Effects of iron complexation and indiscriminate uptake on shoot iron of barley

The free-ion model (FIM) describes iron ( Fe) uptake by barley [ Hordeum vulgare ( L.) 'Grammett'] as being controlled by the activity of the buffered, free, uncomplexed Fe3+ in solution. Chelators' effect on Fe uptake by barley was evaluated and the rate of exchange of Fe between chelators was examined. Barley was grown for two weeks in a low-Fe nutrient solution and transferred to solutions varying in Fe and chelators for 6 h assays. Shoot Fe-59 was higher in barley grown in citrate (7743 and 1928 Fe-59 Bq g(-1)) than in NTA(3220 and 1113 Fe-59 Bq g(-1); P = 0.045) despite similar free-Fe3+ activities. A comparison of Fe uptake by barley from solutions with pFe(3+) activities of 17.1 and 24.6 showed < 5% was from indiscriminate apoplastic-flow uptake (3250 Fe-59 Bq g(-1) vs. 160 Fe-59 Bq g(-1)). Using nutrient solutions from the barley studies but without plants, Fe exchange between chelators and a simulant for the barley phytosiderophore occurred within hours ( for NTA and citrate), or days ( EDTA and HEDTA). Results were similar between the barley and Fe-exchange experiments for the two nutrient-solution treatments where the same Fe3+ activities but different total-Fe concentrations were used: the higher total-Fe treatment resulted in six-fold higher shoot Fe-59, while in the Fe-exchange study that treatment had six-fold more Fe bound to the phytosiderophore simulant after 2 d. Results indicated deviations from the FIM were not explained by indiscriminate-flow uptake, and that sluggish Fe-exchange reactions between chelate and phytosiderophoresimulant, not FIM guidelines, may be more important in explaining Fe uptake from synthetic chelates by Fe-deficient barley.

Effect of curve fitting parameters on quantitative analysis of Fe0.94O and Fe2O3 using XPS

The XPS peaks of Fe 3p for Fe2+ and Fe3+ in FeO and Fe2O3, respectively, have been measured and the effects of curve fitting parameters on interpretation of the data have been analysed. Firstly, the peak fit parameters, i.e. (1) the number of peaks to be deconvoluted, (2) the range of the peak for back ground subtraction, (3) straight line (Li) or the Shirley (Sh) background subtraction method, (4) GL ratio (the ratio of Gaussian and Lorentzian contribution to the peak shape) and (5) asymmetry factor (AS), are manually selected. Secondly, the standard peak fit parameters were systematically investigated. The peak shape was fitted to a Voigt function by changing the peak position, the peak height and the full width at half maximum (FWHM) to minimize the chi(2). The recommended peak positions and peak parameters for Fe2+ and Fe3+ in iron oxides have been determined. (c) 2006 Elsevier B.V. All rights reserved.

Dispersion and size control of layered double hydroxide nanoparticles in aqueous solutions

We report a simple but efficient method to prepare stable homogeneous suspensions containing monodispersed MgAl layered double hydroxide (LDH) nanoparticles that have wide promising applications in cellular drug ( gene) delivery, polymer/LDH nanocomposites, and LDH thin films for catalysis, gas separation, sensing, and electrochemical materials. This new method involves a fast coprecipitation followed by controlled hydrothermal treatment under different conditions and produces stable homogeneous LDH suspensions under variable hydrothermal treatment conditions. Moreover, the relationship between the LDH particle size and the hydrothermal treatment conditions ( time, temperature, and concentration) has been systematically investigated, which indicates that the LDH particle size can be precisely controlled between 40 and 300 nm by adjusting these conditions. The reproducibility of making the identical suspensions under identical conditions has been confirmed with a number of experiments. The dispersion of agglomerated LDH aggregates into individual LDH crystallites during the hydrothermal treatment has been further discussed. This method has also been successfully applied to preparing stable homogeneous LDH suspensions containing various other metal ions such as Ni2+, Fe2+, Fe3+, Co2+, Cd2+, and Gd3+ in the hydroxide layers and many inorganic anions such as Cl-, CO32-, NO3-, and SO42-.

The relationships between microstructure and crystal structure in zincite solid solutions

Single phase (Zn,Fe)(1-x) O zincite solid solution samples have been prepared by high temperature equilibration in air and in reducing atmospheres, followed by quenching to room temperature. The Fe2+/Fe3+ concentrations in the samples have been determined using wet chemical and XPS techniques. Iron is found to be present in zincite predominantly in the form of Fe3+ ions. The transition from an equiaxed grain morphology to plate-like zincite crystals is shown to be associated with increasing Fe3+ concentration, increasing elongation in < 001 > of the hexagonal crystals and increasing anisotropic strain along the c-axis. The plate-like crystals are shown to contain planar defects and zincite polytypes at high iron concentrations.

Siderophore activity of myo-inositol hexakisphosphate in Pseudomonas aeruginosa

Myo-Inositol hexakisphosphate (InsP6), which is found in soil and most, if not all, plant and animal cells, has been estimated to have an affinity for Fe3+ in the range of 10(25) to 10(30) M-1. In this report, we demonstrate that the Fe-InsP6 complex has siderophore activity and is able to reverse the iron-restricted growth inhibition of Pseudomonas aeruginosa by ethylene diamine di(o-hydroxyphenyl)acetic acid. With 55Fe-InsP6 in transport studies, iron uptake is strongly iron regulated, being repressed after growth in iron-replete conditions and inhibited by treatment with potassium cyanide and carbonyl cyanide m-chlorophenylhydrazone. The kinetics of iron transport revealed a Km of 100 nM. Self-displacement of binding of [3H]InsP6 to isolated membranes by InsP6 revealed a single class of binding sites (Kd = 143 +/- 6 nM; Hill coefficient, 1.1 +/- 0.1). The binding of [3H]InsP6 to membranes was not dependent on whether cells had been grown under conditions of high or low iron concentrations. We believe that this is the first report of inositol polyphosphate activity in prokaryotic cells.

Inhibition of iron-catalysed hydroxyl radical formation by inositol polyphosphates: a possible physiological function for myo-inositol hexakisphosphate

1. The ability of myo-inositol polyphosphates to inhibit iron-catalysed hydroxyl radical formation was studied in a hypoxanthine/xanthine oxidase system [Graf, Empson and Eaton (1987) J. Biol. Chem. 262, 11647-11650]. Fe3+ present in the assay reagents supported some radical formation, and a standard assay, with 5 microM Fe3+ added, was used to investigate the specificity of compounds which could inhibit radical generation. 2. InsP6 (phytic acid) was able to inhibit radical formation in this assay completely. In this respect it was similar to the effects of the high affinity Fe3+ chelator Desferral, and dissimilar to the effects of EDTA which, even at high concentrations, still allowed detectable radical formation to take place. 3. The six isomers of InsP5 were purified from an alkaline hydrolysate of InsP6 (four of them as two enantiomeric mixtures) and they were compared with InsP6 in this assay. Ins(1,2,3,4,6)P5 and D/L-Ins(1,2,3,4,5)P5 were similar to InsP6 in that they caused a complete inhibition of iron-catalysed radical formation at > 30 microM. Ins(1,3,4,5,6)P5 and D/L-Ins(1,2,4,5,6)P5, however, were markedly less potent than InsP6, and did not inhibit radical formation completely; even when Ins(1,3,4,5,6)P5 was added up to 600 microM, significant radical formation was still detected. Thus InsP5s lacking 2 or 1/3 phosphates are in this respect qualitatively different from InsP6 and the other InsP5s. 4. scyllo-Inositol hexakisphosphate was also tested, and although it caused a greater inhibition than Ins(1,3,4,5,6)P5, it too still allowed detectable free radical formation even at 600 microM. 5. We conclude that the 1,2,3 (equatorial-axial-equatorial) phosphate grouping in InsP6 has a conformation that uniquely provides a specific interaction with iron to inhibit totally its ability to catalyse hydroxyl radical formation; we suggest that a physiological function of InsP6 might be to act as a 'safe' binding site for iron during its transport through the cytosol or cellular organelles

Characterization of metal ion-induced [3H]inositol hexakisphosphate binding to rat cerebellar membranes

The binding of [3H]inositol hexakisphosphate ([3H] InsP6) to rat cerebellar membranes has been characterized with the objective of establishing the role, if any, of a membrane protein receptor. In the presence of EDTA, we have previously identified an InsP6-binding site with a capacity of approximately 20 pmol/mg protein (Hawkins, P. T., Reynolds, D. J. M., Poyner, D. R., and Hanley, M. R. (1990) Biochem. Biophys. Res. Commun. 167, 819-827). However, in the presence of 1 mM Mg2+, the capacity of [3H]InsP6 binding to membranes was increased approximately 9-fold. This enhancing effect of Mg2+ was reversed by addition of 10 microM of several cation chelators, suggesting that the increased binding required trace quantities of other metal cations. This is supported by experiments where it was possible to saturate binding by addition of excess membranes, despite not significantly depleting radioligand, pointing to removal of some other factor. Removal of endogenous cations from the binding assay by pretreatment with chelex resin also prevents the Mg(2+)-induced potentiation. Consideration of the specificity of the chelators able to abolish this potentiation suggested involvement of Fe3+ or Al3+. Both these ions (but not several others) were able to increase [3H]InsP6 binding to chelex-pretreated membranes at concentrations of 1 microM. It is possible to demonstrate synergy between Fe3+ and Mg2+ under these conditions. We propose that [3H]InsP6 may interact with membranes through non-protein recognition possibly via phospholipids, in a manner dependent upon trace metals. The implications of this for InsP6 biology are considered.

Simplified preparation of human arterial sections for PCR analysis of Chlamydia pneumoniae and human DNA

AIMS: To investigate multiple techniques for the preparation of solid tissue for polymerase chain reaction (PCR) analysis, and to identify the most simple techniques for routine use in the laboratory. METHODS: Techniques for the preparation of arterial tissue samples including homogenisation, ultrafiltration, and treatments involving proteinase K, Gene Clean, lectin, and Fe3+ specific chelators were evaluated using the PCR to amplify both Chlamydia pneumoniae and human DNA. RESULTS: Treatment with either Gene-Clean or lectin and the Fe3+ specific chelator deferoxamine mesylate removed PCR inhibitors from tissue homogenates. Homogenisation followed by GeneClean treatment resulted in the amplification of C pneumoniae DNA from within a section of atherosclerotic carotid artery, implying that C pneumoniae elementary bodies had been disrupted. In eight further clinical samples from patients not known to have C pneumoniae infection, human DNA was amplified and no cross contamination was observed between samples. These samples contained no evidence of C pneumoniae by PCR. CONCLUSIONS: A simple preparation of solid tissue for PCR analysis, involving homogenisation followed by GeneClean treatment has been developed, and is effective for the amplification of both C pneumoniae and human DNA.

Origin of the ESR spectrum in the Prussian Blue analogue RbMn[Fe(CN)6]*H2O

We present an ESR study at excitation frequencies of 9.4 GHz and 222.4 GHz of powders and single crystals of a Prussian Blue analogue (PBA), RbMn[Fe(CN)6]*H2O in which Fe and Mn undergoes a charge transfer transition between 175 and 300 K. The ESR of PBA powders, also reported by Pregelj et al. (JMMM, 316, E680 (2007)) is assigned to cubic magnetic clusters of Mn2+ ions surrounding Fe(CN)6 vacancies. The clusters are well isolated from the bulk and are superparamagnetic below 50 K. In single crystals various defects with lower symmetry are also observed. Spin-lattice relaxation broadens the bulk ESR beyond observability. This strong spin relaxation is unexpected above the charge transfer transition and is attributed to a mixing of the Mn3+ - Fe2+ state into the prevalent Mn2+ - Fe3+ state.

Reactions of directly related tellurium and selenium heterocycic compunds with triiron dodecacarbonyl

The reactions of directly related tellurium and selenium heterocyclic compounds with triiron dodecacarbonyl are described. The reaction of 2-telluraphthalide, C8H8OTe with [Fe3(CO)12 gave [Fe{C6H4(CH2)Te}(CO)3]2, (1). An iron atom has inserted into the telluracyclic ring, and it is probable that one co-ordinated CO ligand arises from the initially organic carbonyl group. X-ray analysis of compound (1) showed that the compound has a Fe2Te2 core, which is achieved by dimerisation. The reaction of telluraphthalic anhydride, C8H402Te with [Fe3(CO)12] gave a known, but unexpected, organic phthalide product, C8H602, which was confirmed by X-ray crystallography. Selenaphthalic anhydride,  C8H4O2Se gave intractable products on reaction with [Fe3(CO)12], 2-selenaphthalide, C8H6OSe, on reaction with [Fe3(CO)12] gave a major product [Fe2{C6H4(CH2)Se}(CO)6], (2) and a minor product [Fe3{C6H4(CH2)Se}(CO)8], (3) which is an intermediate in the formation of (2). X-ray analysis of (2) shows that compound (2) is very similar to (1) except that the 18 electron rule is satisfied by co-ordination of a Fe(CO)3 moiety, rather than dimerisation. Compound (3), also studied by X-ray crystallography, differs from (2) mainly in the addition of an Fe(CO)2 moiety. Telluraphtbalic anhydride, C8H402Te, and selenaphthalic anhydride, C8H402Se, are both monoclinic and crystallise in space group P21/n. 2-Selenaphthalide, C8H402Se, is also monoclinic, space group P21/C. The reactions of the following compounds (l,3-dihydrobenzo[c]selenophene, 1,3,7,9-tetrahydrobenzo[1,2c; 4,5c'] ditellurophene, dibenzoselenophene, phenoxselenine, 3, 5-naphtho-1-telluracyclohexane and 3,5-naphtho-1-selenacyclohexane) with [Fe3lCO)12] are reported. It is unfortunate that the above compounds do not react under the conditions employed; this may be due to differing degrees of ring strain. 1,8-bis(bromomethyl)naphthalene, C12H10Br2 is monoclinic and crystallises in space group C2/c. 1,1-diiodo-3,5-naphthotelluracyclohexane, C12H10TeI2 and 3,5-naphtho-l-telluracyclohexane, C12H10Te are monoclinic and crystallise in space group P21/c. 3,5-naphtho-l-selenacyclohexane, C12H10Se and 2,2,8,8-tetraiodo-1,3,7,9-tetrahydrobenzo[1,2c;4,5c']ditellurophene are also monoclinic, space group P21/a. The syntheses of intramolecular stabilised organo-tellurium and selenium compounds are reported, having a general formula of REX (where R = phenylazophenyl; E = Se, Te; X = electronegative group, for example C1, Br or I). The crystal structures of R'TeBr, RTeI, RSeCI, RSeCI/I and RSeI (where R = phenylazophenyl) are reported. The tellurium containing X-ray structures are triclinic and have a space group P-1. The selenium containing X-ray structures are monoclinic with space group P21/n. The inclusion of nitrogen in selenium heterocycles provides access to an entirely new area of organometallic chemistry. The reaction of 2-methylbenzoselenazole with [Fe3(CO)12] gave [Fe2{C6H4(NCH2CH3)Se}(CO)6]. The reactions of 2-(methyltelluro)benzanilide or 2-(methylseleno)benzanilide with [Fe3(CO)12] gave reaction products [Fe2(μTeMe)2(CO)6] and [Fe2 (μ-SeMe)2(CO)6] respectively, which were confmned by X-ray crystallography. The use of Mossbauer spectroscopy on the products obtained from the reactions of heterocyclic compounds with [Fe3(CO)12] can give useful information, for example the number of iron sites and the environments of these iron sites within the products.

The use of organotellurium heterocycles as precursors for novel organometallic compounds

The reactions of group 16 heterocycles with organometallic reagents are described. Thiophenes have been used as models for organic sulfur in coal and their reactivity towards triiron dodecacarbonyl has been investigated. Reaction of unsubstituted thiophene with Fe3(CO)12 results in desulfurisation of the heterocycle, with the organic fragment being recovered in the form of the ferrole, C4H4.Fe2(CO)6. In addition a novel organometallic compound of iron is isolated, the formula of which is shown to be C4H4.Fe3(CO)8. Bezothiophene reacts with Fe3(CO)12 to yield benzothiaferrole, C8H6S.Fe2(CO)6, in which the sulfur is retained in the heterocycle. Dibenzothiophene, a more accurate model for organic sulfur in coal, displays no reactivity towards the iron carbonyl, suggesting that the more condensed systems will desulfurise less readily. Microwave methodology has been successful in accelerating the reactions of thiophenes with Fe3(CO)12. However, reaction of benzothiophene does not proceed to the desulfurisation stage while dibenzothiophene is unreactive even under microwave conditions. Tellurophenes (Te analogues of thiophenes) are shown to mimic the behaviour of thiophenes towards certain organometallic reagents with the advantage that their greater reactivity enables recovery of products in higher yields. Hence, reaction of tellurophene with Fe3(CO)12 again affords the ferrole but with an almost ten-fold increase in yield over thiophene. More significantly, dibenzotellurophene is also detellurated by the iron carbonyl affording the previously inaccessible dibenzoferrole, C12H8.Fe2(CO)6, thereby demonstrating the mechanistic feasibility of dechalcogenation of the more condensed aromatic molecules. The potential of tellurium heterocycles to act as precursors for novel organometallics is also recognised owing to the relatively facile elimination of the heteroatom from these systems. Thus, 2-telluraindane reacts with Fe3(CO)12 to yield a novel organometallic compound of formula C16H16.Fe(CO)3, arising from the unsymmetric dimerisation of two organic fragments.

A novel and rational approach towards designing aluminium chelators

Aluminium (Al) is known to be neurotoxic and has been associated with the aetiology of Alzheimer's Disease. To date, only desferrioxamine (DFO), a trihydroxamic acid siderophore has been used in the clinical environment for the removal of Al from the body. However, this drug is expensive, orally inactive and is associated with many side effects. These studies employed a theoretical approach, with the use of quantum mechanics (QM) via semi-empirical molecular orbital (MO) calculations, and a practical approach using U87-MG glioblastoma cells as a model for evaluating the influence of potential chelators on the passage of aluminium into cells. Preliminary studies involving the Cambridge Structural Database (CSD) identified that Al prefers binding to bidentate ligands in a 3:1 manner, whereby oxygen was the exclusive donating atom. Statistically significant differences in M-O bond lengths when compared to other trivalent metal ions such as Fe3+ were established and used as an acceptance criterion for subsequent MO calculations. Of the semi-empirical methods parameterised for Al, the PM3 Hamiltonian was found to give the most reliable final optimised geometries of simple 3:1 Al complexes. Consequently the PM3 Hamiltonian was used for evaluating the Hf of 3:1 complexes with more complicated ligands. No correlation exists between published stability constants and individual parameters calculated via PM3 optimisations, although investigation of the dicarboxylates reveals a correlation of 0.961 showing promise for affinity prediction of closely related ligands. A simple and inexpensive morin spectrofluorescence assay has been developed and optimised producing results comparable to atomic absorption spectroscopy methods for the quantitative analysis of Al. This assay was used in subsequent in vitro models, initially on E. coli, which indicated that Al inhibits the antimicrobial action of ciprofloxacin, a potent quinolone antibiotic. Ensuing studies using the second model, U87-MG cells, investigated the influence of chelators on the transmembrane transport of Al, identifying 1,2-diethylhydroxypyridin-4-one as a ligand showing greatest potential for chelating Al in the clinical situation. In conclusion, these studies have explored semi-empirical MO Hamiltonians and an in-vitro U87-MG cell line, both as possible methods for predicting effective chelators of Al.

Crystallographic studies of magnetite and titanomaghemite

The crystal structure of natural magnetite has been investigated on the basis of previously published X-ray intensity data and a newly acquired, more extensive data base. Both investigations show that the structure does not conform to the centrosymmetrical space group Fd3m, as is normally assumed, but the non-centrosymmetrical space group F43m. The structure refinement provides values for the atom positions, anisotropic thermal parameters and bond lengths. A study of Friedel related pairs of X-ray intensities shows that Friedel's law is violated in magnetite, further confirming that the space group is non-centrosymmetrical. It was found that the octahedral site cations in magnetite do not occupy special positions at the centres of the octahedral interstices as they should under the space group Fd3m, but are displaced along <111 > directions leading to F43m symmetry. A mechanism is known for the origin of these displacements and the likelihood of similar displacements occurring in other natural and synthetic spinels is discussed. The crystal structure of a natural titanomaghemite was determined by a combination of X-ray diffraction and Mõssbauer spectroscopy. This was confirmed as possessing a primitive cubic Bravais lattice with the space group P4332 and the structural formula: Fe3+.0.96 0 0.04 [Fe2+0.23 Fe3+0.99 Ti4+0.42 0 0.37 ] 042 - where 0 represents a cation vacancy. As the above formula shows, there are cation vacancies on both tetrahedral arrl octahedral sites, the majority being restricted to octahedral sltes. No tetrahedral site Fe2+ or Ti4+ was observed. Values for the atom positions, anisotropic thermal parameters and bond lengths have been determined for this particular specimen.

Spray dried combinations of lactoferrin with antibiotics appear superior to monotherapy for reducing biofilm formation by pseudomonas aeruginosa

SD Apo Lactoferrin-Tobramycin/Gentamicin Combinations are superior to monotherapy in the eradication of Pseudomonas aeruginosa Biofilm in the lungs Wilson Oguejiofor1, Lindsay J. Marshall1, Andrew J. Ingham1, Robert Price2, Jag. Shur2 1School of Life and Health Sciences, Aston University, Birmingham, UK. 2School of Pharmacy and Pharmacology, University of Bath, Bath, UK. KEYWORDS: lactoferrin, apo lactoferrin, spray drying, biofilm, cystic fibrosis Introduction Chronic lung infections from the opportunistic pathogeen Pseudomonas aeruginosa has been recognised as a major contributor to the incidences of high morbidity and mortality amongst cystic fibrosis (CF) patients (1,2). Currently, strategies for managing lung infections in CF patients involves the aggressive use of aerosolised antibiotics (3), however, increasing evidence suggests that the biofilm component of P. aeruginosa in the lower airway remains unperturbed and is associated with the development of antibiotic resistance. If this is so then, there is an urgent need to suitably adjust the current treatment strategy so that it includes compounds that prevent biofilm formation or disrupt established biofilms. It is well understood that biofilm formation is strongly dependent on iron (Fe3+) availability (4), therefore aerosolised anti-infective formulations which has the ability to chelate iron may essentially be a well suited therapy for eliminating P. aeruginosa biofilms on CF airway epithelial cells (5). In this study, we report the use of combination therapy; an aminoglycosides (tobramycin and gentamicin) and an antimicrobial peptide (lactoferrin) to significantly deplete P. aeruginosa biofilms. We demonstrate that lactoferrin-tobramycin and lactoferrin-gentamicin combinations are superior to the single antibiotic regime currently being employed to combat P. aeruginosa biofilms. MATERIALS AND METHOD Antibiotics: The antibiotics used in this study included gentamicin and tobramycin supplied by Fagron, UK. Bacterial strain and growth conditions: Pseudomonas aeruginosa strain PAO1 was provided by Prof. Peter Lambert of Aston University, Birmingham UK. The Strains were routinely grown from storage in a medium supplemented with magnesium chloride, glucose and casamino acids. Dialysis of lactoferrin: Apo lactoferrin was prepared by dialyzing a suspension of lactoferrin for 24 hrs at 4 °C against 20 mmol/L sodium dihydrogen phosphate, 20 mmol/L sodium acetate and 40 mmol/L EDTA (pH 3.5). Ferric ion (Fe3+) removal was verified by atomic absorption spectroscopy measurements. Spray drying of combinations of lactoferrin and apo lactoferrin with the different aminoglycosides: Combinations of tobramycin and gentamicin with the different preparations of lactoferrin were spray dried (SD) as a 2% (w/v) aqueous suspension. The spray drying parameters utilized for the production of suitable micron-sized particles includes: Inlet temperature, 180°C, spray flow rate, 606 L/hr; pump setting, 10%; aspirator setting, 85% (34m3/hr) to produce various outlet temperatures ranging from 99 - 106°C. Viability assay: To test the bactericidal activity of the various combinations, a viability assay was performed as previously described by Xu, Xiong et al. (6) with some modifications. Briefly, 10µL of ~ c. 6.6 x 107 CFU mL-1 P. aeruginosa strain PAO1 suspension were incubated (37°C, 60 mins) with 90 µL of a 2 µg/mL concentration of the various combinations and sampled every 10 mins. After incubation, the cells were diluted in deionised water and plated in Mueller hinton agar plates. Following 24 h incubation of the plates at 37°C, the percentage of viable cells was determined relative to incubation without added antibiotics. Biofilm assay: To test the susceptibility of the P. aeruginosa strain to various antibiotics in the biofilms mode of growth, overnight cultures of P. aeruginosa were diluted 1:100 into fresh medium supplemented with magnesium chloride, glucose and casamino acids. Aliquots of the dilution were dispensed into a 96 well dish and incubated (37°C, 24 h). Excess broth was removed and the number of colony forming units per milliliter (CFU/mL) of the planktonic bacteria was quantified. The biofilms were then washed and stained with 0.1% (w/v) crystal violet for 15 mins at room temperature. Following vigorous washing with water, the stained biofilms were solubilized in 30% acetic acid and the absorbance at 550nm of a 125 µL aliquot was determined in a microplate reader (Multiskan spectrum, Thermo Scientific) using 30% acetic acid in water as the blank. Aliquots of the broth prior to staining were used as an indicator of the level of planktonic growth. RESULTS AND DISCUSSION Following spray drying, the mean yield, volume weighted mean diameter and moisture content of lactoferrin powder were measured and were as follows (Table 1 and table 2); Table 1: Spray drying parameters FormulationInlet temp (°C)Outlet temp (°C)Airflow rate (L/hr)Mean yield (%)Moisture content (%) SD Lactoferrin18099 - 10060645.2 ±2.75.9 ±0.4 SD Apo Lactoferrin180100 - 10260657.8 ±1.85.7 ±0.2 Tobramycin180102 - 10460682.1 ±2.23.2 ±0.4 Lactoferrin + Tobramycin180104 - 10660687.5 ±1.43.7 ±0.2 Apo Lactoferrin + Tobramycin180103 - 10460676.3 ±2.43.3 ±0.5 Gentamicin18099 - 10260685.4 ±1.34.0 ±0.2 Lactoferrin + Gentamicin180102 - 10460687.3 ±2.13.9 ±0.3 Apo Lactoferrin + Gentamicin18099 -10360680.1±1.93.4 ±0.4 Table 2: Particle size distribution d10 d50d90 SD Lactoferrin1.384.9111.08 SD Apo Lactoferrin1.284.7911.04 SD Tobramycin1.254.9011.29 SD Lactoferrin + Tobramycin1.175.2715.23 SD Apo Lactoferrin + Tobramycin1.115.0614.31 SD Gentamicin1.406.0614.38 SD Lactoferrin + Gentamicin1.476.2314.41 SD Apo Lactoferrin + Gentamicin1.465.1511.53 The bactericidal activity of the various combinations were tested against P. aeruginosa PAO1 following a 60 minute incubation period (Figure 1 and Figure 2). While 2 µg/mL of a 1:1 combination of spray dried apo lactoferrin and Gentamicin was able to completely kill all bacterial cells within 40 mins, the same concentration was not as effective for the other antibiotic combinations. However, there was an overall reduction of bacterial cells by over 3 log units by the other combinations within 60 mins. Figure 1: Logarithmic plot of bacterial cell viability of various combinations of tobramycin and lactoferrin preparations at 2µg/mL (n = 3). Figure 2: Logarithmic plot of bacterial cell viability of various combinations of gentamicin and lactoferrin preparations at 2µg/mL (n = 3). Crystal violet staining showed that biofilm formation by P. aeruginosa PAO1 was significantly (ANOVA, p < 0.05) inhibited in the presence of the different lactoferrin preparations. Interestingly, apo lactoferrin and spray dried lactoferrin exhibited greater inhibition of both biofilm formation and biofilm persistence (Figure 2). Figure 2: Crystal violet staining of residual biofilms of P. aeruginosa following a 24hr incubation with the various combinations of antibiotics and an exposure to 48 hr formed biofilms. CONCLUSION In conclusion, combination therapy comprising of an antimicrobial peptide (lactoferrin) and an aminoglycosides (tobramycin or gentamicin) provides a feasible and alternative approach to monotherapy since the various combinations are more efficient than the respective monotherapy in the eradication of both planktonic and biofilms of P. aeruginosa. ACKNOWLEDGEMENT The authors would like to thank Mr. John Swarbrick and Friesland Campina for their generous donation of the Lactoferrin. REFERENCES 1.Hassett, D.J., Sutton, M.D., Schurr, M.J., Herr, A.B., Caldwell, C.C. and Matu, J.O. (2009), "Pseudomonas aeruginosa hypoxic or anaerobic biofilm infections within cystic fibrosis airways". Trends in Microbiology, 17, 130-138. 2.Trust, C.F. (2009), "Antibiotic treatment for cystic fibrosis". Report of the UK Cystic Fibrosis Trust Antibiotic Working Group. Consensus document. London: Cystic Fibrosis Trust. 3.Garcia-Contreras, L. and Hickey, A.J. (2002), "Pharmaceutical and biotechnological aerosols for cystic fibrosis therapy". Advanced Drug Delivery Reviews, 54, 1491-1504. 4.O'May, C.Y., Sanderson, K., Roddam, L.F., Kirov, S.M. and Reid, D.W. (2009), "Iron-binding compounds impair Pseudomonas aeruginosa biofilm formation, especially under anaerobic conditions". J Med Microbiol, 58, 765-773. 5.Reid, D.W., Carroll, V., O'May, C., Champion, A. and Kirov, S.M. (2007), "Increased airway iron as a potential factor in the persistence of Pseudomonas aeruginosa infection in cystic fibrosis". European Respiratory Journal, 30, 286-292. 6.Xu, G., Xiong, W., Hu, Q., Zuo, P., Shao, B., Lan, F., Lu, X., Xu, Y. and Xiong, S. (2010), "Lactoferrin-derived peptides and Lactoferricin chimera inhibit virulence factor production and biofilm formation in Pseudomonas aeruginosa". J Appl Microbiol, 109, 1311-1318.

Phenol methylation over nanoparticulate Co2FeO4 inverse spinel catalysts:the effect of morphology on catalytic performance

A series of CoFe2O4 nanoparticles have been prepared via co-precipitation and controlled thermal sintering, with tunable diameters spanning 7–50 nm. XRD confirms that the inverse spinel structure is adopted by all samples, while XPS shows their surface compositions depend on calcination temperature and associated particle size. Small (<20 nm) particles expose Fe3+ enriched surfaces, whereas larger (∼50 nm) particles formed at higher temperatures possess Co:Fe surface compositions close to the expected 1:2 bulk ratio. A model is proposed in which smaller crystallites expose predominately (1 1 1) facets, preferentially terminated in tetrahedral Fe3+ surface sites, while sintering favours (1 1 0) and (1 0 0) facets and Co:Fe surface compositions closer to the bulk inverse spinel phase. All materials were active towards the gas-phase methylation of phenol to o-cresol at temperatures as low as 300 °C. Under these conditions, materials calcined at 450 and 750 °C exhibit o-cresol selectivities of ∼90% and 80%, respectively. Increasing either particle size or reaction temperature promotes methanol decomposition and the evolution of gaseous reductants (principally CO and H2), which may play a role in CoFe2O4 reduction and the concomitant respective dehydroxylation of phenol to benzene. The degree of methanol decomposition, and consequent H2 or CO evolution, appears to correlate with surface Co2+ content: larger CoFe2O4 nanoparticles have more Co rich surfaces and are more active towards methanol decomposition than their smaller counterparts. Reduction of the inverse spinel surface thus switches catalysis from the regio- and chemo-selective methylation of phenol to o-cresol, towards methanol decomposition and phenol dehydroxylation to benzene. At 300 °C sub-20 nm CoFe2O4 nanoparticles are less active for methanol decomposition and become less susceptible to reduction than their 50 nm counterparts, favouring a high selectivity towards methylation.