Portrait of Professor Ian Bruce

Professor Ian Bruce

Emeritus Professor of Nanobiotechnology

About

Professor Ian Bruce started his academic life as a geneticist, studying at University College London (UCL) where he also gained his PhD (working with David Wilkie). His first postdoc was also at UCL, followed by the Prince Philip’s Research Laboratory (Paediatric Research Unit), Guy’s Hospital, London (with Francesco Giannelli) and then at PROIMI, Tucuman, Argentina by way of a travelling fellowship from the Royal Society. 

Ian was then appointed a lecturer and rapidly afterwards professor at the University of Greenwich, London. There, he was responsible for Biosciences and the Biosciences Research Centre. After that, he moved as a professor to the Universita` degli Studi di Urbino, Urbino, Italy, subsequently returning to the UK in 2004 as Professor of Nanobiotechnology at the University of Kent. He was Faculty Director for Research and Graduate Studies from 2005 to 2009. 

Ian was elected a Fellow of the Royal Society of Biology in 1995, a Fellow of the Royal Society of Chemistry in 2013 and awarded a DSc from the Universita` degli Studi di Urbino in 2007 in recognition of his work and that of his group in the field of nanomaterial applications in biomedicine. 

Ian retired in December 2011. 

Research interests

Ian's research interests were always pretty diverse and multidisciplinary, including molecular biology, surface science and chemistry, materials science and synthetic organic chemistry. 

His research group carried out work related to ‘bottom-up’ synthesis of complex nanocomposites, nanoparticle surface activation chemistry, especially involving the use of organosilanes in biomolecule/nanoparticle surface conjugation and gene expression and organisation in bacteria. The group gained over £16m of research income over its lifetime, collaborating with many major industries and academic institutes from Europe, Russia, China and Israel, and its work has been used as an indicator of success in European science all over the world 

Publications

Showing 50 of 76 total publications in the Kent Academic Repository. View all publications.

Article

  • Wu, H., Liang, J., Gou, L., Wu, Q., Liang, W., Zhou, X., Bruce, I., Deng, Z. and Wang, Z. (2018). Recycling of overactivated acyls by a type II thioesterase during calcimycin biosynthesis in Streptomyces chartreusis NRRL 3882. Applied and Environmental Microbiology [Online] 84. Available at: http://dx.doi.org/10.1128/AEM.00587-18.
    Type II thioesterases typically function as editing enzymes, removing acyl groups that have been misconjugated to acyl carrier proteins during polyketide secondary metabolite biosynthesis as a consequence of biosynthetic errors. Streptomyces chartreusis NRRL 3882 produces the pyrrole polyether ionophoric antibiotic, and we have identified the presence of a putative type II thioesterase-like sequence, calG, within the biosynthetic gene cluster involved in the antibiotic's synthesis. However, targeted gene mutagenesis experiments in which calG was inactivated in the organism did not lead to a decrease in calcimycin production but rather reduced the strain's production of its biosynthetic precursor, cezomycin. Results from in vitro activity assays of purified, recombinant CalG protein indicated that it was involved in the hydrolysis of cezomycin coenzyme A (cezomycin-CoA), as well as other acyl CoAs, but was not active toward 3-S-N-acetylcysteamine (SNAC; the mimic of the polyketide chain-releasing precursor). Further investigation of the enzyme's activity showed that it possessed a cezomycin-CoA hydrolysis Km of 0.67 mM and a kcat of 17.77 min-1 and was significantly inhibited by the presence of Mn2+ and Fe2+ divalent cations. Interestingly, when S. chartreusis NRRL 3882 was cultured in the presence of inorganic nitrite, NaNO2, it was observed that the production of calcimycin rather than cezomycin was promoted. Also, supplementation of S. chartreusis NRRL 3882 growth medium with the divalent cations Ca2+, Mg2+, Mn2+, and Fe2+ had a similar effect. Taken together, these observations suggest that CalG is not responsible for megasynthase polyketide precursor chain release during the synthesis of calcimycin or for retaining the catalytic efficiency of the megasynthase enzyme complex as is supposed to be the function for type II thioesterases. Rather, our results suggest that CalG is a dedicated thioesterase that prevents the accumulation of cezomycin-CoA when intracellular nitrogen is limited, an apparently new and previously unreported function of type II thioesterases.
  • Wu, H., Liang, J., Wang, J., Liang, W., Gou, L., Wu, Q., Zhou, X., Bruce, I., Deng, Z. and Wang, Z. (2018). Cezomycin is activated by CalC to its ester form for further biosynthesis steps in the production of calcimycin in Streptomyces chartreusis NRRL 3882. Applied and Environmental Microbiology [Online] 84. Available at: http://dx.doi.org/10.1128/AEM.00586-18.
    Calcimycin, N-demethyl calcimycin, and cezomycin are polyether divalent cation ionophore secondary metabolites produced by Streptomyces chartreusis. A thorough understanding of the organization of their encoding genes, biosynthetic pathway(s), and cation specificities is vitally important for their efficient future production and therapeutic use. So far, this has been lacking, as has information concerning any biosynthetic relationships that may exist between calcimycin and cezomycin. In this study, we observed that when a Cal- (calB1 mutant) derivative of a calcimycin-producing strain of S. chartreusis (NRRL 3882) was grown on cezomycin, calcimycin production was restored. This suggested that calcimycin synthesis may have resulted from postsynthetic modification of cezomycin rather than from a de novo process through a novel and independent biosynthetic mechanism. Systematic screening of a number of Cal- S. chartreusis mutants lacking the ability to convert cezomycin to calcimycin allowed the identification of a gene, provisionally named calC, which was involved in the conversion step. Molecular cloning and heterologous expression of the CalC protein along with its purification to homogeneity and negative-staining electron microscopy allowed the determination of its apparent molecular weight, oligomeric forms in solution, and activity. These experiments allowed us to confirm that the protein possessed ATP pyrophosphatase activity and was capable of ligating coenzyme A (CoA) with cezomycin but not 3-hydroxyanthranilic acid. The CalC protein's apparent Km and kcat for cezomycin were observed to be 190 ?M and 3.98 min-1, respectively, and it possessed the oligomeric form in solution. Our results unequivocally show that cezomycin is postsynthetically modified to calcimycin by the CalC protein through its activation of cezomycin to a CoA ester form.
  • Cheraghipour, E., Tamaddon, A., Javadpour, S. and Bruce, I. (2013). PEG conjugated citrate-capped magnetite nanoparticles for biomedical applications. Journal of Magnetism and Magnetic Materials [Online] 328:91-95. Available at: http://dx.doi.org/10.1016/j.jmmm.2012.09.042.
    We aim to develop polyethylene glycol decorated, citric acid capped magnetite nanoparticles (MNPs) with proper physicochemical characteristics including particle size distribution, morphology, magnetic property and stability in a biologic medium. MNP of about 10 nm were synthesized by a biocompatible chemical co-precipitation of Fe2+ and Fe3+ in an ammonia solution. A synthetic methodology has been developed to get a well dispersed and homogeneous aqueous suspension of MNPs. The naked MNPs are often insufficient for their stability, hydrophilicity and further functionalization. In order to overcome these limitations, citric acid was used to stabilize the magnetite particle suspension, which was anchored on the surface of freshly prepared MNPs by a direct addition method. Polyethylene glycol was covalently attached to the carboxylic moieties of citric acid anchored MNPs by carbodiimide chemistry. The microstructure and morphology of the nanoparticles were characterized by X-ray diffraction and transmission electron microscopy, and Fourier transform infrared spectroscopy. Also, the magnetic properties were investigated by vibrating sample magnetometry. It was found that the nanoparticles demonstrated superparamagnetic behavior.
  • Sen, T. and Bruce, I. (2012). Surface engineering of nanoparticles in suspension for particle based bio-sensing. Scientific Reports [Online] 2. Available at: http://dx.doi.org/10.1038/srep00564.
    Surface activation of nanoparticles in suspension using amino organosilane has been carried out via strict control of a particle surface ad-layer of water using a simple but efficient protocol ‘Tri-phasic Reverse Emulsion’ (TPRE). This approach produced thin and ordered layers of particle surface functional groups which allowed the efficient conjugation of biomolecules. When used in bio-sensing applications, the resultant conjugates were highly efficient in the hybrid capture of complementary oligonucleotides and the detection of food borne microorganism. TPRE overcomes a number of fundamental problems associated with the surface modification of particles in aqueous suspension viz. particle aggregation, density and organization of resultant surface functional groups by controlling surface condensation of the aminosilane. The approach has potential for application in areas as diverse as nanomedicine, to food technology and industrial catalysis.
  • Hertz, A., Fitzgerald, V., Pignotti, E., Knowles, J., Sen, T. and Bruce, I. (2012). Preparation and characterisation of porous silica and silica/titania monoliths for potential use in bone replacement. Microporous and Mesoporous Materials [Online] 156:51-61. Available at: http://dx.doi.org/10.1016/j.micromeso.2012.02.004.
    Inorganic materials used in bone regeneration and replacement have developed rapidly over the last 10 years or so with SiO2 as well as TiO2 showing great potential. In this work, porous SiO2 and SiO2/TiO2 monoliths were prepared from mixtures of powdered meso- and macroporous silica and titanium dioxide (anatase) powders. The mixtures were compacted at pressures between 10 and 100 MPa to form monoliths which were then sintered at 700 °C. It was observed that compaction pressure, composition and sintering directly influenced final monolith porosity, density and surface area due to changes in their pore volume and size. Surface area and pore volume of the monoliths decreased with increasing compaction pressure and sintering whilst density increased. The presence of TiO2 was observed to increase the density of monoliths but not their flexural biaxial strength. When the materials were evaluated for their toxicity by the MTS cell proliferation assays against three human cell lines silica monoliths were observed to be nontoxic. A study of the materials ability to promote cell adherence and growth showed that monoliths containing TiO2 did not support cell adhesion but those composed of SiO2 alone did. Finally it was observed that the materials rapidly promoted the nucleation and surface growth of a hydroxyapatite layer when incubated in simulated body fluid. This study indicates that SiO2 and SiO2/TiO2 monolithic bone implant materials with tailored porosities can be prepared on large scale from simple precursors which are biocompatible and able to support cell growth and hydroxyapatite nucleation.
  • Holder, S., Sriskantha, B., Bagshaw, S. and Bruce, I. (2012). Headgroup effects on the krafft temperatures and self-assembly of ?-hydroxy and ?-carboxy hexadecyl quaternary ammonium bromide bolaform amphiphiles: Micelles versus molecular clusters?. Journal of Colloid and Interface Science [Online] 367:293-304. Available at: http://dx.doi.org/10.1016/j.jcis.2011.10.017.
    Eight bolaform amphiphiles were synthesised and characterised; 4 ?,?-hydroxy-alkane trialkyl (and pyridyl) ammonium bromides and 4 ?,?-carboxy-alkane trialkyl (and pyridyl) ammonium bromides where the alkyl groups were methyl, ethyl and propyl. Four of these represented new compounds. Overall the Krafft temperatures (TK) of the eight amphiphiles were high, with 6 of the eight possessing TKs greater than 45 °C. Thus most of the amphiphiles could only expect to find applications at raised temperatures limiting their potential utility. However in addition to the previously reported ?,?-hydroxy-hexadecyl triethylammonium bromide (2b) with a TK of 19.1 °C, another amphiphile, ?,?-carboxy-hexadecyl tripropylammonium bromide (2c) has been identified with a TK near ambient temperatures (TK of 22.1 °C). This provides an acid functional ammonium bolaform amphiphile that micellises at ambient temperatures to complement the hydroxyl derivative. A correlation between TK and the product of the enthalpies and Tms of the compounds was observed for 7 of the eight compounds. No correlation between the amphiphile critical micelle concentrations (cmc) and TKs was observed confirming previous reports that TK values are predominantly determined by crystalline stability rather than solubility. Considerable differences were observed between the various amphiphile TKs at different pHs but no clear trend was apparent for the various compounds (despite the degree to which the compounds’ carboxylic acid and hydroxyl functionalities were likely to be ionised). The cmcs for the amphiphiles were an order of magnitude larger than those for analogous mono-ammonium amphiphiles with little difference in between the hydroxyl- and carboxy-functionalised compounds. The aggregation numbers (Nagg) obtained for all compounds were very low (Nagg < 7) and the apparent micellar diameters for the hydroxyl-bolaforms were in the range 1.0–1.4 nm whereas those for the carboxy-compounds were in the range 2.1–2.4 nm. These results strongly suggest a difference in the packing of the two sets of amphiphiles with loose low density aggregates or ‘molecular clusters’ for the carboxy compounds and denser classical micellar type aggregates for the hydroxyl-compounds. In both cases however the sizes and the low aggregation numbers point suggest that these aggregates are more characteristic of the pre-micellar aggregates observed for many amphiphiles but in particular gemini surfactants.
  • East, D., Mulvihill, D., Todd, M. and Bruce, I. (2011). QD-Antibody conjugates via carbodiimide-mediated coupling; a detailed study of the variables involved and a possible new mechanism for the coupling reaction under basic aqueous conditions. Langmuir [Online] 27:13888-13896. Available at: http://dx.doi.org/10.1021/la203273p.
    A detailed study into the optimization of carbodiimide-mediated coupling of antibodies (Ab) and quantum dots (QD) for use in cellular imaging has been undertaken. This involved the grafting of commercially available carboxyl-modified QDs (Evident Technologies "Lake Placid Blue" Evitag and eBioscience's eflour nanocrystals) with anti-Cdc8 Abs to produce conjugates with specific affinity for fission yeast tropomyosin Cdc8 protein. The water-soluble carbodiimide 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) was used to activate the QDs prior to their incubation with antibody, and a range of QD-carboxyl/EDC/Ab mole ratios were used in the experiments in attempts to optimize fluorescence and bioaffinity of the conjugate products (EDC to QD-carboxyl-600 nmol/15pmol to 0.12 nmol/15 pmol and QD to Ab 120 pmol/24 pmol to 120 pmol/1.2 pmol). It was observed that a specific "optimum" ratio of the three reactants was required to produce the most fluorescent and biologically active product and that it was generated at alkaline pH 10.8. Increasing the ratio of Ab to QD produced conjugate which was less fluorescent while reducing the ratio of EDC to QD in the activation step led to increased fluorescence of product. Conjugates were tested for their possession of antibody by measurement of their absorption at OD(280 nm) and for their fluorescence by assay ?(max(em)) at 495 nm. A quantitative assay of the bioactivity of the conjugates was developed whereby a standardized amount of Cdc8 antigen was spotted onto nylon membranes and reacted with products from conjugation reactions in a sandwich-type colormetric assay The "best" conjugate was used in intracellular imaging of yeast Cdc8 protein and produced brighter, higher definition images of fixed yeast cell actin structure than a fluorescein-Ab conjugate routinely produced in our laboratory. The QD-Ab conjugate was also significantly more resistant to photobleaching than the fluorescein-Ab conjugate. Results from other experiments involving EDC, the water-soluble carbodiimide 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulphonate (CMC), and EDC.HCl have suggested a new reaction mechanism for EDC coupling under basic aqueous conditions. In summary, a robust understanding of commercial QD-COOH surface chemistry and the variables involved in the materials' efficient conjugation with a bioligand using carbidiimide has been obtained along with an optimized approach for Ab-QD conjugate production. A novel assay has been developed for bioassay of QD-Ab conjugates and a new mechanism for EDC coupling under basic aqueous conditions is proposed.
  • Casula, M., Corrias, A., Arosio, P., Lascialfari, A., Sen, T., Floris, P. and Bruce, I. (2011). Design of water-based ferrofluids as contrast agents for magnetic resonance imaging. Journal of Colloid and Interface Science [Online] 357:50-55. Available at: http://dx.doi.org/10.1016/j.jcis.2011.01.088.
    We report the synthesis, characterization and relaxometric study of ferrofluids based on iron oxide, with potential for use as magnetic resonance imaging (MRI) contrast agents (CAs). The effect of different cost-effective, water-based surface modification approaches which can be easily scaled-up for the large scale synthesis of the ferrofluids has been investigated. Surface modification was achieved by silanization, and/or coating with non-toxic commercial dispersants (a lauric polysorbate and a block copolymer with pigment affinic groups, namely Tween 20 and Disperbyk 190) which were added after or during iron oxide nanoparticle synthesis. It was observed that all the materials synthesized functioned as negative contrast agents at physiological temperature and at frequencies covered by clinical imagers. The relaxometric properties of the magnetic nanoparticles were significantly improved after surface coating with stabilizers compared to the original iron oxide nanoparticles, with particular reference to the silica-coated magnetic nanoparticles. The results indicate that the optimization of the preparation of colloidal magnetic ferrofluids by surface modification is effective in the design of novel contrast agents for MRI by enabling better or more effective interaction between the coated iron oxide nanoparticles and protons present in their aqueous environment.
  • Antonelli, A., Sfara, C., Manuali, E., Bruce, I. and Magnani, M. (2011). Encapsulation of superparamagnetic nanoparticles into red blood cells as new carriers of MRI contrast agents. Nanomedicine [Online] 6:211-223. Available at: http://dx.doi.org/10.2217/Nnm.10.163.
    Aims: The half-life of superparamagnetic iron oxide nanoparticles in the bloodstream is very short since they are rapidly taken up by the reticuloendothelial system. In this article, we report the encapsulation of different magnetic nanoparticles into human erythrocytes to increase their blood circulation time. Materials & methods: Newly synthesized and commercially available nanoparticles were evaluated for the encapsulation into red blood cells through the transient opening of membrane pores by controlled hypotonic dialysis and successive isotonic resealing and reannealing of cells. Results: Commercial superparamagnetic iron oxide nanoparticles (SHU 555A, AMI 227 and PMP-50) dextran or carboxydextran coated can be successfully loaded into red blood cells; similarly, some of the new nanomaterials, such as Np-1 nanoparticles dispersed in the Disperbyk®-190 agent, can be efficiently encapsulated into red blood cells. Conclusion: A careful consideration of magnetic nanoparticles parameters, such as size, synthesis protocols, coating and/or dispersant agents, is required in order to obtain efficient loading through the cell membrane pores.
  • Rother, D., Sen, T., East, D. and Bruce, I. (2011). Silicon, silica and its surface patterning/activation with alkoxy- and amino-silanes for nanomedical applications. Nanomedicine [Online] 6:281-300. Available at: http://dx.doi.org/10.2217/nnm.10.159.
    Silica and silicates are widely used in nanomedicine with applications as diverse as medical device coatings to replacement materials in tissue engineering. Although much is known about silica and its synthesis, relatively few biomedical scientists fully appreciate the link that exists between its formulation and its resultant structure and function. This article attempts to provide insight into relevant issues in that context, as well as highlighting their importance in the material’s eventual surface patterning/activation with alkoxy- and organo-silanes. The use of aminosilanes in that context is discussed at some length to permit an understanding of the specific variables that are important in the reproducible and robust aminoactivation of surfaces using such molecules. Recent investigative work is cited to underline the fact that although aminosilanization is a historically accepted mechanism for surface activation, there is still much to be explained about how and why the process works in the way it does. In the last section of this article, there is a detailed discussion of two classical approaches for the use of aminosilanized materials in the covalent immobilization of bioligands, amino-aldehyde and amino-carboxyl coupling. In the former case, the use of the homobifunctional coupler glutaraldehyde is explored, and in the latter, carbodiimides. Although these chemistries have long been employed in bioconjugations, it is apparent that there are still variables to be explored in the processes (as witnessed by continuing investigations into the chemistries concerned). Aspects regarding optimization, standardization and reproducibility of the fabrication of amino functionalized surfaces are discussed in detail and illustrated with practical examples to aid the reader in their own studies, in terms of considerations to be taken into account when producing such materials. Finally, the article attempts to remind readers that although the chemistry and materials involved are ‘old hat’, there is still much to be learnt about the methods involved. The article also reminds readers that although many highly specific and costly conjugation chemistries now exist for bioligands, there still remains a place for these relatively simple and cost-effective approaches in bioligand conjugate fabrication.
  • Bruce, I. (2011). Researching and exploiting nanomaterials useful in forensics and diagnostics. Nanomedicine [Online] 6:185-186. Available at: http://dx.doi.org/10.2217/nnm.10.156.
  • Bruce, I. (2011). Novel and Improved Nanomaterials, Chemistries and Apparatus for Nanobiotechnology: the NACBO Project. Nanomedicine [Online] 6:187-193. Available at: http://dx.doi.org/10.2217/nnm.10.155.
    This article outlines the nature and activities of the recently completed EU Framework Programme 6 Integrated Project, Novel and Improved Nanomaterials, Chemistries and Apparatus for Nanobiotechnology (NACBO). This project was designed to yield new nanomaterials, surface activation and synthetic nucleic acid chemistries, procedures and hardware for applications in forensics and diagnostics. It provides details on the project’s structure and partnership along with its principal objectives and successes in terms of publications and commercial exploitation.
  • Sen, T., Bruce, I. and Mercer, T. (2010). Fabrication of novel hierarchically ordered porous magnetic nanocomposites for bio-catalysis. Chemical Communications [Online] 46:6807-6809. Available at: http://dx.doi.org/10.1039/c0cc01747g.
    Novel hierarchically ordered porous magnetic nanocomposites with interconnecting macroporous windows and meso-microporous walls containing well dispersed magnetic nanoparticles have been fabricated and used as a support to immobilise lipase for the efficient hydrolysis of ester.
  • Amagliani, G., Omiccioli, E., Brandi, G., Bruce, I. and Magnani, M. (2010). A multiplex magnetic capture hybridisation and multiplex Real-Time PCR protocol for pathogen detection in seafood. Food Microbiology [Online] 27:580-585. Available at: http://dx.doi.org/10.1016/j.fm.2010.01.007.
    Seafood could become a source of bacterial pathogens by exposure to contaminated water or through processing practices, thus representing a public health hazard. Conventional culture-based analytical methods take several days to be completed, while the molecular rapid identification of bacterial pathogens is crucial for effective disease control. The developed application consist of a multiplex magnetic capture hybridisation (mMCH) assay for the simultaneous isolation of Salmonella spp. and Listeria monocytogenes DNA from seafood, using paramagnetic amino-modified nanoparticles with capture oligonucleotides, and a triplex Real-Time PCR with an Internal Amplification Control (IAC), in accordance with ISO 22174. The detection probability was 100 with 10 genome equivalents of each target species co-amplified in the same reaction. The complete molecular procedure was tested on raw and smoked salmon fillets artificially contaminated with known amounts of one or both target bacteria (1-103cfu/g), directly or after culture enrichment, and compared for equivalence with the standard methods. Results revealed a complete agreement between the two approaches, with a sensitivity of 1cfu/g, in enriched samples, and higher sensitivity (102-103cfu/g) of the molecular method in samples examined before culture enrichment. The proposed procedure was also able to identify a natural contamination by L. monocytogenes in smoked salmon with a considerable shortening of time.
  • Van De Waterbeemd, M., Sen, T., Biagini, S. and Bruce, I. (2010). Surface functionalisation of magnetic nanoparticles: Quantification of surface to bulk amine density. Micro and Nano Letters [Online] 5:282-285. Available at: http://dx.doi.org/10.1049/mnl.2010.0112.
    Work has been conducted to adapt a colourimetric assay previously used on flat surfaces for the assay of amine group density on nanoparticles silanised with 3-(aminopropyl) triethoxysilane. The new assay was rapid, easy to perform, and linear in the range of optical density (OD282nm) values of 0.080-1.6 for particle suspension densities of between 0.5 and 7.0mg/ml. In addition, the same materials, as well as the ones activated using 3-(aminopropyl) diethoxy methyl silane, were investigated for their elemental compositions by, combustion carbon-hydrogen-nitrogen (CHN) analysis and results from both approaches together have permitted the accurate calculation of the ratio of surface to total amine density for the materials when activated in water. This value can in turn be used as an indication of a surface amino structure (i.e. mono or multilayer). The aminosilanisation processes were further characterised by DNA-binding/elution and zeta potential measurement. This combination of approaches provides a fast, convenient and effective means of measuring surface amine densities on particles and yields information about the structure of the surface aminosilanes layers. © 2010 The Institution of Engineering and Technology.
  • Omiccioli, E., Amagliani, G., Brandi, G., Bruce, I. and Magnani, M. (2009). Simultaneous direct detection of Salmonella spp., Listeria Monocytogenes and Escherichia Coli o157 in milk samples by magnetic extraction and multiplex PCR. Journal of Rapid Methods and Automation in Microbiology [Online] 17:195-213. Available at: http://dx.doi.org/10.1111/j.1745-4581.2009.00170.x.
    The aim of our work was to develop a new molecular method for the simultaneous detection of Salmonella spp., Listeria monocytogenes and Escherichia coli O157 directly in milk samples. Three specific target sequences were chosen for a multiplex polymerase chain reaction (PCR) assay: a 155-bp region of the Salmonella spp. tetrathionate reductase (ttr) locus; a 173-bp region of the L. monocytogenes listeriolysin O gene (hlyA); a 217-bp region of the E. coli O157 lipopolysaccharide gene (rfbE). An internal amplification control was also included to detect PCR inhibition. In addition, a magnetic-based extraction method was also developed to isolate PCR-ready DNA from milk. The assay was able to detect, whether alone or mixed, also with a difference of 2 log units, as few as 102 cells of each pathogen per 10 mL of spiked milk.
  • Sen, T. and Bruce, I. (2009). Mesoporous silica-magnetite nanocomposites: Fabrication, characterisation and applications in biosciences. Microporous and Mesoporous Materials [Online] 120:246-251. Available at: http://dx.doi.org/10.1016/j.micromeso.2008.11.012.
    Template assisted fabrication of magnetic mesoporous silica-magnetite nanocomposites and their performance in binding and elution of Salmon sperm DNA has been reported. The effect of reaction pH (10-2) during the fabrication of nanocomposites has been studied. All materials fabricated at various pH were characterised by XRD, TEM, MIR, nitrogen adsorption and magnetic susceptibility measurements. Fabrication at neutral pH (7) in the presence of a cationic surfactant (cetyl trimethyl ammonium bromide; CTAB) produced core-shell nanocomposites of quasi spherical and rod shaped morphologies with mesoporous (pore size similar to 3.5 nm) silica shell and rhombic magnetite core. Fabrication at alkaline pH (10) produced monolithic mesoporous silica composites with embedded magnetite nanoparticles. Fabrication at acidic pH (4 and 2) produced a biphasic mixture of rhombic magnetite and amorphous silica rods. A similar fabrication at acidic pH (2) in the absence of CTAB produced a biphasic mixture of rhombic magnetite and spherical silica nanoparticles. All materials exhibited a high value of Salmon sperm DNA binding at physiological pH whereas elution value of DNA was observed to be dependent on the extent of silica coating on magnetite nanoparticles.
  • Sebastianelli, A. and Bruce, I. (2008). Tn5530 from Burkholderia cepacia strain 2a encodes a chloride channel protein essential for the catabolism of 2,4-dichlorophenoxyacetic acid (vol 9, pg 256, 2007). Environmental Microbiology [Online] 10:2190-2190. Available at: http://dx.doi.org/10.1111/j.1462-2920.2008.01670.x.
  • Sebastianelli, A., Sen, T. and Bruce, I. (2008). Extraction of DNA from soil using nanoparticles by magnetic bioseparation. Letters in Applied Microbiology [Online] 46:488-491. Available at: http://dx.doi.org/10.1111/j.1472-765X.2008.02343.x.
    Aims: To develop a simple, rapid and inexpensive soil DNA extraction protocol. Methods and Results: The protocol relies on the use of superparamagnetic silica-magnetite nanoparticles for the isolation and purification of DNA from soil samples. DNA suitable for use in molecular biology applications was obtained from a number of soil samples. Conclusions: The DNA extracted using the tested method successfully permitted the PCR amplification of a fragment of the bacterial 16S rDNA gene. The extracted DNA could also be restriction endonuclease digested. Significance and Impact of the Study: The protocol reported here is simple and permits rapid isolation of PCR-ready soil DNA. The method requires only small quantities of soil sample, is scalable and suitable for automation.
  • Bagshaw, S. and Bruce, I. (2008). Rapid calcination of high quality mesostructured MCM-41, MSU-X, and SBA-15 silicate materials: A step towards continuous processing?. Microporous and Mesoporous Materials [Online] 109:199-209. Available at: http://dx.doi.org/10.1016/j.micromeso.2007.04.042.
    Rapid calcination of high quality silicate mesostructures is possible at rates at least up to 100 degrees C min(-1). Some 'fine-tuning' of the mesopore structure is possible by controlling the rate of heating during the calcination process and lower temperatures might be used to calcine the non-ionically templated SBA-15 and MSU-X materials. High calcination heating rates produce a distortion of the Si-MCM-41 hexagonal repeat unit and some form of phase transition in the 3-D disordered Si-MSU-X. At the calcination heating rates used here (2, 5, 10, 20, 100 degrees C min(-1)) the overall structural integrity of siliceous mesostructured materials is maintained while some structural collapse is observed in Al- and Ti-substituted MCM-41. SBA-15 materials appear to benefit from an elevated temperature aging period prior to high temperature calcination. The results suggest that for siliceous mesostructures it might be possible to use continuous processing methods at least in the calcination step.
  • Hertz, A. and Bruce, I. (2007). Inorganic materials for bone repair or replacement applications. Nanomedicine [Online] 2:899-918. Available at: http://www.futuremedicine.com/doi/abs/10.2217/17435889.2.6.899.
    In recent years, excipient systems have been used increasingly in biomedicine in reconstructive and replacement surgery, as bone cements, drug-delivery vehicles and contrast agents. Particularly, interest has been growing in the development and application of controlled pore inorganic ceramic materials for use in bone-replacement and bone-repair roles and, in this context, attention has been focused on calcium-phosphate, bioactive glasses and SiO2- and TiO2-based materials. It has been shown that inorganic materials that most closely mimic bone structure and surface chemistry most closely function best in bone replacement/repair and, in particular, if a substance possesses a macroporous structure (pores and interconnections >100 mu m diameter), then cell infiltration, bone growth and vascularization can all be promoted. The surface roughness and micro/mesoporosity of a material have also been observed to significantly influence its ability to promote apatite nucleation and cell attachment significantly. Pores (where present) can also be packed with pharmaceuticals and biomolecules (e.g., bone morphogenetic proteins [BMPs], which can stimulate bone formation). Finally, the most bio-efficient - in terms of collagen formation and apatite nucleation - materials are those that are able to provide soluble mineralizing species (Si, Ca, PO4) at their implant sites and/or are doped or have been surface-activated with specific functional groups. This article presents the context and latest advances in the field of bone-repair materials, especially with respect to the development of bioactive glasses and micro/mesoporous and macroporous inorganic scaffolds. it deals with the possible methods of preparing porous pure/doped or functionalized silicas or their composites, the studies that have been undertaken to evaluate their abilities to act as bone repair scaffolds and also presents future directions for work in that context.
  • Sebastianelli, A. and Bruce, I. (2007). Tn5530 from Burkholderia cepacia strain 2a encodes a chloride channel protein essential for the catabolism of 2,4-dichlorophenoxyacetic acid. Environmental Microbiology [Online] 9:256-265. Available at: http://dx.doi.org/10.1111/j.1462-2920.2006.01136.x.
    Chloride channel proteins (ClC) are found in living systems where they transport chloride ions across cell membranes. Recently, the structure/function of two prokaryotic ClC has been determined but little is known about the role of these proteins in the microbial metabolism of chlorinated compounds. Here we show that transposon Tn5530 from Burkholderia cepacia strain 2a encodes a ClC protein (BcClC) which is responsible for expelling Cl- ions generated during the catabolism of 2,4-dichlorophenoxyacetic acid (a chlorinated herbicide). We found that BcClC has the ability to transport Cl- ions across reconstituted proteoliposome membranes. We created two mutants in which the intrachannel glutamate residue of the protein, known to be responsible for opening and closing the channel (i.e. gating), was changed in order to create constitutively open and closed forms. We observed that cells carrying the closed-channel protein accumulated Cl- ions intracellularly leading to a decrease in intracellular pH, cell stasis and death. Further, we established that BcClC has the same gating mechanism as that reported for the ClC protein from Salmonella typhimurium. Our results show that the physiological role of ClC is to maintain cellular homeostasis which can be impaired by the catabolism of chlorinated compounds.
  • Galluzzi, L., Magnani, M., Saunders, N., Harms, C. and Bruce, I. (2007). Current molecular techniques for the detection of microbial pathogens. Science Progress 90:29-50.
  • Magnani, M., Galluzzi, L. and Bruce, I. (2006). The use of magnetic nanoparticles in the development of new molecular detection systems. Journal of Nanoscience and Nanotechnology [Online] 6:2302-2311. Available at: http://dx.doi.org/10.1166/jnn.2006.505.
    Magnetic nanoparticles have been widely used in biomolecular separation and discrimination which coincidentally also represents the basis for most current day molecular diagnostic procedures. The specificity, affinity, and binding capacity of magnetic nanoparticles depends on their size, form, dispersion, and surface chemistry. In this review, we will briefly analyze how these factors affect biomolecular separations and focus on the use of magnetic nanoparticles in monitoring the microbial biodiversity in the environment. We found that magnetic nanoparticles are especially effective for biomolecular separations in environmental samples collected and preserved with fixatives. This feature, together with the high sample throughput capability and the generic low cost, makes magnetic nanoparticles particularly suitable for environmental microbial monitoring. Furthermore, key features that permit the optimization of magnetic nanoparticles-based separations and that can be useful in the development of new analytical procedures are also discussed.
  • Sen, T., Magdassi, S., Nizri, G. and Bruce, I. (2006). Dispersion of magnetic nanoparticles in suspension. Micro & Nano Letters [Online] 1:39-42. Available at: http://dx.doi.org/10.1049/mnl:20065033.
    Pure magnetite nanoparticles (Fe3O4) have been synthesised in water : by coprecipitation using two different approaches (from ferrous sulphate and a mixture of ferrous and ferric chlorides). All materials aggregated in aqueous suspension, but their subsequent dispersion on treatment with a variety of agents was observed to be different. Magnetite produced using ferrous sulphate could not be disaggregated, whereas magnetite produced from a mixture of ferrous and ferric chlorides could be disaggregated to a quasi-monodispersed form. The dispersing agents were tetramethyl ammonium hydroxide, Disperbyk 190 and polyacrylic acid. The finding has potentially important implications for the surface activation of superparamagnetic magnetite nanoparticles and their ability to be used in bio/life science applications.
  • Galluzzi, L., Bertozzini, E., Del Campo, A., Penna, A., Bruce, I. and Magnani, M. (2006). Capture probe conjugated to paramagnetic nanoparticles for purification of Alexandrium species (Dinophyceae) DNA from environmental samples. Journal of Applied Microbiology [Online] 101:36-43. Available at: http://dx.doi.org/10.1111/j.1365-2672.2006.02952.x.
    Aims: To develop a rapid, cost-effective and selective Alexandrium DNA extraction procedure from environmental samples in order to provide good-quality template for the downstream PCR-based detection assay. Methods and Results: In this study, we tested a DNA extraction method based on silica-coated, superparamagnetic nanoparticles conjugated to a DNA-capture sequence (probe) complementary to a specific region of 5·8S rDNA of the genus Alexandrium. Cultured Alexandrium catenella cells were used as the harmful algal bloom species for the DNA extraction. Then, a PCR assay was performed with primers specific for the genus Alexandrium to assess the specificity and sensitivity of the nucleic acid extraction method. This method was applied to both cultured and field samples, reaching in both cases a detection limit of one A. catenella cell. Conclusions: The results suggest that the use of probe-conjugated paramagnetic nanoparticles could be effective for the specific purification of microalgal DNA in cultured or environmental samples, ensuring sensitivity and specificity of the subsequent PCR assays. Significance and Impact of the Study: The DNA extraction method optimized in this study represents a progress towards the rapid and efficient direct detection of Alexandrium cells in seawater monitoring. In fact, this method requires no other equipment than a magnet and a hybridization oven and, in principle, can be adapted to different toxic microalgal species and can be automated, allowing the processing of a high number of samples.
  • Amagliani, G., Omiccioli, E., Del Campo, A., Bruce, I., Brandi, G. and Magnani, M. (2006). Development of a magnetic capture hybridization-PCR assay for Listeria monocytogenes direct detection in milk samples. Journal of Applied Microbiology [Online] 100:375-383. Available at: http://dx.doi.org/10.1111/j.1365-2672.2005.02761.x.
    Aims: A rapid and sensitive method for Listeria monocytogenes direct detection from milk was developed. It is based on a magnetic capture hybridization procedure for selective DNA purification, followed by PCR identification. A comparison with two similar commercial systems from Dynal (Dynabeads) was carried out. Methods and Results: The technique used previously developed nanoparticles modified with a 21-mer oligonucleotide. This sequence, sharing homology with all the L. monocytogenes strains, was selected on hlyA gene and located outside the desired specific PCR site to avoid cross-contaminations. Capture probe properties, in term of spacer length and purification, were determined to obtain the highest hybridization efficiency. Its specificity was tested in hybridization experiments with nontarget bacterial species. Any inhibitory effect of the nanoparticles on PCR was also examined. The amplification performed with the purified DNA could reliably identify a 10 CFU ml-1 contamination rate. Conclusions: The optimized purification method showed a high specificity and sensitivity, with a detection level one log more sensitive than PCR carried out with nucleic acids obtained using commercial nanoparticles. Significance and Impact of the Study: The method, avoiding pre-enrichment, provides a rapid alternative to conventional microbiological detection methods. Furthermore, it is suitable for automation and can be proposed for the screening of a large number of samples.
  • Sen, T., Sebastianelli, A. and Bruce, I. (2006). Mesoporous silica-magnetite nanocomposite: fabrication and applications in magnetic bioseparations. Journal of the American Chemical Society [Online] 128:7130-7131. Available at: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=16734444.
    De novo material synthesis and application. Novel material with unique properties produced for Philips Medical Systems Division and others as part of international collaboration on drug delivery, development of SELDI-ToF and contrasting of patients in medical imaging . Competitively funded with EU FP6 IP project grant 12,000,000 Euro.
  • Lellouche, J., Senthil, G., Joseph, A., Buzhansky, L., Bruce, I., Bauminger, E. and Schlesinger, J. (2005). Magnetically responsive carboxylated magnetite-polydipyrrole/polydicarbazole nanocomposites of core-shell morphology. Preparation, characterization, and use in DNA hybridization. Journal of the American Chemical Society [Online] 127:11988-12006. Available at: http://dx.doi.org/10.1021/ja050285l.
    Novel bis-heterocyclic mono- and dicarboxylated dipyrrole and dicarbazole monomers have been synthesized in a modular manner. Their oxidative polymerization around magnetite nanosized particles has been investigated and optimized toward new magnetic magnetite-polydipyrrole/polydicarbazole nanocomposites (NCs) of a core-shell morphology. These NCs were thoroughly characterized by FT-IR, TGA (Thermal Gravimetric Analysis), low- and high-resolution TEM/HR-TEM microscopies, and Mossbauer spectroscopy along with magnetization studies. Exploiting the versatile COOH chemistry (activation by water-soluble diimides) introduced by the polymeric shell, DNA hybridization experiments have been conducted onto NC surfaces using an efficient blue-colored HRP-based enzymatic screening biological system. Highly parallel NC-supported DNA hybridization experimentations revealed that these NCs presented an interesting potential for DNA-based diagnostic applications.
  • Bruce, I. and Sen, T. (2005). Surface modification of magnetic nanoparticles with alkoxysilanes and their application in magnetic bioseparations. Langmuir [Online] 21:7029-7035. Available at: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=16008419.
    A versatile and inexpensive method for the introduction of amine groups onto the surface of silica-coated magnetite composite nanoparticles has been established based on the condensation of (aminopropyl)triethoxysilane (APTS). The process was observed to be sensitive to a range of variables, and a range of silane surface-modified nanoparticles was synthesized under various reaction conditions, that is, solvent systems [water, tetrahydrofuran (THF), ethanol, or 1:1 mixtures of them], reaction times (from 1 to 24 h), and temperatures (18, 50, and 70 degrees C), with water as the catalyst and silane at either 0.2% or 2% (w/v) in an attempt to optimize the process. The products of the various reactions were characterized in terms of their possession of surface -NH2 groups, morphologies, and properties with respect to DNA binding and elution before being modified with a single-stranded oligonucleotide capture sequence. It was observed that careful manipulation of temperature, time, and solvent conditions was important for optimal silanization of the nanoparticles, and in our experiments best results were obtained when silanization of the particles in suspension involved use of water as the solvent and APTS at 0.2% (w/v) and when the reaction was conducted at room temperature for 5 h and was preceded by ultrasonication of the particle suspension. The materials produced were used in experiments to selectively capture complementary nucleic acid sequences by hybridization after grafting with an oligonucleotide. The efficiency of the oligonucleotide-modified particles in the capture experiments was observed to be directly related to the original density of amine groups present at the surface of the support. The results indicate that surface engineering of the nanoparticles was possible by silanization under defined, optimized conditions. This approach could be extended to the activation of such surfaces and other materials with other functional groups.
  • Bertozzini, E., Penna, A., Pierboni, E., Bruce, I. and Magnani, M. (2005). Development of new procedures for the isolation of phytoplankton DNA from fixed samples. Journal of Applied Phycology [Online] 17:223-229. Available at: http://dx.doi.org/10.1007/s10811-005-2130-5.
    Phytoplankton samples collected for routine monitoring programmes have traditionally been preserved with fixatives before subsequent analytical procedures such as microscope-based identification, or simply to permit transport between laboratories. In recent years, to simplify identification and enumeration, the use of DNA or RNA probes coupled with the PCR assay has progressed and now represents a routine procedure for screening cultured and field samples. However, the phytoplankton cells have often still to be treated as fixed samples.
    The extraction of genomic DNA from fixed cultures of Alexandrium minutum cultures was compared using two new methods based on Magnetisable Solid Phase Support (MSPS) techniques with that using three commercial kits. Genomic DNA recovery and PCR amplification were observed and the results obtained from culture samples were validated using field samples. Among the DNA extraction techniques considered, the MSPS methods provided the best results.
  • Del Campo, A., Sen, T., Lellouche, J. and Bruce, I. (2005). Multifunctional magnetite and silica-magnetite nanoparticles: Synthesis, surface activation and applications in life sciences. Journal of Magnetism and Magnetic Materials [Online] 293:33-40. Available at: http://dx.doi.org/10.1016/j.jmmm.2005.01.040.
    A method for the introduction of amine groups onto the surface of magnetite and silica-coated magnetite nanoparticles has been established based on the condensation of aminopropyltriethoxysilane. Amine-modified particles were grafted with an oligonucleotide and used in the capture of a complimentary sequence. The particles' efficiency at capture was observed to correlate directly with amine group surface density.
  • Amagliani, G., Brandi, G., Omiccioli, E., Magnani, M., Bruce, I. and Casiere, A. (2004). Direct detection of Listeria monocytogenes from milk by magnetic based DNA isolation and PCR. Food Microbiology [Online] 21:597-603. Available at: http://dx.doi.org/10.1016/j.fm.2003.10.008.
    Two nucleic acid-based methods for rapid and sensitive detection of the foodborne pathogen Listeria monocytogenes in milk were developed in this study. These methods rely on paramagnetic nanoparticle-based isolation of bacterial DNA directly from milk and subsequent PCR with selective primers for the listeriolysin O (hlyA) gene. The hlyA specific product was reproducibly detected and showed a sensitivity of 10 cfu ml(-1). The magnetic-based system had a sensitivity 10-fold higher than that of commercially available column devices. The detection limit of both methods is sufficient for direct detection of L. monocytogenes DNA in milk avoiding the enrichment culturing step, reducing the time necessary to obtain results from samples to 7 h rather than the 5-day minimum required for the standard procedure. The methods developed are suitable for automation.
  • Sanden, M., Bruce, I., Rahman, M. and Hemre, G. (2004). The fate of transgenic sequences present in genetically modified plant products in fish feed, investigating the survival of GM soybean DNA fragments during feeding trials in Atlantic salmon, Salmo salar L. Aquaculture [Online] 237:391-405. Available at: http://dx.doi.org/10.1016/j.aquaculture.2004.04.004.
    Vegetable protein sources like soybeans, canola and maize gluten are good alternatives to fish meal. However, a large proportion of such products available on the international market may possess genetically modified (GM) components. This report concerns a study to investigate the fate and survival of ingested GM soy DNA fragments (120 and 195 bp) and a 180-bp fragment of the lectin gene of soybean (Glycine max) during feeding trials with Atlantic salmon post-smolt. Specifically, the study focused on the fate of selected GM soy DNA fragments from feed to fish to investigate their survival through the fish gastrointestinal (GI) tract and whether the DNA could be traced in a variety of fish tissues. Fish were fed three experimental diets for 6 weeks, which were formulated from defined components and represented either GM or non-GM materials (17.2% of the fish meal was replaced with either GM or non-GM soy). A control diet composed of fish meal as the only protein source was used for comparison purposes. The transgenic sequences (120 and 195 bp) and the lectin gene (180 bp) could be detected in the GM soy feed. In the fish GI tract, however, only the smaller DNA fragment (120 bp) could be amplified from the content of the stomach, pyloric region, mid intestine and distal intestine. No transgenic or conventional soy DNA fragments could be detected in liver, muscle or brain tissues resected from sacrificed fish. The sensitivity limit of the method was evaluated to be 20 copies. These data indicate that GM soy transgenic sequences may survive passage through the GI tract but that they cannot be traced in fish tissues.
  • Lellouche, J., Perlman, N., Joseph, A., Govindaraji, S., Buzhansky, L., Yakir, A. and Bruce, I. (2004). New magnetically responsive polydicarbazole-magnetite nanoparticles. Chemical Communications [Online]:560-561. Available at: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=14973607.
    Magnetically responsive COOH-polydicarbazole-magnetite nanocomposites have been prepared by chemical oxidation of three COOH-dicarbazole monomers and - in the presence of magnetite nanoparticles. These functionalized nanoparticles have been tested for DNA hybridization experiments.
  • Bruce, I., Taylor, J., Todd, M., Davies, M., Borioni, E., Sangregorio, C. and Sen, T. (2004). Synthesis, characterisation and application of silica magnetite nanocomposites. Journal of Magnetism and Magnetic Materials 284:145-160.
    Novel material with unique properties produced as part of international collaboration on nanocomposite materials with applications in bioseperations. Competitively funded with EU FP5 RTD project grant 5,200,000 Euro. Material has been licensed to numerous companies where it is now produced in-house and forms the basis of many test kits including one major method for GM testing which is listed in the ‘Compendium of Microbiological Methods for the Analysis of Food and Agricultural Products’, AOAC INTERNATIONAL , 481 North Frederick Avenue, Suite 500, Gaithersburg, MD 20877-2417 USA
  • Poh, R., Smith, A. and Bruce, I. (2002). Complete characterisation of Tn5530 from Burkholderia cepacia strain 2a (pIJB1) and studies of 2,4-dichlorophenoxyacetate uptake by the organism. Plasmid [Online] 48:1-12. Available at: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=12206751.
    The complete genetic characterisation of Tn5530 in Burkholderia cepacia strain 2a (pIJB1) has been accomplished, indicating that it is a Tn3-like transposon with a complex structure bearing operons for the catabolism of 2,4-dichlorophenoxyacetate (2,4-D) and malonate. Tn5530 is terminated at both ends by the IS1071::IS1471 element and the 2,4-D- and malonate-dissimilatory operons are separated by a region encoding a putA and lrp gene and a gene encoding a chloride channel protein. The chloride channel protein may have a role in the expulsion of chloride ions liberated by the dissimilation of 2,4-D. In addition, a putative transposase with a high level of sequence similarity to those of plasmid pGH1 from Xanthomonas campestris pv. vesicatoria and Pseudomonas syringae pv. glycinea, and a transcription factor similar to those of the TetR family with low but significant levels of sequence similarity to those identified in a number of other organisms was observed. The entire Tn5530 sequence length, including the IS1071::IS1471 elements, was found to be 40,956bp, and pIJB1 was replicon-typed and otherwise characterised as being of the IncP-1beta subgroup, bearing merA and merD genes conferring resistance to mercuric chloride. The rate of uptake of 2,4-D by B. cepacia strain 2a was observed to proceed more readily at acid pH, suggesting involvement of the undissociated form of the compound. Uptake did not show saturation kinetics, was concentration-dependent, and appeared to occur in two stages; an initial accumulation followed by a linear second phase. Uptake could be inhibited by sodium azide but not by arsenate, N,N(')-dicyclohexylcarbodi-imide (DCCD) or carbonylcyanide m-chlorophenyl-hydrazone (CCCP) suggesting that it is not energy-dependent.
  • Poh, R., Xia, X., Bruce, I. and Smith, A. (2001). 2,4-Dichlorophenoxyacetate/alpha-ketoglutarate dioxygenases from Burkholderia cepacia 2a and Ralstonia eutropha JMP134. Microbios 105:43-63.
    2,4-Dichlorophenoxyacetate (2,4-D)/alpha -ketoglutarate (alpha -KG) dioxygenase has been purified to apparent homogeneity from Burkholderia cepacia strain 2a, which utilizes 2.4-D as sole carbon source. The enzyme required ferrous ions, and was a homodimer composed of subunits having an M-r of similar to 32,000. The reaction catalysed consumed one mol each of 2,4-D, alpha -KG and dioxygen, with the production of one mol each of succinate, 2,4-dichlorophenol and glyoxylate. Maximum activity was exhibited at pH 7.8 and 25 degreesC, and reactivity was enhanced by the presence of ascorbate and cysteine. Mn2+, Zn2+, Cu2+ Fe3+ and Co2+ were inhibitory, and chemical modification of the dioxygenase revealed that thiol groups were essential for activity. The enzyme was active towards other substituted phenoxyacetates, but reacted most rapidly with 2,4-D. The apparent Michaelis constants for 2,4-D and alpha -KG were 109 and 8.9 muM, respectively. The properties of this enzyme are compared with those of the 2,4-D/alpha -KG dioxygenase from Ralstonia eutropha JMP134, which exhibits a differing N-terminal amino-acid sequence, and a different temperature 'optimum', pH optimum, substrate specificity and sensitivity to thiol-binding reagents.
  • Bignell, G., Bruce, I. and Evans, I. (2000). Amylolytic enzymes of Lipomyces starkeyi: Purification and size-determination. Biotechnology Letters [Online] 22:1713-1718. Available at: http://dx.doi.org/10.1023/A:1005692300345.
    ?-Amylase accumulated in the growth medium of the yeast Lipomyces starkeyi (NCYC 1436) to a maximum of 12 units ml-1 after 15 days' batch culture in a soluble starch medium. Extra ?-amylase activity, about 50 of that released into the medium, could be extracted from the cells by a salt wash. ?-Amylase was purified by salt precipitation followed by ion exchange chromatography and gel filtration. Native gel electrophoretic analysis showed the presence of three distinct amylolytic enzymes, with molecular weights of approximately 50, 70 and 80 kDa.
  • Taylor, J., Hurst, C., Davies, M., Sachsinger, N. and Bruce, I. (2000). Application of magnetite and silica-magnetite composites to the isolation of genomic DNA. Journal of Chromatography A [Online] 890:159-166. Available at: http://dx.doi.org/10.1016/S0021-9673(00)00107-2.
    Magnetite and silica-magnetite composites were used as adsorbents for the isolation of genomic DNA from maize kernels. Two methods are described for the preparation of silica-magnetite composites, both of which afford higher yields of genomic DNA than when using magnetite alone, or a commerically available kit. DNA isolated using silica-magnetite was suitable for use in further applications such as polymerase chain reaction amplification and enzyme digestion.
  • Davies, M., Shah, A. and Bruce, I. (2000). Synthesis of fluorescently labelled oligonucleotides and nucleic acids. Chemical Society Reviews [Online] 29:97-107. Available at: http://dx.doi.org/10.1039/a905230e.
    The demand for and application of fluorescently labelled nucleic acid materials are growing, driven by ventures such as the Human Genome Mapping Project and the advent of DNA-based clinical diagnostics. This article surveys the strategies that are currently available for conjugation of fluorescent molecules to oligonucleotides and nucleic acids, and demonstrates how the labelled materials have been used in new and innovative assays for probing nucleic acid structure and properties.
  • Hurst, C., Knight, A. and Bruce, I. (1999). PCR detection of genetically modified soya and maize in foodstuffs. Molecular Breeding [Online] 5:579-586. Available at: http://dx.doi.org/10.1023/A:1009654623025.
    The detection of genetically modified foodstuffs is becoming both a food sales and legal necessity. This study reports a rapid DNA extraction/PCR-based method for the detection of genetically modified soya (GMS) and maize (GMM) in mixed samples of transgenic and unmodified soybeans and maize kernels, and a variety of processed samples including soya flour, soya protein isolates, extruded defatted soya, acid- and alcohol-precipitated soya concentrates, soya lecithin, maize grits, seasoned corn puffs and salted corn chips. The presence of GMS DNA was determined with two pairs of primers directed towards different GMS target sequences and GMM by one primer pain. In addition, a multiplex PCR reaction which utilises an internal positive control was developed for both genetically modified organisms (GMOs). Results indicated that the methods are sensitive and specific enough to detect GMS down to a level of 0.01 dry weight in single-product PCRs and 0.1 in multiplex PCRs and GMM down to 0.001 dry weight in single-product PCRs and 0.01 in multiplex PCR. The methods are considered to represent a viable route for the commercial detection of GMS and GMM in foodstuffs.
  • Hurst, C., Bartlett, S., Davidson, W. and Bruce, I. (1999). The complete mitochondrial DNA sequence of the Atlantic salmon, Salmo salar. Gene [Online] 239:237-242. Available at: http://dx.doi.org/10.1016/S0378-1119(99)00425-4.
    The complete sequence of the Atlantic salmon (Salmo salar) mitochondrial genome has been determined. The entire sequence is 16665 base pairs (bp) in length, with a gene content (13 protein-coding, two ribosomal RNA rRNA and 22 transfer RNA tRNA genes) and order conforming to that observed in most other vertebrates. Base composition and codon usage have been detailed. Nucleotide and derived amino acid sequences of the 13 protein-coding genes from Atlantic salmon have been compared with their counterparts in rainbow trout. A putative structure for the origin of L-strand replication (O(L)) is proposed, and sequence features of the control region (D-loop) are described.

Book section

  • East, D., Todd, M. and Bruce, I. (2014). Quantum dot-antibody conjugates via carbodiimide-mediated coupling for cellular imaging. In: Fontes, A. and Saegesser Santos, B. eds. Quantum Dots: Applications in Biology. Springer, pp. 67-83. Available at: http://dx.doi.org/10.1007/978-1-4939-1280-3_5.
    This chapter describes the processes of antibody (Ab) production, purification, conjugation to quantum dots (QDs), and the use of the conjugates produced in intracellular imaging of cell components and structures. Specifically, information is provided on the conjugation of carboxyl surface-terminated QDs to Abs via a one-step reaction using the water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). The chapter details the process of conjugate optimization in terms of its final fluorescence and biological activity. The method described should guarantee the production of QD-Ab conjugates, which outperform classic organic fluorophore-Ab conjugates in terms of both image definition produced and the longevity of the imaging agent.
  • del Campo, A. and Bruce, I. (2005). Substrate Patterning and Activation Strategies for DNA Chip Fabrication. In: Wittmann, C. ed. Immobilisation of DNA on Chips I. Berlin: Springer, pp. 77-112.
  • del Campo, A. and Bruce, I. (2005). Diagnostics and High Throughput Screening. In: Malsch, N. ed. Biomed Nanotechnology. London: Marcel Dekker, pp. 75-112.
  • Bruce, I., Sen, T. and del Campo, A. (2005). Key for tomorrow: Nanotechnology in food analysis. In: Van Amerongen, A., Barug, D. and Lauwaars, M. eds. Rapid Methods for Biological and Chemical Contaminants in Food and Feed. Wageningen Academic Publishers, pp. 387-408. Available at: https://doi.org/10.3920/978-90-8686-538-3.
  • del Campo, A. and Bruce, I. (2005). Diagnostics and High Throughput Screening. In: Malsch, N. H. ed. Biomedical Nanotechnology. CRC Press, pp. 75-112.
  • Yiu, H. and Bruce, I. (2003). Biological applications of organically functionalised mesoporous molecular sieves and related materials. In: Park, S.-E., Ryoo, R., Ahn, W.-S., Lee, C. W. and Chang, J.-S. eds. Nanotechnology in Mesostructured Materials. Oxford: Elsevier, pp. 581-584.
    The adsorption behaviour of mesoporous molecular sieves SBA-15 and MCF was studied using a family of polysaccharide molecules, dextrans. The results provide some insight into the accessibility of the mesopores to biological molecules. The effect of an amine functionalised surface of the solid was also examined.
  • Yiu, H., Bruce, I., McGuinness, F. and Wright, P. (2003). A novel preparation route for palladium-carbon composite materials - pore filling of SBA-15 mesoporous molecular sieve. In: Park, S.-E., Ryoo, R., Ahn, W.-S., Lee, C. W. and Chang, J.-S. eds. Nanotechnology in Mesostructured Materials. Oxford: Elsevier, pp. 57-60. Available at: http://dx.doi.org/10.1016/S0167-2991(03)80326-9.
    A new, simple preparation route for mesoporous carbon-palladium materials is reported. In this method, a direct decomposition of palladium acetate into palladium metal was applied during carbonisation. The size of the palladium metal particles ranged from 5 to 10 nm in diameter.
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