Gary K. Robinson

Numerical Analysis of a Photonic Crystal Fiber for Biosensing Applications

This paper presents a theoretical study on a photonic crystal fiber (PCF) surface plasmon resonance biosensor. The proposed PCF sensor introduces the concept of simultaneous detection with H E₁₁^x and H E₁₁^x modes, which opens up some possibilities for multianalyte/multichannel sensing. Analysis was performed which considered the operation of the sensor in both amplitude and wavelength interrogation modes. Typical sensor resolutions of 4×10^-5 RIU and 8×10^-5 RIU with respect to H E₁₁^x and H E₁₁^y, respectively, are reported for the amplitude interrogation mode, while resoutions of 5 × 10^-5 RIU and 6×10^-5 RIU are reported for H E₁₁^x and H E₁₁^y, respectively, for the wavelength interrogation mode.

CO2 acts as a signalling molecule in populations of the fungal pathogen Candida albicans.

When colonising host-niches or non-animated medical devices, individual cells of the fungal pathogen Candida albicans expand into significant biomasses. Here we show that within such biomasses, fungal metabolically generated CO2 acts as a communication molecule promoting the switch from yeast to filamentous growth essential for C. albicans pathology. We find that CO2-mediated intra-colony signalling involves the adenylyl cyclase protein (Cyr1p), a multi-sensor recently found to coordinate fungal responses to serum and bacterial peptidoglycan. We further identify Lys 1373 as essential for CO2/bicarbonate regulation of Cyr1p. Disruption of the CO2/bicarbonate receptor-site interferes selectively with C. albicans filamentation within fungal biomasses. Comparisons between the Drosophila melanogaster infection model and the mouse model of disseminated candidiasis, suggest that metabolic CO2 sensing may be important for initial colonisation and epithelial invasion. Our results reveal the existence of a gaseous Candida signalling pathway and its molecular mechanism and provide insights into an evolutionary conserved CO2-signalling system.

Influence of chlorobenzoates on the utilisation of chlorobiphenyls and chlorobenzoate mixtures by chlorobiphenyl/chlorobenzoate-mineralising hybrid bacterial strains

Chlorobenzoates (CBA) arise as intermediates during the degradation of polychlorinated biphenyls (PCBs) and some chlorinated herbicides. Since PCBs were produced as complex mixtures, a range of mono-, di-, and possibly trichloro-substituted benzoates would be formed. Chlorobenzoate degradation has been proposed to be one of the rate-limiting steps in the overall PCB-degradation process. Three hybrid bacteria constructed to have the ability to completely mineralise 2-, 3-, or 4-monochlorobiphenyl respectively, have been studied to establish the range of mono- and diCBAs that can be utilised. The three strains were able to mineralise one or more of the following CBAs: 2-, 3-, and 4-monochlorobenzoate and 3,5-dichlorobenzoate. No utilisation of 2,3-, 2,5-, 2,6-, or 3,4-diCBA was observed, and only a low concentration (0.11 mM) of 2,4-diCBA was mineralised. When the strain with the widest substrate range (Burkholderia cepacia JHR22) was simultaneously supplied with two CBAs, one that it could utilise plus one that it was unable to utilise, inhibitory effects were observed. The utilisation of 2-CBA (2.5 mM) by this strain was inhibited by 2,3-CBA (200 μM) and 3,4-CBA (50 μM). Although 2,5-CBA and 2,6-CBA were not utilised as carbon sources by strain JHR22, they did not inhibit 2-CBA utilisation at the concentrations studied, whereas 2,4-CBA was co-metabolised with 2-CBA. The utilisation of 2-, 3-, and 4-chlorobiphenyl by strain JHR22 was also inhibited by the presence of 2,3- or 3,4-diCBA. We conclude that the effect of the formation of toxic intermediates is an important consideration when designing remediation strategies.

Biocatalysts for clean industrial products and processes.

Biocatalysis inherently offers the prospect of clean industrial processing and has become an accepted technology throughout most sectors. The convergence of biology and chemistry has enabled a plethora of industrial opportunities to be targeted, while discoveries in biodiversity and the impact of molecular biology and computational science are extending the range of natural and engineered biocatalysts that can be customised for clean industrial requirements.

The pancreas-specific protein disulphide-isomerase PDIp interacts with a hydroxyaryl group in ligands

Using a cross-linking approach, we have recently demonstrated that radiolabelled model peptides or misfolded proteins specifically interact in vitro with two members of the protein disulphide- isomerase family, namely PDI and PDIp, in a crude extract from sheep pancreas microsomes. In addition, we have shown that tyrosine and tryptophan residues within a peptide are the recognition motifs for the binding to PDIp. Here we examine non-peptide ligands and present evidence that a hydroxyaryl group is a structural motif for the binding to PDIp; simple constructs containing this group and certain xenobiotics and phytoestrogens, which contain an unmodified hydroxyaryl group, can all efficiently inhibit peptide binding to PDIp. To our knowledge this is the first time that the recognition motif of a molecular chaperone or folding catalyst has been specified as a simple chemical structure.

Passive control of quorum sensing: prevention of Pseudomonas aeruginosa biofilm formation by imprinted polymers.

Here we present the first molecular imprinted polymer (MIP) that is able to attenuate the biofilm formation of the opportunistic human pathogen Pseudomonas aeruginosa through specific sequestration of its signal molecule N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C(12)-AHL). The MIP was rationally designed using computational modeling, and its capacity and specificity and that of a corresponding blank polymer toward signal molecule of P. aeruginosa (3-oxo-C(12)-AHL) and its analogue were tested. The biofilm formation in the presence of polymers and without polymers was studied using scanning confocal laser microscopy. Staining with crystal violet dye was used for the quantification of the biofilm formation. A significant reduction of the biofilm growth was observed in the presence of MIP (>80%), which was superior to that of the resin prepared without template, which showed a reduction of 40% in comparison with biofilm, which was grown without polymer addition. It was shown that 3-oxo-C(12)-AHL-specific MIP prevented the development of quorum-sensing-controlled phenotypes (in this case, biofilm formation) from being up-regulated. The developed MIP could be considered as a new tool for the elimination of life-threatening infections in a multitude of practical applications; it could, for example, be grafted on the surface of medical devices such as catheters and lenses, be a component of paints, or be used as a wound adsorbent.

Attenuation of Vibrio fischeri Quorum Sensing Using Rationally Designed Polymers

A first attempt to attenuate the quorum sensing (QS) of a marine heterotroph microorganism, Vibrio fischeri , using signal molecule-sequestering polymers (SSPs) is presented. A set of rationally designed polymers with affinity toward a signal molecule of V. fischeri , N-(beta-ketocaproyl)-l-homoserine lactone (3-oxo-C6-AHL) was produced. It is reported that computationally designed polymers could sequester a signal molecule of V. fischeri and prevent QS-controlled phenotypes (in this case, bioluminescence) from being up-regulated. It was proven that the attenuation of bioluminescence of V. fischeri was due to sequestration of the signal molecule by specific polymers and not due to the toxicity of polymer or nonspecific depletion of nutrients. The ability to disrupt the bacterial communication using easy to synthesize and chemically inert polymers could provide a new concept for the development of pharmaceuticals and susceptible device coatings such as catheters.

An investigation into the factors influencing lipase-catalyzed intramolecular lactonization in microaqueous systems

Abstract Factors affecting the enzymatic intramolecular lactonization of 16-hydroxyhexadecanoic acid are presented. A screen of 33 enzymes, predominantly lipases, showed that only a proportion were able to catalyze the synthetic reaction in a microaqueous environment. Certain of these enzymes showed no observable formation of the oligolactones by the favored intermolecular esterification reaction, as reported by other workers. Indeed, the immobilized lipase from Candida antarctica showed formation of the intramolecular monolactone product, hexadecanolide, which could be produced continuously for 55 hs. Many variables were considered, including the choice of enzyme (source and preformulation), substrate concentration, product concentration, solvent, temperature, pH, and water content of the system. The effect of these variables on hexadecanolide yield was analyzed statistically using a Plackett-Burman matrix. Significant effects were only demonstrated for the product hexadecanolide (stimulatory) and the substrate 16-hydroxyhexadecanoic acid (inhibitory).

SACRED : A novel catalytic process for the environmental remediation of polychlorinated biphenyls (PCBS)

Abstract The catalytic degradation of polychlorinated biphenyls (PCBs) has been studied in aqueous media and contaminated soils. A novel SAmarium Catalyzed REDuctive dechlorination (SACRED) is demonstrated which is able to operate in a short timescale under mild conditions (ambient temperature; inert atmosphere) and with low energy requirements. Samarium (II) iodide, in the presence of hexamethylphosphoramide in tetrahydrofuran, catalyses hydrogenolytic dechlorination of highly chlorinated PCBs, generating biphenyl, mono- and dichlorinated biphenyls from Aroclor 1242. Catalysis is also effective in co-contaminated soils containing various PCBs and in the presence of up to 5% (w/w) moisture.

Formation of benzaldehyde by Pseudomonas putida ATCC 12633

Abstract Aromatic and heterocyclic aldehydes may be produced by the mandelate pathway of Pseudomonas putida ATCC 12633 via the biotransformation of benzoyl formate and substrate analogues. Under optimised biotransformation conditions (37 °C, pH 5.4) and with benzoyl formate as a substrate, benzaldehyde may be accumulated with yields above 85%. Benzaldehyde is toxic to P. putida ATCC 12633; levels above 0.5 g/l (5 mM) reduce the biotransformation activity. Total activity loss occurs at an aldehyde concentration of 2.1 g/l (20 mM). To overcome this limitation, the rapid removal of the aldehyde is desirable via in situ product removal. The biotransformation of benzoyl formate (working volume 1 l) without in situ product removal accumulates 2.1 g/l benzaldehyde. Benzaldehyde removal by gas stripping produces a total of 3.5 g/l before inhibition. However, the most efficient method is solid-phase adsorption using activated charcoal as the sorbant, this allows the production of over 4.1 g/l benzaldehyde. Addition of bisulphite as a complexing agent causes inhibition of the biotransformation and bisulphite is therefore is not suitable for in situ product removal.