage of a single or double CYP2C9 variant, reduced warfarin dose 18-72%, and of a VKORC1 variant by 65%. Genotype-based modeling explained almost one-half of dose-variance. A quantitative dosing algorithm incorporating genotypes for 2C9 and VKORC1 could substantially improve initial warfarin dose-selection and reduce related complications.

Lipoprotein-associated phospholipase A2 independently predicts the angiographic diagnosis of coronary artery disease and coronary death.

BACKGROUND: Whereas C-reactive protein (CRP) is a nonspecific marker of coronary artery disease (CAD) and cardiovascular (CV) events, Lp-PLA2 may be a nonvariable inflammatory biomarker. We evaluated the independent association of lipoprotein-associated phospholipase A2 (Lp-PLA2) to angiographic CAD and CV events adjusting for standard factors, lipids, and CRP. METHODS: Lipoprotein-associated phospholipase A2 (PLAC test, diaDexus, Inc, San Francisco, CA) and CRP were measured from samples donated by consecutive consenting patients (N = 1493) enrolled in the registry of the Intermountain Heart Collaborative Study. All patients underwent coronary angiography (1996-1998) for CAD determination and were followed for 6.7 +/- 0.5 years (range 5.7-7.9 years) for CV events (death [including all-cause, CAD, and non-CAD CV death], myocardial infarction, and cerebrovascular accident). RESULTS: Lipoprotein-associated phospholipase A2 weakly correlated with lipids (low-density lipoprotein: r = 0.22, P < .001; high-density lipoprotein: r = -0.13, P < .001), but not CRP (r = 0.03, P = .26). Increasing quartile (Q) of Lp-PLA2 predicted greater the presence of CAD (vs Q1) for Q2 (adjusted odds ratio [OR] 1.15, 95% CI 0.78-1.71, P = .48), for Q3 (OR 1.53, 95% CI 1.02-2.31, P = .042), and for Q4 (OR 2.44, 95% CI 1.58-3.79, P < .001), although CRP was also predictive (vs Q1, Q2: OR 1.47, P = .057; Q3: OR 1.93, P = .002; Q4: OR 3.43, P < .001). In Cox regression, Lp-PLA2 predicted CAD death (vs Q1; Q2: adjusted hazard ratio [HR] 1.27, 95% CI 0.58-2.78, P = .55; Q3: HR 2.18, 95% CI 1.04-4.57, P = .04; Q4: HR 1.73, 95% CI 0.84-3.61, P = .14). CONCLUSION: Lipoprotein-associated phospholipase A2 was confirmed to predict the presence of CAD, even among patients undergoing coronary angiography. Uniquely, Lp-PLA2 predicted the risk of CAD death, but not all-cause death, myocardial infarction, or cerebrovascular accident.

High-resolution characterization of linkage disequilibrium structure and selection of tagging single nucleotide polymorphisms: application to the cholesteryl ester transfer protein gene.

Full characterization of intragenic variation may improve candidate gene associations. This study selected tagging (t) single nucleotide polymorphisms (SNPs) to comprehensively represent genetic variability in the cholesteryl ester transfer protein (CETP) gene. Nineteen SNPs were identified in 50 unrelated individuals in the SNP discovery phase, and 13 intronic SNPs were added from the literature. These 32 SNPs were genotyped in 339 apparently healthy individuals and 190 coronary artery disease (CAD) patients. Using phased haplotypes, linkage disequilibrium (LD) structure was characterized and tSNPs selected using a principal component analysis (PCA) method. In healthy individuals, seven LD groups were identified that accounted for 93.4% of the observed genetic variation. These LD groups highlighted a complex LD structure for CETP, including both recombination and mutation, and eleven tSNPs were selected. Among CAD patients the results were essentially the same. Results from PCA using diploid genotype data were reasonably comparable. Finally, the selected tSNPs successfully represented the association evidence discovered for all of the other SNPs studied. This study provides an optimal set of tSNPs for association analyses of CETP. The observed complexity of LD structure highlights the importance of using methods, such as PCA, that allow for multiple dynamics in intragenic LD structure.

Comparison of differing C-reactive protein assay methods and their impact on cardiovascular risk assessment.

A medium-sensitivity assay for C-reactive protein (CRP) was compared with a high-sensitivity, enhanced immunoturbidimetric assay in 803 angiographically studied patients. Different absolute CRP values were found by the assays, but there was a high correlation by quartile rank and similar predictive values for death and myocardial infarction. This suggests that the conclusions of previous studies performed using the medium-sensitivity assay are still valid but that cross-study comparisons should use percentile rank.