Reduction of Staphylococcus aureus and Pseudomonas aeruginosa colonisation on PVC through covalent surface attachment of fluorinated thiols

OBJECTIVES: This study reports the development, characterisation and microbiological testing of surface-modified polyvinylchloride (PVC) films for the purpose of reducing bacterial adherence.

METHODS: Irreversible covalent surface modification was achieved via nucleophilic substitution of fluorinated thiol-terminated compounds onto the polymer backbone. Four fluorinated modifiers, 2,3,5,6-tetrafluorothiophenol (TFTP), 4-(trifluoromethyl)thiophenol (TFMTP), 3,5-bis(trifluoromethyl)benzenethiol (BTFMBT) and 3,3,4,4,5,5,6,6,7, 7,8,8,9,9,10,10,10-heptadecafluoro-decane-1-thiol (HDFDT), were investigated. Modification was confirmed using attenuated total reflectance infrared spectroscopy; Raman mapping demonstrated that modification was homogenous on the macroscopic scale. The influence of fluorination on surface hydrophobicity was studied by contact angle analysis. The effect on microbial adherence was examined using Pseudomonas aeruginosa and Staphylococcus aureus.

KEY FINDINGS: The resultant changes in contact angle relative to control PVC ranged from -4 degrees to +14 degrees . In all cases, adherence of P. aeruginosa and S. aureus was significantly reduced relative to control PVC, with adherence levels ranging from 62% and 51% for TFTP-modified PVC to 32% and 7% for TFMTP-modified PVC.

CONCLUSIONS: These results demonstrate an important method in reducing the incidence of bacterial infection in PVC medical devices without compromising mechanical properties.

Modification of Ag nanoparticles with mixed thiols for improved SERS detection of poorly adsorbing target molecules: detection of MDMA

Here we report an example of a mixed thiol monolayer on the surface of Ag nanoparticles which promotes adsorption and quantitative SERS detection of 3,4-methylenedioxymethamphetamine (MDMA, “Ecstasy”); the thiols in the mixed monolayers act synergistically since MDMA does not adsorb onto colloids modified with either of the thiols separately.

Rates for repair of pBR 322 DNA radicals by thiols as measured by the gas explosion technique: evidence that counter-ion condensation and co-ion depletion are significant at physiological ionic strength.

Rates of rapair of pBR 322 plasmid DNA radicals by thiols of varying net charge (Z) at pH 7 and physiological ionic strength were measured using the oxygen explosion technique. The extent of conversion of supercoiled to relaxed circular plasmid was measured by HPLC as a function of the time of oxygen exposure before or after irradiation, the time-courses being fitted by a pseudo-first-order kinetic expression with k1 = k2[RSH]. Values of k2 (M-1 S-1) were: 2.1 x 10(5) (GSH, Z = -1), 1.4 x 10(6) (2-mercaptoethanol, Z = 0), 1.2 x 10(7) (cysteamine, Z = +1), 6.6 x 10(7) (WR-1065 or N-(2-mercaptoethyl)-1,3-diamino?? propane, Z = +2). The approximately 6-fold increase in rate with each unit increase in Z is attributed to concentration of cationic thiols near DNA as a consequence of counter-ion condensation and reduced levels of anionic thiols near DNA owing to co-ion depletion. The results are quantitatively consistent with chemical repair as a significant mechanism for radioprotection of cells by neutral and cationic thiols under aerobic conditions, but indicate that repair by GSH will compete effectively with oxygen only at low oxygen tension.

Role of charge in the radioprotection of E. coli by thiols.

The role of net charge (Z) of thiols in their ability to radioprotect cells has been investigated in a glutathione (GSH)-deficient strain of E. coli. This strain, 7, is deficient in the enzyme gamma-glutamylcysteine synthetase and allows the effects of added low molecular weight thiols to be studied. Using the gas explosion system it is possible to measure the chemical repair of the free-radical precursors of lethal lesions by thiols in intact cells. The first-order chemical repair rate in strain 7 is 280s(-1) in comparison with 1100s(-1) in the wild-type strain 1157. From the measured difference in the intracellular concentration of GSH between the wild-type and the mutant, this gives a second-order repair rate, k(r)'s of 1.23 +/- 0.3 X 10(5) dm(3)mol(-1)s(-1). Measurement of intracellular thiol levels after addition of various low molecular weight thiols showed that uptake was rapid, leading to stable thiol levels within 1 min. The ratios of the intracellular to extracellular concentrations (C-in/C-out) were 0.74 for 3-mercaptopropionic acid (Z=-1), 0.56 for 2-mercaptoethanol (Z=0), 1.47 for cysteamine (Z=+1) and 1.04 for WR1065 (Z=+2). The k(r)'s for these thiols were 1.3 +/- 0.5 X 10(5) dm(3)mol(-1)s(-1) for 30-mercaptopropionic acid, 3.3 +/- 1.6 x 10(5) dm(3)mol(-1)s(-1) for 2-mercaptoethanol, 3.9 +/- 1.1 X 10(5) dm(3)mol(-1)s(-1) for cysteamine and 2.7 +/- 1.1 X 10(6) dm(3)mol(-1)s(-1) for WR1065. These are lower and increase less with charge than previously published values for chemical repair in isolated pBR322 DNA, probably because of the association of nucleoproteins and polyamines with the cellular DNA of E. coli. However, the approximate three-fold increase in k(r) per unit increase in Z shows that the counter-ion condensation and co-ion depletion are important in determining the effectiveness of charged thiols in the radioprotection of E. coli.

An investigation of clustered radiation-induced lesions in DNA

Recent track structure modelling studies indicate that radiation induced damage to DNA consists of a spectrum of different lesions of varying complexity. There is considerable evidence to suggest that, in repair-proficient systems, it is only the small proportion of more complex forms that is responsible for most of the biological effect. The complex lesions induced consist initially of clustered radical sites and a knowledge of their special chemistry is important in modelling how they react to form the more stable products that are processed by the repair systems. However, much of the current understanding of the chemical stage of radiation has developed from single-radical systems and there is a need to translate this to the more complex reactions that are likely to occur at the important multiple radical sites. With low LET radiation, DNA dsb may derive either from single-radical attack that damages both strands by a transfer mechanism, or from pairs of radical sites induced in close proximity, with one or more radical on each strand. With high LET radiation, modelling studies indicate that there is an increased probability of dsb arising from sites with more than two radical centres, leading to a greater frequency of more complex types of break. The spectrum of these lesions depends on the overall outcome of consecutive physical and chemical processes. The initial pattern of radical damage is determined by the energy depositions on and around the DNA, according to the type of radiation. This pattern is then modified by scavengers that inhibit the formation of radicals on the DNA, and by agents that either chemically repair (e.g. thiols) or fix (e.g. oxygen) a large fraction of these radicals. The reaction kinetics associated with clustered radical sites will differ from those of single sites: (1) because of the opportunities for interactions between the radicals themselves; and (2) because certain endpoints, e.g. a dsb, may require a combination of the products of two or more radicals. Fast response techniques using pulsed low and high LET irradiation have been established to measure the reactions of radical sites on pBR322 plasmid DNA with oxygen and thiols with a view to obtaining information about cluster size. This paper describes experimental approaches to explore the role of the chemical stage of the radiation effect in relation to lesion complexity.

A multiple-radical model for radiation action on DNA and the dependence of OER on LET

We have a developed a multiple-radical model of the chemical modification reactions involving oxygen and thiols relevant to the interactions of ionizing radiations with DNA. The treatment is based on the Alper and Howard-Flanders equation but considers the case where more than one radical may be involved in the production of lesions in DNA. This model makes several predictions regarding the induction of double strand breaks in DNA by ionizing radiation and the role of sensitizers such as oxygen and protectors such as thiols which act at the chemical phase of radiation action via the involvement of free radicals. The model predicts a decreasing OER with increasing LET on the basis that as radical multiplicity increases so will the probability that, even under hypoxia, damage will be fixed and lead to lesion production. The model can be considered to provide an alternative hypothesis to those of 'interacting radicals' or of 'oxygen-in-the-track'.

Label-free detection of nanomolar unmodified single- and double-stranded dna by using surface-enhanced raman spectroscopy on Ag and Au colloids

Unlabelled single- and double-stranded DNA (ssDNA and dsDNA, respectively) has been detected at concentrations =10-9?M by surface-enhanced Raman spectroscopy. Under appropriate conditions the sequences spontaneously adsorbed to the surface of both Ag and Au colloids through their nucleobases; this allowed highly reproducible spectra with good signal-to-noise ratios to be recorded on completely unmodified samples. This eliminated the need to promote absorption by introducing external linkers, such as thiols. The spectra of model ssDNA sequences contained bands of all the bases present and showed systematic changes when the overall base composition was altered. Initial tests also showed that small but reproducible changes could be detected between oligonucleotides with the same bases arranged in a different order. The spectra of five ssDNA sequences that correspond to different strains of the Escherichia coli bacterium were found to be sufficiently composition-dependent so that they could be differentiated without the need for any advanced multivariate data analysis techniques.

Controlling Assembly of Mixed Thiol Monolayers on Silver Nanoparticles to Tune Their Surface Properties

Modifying the surfaces of metal nanoparticles with self-assembled monolayers of functionalized thiols provides a simple and direct method to alter their surface properties. Mixed self-assembled monolayers can extend this approach since, in principle, the surfaces can be tuned by altering the proportion of each modifier that is adsorbed. However, this works best if the composition and microstructure of the monolayers can be controlled. Here, we have modified preprepared silver colloids with binary mixtures of thiols at varying concentrations and modifier ratios. Surface-enhanced Raman spectroscopy was then used to determine the effect of altering these parameters on the composition of the resulting mixed monolayers. The data could be explained using a new model based on a modified competitive Langmuir approach. It was found that the composition of the mixed monolayer only reflected the ratio of modifiers in the feedstock when the total amount of modifier was sufficient for approximately one monolayer coverage. At higher modifier concentrations the thermodynamically favored modifier dominated, but working at near monolayer concentrations allowed the surface composition to be controlled by changing the ratios of modifiers. Finally, a positively charged porphyrin probe molecule was used to investigate the microstructure of the mixed monolayers, i.e., homogeneous versus domains. In this case the modifier domains were found to be <2 nm.

Identification of an arsenic tolerant double mutant with a thiol-mediated component and increased arsenic tolerance in phyA mutants

A genetic screen was performed to isolate mutants showing increased arsenic tolerance using an Arabidopsis thaliana population of activation tagged lines. The most arsenic-resistant mutant shows increased arsenate and arsenite tolerance. Genetic analyses of the mutant indicate that the mutant contains two loci that contribute to arsenic tolerance, designated ars4 and ars5. The ars4ars5 double mutant contains a single T-DNA insertion, ars4, which co-segregates with arsenic tolerance and is inserted in the Phytochrome A (PHYA) gene, strongly reducing the expression of PHYA. When grown under far-red light conditions ars4ars5 shows the same elongated hypocotyl phenotype as the previously described strong phyA-211 allele. Three independent phyA alleles, ars4, phyA-211 and a new T-DNA insertion allele (phyA-t) show increased tolerance to arsenate, although to a lesser degree than the ars4ars5 double mutant. Analyses of the ars5 single mutant show that ars5 exhibits stronger arsenic tolerance than ars4, and that ars5 is not linked to ars4. Arsenic tolerance assays with phyB-9 and phot1/phot2 mutants show that these photoreceptor mutants do not exhibit phyA-like arsenic tolerance. Fluorescence HPLC analyses show that elevated levels of phytochelatins were not detected in ars4, ars5 or ars4ars5, however increases in the thiols cysteine, gamma-glutamylcysteine and glutathione were observed. Compared with wild type, the total thiol levels in ars4, ars5 and ars4ars5 mutants were increased up to 80% with combined buthionine sulfoximine and arsenic treatments, suggesting the enhancement of mechanisms that mediate thiol synthesis in the mutants. The presented findings show that PHYA negatively regulates a pathway conferring arsenic tolerance, and that an enhanced thiol synthesis mechanism contributes to the arsenic tolerance of ars4ars5.

Grain unloading of arsenic species in rice

Rice (Oryza sativa) is the staple food for over half the world's population yet may represent a significant dietary source of inorganic arsenic (As), a nonthreshold, class 1 human carcinogen. Rice grain As is dominated by the inorganic species, and the organic species dimethylarsinic acid (DMA). To investigate how As species are unloaded into grain rice, panicles were excised during grain filling and hydroponically pulsed with arsenite, arsenate, glutathione-complexed As, or DMA. Total As concentrations in flag leaf, grain, and husk, were quantified by inductively coupled plasma mass spectroscopy and As speciation in the fresh grain was determined by x-ray absorption near-edge spectroscopy. The roles of phloem and xylem transport were investigated by applying a +/- stem-girdling treatment to a second set of panicles, limiting phloem transport to the grain in panicles pulsed with arsenite or DMA. The results demonstrate that DMA is translocated to the rice grain with over an order magnitude greater efficiency than inorganic species and is more mobile than arsenite in both the phloem and the xylem. Phloem transport accounted for 90% of arsenite, and 55% of DMA, transport to the grain. Synchrotron x-ray fluorescence mapping and fluorescence microtomography revealed marked differences in the pattern of As unloading into the grain between DMA and arsenite-challenged grain. Arsenite was retained in the ovular vascular trace and DMA dispersed throughout the external grain parts and into the endosperm. This study also demonstrates that DMA speciation is altered in planta, potentially through complexation with thiols.

Investigation into mercury bound to biothiols:structural identification using ESI-ion-trap MS and introduction of a method for their HPLC separation with simultaneous detection by ICP-MS and ESI-MS

Mercury in plants or animal tissue is supposed to occur in the form of complexes formed with biologically relevant thiols (biothiols), rather than as free cation. We describe a technique for the separation and molecular identification of mercury and methylmercury complexes derived from their reactions with cysteine (Cys) and glutathione (GS): Hg(Cys)(2), Hg(GS)(2), MeHgCys, MeHgGS. Complexes were characterised by electrospray mass spectrometry (MS) equipped with an ion trap and the fragmentation pattern of MeHgCys was explained by using MP2 and B3LYP calculations, showing the importance of mercury-amine interactions in the gas phase. Chromatographic baseline separation was performed within 10 min with formic acid as the mobile phase on a reversed-phase column. Detection was done by online simultaneous coupling of ES-MS and inductively coupled plasma MS. When the mercury complexes were spiked in real samples (plant extracts), no perturbation of the separation and detection conditions was observed, suggesting that this method is capable of detecting mercury biothiol complexes in plants.

Arsenate causes differential acute toxicity to two P-deprived genotypes of rice seedlings (Oryza sativa L.)

Significant genotypic difference in response to arsenate toxicity in rice (Oryza sativa) was investigated in root elongation, arsenate uptake kinetics, physiological and biochemical response and arsenic (As) speciation. Uptake kinetics data showed that P-deprived genotype 94D-54 had a little higher As uptake than P-deprived 94D-64, but the difference was not large enough to cause acute toxicity in P-deprived 94D-54. There was no difference in tissue P concentrations between the two genotypes under P deficient conditions. In addition, arsenic speciation in plant tissues (using high performance liquid chromatography-inductively coupled plasma mass spectrometry) was not different between P pretreatments and between genotypes. P-deprived genotype 94D-54 suffered much higher stress induced by arsenate toxicity than P-deprived genotype 94D-64, in terms of lipid peroxidation, tissue H2O2 concentrations and exosmosis of K, P and As. However, P-deprived 94D-54 also had higher overproduction of enzymatic antioxidants (with higher GPX, SOD, CAT) and NPT (non-protein thiols) than P-deprived 94D-64. It appeared that, the higher sensitivity of P-deprived 94D-54 to arsenate toxicity might cause the overproduction of NPT, thus leading to the depletion of GSH and to the accumulation of H2O2. The differential sensitivity of the two genotypes has major implications for breeding rice for As affected paddy soil.

Ionic liquids for efficient hydrogen sulfide and thiol scavenging

Functionalised pyridinium and ammonium ionic liquids bearing a Michael acceptor are shown to scavenge H₂S gas and various thiols, in most cases, without the aid of any added bases. Utilising the effective non-volatility of ionic liquids and ‘tagging’ malodourous substances to an ionic matrix renders them odourless.