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Dr Dominic Hare

Biography

Dominic Hare is an analytical chemist who completed his PhD in 2009 at UTS, and recently returned as a Chancellor’s Postdoctoral Fellow in 2014 after a stint at the Florey Institute of Neuroscience and Mental Health in Melbourne, where he keeps close ties. Originally focused on developing new analytical technology for studying metals in health and disease, he’s now a self-described ‘analytical neurochemist’, using this technology to uncover the root cause of Parkinson’s disease. He’s a founding member of the Elemental Bio-imaging Facility at UTS, which contains one of the finest collections of high-tech analytical equipment in Australia.

Dominic has two major projects at UTS. The first involves trying to isolate the precise reason why the Parkinson’s brain contains more iron than a healthy one, with the goal of contributing to the development of new treatments that target iron in the early stages of the disease. This project builds on observations first made in nearly a century ago that are now coming back to prominence as our search for a treatment for Parkinson’s continues. Dominic’s other project is mapping metals in the healthy brain, providing a unique resource for neuroscientists around the world to integrate ‘metallomic’ data into their research.

Dominic is a Member of the Royal Society of Chemistry, and recently started a new blog devoted to simplifying the complicated world of metallobiology for the everyday web-trawler. He’s passionate about Parkinson’s disease awareness, and is a volunteer for both Parkinson’s New South Wales and Parkinson’s Victoria, where he interacts with Parkinson’s patients to deconvolute the often daunting world of current Parkinson’s research for the people with the greatest interest. He also holds a visiting appointment in the Department of Preventative Medicine at the Icahn School of Medicine at Mount Sinai Hospital in New York, where he’ll be spending several months a year working on building a new, unique resource of Parkinson’s brain tissue, bringing back with a the wealth of experience from working with one of the largest Parkinson’s disease populations in the world.

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Chancellor's Post Doctoral Research Fellow, School of Mathematical and Physical Sciences
BSc App Chem, Ph D
 

Chapters

Bohic, S., Hare, D.J., Daoust, A., Cloetens, P. & Barbier, E.L. 2012, 'Spatially resolved imaging methods to probe metals in the brain: from subcellular to organ level' in Linert, W. & Kozlowski, H. (eds), Metal ions in neurological systems, Springer, Vienna, pp. 211-222.
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Very little is known about the subcellular distribution of metal ions in cells. Some metals such as zinc, copper, and iron are essential and play an important role in the cell metabolism. Dysfunctions in this delicate housekeeping may be at the origin of major diseases. There is also a prevalent use of metals in a wide range of diagnostic agents and drugs for the diagnosis or treatment of a variety of disorders. This is becoming more and more of a concern in the field of nanomedicine with the increasing development and use of nanoparticles, which are suspected of causing adverse effects on cells and organ tissues and particularly the brain. Various analytical methods are developing into well-suited sub-micrometer analytical tools for addressing new problems when studying the role of metals in the brain. In the present review, we describe the possibilities and some current applications offered by three major techniques that cover imaging of metals from subcellular to organ level, namely synchrotron-based X-ray microspectroscopy, laser ablation inductively coupled plasma-mass spectrometry (LA-ICP-MS) and, less thoroughly addressed, the capabilities of magnetic resonance imaging (MRI) for in vivo imaging of metals in the brain

Journal articles

Portbury, S.D., Hare, D.J., Sgambelloni, C., Finkelstein, D.I. & Adlard, P.A. 2016, 'A time-course analysis of changes in cerebral metal levels following a controlled cortical impact.', Metallomics : integrated biometal science, vol. 8, no. 2, pp. 193-200.
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Traumatic brain injury (TBI) is complicated by a sudden and dramatic change in brain metal levels, including iron (Fe), copper (Cu) and zinc (Zn). Specific 'metallo-pathological' features of TBI include increased non-heme bound Fe and the liberation of free Zn ions, both of which may contribute to the pathogenesis of TBI. To further characterise the metal dyshomeostasis that occurs following brain trauma, we performed a quantitative time-course survey of spatial Fe, Cu and Zn distribution in mice receiving a controlled cortical impact TBI. Images of brain metal levels produced using laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) in the upper quadrant of the ipsilateral hemisphere were compared to the corresponding contralateral hemisphere, together with regional areas radiating toward the center of the brain from the site of lesion. Significant regional and time point specific elevations in Fe, Zn and Cu were detected immediately and up to 28 days after TBI. The magnitude and timeframe of many of these changes suggest that TBI results in a pronounced and sustained alteration in normal metal levels within the brain. Such alterations are likely to play a role in both the short- and long-term consequences of head trauma and suggest that pharmacological modulation to normalize these metal levels may be efficacious in improving functional outcome.
Cardoso, B.R., Busse, A.L., Hare, D.J., Cominetti, C., Horst, M.A., McColl, G., Magaldi, R.M., Jacob-Filho, W. & Cozzolino, S.M. 2016, 'Pro198Leu polymorphism affects the selenium status and GPx activity in response to Brazil nut intake.', Food & function, vol. 7, no. 2, pp. 825-833.
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Selenoproteins play important roles in antioxidant mechanisms, and are thus hypothesised to have some involvement in the pathology of certain types of dementia. Mild cognitive impairment (MCI) and Alzheimer's disease (AD) are both thought to involve impaired biological activity of certain selenoproteins. Previously, supplementation with a selenium-rich Brazil nut (Bertholletia excelsa) has shown potential in reducing cognitive decline in MCI patients, and could prove to be a safe and effective nutritional approach early in the disease process to slow decline. Here, we have conducted a pilot study that examined the effects of a range of single nucleotide polymorphisms (SNPs) in genes encoding the selenoproteins glutathione peroxidase (GPX1) and selenoprotein P (SEPP) in response to selenium supplementation via dietary Brazil nuts, including selenium status, oxidative stress parameters and GPX1 and SEPP gene expression. Our data suggest that GPX1 Pro198Leu rs1050450 genotypes may differentially affect the selenium status and GPx activity. Moreover, rs7579 and rs3877899 SNPs in SEPP gene, as well as GPX1 rs1050450 genotypes can influence the expression of GPX1 and SEPP mRNA in response to Brazil nuts intake. This small study gives cause for larger investigations into the role of these SNPs in both the selenium status and response to selenium dietary intake, especially in chronic degenerative conditions like MCI and AD.
Hare, D.J., Jones, M.W., Wimmer, V.C., Jenkins, N.L., de Jonge, M.D., Bush, A.I. & McColl, G. 2016, 'High-resolution complementary chemical imaging of bio-elements in Caenorhabditis elegans.', Metallomics : integrated biometal science, vol. 8, no. 2, pp. 156-160.
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Here, we present a sub-m multimodal approach to image essential elements in Caenorhabditis elegans. A combination of chemical imaging technologies reveals total metal concentration, chemical state and the protein to which an element is associated. This application of distinct yet complementary chemical imaging techniques provided unique insight into essential and trace elements at the subcellular level.
Finkelstein, D.I., Hare, D.J., Billings, J.L., Sedjahtera, A., Nurjono, M., Arthofer, E., George, S., Culvenor, J.G., Bush, A.I. & Adlard, P.A. 2016, 'Clioquinol Improves Cognitive, Motor Function, and Microanatomy of the Alpha-Synuclein hA53T Transgenic Mice.', ACS chemical neuroscience, vol. 7, no. 1, pp. 119-129.
The abnormal accumulation of alpha-synuclein (-syn) has been linked to a number of neurodegenerative disorders, the most noteworthy of which is Parkinson's disease. Alpha-synuclein itself is not toxic and fulfills various physiological roles in the central nervous system. However, specific types of aggregates have been shown to be toxic, and metals have been linked to the assembly of these toxic aggregates. In this paper, we have characterized a transgenic mouse that overexpresses the A53T mutation of human -syn, specifically assessing cognition, motor performance, and subtle anatomical markers that have all been observed in synucleinopathies in humans. We hypothesized that treatment with the moderate-affinity metal chelator, clioquinol (CQ), would reduce the interaction between metals and -syn to subsequently improve the phenotype of the A53T animal model. We showed that CQ prevents an iron-synuclein interaction, the formation of urea-soluble -syn aggregates, -syn-related substantia nigra pars compacta cell loss, reduction in dendritic spine density of hippocampal and caudate putamen medium spiny neurons, and the decline in motor and cognitive function. In conclusion, our data suggests that CQ is capable of mitigating the pathological metal/-syn interactions, suggesting that the modulation of metal ions warrants further study as a therapeutic approach for the synucleinopathies.
Bishop, D.P., Clases, D., Fryer, F., Williams, E., Wilkins, S., Hare, D.J., Cole, N., Karst, U. & Doble, P.A. 2016, 'Elemental bio-imaging using laser ablation-triple quadrupole-ICP-MS', Journal of Analytical Atomic Spectrometry, vol. 31, no. 1, pp. 197-202.
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© 2016 The Royal Society of Chemistry. Elemental bio-imaging (EBI) of trace metal distributions in tissue sections is typically performed by laser ablation-single quadrupole-inductively coupled plasma-mass spectrometry (LA-ICP-SQ-MS) in which the sensitivity of biologically relevant elements such as Fe may be inhibited by polyatomic interferences. The triple quadrupole ICP-MS (ICP-QQQ-MS) is designed to reduce interferences and was examined for its potential application to EBI. Three tune modes; a no-gas, an H2, and an O2/H2 tune were developed and investigated for their suitability. All three performed well with high signal to noise ratios and low limits of detection. The O2/H2 tune was applied to a prostate cancer biopsy sample, with high levels of Zn found in the area correlating with the cancer. The oxygen mass shift was applied to image P and Se in a mouse brain. The unique focussing properties of the ICP-QQQ-MS and the ability to use mixed cell gases provided sensitive analyses of all measured isotopes with the O2/H2 tune, including those unaffected by mass shift reactions.
Callahan, D.L., Hare, D.J., Bishop, D.P., Doble, P.A. & Roessner, U. 2016, 'Elemental imaging of leaves from the metal hyperaccumulating plant Noccaea caerulescens shows different spatial distribution of Ni, Zn and Cd', RSC Advances, vol. 6, no. 3, pp. 2337-2344.
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© The Royal Society of Chemistry 2016. Elemental imaging using laser ablation inductively coupled plasma mass spectrometry was performed on whole leaves of the hyperaccumulating plant Noccaea caerulescens after treatments with either Ni, Zn or Cd. These detailed elemental images reveal differences in the spatial distribution of these three elements across the leaf and provide new insights in the metal ion homeostasis within hyperaccumulating plants. In the Zn treated plants, Zn accumulated in the leaf tip while Mn was co-localised with Zn suggesting similar storage mechanisms for these two metals. These data show a Zn concentration difference of up to 13-fold higher in the distal part of the leaf. Also, there was no correlation between the S and Zn concentrations providing further evidence against S-binding ligands. In contrast, Ni was more evenly distributed while a more heterogeneous distribution of Cd was present with some high levels on leaf edges, suggesting that different storage and transport mechanisms are used for the hyperaccumulation of these two metals. These results show the importance of correct sampling when carrying out subcellular localisation studies as the hyperaccumulated elements are not necessarily homogenously distributed over the entire leaf area. The results also have great implications for biotechnological applications of N. caerulescens showing that it may be possible to use the mechanisms employed by N. caerulescens to increase the Zn concentration in nutrient poor crops without increasing the risk of accumulating other toxic elements such as Ni and Cd.
Billings, J.L., Hare, D.J., Nurjono, M., Volitakis, I., Cherny, R.A., Bush, A.I., Adlard, P.A. & Finkelstein, D.I. 2016, 'Effects of Neonatal Iron Feeding and Chronic Clioquinol Administration on the Parkinsonian Human A53T Transgenic Mouse.', ACS chemical neuroscience, vol. 7, no. 3, pp. 360-366.
Increased nigral iron (Fe) is a cardinal feature of Parkinson's disease, as is the accumulation of aggregates comprising -synuclein. We used wild-type mice and transgenic mice overexpressing the human A53T mutation to -synuclein to examine the influence of increased Fe (days 10-17 postpartum) on the parkinsonian development phenotype of these animals (including abnormal nigral Fe levels and deficits in both cell numbers and locomotor activity), and to explore the impact of the Fe chelator clioquinol in the model. Both untreated and Fe-loaded A53T mice showed similar levels of nigral cell loss, though 5 months of clioquinol treatment was only able to prevent the loss in the non-Fe-loaded A53T group. Iron levels in the Fe-loaded A53T mice returned to normal at 8 months, though effects of dopamine denervation remained, demonstrated by limited locomotor activity and sustained neuron loss. These data suggest that Fe exposure during a critical developmental window, combined with the overexpression mutant -synuclein, presents a disease phenotype resistant to intervention using clioquinol later in life.
Ganio, K., James, S.A., Hare, D.J., Roberts, B.R. & McColl, G. 2016, 'Accurate biometal quantification per individual Caenorhabditis elegans.', The Analyst, vol. 141, no. 4, pp. 1434-1439.
In the life sciences, small model-organisms are an established research platform. Due to the economy of culturing and maintenance animals such as the roundworm Caenorhabditis elegans, and the fly Drosophila melanogaster, have been instrumental for investigating key genetic pathways, early development, neuronal function, as well as disease pathogenesis and toxicology. Small model organisms have also found utility in the study of inorganic biochemistry, where the role of metal ion cofactors are investigated for numerous fundamental cellular processes. The metabolism and homeostasis of metal ions is also central to many aspects of biology and disease. Accurate quantification of endogenous metal ion content is an important determinant for many biological questions. There is currently no standardised method for quantifying biometal content in individual C. elegans or estimating the variation between individuals within clonal populations. Here, we have determined that ten or more adults are required to quantify physiologically important metals via inductively coupled plasma mass spectrometry (ICP-MS). The accuracy and precision of this method was then compared to synchrotron-based X-ray fluorescence microscopy (XFM) to determine the variation between isogenic, developmentally synchronous C. elegans adults.
James, S.A., Hare, D.J., Jenkins, N.L., de Jonge, M.D., Bush, A.I. & McColl, G. 2016, 'XANES: In vivo imaging of metal-protein coordination environments.', Scientific reports, vol. 6, p. 20350.
We have developed an X-ray absorption near edge structure spectroscopy method using fluorescence detection for visualizing in vivo coordination environments of metals in biological specimens. This approach, which we term fluorescence imaging XANES (XANES), allows us to spatially depict metal-protein associations in a native, hydrated state whilst avoiding intrinsic chemical damage from radiation. This method was validated using iron-challenged Caenorhabditis elegans to observe marked alterations in redox environment.
Hare, D.J., Faux, N.G., Roberts, B.R., Volitakis, I., Martins, R.N. & Bush, A.I. 2016, 'Lead and manganese levels in serum and erythrocytes in Alzheimer's disease and mild cognitive impairment: results from the Australian Imaging, Biomarkers and Lifestyle Flagship Study of Ageing.', Metallomics : integrated biometal science, vol. 8, no. 6, pp. 628-632.
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We examined serum and erythrocyte lead and manganese levels in the Australian Imaging, Biomarkers and Lifestyle Flagship Study of Ageing (AIBL), which contains over 1000 registrants including over 200 cases of Alzheimer's disease (AD) and 100 mildly cognitively impaired (MCI) individuals. After correcting for confounding effects of age, collection site and sex, we found a significant decrease in serum manganese levels in AD subjects compared to healthy controls. Analysis of smaller subset of erythrocytes revealed no difference in either lead or manganese levels in AD. Although lead and manganese have neurotoxic effects and may be involved in AD pathology, our results showed that neither metal in serum nor erythrocytes are suitable biomarkers in our cohort. However, prospective studies might reveal whether the burden of either metal modifies disease outcomes.
Hare, D.J. & Double, K.L. 2016, 'Iron and dopamine: a toxic couple.', Brain : a journal of neurology.
Iron accumulation is a cardinal feature of degenerating regions in the Parkinson's disease brain. As a potent pro-oxidant, redox-active iron may be a key player in upstream mechanisms that precipitate cell death in this disorder. Although an elevation in brain iron levels is a normal feature of ageing, the increase is greater in Parkinson's disease; on the other hand, the effects of the disease are most marked in the nigrostriatal dopaminergic system. In this Update, we explain that neurodegeneration in the affected regions may result from the potent redox couple formed by iron and dopamine itself, and discuss the clinical implications of this molecular trait in this dynamic and rapidly moving area of Parkinson's disease research.
Hare, D.J. & New, E.J. 2016, 'On the outside looking in: redefining the role of analytical chemistry in the biosciences.', Chemical communications (Cambridge, England).
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Biomedical research has moved on from the study of the structure of organs, cells and organelles. Today, the key questions that must be addressed to understand the body in health and disease are related to fundamental biochemistry: the distribution and speciation of chemicals, the regulation of chemical reactions, and the control of chemical environments. To see advances in this field, it is essential for analytical chemists to actively engage in this process, from beginning to end. In this Feature Article, we review the progress that has been made towards gaining an understanding of the chemistry of the body, while commenting on the intrinsic disconnect between new innovations in the field of analytical chemistry and practical application within the biosciences. We identify the challenges that prevent chemists from making a greater impact in this field, and highlight key steps for moving forward.
James, S.A., Hare, D.J., Jenkins, N.L., De Jonge, M.D., Bush, A.I. & McColl, G. 2016, 'Erratum: †xANES: In vivo imaging of metal-protein coordination environments (Scientific Reports (2016) 6 (20350) DOI: 10.1038/srep20350)', Scientific Reports, vol. 6.
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Hare, D.J., Raven, E.P., Roberts, B.R., Bogeski, M., Portbury, S.D., McLean, C.A., Masters, C.L., Connor, J.R., Bush, A.I., Crouch, P.J. & Doble, P.A. 2016, 'Laser ablation-inductively coupled plasma-mass spectrometry imaging of white and gray matter iron distribution in Alzheimer's disease frontal cortex', NeuroImage, vol. 137, pp. 124-131.
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© 2016.Iron deposition in the brain is a feature of normal aging, though in several neurodegenerative disorders, including Alzheimer's disease, the rate of iron accumulation is more advanced than in age-matched controls. Using laser ablation-inductively coupled plasma-mass spectrometry imaging we present here a pilot study that quantitatively assessed the iron content of white and gray matter in paraffin-embedded sections from the frontal cortex of Alzheimer's and control subjects. Using the phosphorus image as a confirmed proxy for the white/gray matter boundary, we found that increased intrusion of iron into gray matter occurs in the Alzheimer's brain compared to controls, which may be indicative of either a loss of iron homeostasis in this vulnerable brain region, or provide evidence of increased inflammatory processes as a response to chronic neurodegeneration. We also observed a trend of increasing iron within the white matter of the frontal cortex, potentially indicative of disrupted iron metabolism preceding loss of myelin integrity. Considering the known potential toxicity of excessive iron in the brain, our results provide supporting evidence for the continuous development of novel magnetic resonance imaging approaches for assessing white and gray matter iron accumulation in Alzheimer's disease.
Roberts, B.R., Hare, D.J., McLean, C.A., Conquest, A., Lind, M., Li, Q.-.X., Bush, A.I., Masters, C.L., Morganti-Kossmann, M.-.C. & Frugier, T. 2015, 'Traumatic brain injury induces elevation of Co in the human brain', METALLOMICS, vol. 7, no. 1, pp. 124-128.
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Hare, D.J., Doecke, J.D., Faux, N.G., Rembach, A., Volitakis, I., Fowler, C.J., Grimm, R., Doble, P.A., Cherny, R.A., Masters, C.L., Bush, A.I. & Roberts, B.R. 2015, 'Decreased Plasma Iron in Alzheimer's Disease Is Due to Transferrin Desatu ration', ACS CHEMICAL NEUROSCIENCE, vol. 6, no. 3, pp. 398-402.
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Adlard, P.A., Parncutt, J., Lal, V., James, S., Hare, D., Doble, P., Finkelstein, D.I. & Bush, A.I. 2015, 'Metal chaperones prevent zinc-mediated cognitive decline', NEUROBIOLOGY OF DISEASE, vol. 81, pp. 196-202.
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Ryan, T.M., Roberts, B.R., McColl, G., Hare, D.J., Doble, P.A., Li, Q.X., Lind, M., Roberts, A.M., Mertens, H.D., Kirby, N., Pham, C.L., Hinds, M.G., Adlard, P.A., Barnham, K.J., Curtain, C.C. & Masters, C.L. 2015, 'Stabilization of nontoxic A-oligomers: insights into the mechanism of action of hydroxyquinolines in Alzheimer's disease.', The Journal of neuroscience : the official journal of the Society for Neuroscience, vol. 35, no. 7, pp. 2871-2884.
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The extracellular accumulation of amyloid (A) peptides is characteristic of Alzheimer's disease (AD). However, formation of diffusible, oligomeric forms of A, both on and off pathways to amyloid fibrils, is thought to include neurotoxic species responsible for synaptic loss and neurodegeneration, rather than polymeric amyloid aggregates. The 8-hydroxyquinolines (8-HQ) clioquinol (CQ) and PBT2 were developed for their ability to inhibit metal-mediated generation of reactive oxygen species from A:Cu complexes and have both undergone preclinical and Phase II clinical development for the treatment of AD. Their respective modes of action are not fully understood and may include both inhibition of A fibrillar polymerization and direct depolymerization of existing A fibrils. In the present study, we find that CQ and PBT2 can interact directly with A and affect its propensity to aggregate. Using a combination of biophysical techniques, we demonstrate that, in the presence of these 8-HQs and in the absence of metal ions, A associates with two 8-HQ molecules and forms a dimer. Furthermore, 8-HQ bind A with an affinity of 1-10 m and suppress the formation of large (>30 kDa) oligomers. The stabilized low molecular weight species are nontoxic. Treatment with 8-HQs also reduces the levels of in vivo soluble oligomers in a Caenorhabditis elegans model of A toxicity. We propose that 8-HQs possess an additional mechanism of action that neutralizes neurotoxic A oligomer formation through stabilization of small (dimeric) nontoxic A conformers.
Bishop, D.P., Hare, D.J., Fryer, F., Taudte, R.V., Cardoso, B.R., Cole, N. & Doble, P.A. 2015, 'Determination of selenium in serum in the presence of gadolinium with ICP-QQQ-MS.', The Analyst, vol. 140, no. 8, pp. 2842-2846.
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Gadolinium (Gd)-based magnetic resonance imaging (MRI) contrasting agents interfere with the determination of selenium (Se) when analysed by single quadrupole inductively coupled plasma-mass spectrometry (ICP-MS). This paper demonstrates that an ICP-triple quadrupole-MS (ICP-QQQ-MS) with oxygen mass shift overcomes Gd(++) interference on Se(+) and mitigates typically encountered matrix and spectral based interferences. Normal human serum was diluted in a solution containing isopropanol, EDTA, NH4OH and Triton X-100. Samples were unspiked (control) serum; serum spiked with 0.127 mol L(-1) Se or 127 mol L(-1) Gd; and serum spiked with both 0.127 mol L(-1) Se and 127 mol L(-1) Gd. Consideration of collision/reaction gases and conditions for interference mitigation included helium (He); a 'low' and 'high' hydrogen (H2) flow, and oxygen (O2). The instrument tune for O2 was optimised for effective elimination of interferences via a mass shift reaction of Se(+) to SeO(+). The ICP-QQQ-MS was capable of detecting trace (>9.34 nmol L(-1)) levels of Se in serum in the presence of Gd in our simulated post-MRI serum sample. The multi-tune capabilities of the ICP-QQQ-MS may be adapted to eliminate other specific isobaric interferences that cause false positive results in other analyses where the analyte is confounded by doubly charged and/or polyatomic species.
Ayton, S., Lei, P., Hare, D.J., Duce, J.A., George, J.L., Adlard, P.A., McLean, C., Rogers, J.T., Cherny, R.A., Finkelstein, D.I. & Bush, A.I. 2015, 'Parkinson's Disease Iron Deposition Caused by Nitric Oxide-Induced Loss of beta-Amyloid Precursor Protein', JOURNAL OF NEUROSCIENCE, vol. 35, no. 8, pp. 3591-3597.
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James, S.A., Roberts, B.R., Hare, D.J., de Jonge, M.D., Birchall, I.E., Jenkins, N.L., Cherny, R.A., Bush, A.I. & McColl, G. 2015, 'Direct in vivo imaging of ferrous iron dyshomeostasis in ageing Caenorhabditis elegans', CHEMICAL SCIENCE, vol. 6, no. 5, pp. 2952-2962.
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Davies, K.M., Hare, D.J., Bohic, S., James, S.A., Billings, J.L., Finkelstein, D.I., Doble, P.A. & Double, K.L. 2015, 'Comparative Study of Metal Quantification in Neurological Tissue Using Laser Ablation-Inductively Coupled Plasma-Mass Spectrometry Imaging and X-ray Fluorescence Microscopy.', Analytical chemistry, vol. 87, no. 13, pp. 6639-6645.
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Redox-active metals in the brain mediate numerous biochemical processes and are also implicated in a number of neurodegenerative diseases. A number of different approaches are available for quantitatively measuring the spatial distribution of biometals at an image resolution approaching the subcellular level. Measured biometal levels obtained using laser ablation-inductively coupled plasma mass spectrometry (LA-ICPMS; spatial resolution 15 m 15 m) were within the range of those obtained using X-ray fluorescence microscopy (XFM; spatial resolution 2 m 7 m) and regional changes in metal concentration across discrete brain regions were replicated to the same degree. Both techniques are well suited to profiling changes in regional biometal distribution between healthy and diseased brain tissues, but absolute quantitation of metal levels varied significantly between methods, depending on the metal of interest. Where all possible variables affect metal levels, independent of a treatment/phenotype are controlled, either method is suitable for examining differences between experimental groups, though, as with any method for imaging post mortem brain tissue, care should be taken when interpreting the total metal levels with regard to physiological concentrations.
Hare, D.J., Arora, M., Jenkins, N.L., Finkelstein, D.I., Doble, P.A. & Bush, A.I. 2015, 'Is early-life iron exposure critical in neurodegeneration?', NATURE REVIEWS NEUROLOGY, vol. 11, no. 9, pp. 536-544.
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Bishop, D.P., Hare, D.J., de Grazia, A., Fryer, F. & Doble, P.A. 2015, 'Speciation and quantification of organotin compounds in sediment and drinking water by isotope dilution liquid chromatography-inductively coupled plasma-mass spectrometry', ANALYTICAL METHODS, vol. 7, no. 12, pp. 5012-5018.
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Martens, I.B., Cardoso, B.R., Hare, D.J., Niedzwiecki, M.M., Lajolo, F.M., Martens, A. & Cozzolino, S.M. 2015, 'Selenium status in preschool children receiving a Brazil nut-enriched diet.', Nutrition (Burbank, Los Angeles County, Calif.), vol. 31, no. 11-12, pp. 1339-1343.
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The Brazilian Amazon region has selenium (Se)-rich soil, which is associated with higher Se levels in populations fed locally grown produce. Brazil nuts are a major source of dietary Se and are included with meals offered to children enrolled in public preschool in Macapá. The aim of this study was to examine Se intake and status of these children.The Macapá group consisted of 41 children from a public preschool who received 15 to 30 g of Brazil nuts 3 d/wk. The control group included 88 children from the nearby city of Belém who did not receive Brazil nut-enriched meals. In both groups, school meals comprised 90% of the children's total food consumption. Selenium was assessed using hydride generation quartz tube atomic absorption spectroscopy in plasma, erythrocytes, nails, hair and urine. Dietary intakes (macronutrients and Se) were evaluated using the duplicate-portion method.Both groups received inadequate intakes of energy and macronutrients. Selenium intake was excessive in both groups (155.30 and 44.40 g/d, in Macapá and Belém, respectively). Intake was potentially toxic in Macapá on days when Brazil nuts were added to meals. Although biomarkers of Se exposure exceeded reference levels in the Macapá group, no clinical symptoms of Se overload (selenosis) were observed.The inclusion of Brazil nuts in school meals provided to children with already high dietary Se intakes increased Se levels and may result in an increased risk for toxicity. As selenosis is associated with some chronic diseases, we recommend continued monitoring of Se intake and status in this population.
Hare, D.J., New, E.J., de Jonge, M.D. & McColl, G. 2015, 'Imaging metals in biology: balancing sensitivity, selectivity and spatial resolution.', Chemical Society reviews, vol. 44, pp. 5941-5958.
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Metal biochemistry drives a diverse range of cellular processes associated with development, health and disease. Determining metal distribution, concentration and flux defines our understanding of these fundamental processes. A comprehensive analysis of biological systems requires a balance of analytical techniques that inform on metal quantity (sensitivity), chemical state (selectivity) and location (spatial resolution) with a high degree of certainty. A number of approaches are available for imaging metals from whole tissues down to subcellular organelles, as well as mapping metal turnover, protein association and redox state within these structures. Technological advances in micro- and nano-scale imaging are striving to achieve multi-dimensional and in vivo measures of metals while maintaining the native biochemical environment and physiological state. This Tutorial Review discusses state-of-the-art imaging technology as a guide to obtaining novel insight into the biology of metals, with sensitivity, selectivity and spatial resolution in focus.
Cardoso, B.R., Roberts, B.R., Bush, A.I. & Hare, D.J. 2015, 'Selenium, selenoproteins and neurodegenerative diseases', Metallomics, vol. 7, no. 8, pp. 1213-1228.
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© The Royal Society of Chemistry 2015. It is unsurprising that our understanding of the role of selenium in neurological function is somewhat immature, considering its relatively recent discovery as an essential element to human health. Selenocysteine, the 21st amino acid, is the defining feature of the 25 selenoprotein-encoding genes so far discovered within the human genome. The low abundance of these proteins in the brain belies the integral role they play in normal neurological function, from well-characterised antioxidant activity in the periphery to poorly understood mechanisms that modulate mitochondrial function and response to brain pathology. Selenium has been identified as playing a role in several neurodegenerative disorders, including Alzheimer's and Parkinson's disease, though its function as a 'cause or effect' of disease process remains unclear. This review discusses selenium metabolism in detail, specifically with regard to the role it plays within the central nervous system, and examines the most current literature investigating how selenium may be involved in chronic diseases of the central nervous system.
Paul, B., Hare, D.J., Bishop, D.P., Paton, C., Nguyen, V.T., Cole, N., Niedwiecki, M.M., Andreozzi, E., Vais, A., Billings, J.L., Bray, L., Bush, A.I., McColl, G., Roberts, B.R., Adlard, P.A., Finkelstein, D.I., Hellstrom, J., Hergt, J.M., Woodhead, J.D. & Doble, P.A. 2015, 'Visualising mouse neuroanatomy and function by metal distribution using laser ablation-inductively coupled plasma-mass spectrometry imaging', Chemical Science, vol. 6, no. 10, pp. 5383-5393.
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Cho, H.R., Lee, Y., Doble, P., Bishop, D., Hare, D., Kim, Y.-.J., Kim, K.G., Jung, H.S., Park, K.S., Choi, S.H. & Moon, W.K. 2015, 'Magnetic resonance imaging of the pancreas in streptozotocin-induced diabetic rats: Gadofluorine P and Gd-DOTA', WORLD JOURNAL OF GASTROENTEROLOGY, vol. 21, no. 19, pp. 5831-5842.
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Arora, M. & Hare, D.J. 2015, 'Tooth lead levels as an estimate of lead body burden in rats following pre- and neonatal exposure', RSC Advances, vol. 5, no. 82, pp. 67308-67314.
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© The Royal Society of Chemistry. Accurate assessment of early life lead exposure requires an accessible and reliable biomarker. Blood lead levels alone are not a suitable measure of chronic lead exposure, due to the rapid turnover and proportionally smaller amount compared to calcified tissues, such as bone and teeth. To further validate and standardise tooth lead levels as an appropriate biomarker of exposure, we designed a series of experiments where Wistar rats were fed, through drinking water, a regime of lead dosage that included stable, increasing and decreasing exposures, as well as prenatal exposure via the mother. At 15 days of age the animals were culled and blood, kidney, liver, brain, bone and teeth lead levels were assessed using inductively coupled plasma-mass spectrometry. Bone and tooth lead levels were by far the highest, and were reflective of the dosing parameters used. Brain lead levels were the lowest, suggesting that the blood-brain barrier provided some protection against lead uptake. These results give further support to the use of tooth lead levels as a biomarker of environmental lead exposure, particularly during early life periods.
Paul, B., Hare, D.J., Bishop, D.P., Paton, C., Nguyen, V.T., Cole, N., Niedzwiecki, M.M., Andreozzi, E., Vais, A., Billings, J.L., Bray, L., Bush, A.I., McColl, G., Roberts, B.R., Adlard, P.A., Finkelstein, D.I., Hellstrom, J., Hergt, J.M., Woodhead, J.D. & Doble, P.A. 2015, 'Erratum: Visualising mouse neuroanatomy and function by metal distribution using laser ablation-inductively coupled plasma-mass spectrometry imaging (Chem. Sci. (2015) DOI:10.1039/c5sc02231b)', Chemical Science, vol. 6, no. 11, p. 6677.
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This journal is © The Royal Society of Chemistry. Correction for 'Visualising mouse neuroanatomy and function by metal distribution using laser ablation-inductively coupled plasma-mass spectrometry imaging' by Bence Paul et al., Chem. Sci., 2015, DOI: 10.1039/c5sc02231b.
Hare, D.J., Rembach, A. & Roberts, B.R. 2015, 'The emerging role of metalloproteomics in Alzheimer's disease research', pp. 379-389.
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© Springer Science+Business Media New York 2015. All right reserved. Metals are increasingly recognized to have an important role in molecular processes underlying Alzheimer's disease (AD). This chapter discusses the current role of metals in AD and expands on the development of metalloproteomics and how the recent advances in analytical technology will allow detailed investigation of metalloproteins. Investigation of individual metalloproteins will yield new mechanistic details about the role of metals in AD.
Hare, D.J. 2014, 'Copper pathology in vulnerable brain regions in Parkinsons disease', Neurobiology of Aging, vol. 35, pp. 858-866.
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Hare, D.J., George, J., Bray, L., Volitakis, I., Vais, A., Ryan, T., Cherny, R., Bush, A., Masters, C., Adlard, P., Doble, P.A. & Finkelstein, D. 2014, 'The effect of paraformaldehyde fixation and sucrose cryoprotection on metal concentration in murine neurological tissue', Journal of Analytical Atomic Spectrometry, vol. 29, pp. 565-570.
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Aldehyde preservation of tissue and cryoprotection by sugars and polyols has traditionally been used in histology to preserve structural integrity of samples. The effect of paraformaldehyde (PFA) fixation and sucrose cryoprotection on murine brain metals was studied by examining the changes in total brain metal levels throughout a complete fixation/cryoprotection protocol. Blank-corrected metal levels in 4% PFA and two changes of 30% sucrose solutions following brain immersion were determined by ICP-MS. Fixed and cryoprotected tissue was lyophilised, digested in HNO3/H2O2 and analysed using the same ICP-MS protocol. The sum of metal loss or gain in each solution and total digest was used to determine the percentage of total brain metal present in fixed, cryoprotected tissue. Almost all K (99.78%) and Mg (78.01%) leached from brain tissue during fixation and cryoprotection, whilst Ca and Sr either retained or absorbed additional metal ions from the sucrose solution. Less water-soluble transition metals displayed smaller degrees of metal leaching, with Fe, Cu and Zn losing 26.6131.62% of their total metal content during preparation for cryosectioning. These results suggest that metal leaching is highly metal specific, and that preparatory steps for cryosectioning of brain tissue should be approached according to this specificity.
Adlard, P.A., Sedjahtera, A., Gunawan, L., Bray, L., Hare, D.J., Lear, J., Doble, P.A., Bush, A., Finkelstein, D. & Cherny, R. 2014, 'A novel approach to rapidly prevent age-related cognitive decline', Aging Cell, vol. 13, no. 2, pp. 351-359.
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The loss of cognitive function is a pervasive and often debilitating feature of the aging process for which there are no effective therapeutics. We hypothesized that a novel metal chaperone (PBT2; Prana Biotechnology, Parkville, Victoria, Australia) would enhance cognition in aged rodents. We show here that PBT2 rapidly improves the performance of aged C57Bl/6 mice in the Morris water maze, concomitant with increases in dendritic spine density, hippocampal neuron number and markers of neurogenesis. There were also increased levels of specific glutamate receptors (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and N-methyl-d-aspartate), the glutamate transporter (VGLUT1) and glutamate itself. Markers of synaptic plasticity [calmodulin-dependent protein kinase II (CaMKII) and phosphorylated CaMKII, CREB, synaptophysin] were also increased following PBT2 treatment. We also demonstrate that PBT2 treatment results in a subregion-specific increase in hippocampal zinc, which is increasingly recognized as a potent neuromodulator. These data demonstrate that metal chaperones are a novel approach to the treatment of age-related cognitive decline.
cho, H., kim, D., Kim, D., Doble, P.A., Bishop, D., Hare, D.J., Park, C., Moon, W.K., Han, M.H. & Choi, S.H. 2014, 'Malignant Glioma: MR imaging by using 5-Aminolevulinic acid in an animal model', Radiology, vol. 272, no. 3, pp. 720-730.
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Roberts, B., Lim, N., McAllum, E., Donnelly, P., Hare, D.J., Doble, P.A., Turner, B., Price, K., Lim, S.C., Paterson, B., Hickey, J., Rhoads, T., Williams, J., Kunninen, K., Hung, L., Liddell, J., Grubman, A., Monty, J., Llanos, R., Kamer, D., Mercer, J., Bush, A., Masters, C., Duce, J., Li, Q., Beckman, J., Barnham, K., White, A. & Crouch, P. 2014, 'Oral Treatment with Cu(II)(atsm) Increases Mutant SOD1 In Vivo but Protects Motor Neurons and Improves the Phenotype of a Transgenic Mouse Model of Amyotrophic Lateral Sclerosis', The Journal of Neuroscience, vol. 34, no. 23, pp. 8021-8031.
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Mutations in the metallo-protein Cu/Zn-superoxide dismutase (SOD1) cause amyotrophic lateral sclerosis (ALS) in humans and an expression level-dependent phenotype in transgenic rodents. We show that oral treatment with the therapeutic agent diacetyl-bis(4- methylthiosemicarbazonato)copperII [CuII(atsm)] increased the concentration of mutant SOD1 (SOD1G37R) in ALS model mice, but paradoxically improved locomotor function and survival of the mice. To determine why the mice with increased levels of mutant SOD1 had an improved phenotype, we analyzed tissues by mass spectrometry. These analyses revealed most SOD1 in the spinal cord tissue of the SOD1G37R mice was Cu deficient. Treating with CuII(atsm) decreased the pool of Cu-deficient SOD1 and increased the pool of fully metallated (holo) SOD1. Tracking isotopically enriched 65CuII(atsm) confirmed the increase in holo-SOD1 involved transfer of Cu from CuII(atsm) to SOD1, suggesting the improved locomotor function and survival of the CuII(atsm)-treated SOD1G37R mice involved, at least in part, the ability of the compound to improve the Cu content of the mutant SOD1. This was supported by improved survival of SOD1G37Rmice that expressed thehumangene for theCuuptake protein CTR1. Improving the metal content of mutantSOD1in vivo with CuII(atsm) did not decrease levels of misfolded SOD1. These outcomes indicate the metal content of SOD1 may be a greater determinant of the toxicity of the protein in mutant SOD1-associated forms of ALS than the mutations themselves. Improving the metal content of SOD1 therefore represents a valid therapeutic strategy for treating ALS caused by SOD1.
Hare, D.J., Lei, P., Ayton, S., Roberts, B., Grimm, R., George, J., Bishop, D., Beavis, A.B., Donovan, S., McColl, G., Volitakis, I., Masters, C., Adlard, P., Cherny, R., Bush, A., Finkelstein, D. & Doble, P.A. 2014, 'An iron-dopamine index predicts risk of parkinsonian neurodegeneration in the substantia nigra pars compacta', Chemical Science, vol. 5, pp. 2160-2169.
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The co-localization of iron and dopamine raises the risk of a potentially toxic reaction. Disturbance of the balance in this unique chemical environment makes neurons in the substantia nigra pars compacta (SNc) particularly vulnerable to parkinsonian neurodegeneration in the aging brain. In Parkinson's disease, these neurons degenerate coincident with an elevation in brain iron levels, yet relatively little is known about specific regional iron distribution with respect to dopamine. To directly appraise the irondopamine redox couple, we applied immuno-assisted laser ablation-inductively coupled plasma-mass spectrometry imaging to co-localize iron with the dopamine-producing enzyme tyrosine hydroxylase at the coronal level of the substantia nigra. We found that in the healthy brain the SNc does not contain the greatest concentration of iron within the midbrain, while the dopamine-rich environment in this region reflects an increased oxidative load. The product of iron and dopamine was significantly greater in the SNc than the adjacent ventral tegmental area, which is less susceptible to neuron loss in Parkinson's disease. Accordingly, this `risk factor was elevated further following 6-hydroxydopamine (6-OHDA) lesioning. Considering mounting evidence that brain iron increases with age, this measurable irondopamine index provides direct experimental evidence of a relationship between these two redox-active chemicals in degenerating dopaminergic neurons.
McGowan, N., Fowler, A., Parkinson, K., Bishop, D., Ganio, K., Doble, P.A., Booth, D.J. & Hare, D.J. 2014, 'Beyond the transect: An alternative microchemical imaging method for fine scale analysis of trace elements in fish otoliths during early life', The Science of the Total Environment, vol. 494-495, pp. 177-186.
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Microchemical analysis of otolith (calcified `ear stones used for balance and orientation) of fishes is an important tool for studying their environmental history and management. However, the spatial resolution achieved is often too coarse to examine short-termevents occurring in early life. Current methods rely on single points or transects across the otolith surface, which may provide a limited viewof elemental distributions, a matter that has not previously been investigated. Imaging by laser ablation-inductively coupled plasma-mass spectrometry (LA-ICPMS) permits microchemical analyses of short-term events in early life with high (b10 ìm) resolution, twodimensional (2D) visualization of elemental distributions. To demonstrate the potential of this method, we mapped the concentrations of Sr and Ba, two key trace elements, in a small number of juvenile otoliths of neon damselfish (Pomacentrus coelestis) using an 8 ìm beam diameter (laser fluence of 13.8 ± 3.5 J cm.2). Quantification was performed using the established method by Longerich et al. (1996), which is applied to 2D imaging of a biological matrix here for the first time. Accuracy of N97% was achieved using a multi-point non matrix-matched calibration of National Institute of Standards and Technology (NIST) 610 and 612 (trace elements in glass) using Longerich's calculation method against the matrix-matched standard FEBS-1 (powdered red snapper [Lutjanus campechanus] otolith). The spatial resolution achieved in the otolith corresponded to a time period of 2 ± 1 days during the larval phase, and 4 ± 1 days during the post-settlement juvenile phase. This method has the potential to improve interpretations of early life-history events at scales corresponding to specific events. While the images showed gradients in Sr and Ba across the larval settlement zone more clearly.
Hare, D.J., George, J.L., Bray, L., Volitakis, I., Vais, A., Ryan, T.M., Cherny, R.A., Bush, A.I., Masters, C.L., Adlard, P.A., Doble, P.A. & Finkelstein, D.I. 2014, 'The effect of paraformaldehyde fixation and sucrose cryoprotection on metal concentration in murine neurological tissue (vol 29, pg 565, 2014)', JOURNAL OF ANALYTICAL ATOMIC SPECTROMETRY, vol. 29, no. 9, pp. 1726-1726.
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Rembach, A., Hare, D.J., Doecke, J.D., Burnham, S.C., Volitakis, I., Fowler, C.J., Cherny, R.A., McLean, C., Grimm, R., Martins, R., Ames, D., Masters, C.L., Bush, A.I. & Roberts, B.R. 2014, 'Decreased serum zinc is an effect of ageing and not Alzheimer's disease', METALLOMICS, vol. 6, no. 7, pp. 1216-1219.
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Davies, K.M., Hare, D.J., Cottam, V., Chen, N., Hilgers, L., Halliday, G., Mercer, J.F. & Double, K.L. 2013, 'Localization of copper and copper transporters in the human brain', Metallomics, vol. 5, no. 1, pp. 43-51.
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Disturbances in brain copper result in rare and severe neurological disorders and may play a role in the pathogenesis and progression of multiple neurodegenerative diseases. Our current understanding of mammalian brain copper transport is based on model systems outside the central nervous system and no data are available regarding copper transport systems in the human brain. To address this deficit, we quantified regional copper concentrations and examined the distribution and cellular localization of the copper transport proteins Copper transporter 1, Atox1, ATP7A, and ATP7B in multiple regions of the human brain using inductively coupled plasma-mass spectrometry, Western blot and immunohistochemistry. We identified significant relationships between copper transporter levels and brain copper concentrations, supporting a role for these proteins in copper transport in the human brain. Interestingly, the substantia nigra contained twice as much copper than that in other brain regions, suggesting an important role for copper in this brain region. Furthermore, ATP7A levels were significantly greater in the cerebellum, compared with other brain regions, supporting an important role for ATP7A in cerebellar neuronal health. This study provides novel data regarding copper regulation in the human brain, critical to understand the mechanisms by which brain copper levels can be altered, leading to neurological disease.
Austin, C.E., Smith, T.M., Bradman, A., Hinde, K., Joannes-Boyau, R., Bishop, D.P., Hare, D.J., Doble, P.A., Eskenazi, B. & Arora, M. 2013, 'Barium distributions in teeth reveal early-life dietary transitions in primates', Nature, vol. 498, pp. 216-219.
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Early-life dietary transitions reflect fundamental aspects of primate evolution and are important determinants of health in contemporary human populations1, 2. Weaning is critical to developmental and reproductive rates; early weaning can have detrimental health effects but enables shorter inter-birth intervals, which influences population growth3. Uncovering early-life dietary history in fossils is hampered by the absence of prospectively validated biomarkers that are not modified during fossilization4. Here we show that large dietary shifts in early life manifest as compositional variations in dental tissues. Teeth from human children and captive macaques, with prospectively recorded diet histories, demonstrate that barium (Ba) distributions accurately reflect dietary transitions from the introduction of mothers milk through the weaning process. We also document dietary transitions in a Middle Palaeolithic juvenile Neanderthal, which shows a pattern of exclusive breastfeeding for seven months, followed by seven months of supplementation.
Chen, H., Dorrigan, A., Saad, S., Hare, D.J., Cortie, M.B. & Valenzuela, S. 2013, 'In Vivo Study of Spherical Gold Nanoparticles: Inflammatory Effects and Distribution in Mice', PLoS One, vol. 8, no. 2, pp. 1-8.
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Objectives Gold nanoparticles (AuNPs) of 21 nm have been previously well characterized in vitro for their capacity to target macrophages via active uptake. However, the short-term impact of such AuNPs on physiological systems, in particular resident macrophages located in fat tissue in vivo, is largely unknown. This project investigated the distribution, organ toxicity and changes in inflammatory cytokines within the adipose tissue after mice were exposed to AuNPs. Methods Male C57BL/6 mice were injected intraperitoneally (IP) with a single dose of AuNPs (7.85 µg AuNPs/g). Body weight and energy intake were recorded daily. Tissues were collected at 1 h, 24 h and 72 h post-injection to test for organ toxicity. AuNP distribution was examined using electron microscopy. Proinflammatory cytokine expression and macrophage number within the abdominal fat pad were determined using real-time PCR.
Veasey, S.C., Lear, J., Zhu, Y., Grinspan, J.B., Hare, D.J., Wang, S., Bunch, D., Doble, P.A. & Robinson, S.R. 2013, 'Long-Term Intermittent Hypoxia Elevates Cobalt Levels in the Brain and Injures White Matter in Adult Mice', Sleep, vol. 36, no. 10, pp. 1471-1481.
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Study Objectives: Exposure to the variable oxygenation patterns in obstructive sleep apnea (OSA) causes oxidative stress within the brain. We hypothesized that this stress is associated with increased levels of redox-active metals and white matter injury. Design: Participants were randomly allocated to a control or experimental group (single independent variable). Setting: University animal house. Participants: Adult male C57BL/6J mice. Interventions: To model OSA, mice were exposed to long-term intermittent hypoxia (LTIH) for 10 hours/day for 8 weeks or sham intermittent hypoxia (SIH).
Hare, D.J., Lear, J., Bishop, D.P., Beavis, A.B. & Doble, P.A. 2013, 'Protocol for production of matrix-matched brain tissue standards for imaging by laser ablation-inductively coupled plasma-mass spectrometry', Analytical Methods, vol. 5, no. 8, pp. 1915-1921.
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Calibration with matrix-matched standards remains the most practical means for producing quantitative images of trace metal distribution in tissue sections by laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS). A general guide for producing matrix-matched standards for assay of trace metals in brain tissue is presented. Cortical tissue was taken from pooled sheep brains and spiked with varying approximate concentrations of standard solutions of Co, Cu, Fe, Mg, Mn, Sr, Se and Zn. The tissue was homogenised and the total analyte amount accurately determined by solution nebulisation ICP-MS. The analytical ?gures of merit for LA-ICP-MS imaging were determined from these tissue standards cryosectioned at 30 mm. Repeated 8-point calibration curves were reproducibly linear, with correlation coe?cients ranging from 0.9874 (Mg) to 0.9991 (Sr). Limits of analysis were suitable for quantifying most analytes in a sample mouse brain, with the exception of Co and Se
Hare, D.J., Grubman, A., Ryan, T., Lothian, A., Liddell, J., Grimm, R., Matsuda, T., Doble, P.A., Cherny, R., Bush, A., White, A., Masters, C. & Roberts, B.R. 2013, 'Profiling the iron, copper and zinc content in primary neuron and astrocyte cultures by rapid online quantitative size exclusion chromatography-inductively coupled plasma-mass spectrometry', Metallomics, vol. 5, no. 12, pp. 1656-1662.
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Metals often determine the chemical reactivity of the proteins to which they are bound. Each cell in the body tightly maintains a unique metalloproteomic profile, mostly dependent on function. This paper describes an analytical online flow injection quan
Hare, D.J., Adlard, P.A., Doble, P.A. & Finkelstein, D.I. 2013, 'Metallobiology of 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine neurotoxicity', Metallomics, vol. 5, no. 2, pp. 91-109.
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1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is a potent toxin used to selectively destroy dopaminergic neurons in the substantia nigra and induce parkinsonism. MPTP is metabolised to the 1-methyl-4-phenylpyridinium ion (MPP(+)) in glia, after which it enters the neuron via the dopamine transporter and results in elevated levels of oxidative stress. The mechanism through which MPP(+) causes cell death is thought to involve redox-active metals, particularly iron (Fe). This review will examine how cellular metal metabolism is altered following MPTP insult, and how this relates to metal dyshomeostasis in idiopathic Parkinson's disease. This includes both cell damage arising from increased metal concentration, and how metal-binding proteins respond to MPTP-induced neurotoxicity. Implications for using MPTP as a model for human Parkinson's disease will be discussed in terms of cell metallobiology.
Finkelstein, D.I., George, J.L., Adlard, P.A., Masters, C.L., Hare, D.J., Doble, P.A., Gautier, E., Parsons, J., Kok, G., Huggins, P., Barnham, K.J., Bush, A.I. & Cherny, R.A. 2013, 'PBT434, a novel 8-hydroxyquinazolinone, preserves nigro-striatal circuitry, improves motor performance and inhibits alpha synuclein accumulation in animal models of Parkinson's disease by modulation of iron homeostasis', MOVEMENT DISORDERS, vol. 28, pp. S369-S369.
Lothian, A., Hare, D.J., Grimm, R., Ryan, T.M., Masters, C.L. & Roberts, B.R. 2013, 'Metalloproteomics: principles, challenges, and applications to neurodegeneration', FRONTIERS IN AGING NEUROSCIENCE, vol. 5.
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Hare, D., Ayton, S., Bush, A. & Lei, P. 2013, 'A delicate balance: Iron metabolism and diseases of the brain', FRONTIERS IN AGING NEUROSCIENCE, vol. 5.
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Rembach, A., Hare, D.J., Lind, M., Fowler, C.J., Cherny, R.A., McLean, C., Bush, A.I., Masters, C.L. & Roberts, B.R. 2013, 'Decreased copper in Alzheimer's disease brain is predominantly in the soluble extractable fraction.', International journal of Alzheimer's disease, vol. 2013, p. 623241.
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Alzheimer's disease (AD) is the leading cause of dementia and represents a significant burden on the global economy and society. The role of transition metals, in particular copper (Cu), in AD has become of significant interest due to the dyshomeostasis of these essential elements, which can impart profound effects on cell viability and neuronal function. We tested the hypothesis that there is a systemic perturbation in Cu compartmentalization in AD, within the brain as well as in the periphery, specifically within erythrocytes. Our results showed that the previously reported decrease in Cu within the human frontal cortex was confined to the soluble (P < 0.05) and total homogenate (P < 0.05) fractions. No differences were observed in Cu concentration in erythrocytes. Our data indicate that there is a brain specific alteration in Cu levels in AD localized to the soluble extracted material, which is not reflected in erythrocytes. Further studies using metalloproteomics approaches will be able to elucidate the metabolic mechanism(s) that results in the decreased brain Cu levels during the progression of AD.
Davies, K.M., Bohic, S., Ortega, R., Cottam, V., Hare, D.J., Finberg, J.P.M., Halliday, G., Mercer, J.F.B. & Double, K.L. 2013, 'Copper pathology in the vulnerable substantia nigra in Parkinson's disease', MOVEMENT DISORDERS, vol. 28, pp. S366-S366.
Hare, D.J., Lee, J.K., Beavis, A.B., Van Gramberg, A.A., George, J., Adlard, P.A., Finkelstein, D.I. & Doble, P.A. 2012, 'Three-dimensional atlas of iron, copper, and zinc in the mouse cerebrum and brainstem', Analytical Chemistry, vol. 84, no. 9, pp. 3990-3997.
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Atlases depicting molecular and functional features of the brain are becoming an integral part of modern neuroscience. In this study we used laser ablation-inductively coupled plasma-mass spectrometry (LA-ICPMS) to quantitatively measure iron (Fe), copper (Cu), and zinc (Zn) levels in a serially sectioned C57BL/6 mouse brain (cerebrum and brainstem). Forty-six sections were analyzed in a single experiment of approximately 158 h in duration. We constructed a 46-plate reference atlas by aligning quantified images of metal distribution with corresponding coronal sections from the Allen Mouse Brain Reference Atlas. The 46 plates were also used to construct three-dimensional models of Fe, Cu, and Zn distribution. This atlas represents the first reconstruction of quantitative trace metal distribution through the brain by LA-ICPMS and will facilitate the study of trace metals in the brain and help to elucidate their role in neurobiology.
Lear, J., Hare, D.J., Adlard, P., Finkelstein, D. & Doble, P.A. 2012, 'Improving acquisition times of elemental bio-imaging for quadrupole-based LA-ICP-MS', Journal of Analytical Atomic Spectrometry, vol. 27, no. 1, pp. 159-164.
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Elemental bio-imaging experiments by quadrupole-based LA-ICP-MS normally employ scan speeds where the distance traversed in one second is equal to or less than the diameter of the laser beam. Consequently, data for a higher-resolution (pixel size = 15 &micro;m2) image of a 5 mm2 tissue section can take upwards of 30 h to acquire. Appropriate laser scan speeds may be calculated by consideration of the relationship between laser scan speed, laser spot diameter and the total scan cycle of the quadrupole mass analyser. This paper presents a simple method to calculate the laser scan speeds capable of reducing the acquisition time by up to a factor of 5 whilst maintaining dimensional integrity of the image.
Hare, D.J., Gerlach, M. & Riederer, P. 2012, 'Considerations for measuring iron in post-mortem tissue of Parkinson's disease patients', Journal of Neural Transmission, vol. 119, no. 12, pp. 1515-1521.
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Redox-active iron is considered to be an important factor in the pathology and progression of several neurodegenerative disorders, including Parkinsons disease. The various roles of iron in normal physiology and its prevalence in the wider environment present numerous challenges to both accurate measurement and interpretation of brain iron levels. This review will discuss considerations for the analysis of iron in post-mortem samples, including how contamination, sample preparation and methods of analysis may influence results. In addition, several important factors influencing interpretation of iron levels will be considered.
Lear, J., Hare, D.J., Fryer, F., Adlard, P.A., Finkelstein, D.I. & Doble, P.A. 2012, 'High-resolution elemental bioimaging of Ca, Mn, Fe, Co, Cu, and Zn employing LA-ICP-MS and hydrogen reaction gas', Analytical Chemistry, vol. 84, no. 15, pp. 6707-6714.
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Imaging of trace metal distribution in tissue sections by laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) is typically performed using spatial resolutions of 30 &micro;m2 and above. Higher resolution imaging is desirable for many biological applications in order to approach the dimensions of a single cell. The limiting factor for increasing resolution is sensitivity, where signal-to-noise ratios are poor due to inherent background spectral interferences and reduced sample volume with decreasing laser beam diameter. Several prominent spectral interferences are present for a number of biologically relevant isotopes, including the 40Ar16O+ spectral interference on 56Fe+. We examined if H2 as a reaction gas could improve the analytical performance of imaging experiments for a range of masses with spectral interferences. At low (<1 mL min1) H2 flow rates, greater spectral interference due to H+ adducts was observed for 55Mn, 57Fe, and 59Co. At higher flow rates of up to 3 mL H2 per minute, the spectral interferences were reduced leading to improvement in limits of analysis for masses with O- and N-based polyatomic interferences. Enhanced sensitivity with the reaction cell allowed construction of high resolution (6 &micro;m2) imaging of 56Fe in the mouse brain that approached the dimensions of single cells.
Hare, D.J., Austin, C.E. & Doble, P.A. 2012, 'Quantification strategies for elemental imaging of biological samples using laser ablation-inductively coupled plasma-mass spectrometry', The Analyst, vol. 137, pp. 1527-1537.
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This review provides analysts with critical insights of current approaches for quantification by laser ablation-inductively coupled plasma-mass spectrometry in the field of elemental imaging. This encompasses both calibration strategies that have been used with success in imaging biological samples, as well as those with potential to improve analytical accuracy and precision if applied to imaging. Methods reviewed include the use of CRMs, laboratory prepared matrix matched standards, internal standardisation, online standard addition and a variety of novel approaches that makes elemental imaging accessible to a wider base of analysts. The importance of quantification and factors affecting its use in imaging will also be considered.
Hare, D., Austin, C. & Doble, P. 2012, 'Quantification strategies for elemental imaging of biological samples using laser ablation-inductively coupled plasma-mass spectrometry', ANALYST, vol. 137, no. 7, pp. 1527-1537.
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Arora, M., Hare, D.J., Austin, C.E., Smith, D.B. & Doble, P.A. 2011, 'Spatial Distribution Of Manganese In Enamel And Coronal Dentine Of Human Primary Teeth', Science Of The Total Environment, vol. 409, no. 7, pp. 1315-1319.
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Emerging evidence indicates that excessive exposure to manganese (Mn) during the prenatal period and early childhood may result in neurodevelopmental deficits. However, accurate exposure biomarkers are not well established, limiting our understanding of exposureresponse relationships over these susceptible periods of development. Naturally shed deciduous teeth are potentially a useful biomarker of environmental exposure to Mn. However, the uptake and distribution of Mn in human teeth has not been studied in detail. Mn distribution was measured at high resolution (~20 &igrave;m) in eight human primary teeth using laser ablationinductively coupled plasma-mass spectrometry. A bio-imaging methodology was applied to construct detailed elemental maps of three incisors, and bone meal (NIST SRM 1486) was used to validate the analyses. The distribution of Mn in enamel and coronal dentine showed a distinct and reproducible pattern. In enamel, the 55Mn:43Ca ratio was highest at the outer edge of enamel (range=0.57 to 4.74) for approximately 2040 &igrave;m butwas substantially lower in deeper layers (range=0.005 to 0.013). The highest levels ofMnwere observed in dentine immediately adjacent the pulpal margin (55Mn:43Ca range=2.27 to 6.95). Importantly, a clearly demarcated high Mn zone was observed in dentine at the incisal end of the teeth. Using confocal laser scanning microscopy to visualize the neonatal line, this region was identified as being in the prenatally formed dentine. The high-resolution map of the spatial distribution of Mn in human primary teeth highlighted specific reproducible patterns of Mn distribution in enamel and coronal dentine.
Hare, D.J., Austin, C.E., Doble, P.A. & Arora, M. 2011, 'Elemental Bio-imaging Of Trace Elements In Teeth Using Laser Ablation-inductively Coupled Plasma-mass Spectrometry', Journal of Dentistry, vol. 39, no. 5, pp. 397-403.
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Objectives: In this study we present the application of a novel laboratory method that employs laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) to construct two-dimensional maps of trace elements in teeth. Methods: Teeth were secti
Austin, C.E., Fryer, F.I., Lear, J., Bishop, D.P., Hare, D.J., Rawling, T., Kirkup, L., McDonagh, A.M. & Doble, P.A. 2011, 'Factors Affecting Internal Standard Selection For Quantitative Elemental Bio-Imaging Of Soft Tissues By LA-ICP-MS', Journal of Analytical Atomic Spectrometry, vol. 26, no. 7, pp. 1494-1501.
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Element response variations under different laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) operating conditions were investigated to identify important factors for selecting an internal standard (IS) for quantitative elemental bi
Tolmachev, S.Y., Ketterer, E., Hare, D.J., Doble, P.A. & James, A.C. 2011, 'The US Transuranium and Uranium Registries: forty years' experience and new directions in the analysis of actinides in human tissues', Radiochimica Acta, vol. 1, pp. 173-181.
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The US Transuranium and Uranium Registries (USTUR) studies the distribution, biokinetics and tissue dosimetry of actinide elements through radiochemical analysis of autopsy tissues voluntarily donated by occupationally exposed persons. The paper provides an overview of the analytical methods for plutonium (Pu), americium (Am) and uranium (U) isotopic determination in human tissues currently applied at USTUR. The results of inter-comparing 239+240Pu, 241Am and 234,235,238U determinations by sector ?eld inductively coupled mass spectrometry (SF-ICP-MS), a-spectrometry (AS) and kinetic phosphorescence analysis (KPA) are discussed. SFICP-MS is a major advance over AS and KPA in enabling the measurement of the 240Pu/ 239Pu atom ratio, the short-lived -emitter 241Pu, and long-lived 236U. For the ?rst time, 241Am and 241Pu were measured in human tissues using SF-ICP-MS. The paper also presents a new avenue of USTUR research in the application of laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) to elemental bio-imaging (EBI) of the actinides in human tissues.
Hare, D.J., Tolmachev, S., James, A., Bishop, D.P., Austin, C.E., Fryer, F.I. & Doble, P.A. 2010, 'Elemental bio-imaging of thorium, uranium, and plutonium in tissues from occupationally exposed former nuclear workers', Analytical Chemistry, vol. 82, no. 8, pp. 3176-3182.
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Internal exposure from naturally occurring radionuclides (including the inhaled long-lived actinides 232Th and 238U) is a component of the ubiquitous background radiation dose (National Council on Radiation Protection and Measurements. Ionizing radiation exposure of the population of the United States; NCRP Report No. 160; NCRP: Bethesda, MD, 2009). It is of interest to compare the concentration distribution of these natural ?-emitters in the lungs and respiratory lymph nodes with those resulting from occupational exposure, including exposure to anthropogenic plutonium and depleted and enriched uranium. This study examines the application of laser ablation-inductively coupled plasma-mass spectrometry (LA-ICPMS) to quantifying and visualizing the mass distribution of uranium and thorium isotopes from both occupational and natural background exposure in human respiratory tissues and, for the first time, extends this application to the direct imaging of plutonium isotopes. Sections of lymphatic and lung tissues taken from deceased former nuclear workers with a known history of occupational exposure to specific actinide elements (uranium, plutonium, or americium) were analyzed by LA-ICPMS.
Austin, C.E., Hare, D.J., Rawling, T., McDonagh, A.M. & Doble, P.A. 2010, 'Quantification method for elemental bio-imaging by LA-ICP-MS using metal spiked PMMA films', Journal of Analytical Atomic Spectrometry, vol. 25, no. 5, pp. 722-725.
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A method for quantitative analysis of biological soft tissues by laser ablation-inductively coupled plasma-mass spectrometry has been developed. Polymer film standards were produced by spin coating spiked solutions of poly methyl methacrylate onto quartz substrates. Calibration curves throughout the range of 0--400"llg g-l yielded correlation coefficients better than 0.999 for 66Zn and 63CU. Spiked, homogenised soft tissue standards were quantified by LA-ICP-MS against the thin film standards. The results agreed with values calculated from solution nebulisation rCP-MS. A procedure for internal standardisation by employing ruthenium or yttrium in the underlying thin film was also assessed.
Hare, D.J., George, J.L., Grimm, R., Wilkins, S.W., Adlard, P., Cherny, R., Bush, A., Finkelstein, D. & Doble, P.A. 2010, 'Three-Dimensional Elemental Bio-Imaging Of Fe, Zn, Cu, Mn And P In A 6-Hydroxydopamine Lesioned Mouse Brain', Metallomics, vol. 2, no. 11, pp. 745-753.
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Three dimensional maps of iron (Fe), zinc (Zn), copper (Cu), manganese (Mn) and phosphorous (P) in a 6-hydroxydopamine (6-OHDA) lesioned mouse brain were constructed employing a novel quantitative laser ablation-inductively coupled plasma-mass spectromet
Austin, C.E., Hare, D.J., Rozelle, A., Robinson, W., Grimm, R. & Doble, P.A. 2009, 'Elemental bio-imaging of calcium phosphate crystal deposits in knee samples from arthritic patients', Metallomics, vol. 1, no. 2, pp. 142-147.
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Laser ablation inductively coupled plasma mass spectrometry (LA ICP-MS) was employed to image deposits of calcium phosphate based crystals in knee cartilage and synovial fluid from arthritic patients. A reaction/collision cell containing hydrogen minimised plasma interferences on calcium and also improved the image quality without significant sensitivity reduction. Areas of high calcium and phosphorus intensities consistent with crystal deposits were observed for both the cartilage and synovial fluid samples. These areas were also characterised by high magnesium and strontium intensities. Distribution patterns of other elements such as copper and sulfur did not correlate with the crystal deposits. Filtered and non-filtered solutions of calcium phosphate crystals grown in synthetic synovial fluid were also imaged as further evidence of crystal deposits. The crystal deposits were detected in the unfiltered Solution, and were absent from the filtered solutions.
Rawling, T., Austin, C.E., Hare, D.J., Doble, P.A., Zareie, H.M. & McDonagh, A.M. 2009, 'Thin films of ruthenium phthalocyanine complexes', Nano Research, vol. 2, no. 9, pp. 678-687.
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Four new ruthenium phthalocyanine complexes bearing axial ligands with thioacetate groups that facilitate thin film formation on gold surfaces are presented. Scanning tunnelling microscopy (STM) images and surface coverage data obtained by solution inductively coupled plasma mass spectrometry (ICP-MS) experiments show that peripheral and axial ligand substituents on the complexes have a significant effect on their surface coverage. A laser ablation ICP-MS technique that provides information about thin films across macro-sized areas is described here for the first time. Using the technique, the maximum surface coverage of a ruthenium phthalocyanine complex was found to occur within one minute of gold substrate immersion in the complex-containing solution.
Hare, D.J., Haarburger, D., Austin, C.E., Fryer, F.I., Grimm, R., Reedy, B.J., Scolyer, R.A., Thompson, J.F. & Doble, P.A. 2009, 'Elemental bio-imaging of melanoma in lymph node biopsies', The Analyst, vol. 134, no. 3, pp. 450-453.
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The spatial distribution of trace elements in human lymph nodes partially infiltrated by melanoma cells was determined by elemental bio-imaging. Imaging of 31P within the nodal capsule and normal lymph node tissue showed a clear demarcation of the tumour boundary, with a significant decrease in relative 31P concentration within the tumour. The location of the tumour boundary was confirmed by haematoxylin and eosin staining of serial sections and observation by light microscopy. Further enhancement of the tumour boundary was achieved by imaging the 31P/34S ratio. 31P/66Zn ratio images showed a decreasing ratio beyond the tumour boundary that extended into peritumour normal lymph node tissue.
Rawling, T., Austin, C.E., Hare, D.J., Doble, P.A., Zareie, H.M. & McDonagh, A.M. 2009, 'Thin films of Ruthenium Phthalocyanine complexes', Nano Research, vol. 2, pp. 678-687.
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Four new ruthenium phthalocyanine complexes bearing axial ligands with thioacetate groups that facilitate thin film formation on gold surfaces are presented. Scanning tunnelling microscopy (STM) images and surface coverage data obtained by solution inductively coupled plasma mass spectrometry (ICP-MS) experiments show that peripheral and axial ligand substituents on the complexes have a significant effect on their surface coverage. A laser ablation ICP-MS technique that provides information about thin films across macro-sized areas is described here for the first time. Using the technique, the maximum surface coverage of a ruthenium phthalocyanine complex was found to occur within one minute of gold substrate immersion in the complexcontaining solution.
Hare, D.J., Reedy, B.J., Grimm, R., Wilkins, S., Volitakis, I., George, J.L., Cherny, R., Bush, A., Finkelstein, D. & Doble, P.A. 2009, 'Quantitative elemental bio-imaging of Mn, Fe, Cu and Zn in 6-hydroxydopamine induced Parkinsonism mouse models', metallomics, vol. 1, no. 1, pp. 53-58.
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This study demonstrates the application of quantitative elemental bio-imaging for the determination of the distribution Cu, Mn, Fe and Zn in Parkinsonism mouse model brains. Elevated concentrations of these metals within the substantia nigra (SN) are suspected to play a role on the development of Parkinsons disease. Elemental bio-imaging employs laser ablation inductively coupled mass spectrometry (LA-ICP-MS) to construct images of trace element distribution. Quantitative data was produced by ablating the standard tissue sections and recording the mean signal intensity calibrated against multi level matrix matched tissue standards. The concentrations of Fe within the substantia nigra of the lesioned animals increased significantly when compared against control animals. Furthermore, the data was compared against solution nebulisation ICP-MS in which the whole substantia nigra was excised. The trends were the same for both methods; however the elemental bio-imaging method returned significantly higher concentrations. This was caused by dilution from inclusion of surrounding tissue of the SN during the excision procedure.
Hare, D., Bishop, D., Austin, C. & Doble, P. 2009, 'The answer is elemental', Biochemist, vol. 31, no. 1, p. 46.
Trace elements play a key role in a wide range of biological processes. A full understanding of those processes requires a knowledge not only of the genomics and proteomics of the organism, but also the distribution and concentration of trace elements. A new application of inductively coupled plasma mass spectrometry (ICP-MS) provides the capability to directly visualize trace elements in soft tissue, offering the promise of improved diagnosis and monitoring of disease. &copy; 2009 The Biochemical Society.