(Table T1) Age determination of seven ODP Leg 201 sites

As part of a wider paleoclimate and paleoceanographic study of Holocene-upper Pleistocene laminated sediments from the eastern equatorial Pacific and Peru continental margin, we completed 32 accelerator mass spectrometry (AMS) 14C dates from cores recovered during Ocean Drilling Program (ODP) Leg 201. Sample preparation and measurement were carried out at the ANTARES AMS facility, Australian Nuclear Science and Technology Organisation (ANSTO), in Sydney, Australia (Lawson et al., 2000, doi:10.1016/S0168-583X(00)00276-7; Fink et al., 2004, doi:10.1016/j.nimb.2004.04.025). Although the sediments are predominantly diatomaceous oozes (D'Hondt, Jørgensen, Miller, et al., 2003, doi:10.2973/odp.proc.ir.201.2003), they contain sufficient inorganic (e.g., foraminifer tests and nannofossil plates) and organic (Meister et al., 2005, doi:10.2973/odp.proc.sr.201.105.2005) carbon to allow 14C dating. These dates permitted us to reconstruct a history of sediment accumulation over the past 20 k.y., particularly on the Peru continental margin. In this report we present 14C AMS dates and other pertinent data from cores from Sites 1227, 1228, and 1229 collected during Leg 201 at the Peru continental margin.

Sediment geochemistry of ODP Holes 206-1256B and 206-1256C

We measured the chemical composition of 100 samples from the 250-m sediment sequence retrieved from Ocean Drilling Program Site 1256 in the Guatemala Basin using a newly developed microwave-assisted acid digestion protocol followed by inductively coupled plasma-atomic emission spectroscopy (ICP-AES) analysis. We compared these data gathered onshore to the results from the flux fusion prepared samples analyzed by shipboard ICP-AES during the leg and published in the Leg 206 Initial Reports volume, as well as to 35 randomly selected samples that were prepared by flux fusion at Boston University and analyzed by ICP-AES. Comparison of the newly developed acid digestion protocol to shore-based flux fusion demonstrates that the microwave-assisted acid technique yields a complete digestion, and because this procedure includes boric acid, it is safe for use with HF acid as boric acid neutralizes excess HF. The precision for nearly all elements in shore-based acid digestions is better than 3% of the measured values, including for elements such as Ni, Cr, and V, which are typically difficult to measure in biogenic-rich sediments. The shore-based flux fusions, while better than shipboard reported precision values (as expected), has precision better than 3% of their respective measured values for all major elements (Si, Al, Ti, Fe, Mn, Ca, Mg, Na, and K) and several trace elements (Ba and Sr). Results for P, Cr, Ni, V, Sc, and Zr are better than 5% of their measured values. Not only does the newly developed acid digestion provide better analytical results than the typical flux fusion method, the shore-based acid procedure also exhibits downhole lithologic and chemical characteristics similar to the shipboard flux fusion prepared results. These results confirm that the current shipboard methods are adequate for first-order geochemical interpretations and that the microwave-assisted acid digestion holds great potential to be the primary technique of preparing sediments on future Integrated Ocean Drilling Program expeditions.

Chemical analysis of powdered metallurgical slags by X-ray fluorescence spectrometry

Slag composition determines the physical and chemical properties as well as the application performance of molten oxide mixtures. Therefore, it is necessary to establish a routine instrumental technique to produce accurate and precise analytical results for better process and production control. In the present paper, a multi-component analysis technique of powdered metallurgical slag samples by X-ray Fluorescence Spectrometer (XRFS) has been demonstrated. This technique provides rapid and accurate results, with minimum sample preparation. It eliminates the requirement for a fused disc, using briquetted samples protected by a layer of Borax(R). While the use of theoretical alpha coefficients has allowed accurate calibrations to be made using fewer standard samples, the application of pseudo-Voight function to curve fitting makes it possible to resolve overlapped peaks in X-ray spectra that cannot be physically separated. The analytical results of both certified reference materials and industrial slag samples measured using the present technique are comparable to those of the same samples obtained by conventional fused disc measurements.

Aluminium phosphide as a eutectic grain nucleus in hypoeutectic Al-Si alloys

Aluminium phosphide (AlP) particles arc often suggested to be the nucleation site for eutectic silicon in Al-Si alloys, since both the crystal structure and lattice parameter of AlP (crystal structure: cubic K(4) over bar m; lattice parameter: 5.421 Angstrom) are close to that of silicon (cubic Fd3m, 5.431 Angstrom), and the melting point is higher than the Al-Si eutectic temperature. However, the crystallographic relationships between AlP particles and the surrounding eutectic silicon are seldom reported due to the difficulty in analysing the AlP particles, which react with water during sample preparation for polishing. in this study, the orientation relationships between AlP and Si are analysed by transmission electron microscopy using focused ion-beam milling for sample preparation to investigate the nucleation mechanism of eutectic silicon on AlP. The results show a clear and direct lattice relationship between centrally located AlP particles and the surrounding silicon in the hypoeutectic Al-Si alloy.

Conventional detection methodology is limiting our ability to understand the roles and functions of fine roots

We lack a thorough conceptual and functional understanding of fine roots. Studies that have focused on estimating the quantity of fine roots provide evidence that they dominate overall plant root length. We need a standard procedure to quantify root length/biomass that takes proper account of fine roots. Here we investigated the extent to which root length/biomass may be underestimated using conventional methodology, and examined the technical reasons that could explain such underestimation. Our discussion is based on original X-ray-based measurements and on a literature review spanning more than six decades. We present evidence that root-length recovery depends strongly on the observation scale/spatial resolution at which measurements are carried out; and that observation scales/resolutions adequate for fine root detection have an adverse impact on the processing times required to obtain precise estimates. We conclude that fine roots are the major component of root systems of most (if not all) annual and perennial plants. Hence plant root systems could be much longer, and probably include more biomass, than is widely accepted.

Matrix effects: The Achilles heel of quantitative high-performance liquid chromatography-electrospray-tandem mass spectrometry

High-performance liquid chromatography coupled by an electrospray ion source to a tandem mass spectrometer (HPLC-EST-MS/ MS) is the current analytical method of choice for quantitation of analytes in biological matrices. With HPLC-ESI-MS/MS having the characteristics of high selectivity, sensitivity, and throughput, this technology is being increasingly used in the clinical laboratory. An important issue to be addressed in method development, validation, and routine use of HPLC-ESI-MS/MS is matrix effects. Matrix effects are the alteration of ionization efficiency by the presence of coeluting substances. These effects are unseen in the chromatograrn but have deleterious impact on methods accuracy and sensitivity. The two common ways to assess matrix effects are either by the postextraction addition method or the postcolumn infusion method. To remove or minimize matrix effects, modification to the sample extraction methodology and improved chromatographic separation must be performed. These two parameters are linked together and form the basis of developing a successful and robust quantitative HPLC-EST-MS/MS method. Due to the heterogenous nature of the population being studied, the variability of a method must be assessed in samples taken from a variety of subjects. In this paper, the major aspects of matrix effects are discussed with an approach to address matrix effects during method validation proposed. (c) 2004 The Canadian Society of Clinical Chemists. All rights reserved.

An automatable screen for the rapid identification of proteins amenable to refolding

Insoluble expression of heterologous proteins in Escherichia coli is a major bottleneck of many structural genomics and high-throughput protein biochemistry projects. Many of these proteins may be amenable to refolding, but their identification is hampered by a lack of high-throughput methods. We have developed a matrix-assisted refolding approach in which correctly folded proteins are distinguished from misfolded proteins by their elution from affinity resin under nondenaturing conditions. Misfolded proteins remain adhered to the resin, presumably via hydrophobic interactions. The assay can be applied to insoluble proteins on an individual basis but is particularly well suited for high-throughput applications because it is rapid, automatable and has no rigorous sample preparation requirements. The efficacy of the screen is demonstrated on small-scale expression samples for 15 proteins. Refolding is then validated by large-scale expressions using SEC and circular dichroism.

Microscopy imaging of liposomes:from coverslips to environmental SEM

A plethora of techniques for the imaging of liposomes and other bilayer vesicles are available. However, sample preparation and the technique chosen should be carefully considered in conjunction with the information required. For example, larger vesicles such as multilamellar and giant unilamellar vesicles can be viewed using light microscopy and whilst vesicle confirmation and size prior to additional physical characterisations or more detailed microscopy can be undertaken, the technique is limited in terms of resolution. To consider the options available for visualising liposome-based systems, a wide range of microscopy techniques are described and discussed here: these include light, fluorescence and confocal microscopy and various electron microscopy techniques such as transmission, cryo, freeze fracture and environmental scanning electron microscopy. Their application, advantages and disadvantages are reviewed with regard to their use in analysis of lipid vesicles.

Environmental scanning electron microscope imaging of vesicle systems

The structural characteristics of liposomes have been widely investigated and there is certainly a strong understanding of their morphological characteristics. Imaging of these systems, using techniques such as freeze-fracturing methods, transmission electron microscopy, and cryo-electron imaging, has allowed us to appreciate their bilayer structures and factors that influence this. However, there are a few methods that study these systems in their natural hydrated state; commonly, the liposomes are visualized after drying, staining and/or fixation of the vesicles. Environmental scanning electron microscopy (ESEM) offers the ability to image a liposome in its hydrated state without the need for prior sample preparation. We were the first to use ESEM to study the liposomes and niosomes, and have been able to dynamically follow the hydration of lipid films and changes in liposome suspensions as water condenses onto, or evaporates from, the sample in real-time. This provides an insight into the resistance of liposomes to coalescence during dehydration, thereby providing an alternative assay for liposome formulation and stability.

Mass spectrometry imaging of pharmacological compounds in tissue sections

The use of MS imaging (MSI) to resolve the spatial and pharmacodynamic distributions of compounds in tissues is emerging as a powerful tool for pharmacological research. Unlike established imaging techniques, only limited a priori knowledge is required and no extensive manipulation (e.g., radiolabeling) of drugs is necessary prior to dosing. MS provides highly multiplexed detection, making it possible to identify compounds, their metabolites and other changes in biomolecular abundances directly off tissue sections in a single pass. This can be employed to obtain near cellular, or potentially subcellular, resolution images. Consideration of technical limitations that affect the process is required, from sample preparation through to analyte ionization and detection. The techniques have only recently been adapted for imaging and novel variations to the established MSI methodologies will further enhance the application of MSI for pharmacological research.

A solvent-free matrix application method for matrix-assisted laser desorption/ionization imaging of small molecules

Matrix application continues to be a critical step in sample preparation for matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI). Imaging of small molecules such as drugs and metabolites is particularly problematic because the commonly used washing steps to remove salts are usually omitted as they may also remove the analyte, and analyte spreading is more likely with conventional wet matrix application methods. We have developed a method which uses the application of matrix as a dry, finely divided powder, here referred to as dry matrix application, for the imaging of drug compounds. This appears to offer a complementary method to wet matrix application for the MALDI-MSI of small molecules, with the alternative matrix application techniques producing different ion profiles, and allows the visualization of compounds not observed using wet matrix application methods. We demonstrate its value in imaging clozapine from rat kidney and 4-bromophenyl-1,4-diazabicyclo(3.2.2)nonane-4-carboxylic acid from rat brain. In addition, exposure of the dry matrix coated sample to a saturated moist atmosphere appears to enhance the visualization of a different set of molecules.

Matrix-free mass spectrometric imaging using laser desorption ionisation Fourier transform ion cyclotron resonance mass spectrometry

Mass spectrometry imaging (MSI) is a powerful tool in metabolomics and proteomics for the spatial localization and identification of pharmaceuticals, metabolites, lipids, peptides and proteins in biological tissues. However, sample preparation remains a crucial variable in obtaining the most accurate distributions. Common washing steps used to remove salts, and solvent-based matrix application, allow analyte spreading to occur. Solvent-free matrix applications can reduce this risk, but increase the possibility of ionisation bias due to matrix adhesion to tissue sections. We report here the use of matrix-free MSI using laser desorption ionisation performed on a 12 T Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. We used unprocessed tissue with no post-processing following thaw-mounting on matrix-assisted laser desorption ionisation (MALDI) indium-tin oxide (ITO) target plates. The identification and distribution of a range of phospholipids in mouse brain and kidney sections are presented and compared with previously published MALDI time-of-flight (TOF) MSI distributions.

Environmental scanning electron microscopy offers real-time morphological analysis of liposomes and niosomes

Liposomes have been imaged using a plethora of techniques. However, few of these methods offer the ability to study these systems in their natural hydrated state without the requirement of drying, staining, and fixation of the vesicles. However, the ability to image a liposome in its hydrated state is the ideal scenario for visualization of these dynamic lipid structures and environmental scanning electron microscopy (ESEM), with its ability to image wet systems without prior sample preparation, offers potential advantages to the above methods. In our studies, we have used ESEM to not only investigate the morphology of liposomes and niosomes but also to dynamically follow the changes in structure of lipid films and liposome suspensions as water condenses on to or evaporates from the sample. In particular, changes in liposome morphology were studied using ESEM in real time to investigate the resistance of liposomes to coalescence during dehydration thereby providing an alternative assay of liposome formulation and stability. Based on this protocol, we have also studied niosome-based systems and cationic liposome/DNA complexes. Copyright © Informa Healthcare.

Stabilisation of isocyanate cross linked polybutadiene binder

Isocyanate cross-linked hydroxy terminated polybutadiene is used as a binder for solid rocket propellant. Rocket motors containing this propellant require a storage life of at least 20 years. During storage it has been found that the important rubbery properties of the binder can be lost due to oxidative cross-linking of the polybutadiene chains. This could cause catastrophic failure when the rocket motor is required. At present the bis-hindered phenol Calco 2246 is used as a thermal oxidative stabiliser, but it's performance is only adequate. This has led to the search for a more efficient stabiliser system. To hasten the evaluation of new antioxidant systems the use of dynamic thermal analysis was investigated. Results showed that a tentative relationship existed between predictions by thermal analysis and the long term oven ageing for simple single antioxidant systems. But for more complex systems containing either autosynergistic or mixed antioxidants no relationship was observed suggesting that results for such an "accelerated" technique cannot be used for the purpose of extrapolation for long term performance. This was attributed to the short time and more aggressive condition used (hjgher temperature and oxygen rich atmosphere in thermal analysis) altering the mechanism of action of the antioxidants and not allowing time for co-operative effect of the combined antioxidant system to form. One potential problem for the binder system is the use of an diisocyanate as a cross-linking agent. This reacts with the hydroxyl hydrogen on the polymer as well as other active hydrogens such as those contained in a number of antioxidants, affecting both cross-linking and antioxidant effectiveness. Studies in this work showed that only antioxidants containing amine moieties have a significant affect on binder preparation, with the phenolic antioxidants not reacting. This is due to the greater nucleophilicity of the amines. Investigation of a range of antioxidant systems, including potentially homo, hetero and autosynergistic systems, has highlighted a number of systems which show considerably greater effectiveness than the currently used antioxidant Calco 2246. The only single antioxidant which showed improvement was the partially unhindered phenol y-Tocopherol. Of the mixed systems combinations of the sulphur containing antioxidants e.g. DLTP with higher levels of chain-breaking antioxidants, especially Calco 2246, were the most promising. Also the homosynergistic mix of an aromatic amine and a phenol was seen to be very effective but the results were inconsistent. This inconsistency could be explained by the method of sample preparation used. It was shown that the efficiency of a number of antioxidant.s could be dramatically improved by the use of ultrasound during the mixing stage of preparation. The reason for this increase in performance is unclear but in the case of the homosynergistic amine/phenol mix both more efficient mixing and/or the production of a novel mechanism of action are suggested

Chemical synthesis of isotopically labelled (M+4) purine nucleosides and their incorporation into DNA oligomers

Development of accurate and sensitive analytical methods to measure the level of biomarkers, such as 8-oxo-guanine or its corresponding nucleoside, 8-oxo-2’-deoxyguanosine, has become imperative in the study of DNA oxidative damage in vivo. Of the most promising techniques, HPLC-MS/MS, has many attractive advantages. Like any method that employs the MS technique, its accuracy depends on the use of multiply, isotopically-labelled internal standards. This project is aimed at making available such internal standards. The first task was to synthesise the multiply, isotopically-labelled bases (M+4) guanine and (M+4) 8-oxo-guanine. Synthetic routes for both (M+4) guanine and (M+4) 8-oxo-guanine were designed and validated using the unlabelled compounds. The reaction conditions were also optimized during the “dry runs”. The amination of the 4-hydroxy-2,6-dichloropyrimidine, appeared to be very sensitive to the purity of the commercial [15]N benzylamine reagent. Having failed, after several attempts, to obtain the pure reagent from commercial suppliers, [15]N benzylamine was successfully synthesised in our laboratory and used in the first synthesis of (M+4) guanine. Although (M+4) bases can be, and indeed have been used as internal standards in the quantitative analysis of oxidative damage, they can not account for the errors that may occur during the early sample preparation stages. Therefore, internal standards in the form of nucleosides and DNA oligomers are more desirable. After evaluating a number of methods, an enzymatic transglycolization technique was adopted for the transfer of the labelled bases to give their corresponding nucleosides. Both (M+4) 2-deoxyguanosine and (M+4) 8-oxo-2’-deoxyguanosine can be purified on micro scale by HPLC. The challenge came from the purification of larger scale (>50 mg) synthesis of nucleosides. A gel filtration method was successfully developed, which resulted in excellent separation of (M+4) 2’-deoxyguanosine from the incubation mixture. The (M+4) 2’-deoxyguanosine was then fully protected in three steps and successfully incorporated, by solid supported synthesis, into a DNA oligomer containing 18 residues. Thus, synthesis of 8-oxo-deoxyguanosine on a bigger scale for its future incorporation into DNA oligomers is now a possibility resulting from this thesis work. We believe that these internal standards can be used to develop procedures that can make the measurement of oxidative DNA damage more accurate and sensitive.