Search

The insidious and deadly nature of mercury's organometallic compounds is informed by two large scale poisonings due to industrial mercury pollution that occurred decades ago in Minamata and Niigata, Japan. The present study examined chemical speciation for both mercury and selenium in a historic umbilical cord sample from a child born to a mother who lived near the Agano River in Niigata. The mother had experienced mercury exposure leading to more than 50 ppm mercury measured in her hair and was symptomatic 9 years prior to the birth. We sought to determine the mercury and selenium speciation in the child's cord using Hg Lα1 and Se Kα1 high-energy resolution fluorescence detected X-ray absorption spectroscopy, the chemical speciation of mercury was found to be predominantly organometallic and coordinated to a thiolate. The selenium was found to be primarily in an organic form and at levels higher than those of mercury, with no evidence of mercury-selenium chemical species. Our results are consistent with mercury exposure at Niigata being due to exposure to organometallic mercury species., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Graham George reports financial support was provided by Natural Sciences and Engineering Research Council of Canada. Ingrid Pickering reports financial support was provided by Canada Research Chairs. Ingrid Pickering reports financial support was provided by Natural Sciences and Engineering Research Council of Canada. Graham George reports financial support was provided by Canada Research Chairs. Graham George reports financial support was provided by New Frontiers in Research Fund. Ingrid Pickering reports financial support was provided by Canada Foundation For Innovation. Gary Myers reports financial support was provided by National Institute of Environmental Health Sciences., (Copyright © 2023. Published by Elsevier B.V.)

In situ methods are valuable in all fields of research. In toxicology, the importance of dose is well known, elevating the need for in situ techniques to measure levels of toxicants and their byproducts in precise anatomically identifiable locations. More recently, additional emphasis has been placed on the value of techniques which can detect chemical form or speciation, which is equally important in the toxicology of a chemical compound. Many important but conventional methods risk losing valuable information due to extractions, digestions, or the general reliance on mobile phases. Few analytical tools possess the power and diversity of X-ray methods as in-situ methods. Here we present an overview, intended for toxicologists and pathologists, of a variety of synchrotron X-ray methods for determining in situ chemical form and distribution of heavier elements. The versatility and range of these synchrotron techniques, which are both established and emerging, is demonstrated in the context of the study of neurotoxicology of mercury, a global pollutant with the ability to harm both human health and the environment., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The sulfones are a widespread group of organo-sulfur compounds, which contain the sulfonyl SO 2 group attached to two carbons and have a formal sulfur oxidation state of +2. We have examined the sulfur K near-edge X-ray absorption spectroscopy (XAS) of a range of different sulfones and find substantial spectroscopic variability depending upon the nature of the coordination to the sulfonyl group. We have also examined the sulfur Kβ X-ray emission spectroscopy (XES) of selected representative sulfones. Density functional theory simulations show satisfactory reproduction of both absorption and emission spectra while enabling assignment of the various transitions comprising the spectra. The correspondence between observed and simulated spectra shows promise for ab initio prediction of sulfur X-ray absorption and emission spectra of sulfones of any substituent. The absorption spectra and, to a lesser extent, the emission spectra are sensitive to the nature of the organic groups bound to the sulfonyl (SO 2 ) moiety, clearly showing the potential of X-ray spectroscopy as an in situ probe of sulfone chemistry.

The exposure of humans to Cd exerts adverse human health effects at low chronic exposure doses, but the underlying biomolecular mechanisms are incompletely understood. To gain insight into the toxicologically relevant chemistry of Cd 2+ in the bloodstream, we employed an anion-exchange HPLC coupled to a flame atomic absorption spectrometer (FAAS) using a mobile phase of 100 mM NaCl with 5 mM Tris-buffer (pH 7.4) to resemble protein-free blood plasma. The injection of Cd 2+ onto this HPLC-FAAS system was associated with the elution of a Cd peak that corresponded to [CdCl 3 ] - /[CdCl 4 ] 2- complexes. The addition of 0.1-10 mM L-cysteine (Cys) to the mobile phase significantly affected the retention behavior of Cd 2+ , which was rationalized by the on-column formation of mixed CdCys x Cl y complexes. From a toxicological point of view, the results obtained with 0.1 and 0.2 mM Cys were the most relevant because they resembled plasma concentrations. The corresponding Cd-containing (~30 μM) fractions were analyzed by X-ray absorption spectroscopy and revealed an increased sulfur coordination to Cd 2+ when the Cys concentration was increased from 0.1 to 0.2 mM. The putative formation of these toxicologically relevant Cd species in blood plasma was implicated in the Cd uptake into target organs and underscores the notion that a better understanding of the metabolism of Cd in the bloodstream is critical to causally link human exposure with organ-based toxicological effects.

Desferrioxamine (DFO) has long been considered the gold standard chelator for incorporating [ 89 Zr]Zr 4+ in radiopharmaceuticals for positron emission tomography (PET) imaging. To improve the stability of DFO with zirconium-89 and to expand its coordination sphere to enable binding of large therapeutic radiometals, we have synthesized the highest denticity DFO derivatives to date: dodecadentate DFO2 and DFO2p. In this study, we describe the synthesis and characterization of a novel DFO-based chelator, DFO2p, which is comprised of two DFO strands connected by an p -NO 2 -phenyl linker and therefore contains double the chelating moieties of DFO (potential coordination number up to 12 vs 6). The chelator DFO2p offers an optimized synthesis comprised of only a single reaction step and improves water solubility relative to DFO2, but the shorter linker reduces molecular flexibility. Both DFO2 and DFO2p, each with 6 potential hydroxamate ligands, are able to reach a more energetically favorable 8-coordinate environment for Zr(IV) than DFO. The zirconium(IV) coordination environment of these complexes were evaluated by a combination of density functional theory (DFT) calculations and synchrotron spectroscopy (extended X-ray absorption fine structure), which suggest the inner-coordination sphere of zirconium(IV) to be comprised of the outermost four hydroxamate ligands. These results also confirm a single Zr(IV) in each chelator, and the hydroxide ligands which complete the coordination sphere of Zr(IV)-DFO are absent from Zr(IV)-DFO2 and Zr(IV)-DFO2p. Radiochemical stability studies with zirconium-89 revealed the order of real-world stability to be DFO2 > DFO2p ≫ DFO. The zirconium-89 complexes of these new high-denticity chelators were found to be far more stable than DFO, and the decreased molecular flexibility of DFO2p, relative to DFO2, could explain its decreased stability, relative to DFO2.

Although Alzheimer's disease (AD) was first described over a century ago, it remains the leading cause of age-related dementia. Innumerable changes have been linked to the pathology of AD; however, there remains much discord regarding which might be the initial cause of the disease. The "amyloid cascade hypothesis" proposes that the amyloid β (Aβ) peptide is central to disease pathology, which is supported by elevated Aβ levels in the brain before the development of symptoms and correlations of amyloid burden with cognitive impairment. The "metals hypothesis" proposes a role for metal ions such as iron, copper, and zinc in the pathology of AD, which is supported by the accumulation of these metals within amyloid plaques in the brain. Metals have been shown to induce aggregation of Aβ, and metal ion chelators have been shown to reverse this reaction in vitro . 8-Hydroxyquinoline-based chelators showed early promise as anti-Alzheimer's drugs. Both 5-chloro-7-iodo-8-hydroxyquinoline (CQ) and 5,7-dichloro-2-[(dimethylamino)methyl]-8-hydroxyquinoline (PBT2) underwent unsuccessful clinical trials for the treatment of AD. To gain insight into the mechanism of action of 8HQs, we have investigated the potential interaction of CQ, PBT2, and 5,7-dibromo-8-hydroxyquinoline (B2Q) with Cu(II)-bound Aβ(1-42) using X-ray absorption spectroscopy (XAS), high energy resolution fluorescence detected (HERFD) XAS, and electron paramagnetic resonance (EPR). By XAS, we found CQ and B2Q sequestered ∼83% of the Cu(II) from Aβ(1-42), whereas PBT2 sequestered only ∼59% of the Cu(II) from Aβ(1-42), suggesting that CQ and B2Q have a higher relative Cu(II) affinity than PBT2. From our EPR, it became clear that PBT2 sequestered Cu(II) from a heterogeneous mixture of Cu(II)Aβ(1-42) species in solution, leaving a single Cu(II)Aβ(1-42) species. It follows that the Cu(II) site in this Cu(II)Aβ(1-42) species is inaccessible to PBT2 and may be less solvent-exposed than in other Cu(II)Aβ(1-42) species. We found no evidence to suggest that these 8HQs form ternary complexes with Cu(II)Aβ(1-42).

Copper(II) coordination by bis(cyclohexanone)oxalyldihydrazone (also known as cuprizone), resulting in the formation of an intensely coloured blue complex, was first reported over 70 years ago. The cuprizone reaction has been employed in colourimetric tests for the presence of trace levels of copper. Cuprizone administration in C57BL/6 mice also leads to demyelination over time - a consequence that appears to be due to copper dyshomeostasis - and this has led to use of cuprizone as the leading method for toxicant-induced generation of an animal model of demyelination since its first use in the 1960s. Despite broad interest in cuprizone and its ability to bind copper there have been relatively few studies to structurally characterize the copper coordination properties of this ligand. In the absence of an aqueous medium, such as neat alcohol, copper and cuprizone exclusively form an amorphous green precipitate. Under aqueous conditions, where a large excess of cuprizone (relative to copper) is present, the blue complex that is synonymous with copper-cuprizone coordination is predominantly formed. The blue and green copper-cuprizone products demonstrate poor solubility and present challenges for conventional structure characterization methods, such as X-ray crystallography or nuclear magnetic resonance spectroscopy. By combining mass spectrometry, X-ray absorption spectroscopy, computational chemistry, and other techniques, a self-consistent picture of the copper coordination structures of the blue and green complexes is revealed - confirming that the blue complex is in the Cu(III) state, containing two hydrolyzed cuprizone ligands per metal centre, while the green complex represents an extended oligomeric complex, comprised of repeating Cu(II) centres that lie 4.8 Å apart and are bridged by unhydrolyzed cuprizone donors.

Mercury is ubiquitous in the environment, with rising levels due to pollution and climate change being a current global concern. Many mercury compounds are notorious for their toxicity, with the potential of organometallic mercury compounds for devastating effects on the structures and functions of the central nervous system being of particular concern. Chronic exposure of human populations to low levels of methylmercury compounds occurs through consumption of fish and other seafood, although the health consequences, if any, from this exposure remain controversial. We have used high energy resolution fluorescence detected X-ray absorption spectroscopy to determine the speciation of mercury and selenium in human brain tissue. We show that the molecular fate of mercury differs dramatically between individuals who suffered acute organometallic mercury exposure (poisoning) and individuals with chronic low-level exposure from a diet rich in marine fish. For long-term low-level methylmercury exposure from fish consumption, mercury speciation in brain tissue shows methylmercury coordinated to an aliphatic thiolate, resembling the coordination environment observed in marine fish. In marked contrast, for short-term high-level exposure, we observe the presence of biologically less available mercuric selenide deposits, confirmed by X-ray fluorescence imaging, as well as mercury(II)-bis-thiolate complexes, which may be signatures of severe poisoning in humans. These differences between low-level and high-level exposures challenge the relevance of studies involving acute exposure as a proxy for low-level chronic exposure.

Mercury is in some sense an enigmatic element. The element and some of its compounds are a natural part of the biogeochemical cycle; while many of these can be deadly poisons at higher levels, environmental levels in the absence of anthropogenic contributions would generally be below the threshold for concern. However, mercury pollution, particularly from burning fossil fuels such as coal, is providing dramatic and increasing emissions into the environment. Because of this, the environmental chemistry and toxicology of mercury are of growing importance, with the fate of mercury being vitally dependent upon its speciation. X-ray absorption spectroscopy (XAS) provides a powerful tool for in situ chemical speciation, but is severely limited by poor spectroscopic energy resolution. Here, we provide a systematic examination of mercury Lα1 high energy resolution fluorescence detected XAS (HERFD-XAS) as an approach for chemical speciation of mercury, in quantitative comparison with conventional Hg L III -edge XAS. We show that, unlike some lighter elements, chemical shifts in the Lα1 X-ray fluorescence energy can be safely neglected, so that mercury Lα1 HERFD-XAS can be treated simply as a high-resolution version of conventional XAS. We present spectra of a range of mercury compounds that may be relevant to the environmental and life science research and show that density functional theory can produce adequate simulations of the spectra. We discuss strengths and limitations of the method and quantitatively demonstrate improvements both in speciation for complex mixtures and in background rejection for low concentrations.

The current coronavirus pandemic is exerting a tremendously detrimental impact on global health. The Spike proteins of coronaviruses, responsible for cell receptor binding and viral internalization, possess multiple and frequently conserved disulfide bonds raising the question about their role in these proteins. Here, we present a detailed structural and functional investigation of the disulfide bonds of the SARS-CoV-2 Spike receptor-binding domain (RBD). Molecular dynamics simulations of the RBD predict increased flexibility of the surface loops when the four disulfide bonds of the domain are reduced. This flexibility is particularly prominent for the disulfide bond-containing surface loop (residues 456-490) that participates in the formation of the interaction surface with the Spike cell receptor ACE2. In vitro, disulfide bond reducing agents affect the RBD secondary structure, lower its melting temperature from 52 °C to 36-39 °C and decrease its binding affinity to ACE2 by two orders of magnitude at 37 °C. Consistent with these in vitro findings, the reducing agents tris(2-carboxyethyl)phosphine (TCEP) and dithiothreitol (DTT) were able to inhibit viral replication at low millimolar levels in cell-based assays. Our research demonstrates the mechanism by which the disulfide bonds contribute to the molecular structure of the RBD of the Spike protein, allowing the RBD to execute its viral function., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Oxygen K-edge X-ray absorption spectroscopy is used routinely to study a range of solid materials. However, liquid samples are studied less frequently at the oxygen K-edge due to the combined challenges of high-vacuum conditions and oxygen contamination of window materials. A modular sample holder design with a twist-seal sample containment system that provides a simple method to encapsulate liquid samples under high-vacuum conditions is presented. This work shows that pure silicon nitride windows have lower oxygen contamination than both diamond- and silicon-rich nitride windows, that the levels of oxygen contamination are related to the age of the windows, and provides a protocol for minimizing the background oxygen contamination. Acid-washed 100 nm-thick silicon nitride windows were found to give good quality oxygen K-edge data on dilute liquid samples.

Recent improvements in both X-ray detectors and readout speeds have led to a substantial increase in the volume of X-ray fluorescence data being produced at synchrotron facilities. This in turn results in increased challenges associated with processing and fitting such data, both temporally and computationally. Herein an abridging approach is described that both reduces and partially integrates X-ray fluorescence (XRF) data sets to obtain a fivefold total improvement in processing time with negligible decrease in quality of fitting. The approach is demonstrated using linear least-squares matrix inversion on XRF data with strongly overlapping fluorescent peaks. This approach is applicable to any type of linear algebra based fitting algorithm to fit spectra containing overlapping signals wherein the spectra also contain unimportant (non-characteristic) regions which add little (or no) weight to fitted values, e.g. energy regions in XRF spectra that contain little or no peak information.

Selenium is in many ways an enigmatic element. It is essential for health but toxic in excess, with the difference between the two doses being narrower than for any other element. Environmentally, selenium is of concern due to its toxicity. As the rarest of the essential elements, its low levels often provide challenges to the analytical chemist. X-ray absorption spectroscopy (XAS) provides a powerful tool for in situ chemical speciation but is severely limited by poor spectroscopic resolution arising from core-hole lifetime broadening. Here we explore selenium Kα1 high energy resolution fluorescence detected XAS (HERFD-XAS) as a novel approach for chemical speciation of selenium, in comparison with conventional Se K-edge XAS. We present spectra of a range of selenium species relevant to environmental and life science studies, including spectra of seleno-amino acids, which show strong similarities with S K-edge XAS of their sulfur congeners. We discuss strengths and limitations of HERFD-XAS, showing improvements in both speciation performance and low concentration detection. We also develop a simple method to correct fluorescence self-absorption artifacts, which is generally applicable to any HERFD-XAS experiment.

The compounds of mercury can be highly toxic and can interfere with a range of biological processes, although many aspects of the mechanism of toxicity are still obscure or unknown. One especially intriguing property of Hg(II) is its ability to bind DNA directly, making interstrand cross-links between thymine nucleobases in AT-rich sequences. We have used a combination of small molecule X-ray diffraction, X-ray spectroscopies, and computational chemistry to study the interactions of Hg(II) with thymine. We find that the energetically preferred mode of thymine binding in DNA is to the N3 and predict only minor distortions of the DNA structure on binding one Hg(II) to two cross-adjacent thymine nucleotides. The preferred geometry is predicted to be twisted away from coplanar through a torsion angle of between 32 and 43°. Using 1-methylthymine as a model, the bis -thymine coordination of Hg(II) is found to give a highly characteristic X-ray spectroscopic signature that is quite distinct from other previously described biological modes of binding of Hg(II). This work enlarges and deepens our view of significant biological targets of Hg(II) and demonstrates tools that can provide a characteristic signature for the binding of Hg(II) to DNA in more complex matrices including intact cells and tissues, laying the foundation for future studies of mechanisms of mercury toxicity.

Until recently, sulfur was known as a "spectroscopically silent" element because of a paucity of convenient spectroscopic probes suitable for in situ chemical speciation. In recent years the technique of sulfur K-edge X-ray absorption spectroscopy (XAS) has been used extensively in sulfur speciation in a variety of different fields. With an initial focus on reduced forms of organic sulfur, we have explored a complementary X-ray based spectroscopy - sulfur Kβ X-ray emission spectroscopy (XES) - as a potential analytical tool for sulfur speciation in complex samples. We compare and contrast the sensitivity of sulfur Kβ XES with that of sulfur K-edge XAS, and find differing sensitivities for the two techniques. In some cases an approach involving both sulfur K-edge XAS and sulfur Kβ XES may be a powerful combination for deducing sulfur speciation in samples containing complex mixtures.

8-Hydroxyquinolines (8HQs) comprise a family of metal-binding compounds that have been used or tested for use in numerous medicinal applications, including as treatments for bacterial infection, Alzheimer's disease, and cancer. Two key 8HQs, CQ (5-chloro-7-iodo-8-hydroxyquinoline) and PBT2 (2-(dimethylamino)methyl-5,7-dichloro-8-hydroxyquinoline), have drawn considerable interest and have been the focus of many studies investigating their in vivo properties. These drugs have been described as copper and zinc ionophores because they do not cause metal depletion, as would be expected for a chelation mechanism, but rather cellular accumulation of these ions. In studies of their anti-cancer properties, CQ has been proposed to elicit toxic intracellular copper accumulation and to trigger apoptotic cancer cell death through several possible pathways. In this study we used synchrotron X-ray fluorescence imaging, in combination with biochemical assays and light microscopy, to investigate 8HQ-induced alterations to metal ion homeostasis, as well as cytotoxicity and cell death. We used the bromine fluorescence from a bromine labelled CQ congener (5,7-dibromo-8-hydroxyquinoline; B2Q) to trace the intracellular localization of B2Q following treatment and found that B2Q crosses the cell membrane. We also found that 8HQ co-treatment with Cu(ii) results in significantly increased intracellular copper and significant cytotoxicity compared with 8HQ treatments alone. PBT2 was found to be more cytotoxic, but a weaker Cu(ii) ionophore than other 8HQs. Moreover, treatment of cells with copper in the presence of CQ or B2Q resulted in copper accumulation in the nuclei, while PBT2-guided copper was distributed near to the cell membrane. These results suggest that PBT2 may be acting through a different mechanism than that of other 8HQs to cause the observed cytotoxicity.

Positron emission tomography (PET) using radiolabeled, monoclonal antibodies has become an effective, noninvasive method for tumor detection and is a critical component of targeted radionuclide therapy. Metal ion chelator and bacterial siderophore desferrioxamine (DFO) is the gold standard compound for incorporation of zirconium-89 in radiotracers for PET imaging because it is thought to form a stable chelate with [ 89 Zr]Zr 4+ . However, DFO may not bind zirconium-89 tightly in vivo , with free zirconium-89 reportedly liberated into the bones of experimental mouse models. Although high bone uptake has not been observed to date in humans, this potential instability has been proposed to be related to the unsaturated coordination sphere of [ 89 Zr]Zr-DFO, which is thought to consist of the 3 hydroxamate groups of DFO and 1 or 2 water molecules. In this study, we have used a combination of X-ray absorption spectroscopy and density functional theory (DFT) geometry optimization calculations to further probe the coordination chemistry of this complex in solution. We find the extended X-ray absorption fine structure (EXAFS) curve fitting of an aqueous solution of Zr(IV)-DFO to be consistent with an 8-coordinate Zr with oxygen ligands. DFT calculations suggest that the most energetically favorable Zr(IV) coordination environment in DFO likely consists of the 3 hydroxamate ligands from DFO, each with bidentate coordination, and 2 hydroxide ligands. Further EXAFS curve fitting provides additional support for this model. Therefore, we propose that the coordination sphere of Zr(IV)-DFO is most likely completed by 2 hydroxide ligands rather than 2 water molecules, forming Zr(DFO)(OH) 2 .

PBT2 (5,7-dichloro-2-[(dimethylamino)methyl]-8-hydroxyquinoline) is a small Cu(II)-binding drug that has been investigated in the treatment of neurodegenerative diseases, namely, Alzheimer's disease (AD). PBT2 is thought to be highly effective at crossing the blood-brain barrier and has been proposed to exert anti-Alzheimer's effects through the modulation of metal ion concentrations in the brain, specifically the sequestration of Cu(II) from amyloid plaques. However, despite promising initial results in animal models and in clinical trials where PBT2 was shown to improve cognitive function, larger-scale clinical trials did not find PBT2 to have a significant effect on the amyloid plaque burden compared with controls. We propose that the results of these clinical trials likely point to a more complex mechanism of action for PBT2 other than simple Cu(II) sequestration. To this end, herein we have investigated the solution chemistry of Cu(II) coordination by PBT2 primarily using X-ray absorption spectroscopy (XAS), high-energy-resolution fluorescence-detected XAS, and electron paramagnetic resonance. We propose that a novel bis -PBT2 Cu(II) complex with asymmetric coordination may coexist in solution with a symmetric four-coordinate Cu(II)- bis -PBT2 complex distorted from coplanarity. Additionally, PBT2 is a more flexible ligand than other 8HQs because it can act as both a bidentate and a tridentate ligand as well as coordinate Cu(II) in both 1:1 and 2:1 PBT2/Cu(II) complexes.

Transition metal ions (Fe, Mn, Cu, Zn) are essential for healthy brain function, but altered concentration, distribution, or chemical form of the metal ions has been implicated in numerous brain pathologies. Currently, it is not possible to image the cellular or sub-cellular distribution of metal ions in vivo and therefore, studying brain-metal homeostasis largely relies on ex vivo in situ elemental mapping. Sample preparation methods that accurately preserve the in vivo elemental distribution are essential if one wishes to translate the knowledge of elemental distributions measured ex vivo toward increased understanding of chemical and physiological pathways of brain disease. The choice of sample preparation is particularly important for metal ions that exist in a labile or mobile form, for which the in vivo distribution could be easily distorted by inappropriate sample preparation. One of the most widely studied brain structures, the hippocampus, contains a large pool of labile and mobile Zn. Herein, we describe how sucrose cryoprotection, the gold standard method of preparing tissues for immuno-histochemistry or immuno-fluorescence, which is also often used as a sample preparation method for elemental mapping studies, drastically alters hippocampal Zn distribution. Based on the results of this study, in combination with a comparison against the strong body of published literature that has used either rapid plunge freezing of brain tissue, or sucrose cryo-protection, we strongly urge investigators in the future to cease using sucrose cryoprotection as a method of sample preparation for elemental mapping, especially if Zn is an analyte of interest.

8-Hydroxyquinolines (8HQs) are a family of lipophilic metal ion chelators that have been used in a range of analytical and pharmaceutical applications over the last 100 years. More recently, CQ (clioquinol; 5-chloro-7-iodo-8-hydroxyquinoline) and PBT2 (5,7-dichloro-2-[(dimethylamino)methyl]-8-hydroxyquinoline) have undergone clinical trials for the treatment of Alzheimer's disease and Huntington's disease. Because CQ and PBT2 appear to redistribute metals into cells, these compounds have been redefined as copper and zinc ionophores. Despite the attention surrounding the clinical trials and the clear link between 8HQs and metals, the fundamental solution chemistry of how these compounds bind divalent metals such as copper and zinc, as well as their mechanism(s) of action in mammalian systems, remains poorly understood. In this study, we used a combination of X-ray absorption spectroscopy (XAS), high-energy resolution fluorescence detected (HERFD) XAS, electron paramagnetic resonance (EPR), and UV-visible absorption spectroscopies to investigate the aqueous solution chemistry of a range of 8HQ derivatives. To circumvent the known solubility issues with 8HQ compounds and their complexes with Cu(II), and to avoid the use of abiological organic solvents, we have devised a surfactant buffer system to investigate these Cu(II) complexes in aqueous solution. Our study comprises the first comprehensive investigation of the Cu(II) complexes formed with many 8HQs of interest in aqueous solution, and it provides the first structural information on some of these complexes. We find that halogen substitutions in 8HQ derivatives appear to have little effect on the Cu(II) coordination environment; 5,7-dihalogenated 8HQ conformers all have a pseudo square planar Cu(II) bound by two quinolin-8-olate anions, in agreement with previous studies. Conversely, substituents in the 2-position of the 8HQ moiety appear to cause significant distortions from the typical square-planar-like coordination of most Cu(II)-bis-8HQ complexes, such that the 8HQ moieties in the Cu(II)-bis-8HQ complex are rotated approximately 30-40° apart in a "propeller-like" arrangement.

Over 200 million people worldwide are exposed to the human carcinogen, arsenic, in contaminated drinking water. In laboratory animals, arsenic and the essential trace element, selenium, can undergo mutual detoxification through the formation of the seleno-bis(S-glutathionyl) arsinium ion [(GS) 2 AsSe] - , which undergoes biliary and fecal elimination. [(GS) 2 AsSe] - , formed in animal red blood cells (RBCs), sequesters arsenic and selenium, and slows the distribution of both compounds to peripheral tissues susceptible to toxic effects. In human RBCs, the influence of arsenic on selenium accumulation, and vice versa, is largely unknown. The study aims were to characterize arsenite (As III ) and selenite (Se IV ) uptake by human RBCs, to determine if Se IV and As III increase the respective accumulation of the other in human RBCs, and ultimately to determine if this occurs through the formation and sequestration of [(GS) 2 AsSe] - . 75 Se IV accumulation was temperature and Cl - -dependent, inhibited by 4,4'-diisothiocyanatodihydrostilbene-2,2'-disulfonic acid (H 2 DIDS) (IC 50 1 ± 0.2 µM), and approached saturation at 30 µM, suggesting uptake is mediated by the erythrocyte anion-exchanger 1 (AE1 or Band 3, gene SLC4A1). HEK293 cells overexpressing AE1 showed concentration-dependent 75 Se IV uptake. 73 As III uptake by human RBCs was temperature-dependent, partly reduced by aquaglyceroporin 3 inhibitors, and not saturated. As III increased 75 Se IV accumulation (in the presence of albumin) and Se IV increased 73 As III accumulation in human RBCs. Near-edge X-ray absorption spectroscopy revealed the formation of [(GS) 2 AsSe] - in human RBCs exposed to both As III and Se IV . The sequestration of [(GS) 2 AsSe] - in human RBCs potentially slows arsenic distribution to susceptible tissues and could reduce arsenic-induced disease., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Hundreds of millions of people worldwide are exposed to unacceptable levels of carcinogenic inorganic arsenic. Animal models have shown that selenium and arsenic are mutually protective through the formation and elimination of the seleno-bis(S-glutathionyl) arsinium ion [(GS) 2 AsSe] - . Consistent with this, human selenium deficiency in arsenic-endemic regions is associated with arsenic-induced disease, leading to the initiation of human selenium supplementation trials. In contrast to the protective effect observed in vivo, in vitro studies have suggested that selenite increases arsenite cellular retention and toxicity. This difference might be explained by the rapid conversion of selenite to selenide in vivo. In the current study, selenite did not protect the human hepatoma (HepG2) cell line against the toxicity of arsenite at equimolar concentrations, however selenide increased the IC 50 by 2.3-fold. Cytotoxicity assays of arsenite + selenite and arsenite + selenide at different molar ratios revealed higher overall mutual antagonism of arsenite + selenide toxicity than arsenite + selenite. Despite this protective effect, in comparison to 75 Se-selenite, HepG2 cells in suspension were at least 3-fold more efficient at accumulating selenium from reduced 75 Se-selenide, and its accumulation was further increased by arsenite. X-ray fluorescence imaging of HepG2 cells also showed that arsenic accumulation, in the presence of selenide, was higher than in the presence of selenite. These results are consistent with a greater intracellular availability of selenide relative to selenite for protection against arsenite, and the formation and retention of a less toxic product, possibly [(GS) 2 AsSe] - ., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Organic sulfoxides, a group of compounds containing the sulfinyl S[double bond, length as m-dash]O group, are widespread in nature, important in health and disease, and used in a variety of applications in the pharmaceutical industry. We have examined the sulfur K-edge X-ray absorption near-edge spectra of a range of different sulfoxides and find that their spectra are remarkably similar. Spectra show an intense absorption peak that is comprised of two transitions; a S 1s → (S-O)σ* and a S 1s → [(S-O)π* + (S-C)σ*] transition. In most cases these are sufficiently close in energy that they are not properly resolved; however for dimethylsulfoxide the separation between these transitions increases in aqueous solution due to hydrogen bonding to the sulfinyl oxygen. We also examined tetrahydrothiophene sulfoxide using both the sulfur and oxygen K-edge. This compound has a mild degree of ring strain at the sulfur atom, which changes the energies of the two transitions so that the S 1s → [(S-O)π* + (S-C)σ*] is below the S 1s → (S-O)σ*. A comparison of the oxygen K-edge X-ray absorption near-edge spectra of tetrahydrothiophene sulfoxide with that of an unhindered sulfoxide shows little change, indicating that the electronic environment of oxygen is very similar., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

Benzeneperoxyseleninic acid has been proposed as the key intermediate in the widely used epoxidation of alkenes with benzeneseleninic acid and hydrogen peroxide. However, it reacts sluggishly with cyclooctene and instead rapidly decomposes in solution to a mixed selenonium-selenonate salt that was identified by X-ray absorption and 77 Se NMR spectroscopy, as well as by single crystal X-ray diffraction. This process includes a selenoxide elimination of the peroxyseleninic acid with liberation of oxygen and additional redox steps. The salt is relatively stable in the solid state, but generates the corresponding selenonic acid in the presence of hydrogen peroxide. The selenonic acid is inert towards cyclooctene on its own; however, rapid epoxidation occurs when hydrogen peroxide is added. This shows that the selenonic acid must first be activated through further oxidation, presumably to the heretofore unknown benzeneperoxyselenonic acid. The latter is the principal oxidant in this epoxidation., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Industrial release of mercury into the local Minamata environment with consequent poisoning of local communities through contaminated fish and shellfish consumption is considered the classic case of environmental mercury poisoning. However, the mercury species in the factory effluent has proved controversial, originally suggested as inorganic, and more recently as methylmercury species. We used newly available methods to re-examine the cerebellum of historic Cat 717, which was fed factory effluent mixed with food to confirm the source. Synchrotron high-energy-resolution fluorescence detection-X-ray absorption spectroscopy revealed sulfur-bound organometallic mercury with a minor β-HgS phase. Density functional theory indicated energetic preference for α-mercuri-acetaldehyde as a waste product of aldehyde production. The consequences of this alternative species in the "classic" mercury poisoning should be re-evaluated.

Selenoenzymes, containing a selenocysteine (Sec) residue, fulfill important roles in biology. The mammalian thioredoxin reductase selenoenzymes are key regulators of antioxidant defense and redox signaling and are inhibited by methylmercury species and by the gold-containing drug auranofin. It has been proposed that such inhibition is mediated by metal binding to Sec in the enzyme. However, direct structural observations of these classes of inhibitors binding to selenoenzymes have been few to date. Here we therefore have used extended X-ray absorption fine structure as a direct structural probe to investigate binding to the selenium site in recombinant rat thioredoxin reductase 1 (TrxR1). The results demonstrate for the first time the direct and complete binding of the metal atom of the inhibitors to the selenium atom in TrxR1 for both methylmercury and auranofin, indicating that TrxR1 inhibition indeed can be attributed to such direct metal-selenium binding.

A new energy dispersive X-ray absorption spectroscopy (EDXAS) method is presented for simultaneous wide-field imaging and transmission X-ray absorption spectroscopy (XAS) to enable rapid imaging and speciation of elements. Based on spectral K-Edge Subtraction imaging (sKES), a bent Laue imaging system diffracting in the vertical plane was developed on a bend magnet beamline for selenium speciation. The high flux and small vertical focus, forming a wide horizontal line beam for projection imaging and computed tomography applications, is achieved by precise matching of lattice plane orientation and crystal surface (asymmetry angle). The condition generating a small vertical focus for imaging also provides good energy dispersion. Details for achieving sufficient energy and spatial resolution are demonstrated for both full field imaging and computed tomography in quantifying selenium chemical species. While this system has lower sensitivity as it uses transmission and may lack the flux and spatial resolution of a dedicated focused beamline system, it has significant potential in rapid screening of heterogeneous biomedical or environmental systems to correlate metal speciation with function.

Alzheimer's disease (AD) is the main cause of age-related dementia and currently affects approximately 5.7 million Americans. Major brain changes associated with AD pathology include accumulation of amyloid beta (Aβ) protein fragments and formation of extracellular amyloid plaques. Redox-active metals mediate oligomerization of Aβ, and the resultant metal-bound oligomers have been implicated in the putative formation of harmful, reactive species that could contribute to observed oxidative damage. In isolated plaque cores, Cu(II) is bound to Aβ via histidine residues. Despite numerous structural studies of Cu(II) binding to synthetic Aβ in vitro, there is still uncertainty surrounding Cu(II) coordination in Aβ. In this study, we used X-ray absorption spectroscopy (XAS) and high energy resolution fluorescence detected (HERFD) XAS to investigate Cu(II) coordination in Aβ(1-42) under various solution conditions. We found that the average coordination environment in Cu(II)Aβ(1-42) is sensitive to X-ray photoreduction, changes in buffer composition, peptide concentration, and solution pH. Fitting of the extended X-ray absorption fine structure (EXAFS) suggests Cu(II) is bound in a mixture of coordination environments in monomeric Aβ(1-42) under all conditions studied. However, it was evident that on average only a single histidine residue coordinates Cu(II) in monomeric Aβ(1-42) at pH 6.1, in addition to 3 other oxygen or nitrogen ligands. Cu(II) coordination in Aβ(1-42) at pH 7.4 is similarly 4-coordinate with oxygen and nitrogen ligands, although an average of 2 histidine residues appear to coordinate at this pH. At pH 9.0, the average Cu(II) coordination environment in Aβ(1-42) appears to be 5-coordinate with oxygen and nitrogen ligands, including two histidine residues.

Aryl and mixed aryl-alkyl organic sulfides are important species in a variety of fields, including the drug and food industries. They also are present in fossil fuels, where they contribute to the range of sulfur compounds that must be removed by the fuel industry. We have used sulfur K-edge X-ray absorption spectroscopy, in combination with density functional theory calculations, to study the aryl sulfide diphenyl sulfide and two different aryl-alkyl sulfides. The sulfur K near-edge X-ray absorption spectra are strongly affected by the coordination of the phenyl ring and are distinct from spectra of the alkyl sulfides. For diphenyl sulfide the spectra are predicted to be sensitive to rotation about the S-C bonds, with experimental spectra corresponding to a sum of thermally accessible conformations. We also have investigated the vapor-phase spectrum of diphenyl sulfide, which is found to be very similar to that of toluene solutions of the compound.

Mercury is one of the most toxic elements threatening the biosphere, with levels steadily rising due to both natural and human activities. Selenium is an essential micronutrient, required for normal development and functioning of many organisms. While selenium is known to counteract mercury's toxicity under some conditions, to date information about the mercury-selenium relationship is fragmented and often controversial. As part of a systematic study of mercury and selenium interactions, zebrafish (Danio rerio) larvae (a model verterbrate) were exposed to methylmercury chloride or mercuric chloride. The influence of pre- and post-treatment of selenomethionine on the level and distribution of mercury and selenium in the brain and eye sections, as well as on toxicity, were examined. Selenomethionine treatment decreased the amount of maternally transfered mercury in the larval brain. Selenomethionine treatment prior to exposure to mercuric chloride increased both mercury and selenium levels in the brain but decreased their toxic effects. Conversely, methylmercury levels were not changed as a result of selenium pre-treatment, while toxicity was increased. Strikingly, both forms of mercury severely disrupted selenium metabolism, not only by depleting selenium levels due to formation of Hg-Se complexes, but also by blocking selenium transport into and out of tissues, suggesting that restoring normal selenium levels by treating the organism with selenium after mercury exposure may not be possible. Disruption of selenium metabolism by mercury may lead to disruption in function of selenoproteins. Indeed, the production of thyroid hormones by selenoprotein deiodinases was found to be severely impaired as a result of mercury exposure, with selenomethionine not always being a suitable source of selenium to restore thyroid hormone levels.

A unique combination of sensitivity, resolution, and penetration make X-ray fluorescence imaging (XFI) ideally suited to investigate trace elemental distributions in the biological context. XFI has gained widespread use as an analytical technique in the biological sciences, and in particular enables exciting new avenues of research in the field of neuroscience. In this study, elemental mapping by XFI was applied to characterise the elemental content within neuronal cell layers of hippocampal sub-regions of mice and rats. Although classical histochemical methods for metal detection exist, such approaches are typically limited to qualitative analysis. Specifically, histochemical methods are not uniformly sensitive to all chemical forms of a metal, often displaying variable sensitivity to specific "pools" or chemical forms of a metal. In addition, histochemical methods require fixation and extensive chemical treatment of samples, creating the strong likelihood for metal redistribution, leaching, or contamination. Direct quantitative elemental mapping of total elemental pools, in situ within ex vivo tissue sections, without the need for chemical fixation or addition of staining reagents is not possible with traditional histochemical methods; however, such a capability, which is provided by XFI, can offer an enormous analytical advantage. The results we report herein demonstrate the analytical advantage of XFI elemental mapping for direct, label-free metal quantification, in situ within ex vivo brain tissue sections. Specifically, we definitively characterise for the first time, the abundance of Fe within the pyramidal cell layers of the hippocampus. Localisation of Fe to this cell layer is not reproducibly achieved with classical Perls histochemical Fe stains. The ability of XFI to directly quantify neuronal elemental (P, S, Cl, K, Ca, Fe, Cu, Zn) distributions, revealed unique profiles of Fe and Zn within anatomical sub-regions of the hippocampus i.e., cornu ammonis 1, 2 or 3 (CA1, CA2 or CA3) sub-regions. Interestingly, our study reveals a unique Fe gradient across neuron populations within the non-degenerating and pathology free rat hippocampus, which curiously mirrors the pattern of region-specific vulnerability of the hippocampus that has previously been established to occur in various neurodegenerative diseases.

Synchrotron X-ray fluorescence imaging enables visualization and quantification of microscopic distributions of elements. This versatile technique has matured to the point where it is used in a wide range of research fields. The method can be used to quantitate the levels of different elements in the image on a pixel-by-pixel basis. Two approaches to X-ray fluorescence image analysis are commonly used, namely, (i) integrative analysis, or window binning, which simply sums the numbers of all photons detected within a specific energy region of interest; and (ii) parametric analysis, or fitting, in which emission spectra are represented by the sum of parameters representing a series of peaks and other contributing factors. This paper presents a quantitative comparison between these two methods of image analysis using X-ray fluorescence imaging of mouse brain-tissue sections; it is shown that substantial errors can result when data from overlapping emission lines are binned rather than fitted. These differences are explored using two different digital signal processing data-acquisition systems with different count-rate and emission-line resolution characteristics. Irrespective of the digital signal processing electronics, there are substantial differences in quantitation between the two approaches. Binning analyses are thus shown to contain significant errors that not only distort the data but in some cases result in complete reversal of trends between different tissue regions.

Background: Selenium is an essential element with a rich and varied chemistry in living organisms. It plays a variety of important roles ranging from being essential in enzymes that are critical for redox homeostasis to acting as a deterrent for herbivory in hyperaccumulating plants. Despite its importance there are many open questions, especially related to its chemistry in situ within living organisms., Scope of Review: This review discusses X-ray spectroscopy and imaging of selenium in biological samples, with an emphasis on the methods, and in particular the techniques of X-ray absorption spectroscopy (XAS) and X-ray fluorescence imaging (XFI). We discuss the experimental methods and capabilities of XAS and XFI, and review their advantages and their limitations. A perspective on future possibilities and next-generation of experiments is also provided., Major Conclusions: XAS and XFI provide powerful probes of selenium chemistry, together with unique in situ capabilities. The opportunities and capabilities of the next generation of advanced X-ray spectroscopy experiments are particularly exciting., General Significance: XAS and XFI provide versatile tools that are generally applicable to any element with a convenient X-ray absorption edge, suitable for investigating complex systems essentially without pre-treatment., (Copyright © 2018. Published by Elsevier B.V.)

Stereoisomers of 5-(2-allylsulfinyl)-3,4-dimethylthiolane-2-ol, a family of 3,4-dimethylthiolanes of formula C 9 H 16 O 2 S 2 we name ajothiolanes, were isolated from garlic ( Allium sativum) macerates and characterized by a variety of analytical and spectroscopic techniques, including ultraperformance liquid chromatography (UPLC), direct analysis in real time-mass spectrometry (DART-MS), and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Ajothiolanes were found to be spectroscopically identical to a family of previously described compounds named garlicnins B 1-4 (C 9 H 16 O 2 S 2 ), whose structures we demonstrate have been misassigned. 2D 13 C- 13 C NMR incredible natural abundance double quantum transfer experiments (INADEQUATE) were used to disprove the claim of nine contiguous carbons in these compounds, while X-ray absorption spectroscopy (XAS) along with computational modeling was used to disprove the claim that these compounds were thiolanesulfenic acids. On the basis of the similarity of their NMR spectra to those of the ajothiolanes, we propose that the structures of previously described, biologically active onionins A 1-3 (C 9 H 16 O 2 S 2 ), from extracts of onion ( Allium cepa) and Allium fistulosum, and garlicnin A (C 12 H 20 O 2 S 4 ), from garlic extracts, should also be reassigned, in each case as isomeric mixtures of 5-substituted-3,4-dimethylthiolane-2-ols. We conclude that 3,4-dimethylthiolanes may be a common motif in Allium chemistry. Finally, we show that another garlic extract component, garlicnin D (C 7 H 12 O 2 S 3 ), claimed to have an unprecedented structure, is in fact a known compound from garlic with a structure different from that proposed, namely, 2( E)-3-(methylsulfinyl)-2-propenyl 2-propenyl disulfide.

Selenium-based selenyl free radicals are chemical entities that may be involved in a range of biochemical processes. We report the first X-ray spectroscopic observation of a selenyl radical species generated photochemically by X-ray irradiation of low-temperature solutions of l-selenocysteine. We have employed high energy resolution fluorescence detected X-ray absorption spectroscopy (HERFD-XAS) and electron paramagnetic resonance (EPR) spectroscopy, coupled with density functional theory calculations, to characterize and understand the species. The HERFD-XAS spectrum of the selenyl radical is distinguished by a uniquely low-energy transition with a peak energy at 12 659.0 eV, which corresponds to a 1s → 4p transition to the singly occupied molecular orbital of the free radical. The EPR spectrum shows the broad features and highly anisotropic g-values that are expected for a selenium free radical species. The availability of spectroscopic probes for selenyl radicals may assist in understanding why life chooses selenium over sulfur in selected biochemical processes.

We use X-ray-induced photochemistry, which is well known to cause changes in a number of systems, to reduce Hg(II) to Hg(0) in frozen aqueous solution with added glycerol maintained at 10 K. X-ray absorption spectroscopy was used to monitor the extent of the reaction and to characterize the species. An analysis of the extended X-ray absorption fine structure (EXAFS) of the photochemical product indicated a nearly monatomic Hg(0) species bound only by long, weak bonds to oxygens at ∼3.5 Å. The results of the EXAFS analysis agree quantitatively with the results of density functional theory calculations using the meta-GGA approximation with the M11-L functional. This is the first structural characterization of nearly monatomic Hg(0) bound by hard ligands similar to those expected in aqueous environmental systems. We conclude that Hg(0) is expected to exist in solution as a nearly monatomic entity.

Approximately 11% of enzymes contain a transition metal ion that is essential for catalytic function. Such metalloenzymes catalyze much of the most chemically challenging and biologically essential chemistry carried out by life. X-ray-based methods, predominantly macromolecular crystallography (MX) and also X-ray absorption spectroscopy (XAS), have proved essential for determining structures of transition metal ion-containing active sites in order to deduce enzyme catalytic mechanisms. However, X-ray irradiation can induce change in both the oxidation state and structure of the metal, which is problematic in structure determination. We present an XAS study of whether cryoprotectants such as polyethylene glycol (PEG) or glycerol, routinely added to MX or XAS samples to improve data quality, affect photoreduction. Our data demonstrate a remarkable 10-fold exacerbation in rate of photoreduction of Cu(II) to Cu(I) when alcohol or ether cryoprotectants are present. Our results suggest that widespread use of cryoprotectants may increase the potential for erroneous structures.

Arsenicosis, a syndrome caused by ingestion of arsenic contaminated drinking water, currently affects millions of people in South-East Asia and elsewhere. Previous animal studies revealed that the toxicity of arsenite essentially can be abolished if selenium is co-administered as selenite. Although subsequent studies have provided some insight into the biomolecular basis of this striking antagonism, many details of the biochemical pathways that ultimately result in the detoxification and excretion of arsenic using selenium supplements have yet to be thoroughly studied. To this end and in conjunction with the recent Phase III clinical trial "Selenium in the Treatment of Arsenic Toxicity and Cancers", we have applied synchrotron X-ray techniques to elucidate the mechanisms of this arsenic-selenium antagonism at the tissue and organ levels using an animal model. X-ray fluorescence imaging (XFI) of cryo-dried whole-body sections of laboratory hamsters that had been injected with arsenite, selenite, or both chemical species, provided insight into the distribution of both metalloids 30 minutes after treatment. Co-treated animals showed strong co-localization of arsenic and selenium in the liver, gall bladder and small intestine. X-ray absorption spectroscopy (XAS) of freshly frozen organs of co-treated animals revealed the presence in liver tissues of the seleno bis-(S-glutathionyl) arsinium ion, which was rapidly excreted via bile into the intestinal tract. These results firmly support the previously postulated hepatobiliary excretion of the seleno bis-(S-glutathionyl) arsinium ion by providing the first data pertaining to organs of whole animals.

Organic sulfides, sometimes called thioethers, are important in a variety of materials with diverse roles in biology and the environment. They also contribute a significant proportion of the sulfur in fossil fuels. We have studied a range of aliphatic sulfides using a combination of sulfur K-edge X-ray absorption spectroscopy and density functional theory calculations. We show that the sulfur K-edge near-edge X-ray absorption spectra of aliphatic organic sulfides comprise two intense transitions in the near-edge spectrum, which can be assigned as 1s → (S-C)σ* and 1s → (S-C)π* transitions. These transitions are found to change in a systematic manner in sterically hindered sulfides composed of four-, five- and six-membered rings. Both the 1s → (S-C)σ* and 1s → (S-C)π* transitions are sensitive to the presence of strain in the C-S-C angle, shifting to lower values with more strained ring systems. Steric effects can give obtuse C-S-C angles, which are predicted to cause the two transitions to converge to the same energy and even cross over at very obtuse angles.

Sulfur-based thiyl radicals are known to be involved in a wide range of chemical and biological processes, but they are often highly reactive, which makes them difficult to observe directly. We report herein X-ray absorption spectra and analysis that support the direct observation of two different thiyl species generated photochemically by X-ray irradiation. The thiyl radical sulfur K-edge X-ray absorption spectra of both species are characterized by a uniquely low energy transition at about 2465 eV, which occurs at a lower energy than any previously observed feature at the sulfur K-edge and corresponds to a 1s→3p transition to the singly occupied molecular orbital of the free radical. Our results constitute the first observation of substantial levels of thiyl radicals generated by X-ray irradiation and detected by sulfur K-edge X-ray absorption spectroscopy.

Alzheimer's disease (AD) is a major international health and economic concern. A key pathological feature of AD is so-called "amyloid-β-plaques", or "Aβ-plaques", which are deposits of aggregated protein, enriched with the Aβ fragment of amyloid precursor protein. Despite their name, the deposits are not pure Aβ and have a heterogeneous, chemically complex composition that can include multiple proteins, lipids, and metal ions (Fe, Cu, or Zn). Despite extensive research, it is still uncertain whether Aβ-plaques are a cause or a consequence of AD pathology. Further characterization of the elemental and biochemical composition within and surrounding Aβ-plaques, and knowledge of how composition varies with disease state or progression, may provide important insight into the relationship between Aβ-plaques and AD pathology. With this aim in mind, herein we demonstrate a multimodal spectroscopic imaging workflow to better characterize the complex composition of Aβ-plaques. Our approach incorporates several spectroscopic imaging techniques, such as Fourier transform infrared spectroscopic imaging (FTIR), Raman microscopy, and X-ray fluorescence microscopy (XFM). While FTIR, Raman, and XFM have been used previously, mostly in isolation, to study Aβ-plaques, application of all three techniques, in combination with histology and fluorescence microscopy, has not been reported previously. We demonstrate that a multimodal workflow, incorporating all three methods on adjacent or serial tissue sections, can reveal substantial complementary information about the biochemical and elemental composition of Aβ-plaques. Information revealed by the method includes the relative content and distribution of aggregated protein, total lipid, lipid esters, cholesterol, and metals (Fe, Cu, or Zn).

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system (CNS) in which oligodendrocytes, the CNS cells that stain most robustly for iron and myelin are the targets of injury. Metals are essential for normal CNS functioning, and metal imbalances have been linked to demyelination and neurodegeneration. Using a multidisciplinary approach involving synchrotron techniques, iron histochemistry and immunohistochemistry, we compared the distribution and quantification of iron and zinc in MS lesions to the surrounding normal appearing and periplaque white matter, and assessed the involvement of these metals in MS lesion pathogenesis. We found that the distribution of iron and zinc is heterogeneous in MS plaques, and with few remarkable exceptions they do not accumulate in chronic MS lesions. We show that brain iron tends to decrease with increasing age and disease duration of MS patients; reactive astrocytes organized in large astrogliotic areas in a subset of smoldering and inactive plaques accumulate iron and safely store it in ferritin; a subset of smoldering lesions do not contain a rim of iron-loaded macrophages/microglia; and the iron content of shadow plaques varies with the stage of remyelination. Zinc in MS lesions was generally decreased, paralleling myelin loss. Iron accumulates concentrically in a subset of chronic inactive lesions suggesting that not all iron rims around MS lesions equate with smoldering plaques. Upon degeneration of iron-loaded microglia/macrophages, astrocytes may form an additional protective barrier that may prevent iron-induced oxidative damage.

Copper is an essential nutrient required for many biological processes involved in primary metabolism, but free copper is toxic due to its ability to catalyze formation of free radicals. To prevent toxic effects, in the cell copper is bound to proteins and low molecular weight compounds, such as glutathione, at all times. The widely used chemotherapy agent cisplatin is known to bind to copper-transporting proteins, including copper chaperone Atox1. Cisplatin interactions with Atox1 and other copper transporters are linked to cancer resistance to platinum-based chemotherapy. Here we analyze the binding of copper and cisplatin to Atox1 in the presence of glutathione under redox conditions that mimic intracellular environment. We show that copper(I) and glutathione form large polymers with a molecular mass of approximately 8 kDa, which can transfer copper to Atox1. Cisplatin also can form polymers with glutathione, albeit at a slower rate. Analysis of simultaneous binding of copper and cisplatin to Atox1 under physiological conditions shows that both metals are bound to the protein through copper-sulfur-platinum bridges.

A method to image taurine distributions within the central nervous system and other organs has long been sought. Since taurine is small and mobile, it cannot be chemically "tagged" and imaged using conventional immuno-histochemistry methods. Combining numerous indirect measurements, taurine is known to play critical roles in brain function during health and disease and is proposed to act as a neuro-osmolyte, neuro-modulator, and possibly a neuro-transmitter. Elucidation of taurine's neurochemical roles and importance would be substantially enhanced by a direct method to visualize alterations, due to physiological and pathological events in the brain, in the local concentration of taurine at or near cellular spatial resolution in vivo or in situ in tissue sections. We thus have developed chemically specific X-ray fluorescence imaging (XFI) at the sulfur K-edge to image the sulfonate group in taurine in situ in ex vivo tissue sections. To our knowledge, this represents the first undistorted imaging of taurine distribution in brain at 20 μm resolution. We report quantitative technique validation by imaging taurine in the cerebellum and hippocampus regions of the rat brain. Further, we apply the technique to image taurine loss from the vulnerable CA1 (cornus ammonis 1) sector of the rat hippocampus following global brain ischemia. The location-specific loss of taurine from CA1 but not CA3 neurons following ischemia reveals osmotic stress may be a key factor in delayed neurodegeneration after a cerebral ischemic insult and highlights the significant potential of chemically specific XFI to study the role of taurine in brain disease.

Numerous in vivo studies have shown that the severe toxic side-effects of intravenously administered cisplatin can be significantly reduced by the co-administration of sulfur-containing 'chemoprotective agents'. Using a metallomics approach, a likely biochemical basis for these potentially useful observations was only recently uncovered and appears to involve the reaction of chemoprotective agents with cisplatin-derived Pt-species in human plasma to form novel platinum-sulfur complexes (PSC's). We here reveal aspects of the structure of two PSC's and establish the identification of an optimal chemoprotective agent to ameliorate the toxic side-effects of cisplatin, while leaving its antineoplastic activity largely intact, as a feasible research strategy to transform cisplatin into a safer and more effective anticancer drug.

Selenium (Se) is an element of growing environmental concern, because low aqueous concentrations can lead to biomagnification through the aquatic food web. Biofilms, naturally occurring microbial consortia, play numerous important roles in the environment, especially in biogeochemical cycling of toxic elements in aquatic systems. The complexity of naturally forming multispecies biofilms presents challenges for characterization because conventional microscopic techniques require chemical and physical modifications of the sample. Here, multispecies biofilms biotransforming selenium oxyanions were characterized using X-ray fluorescence imaging (XFI) and scanning transmission X-ray microscopy (STXM). These complementary synchrotron techniques required minimal sample preparation and were applied correlatively to the same biofilm areas. Sub-micrometer XFI showed distributions of Se and endogenous metals, while Se K-edge X-ray absorption spectroscopy indicated the presence of elemental Se (Se 0 ). Nanoscale carbon K-edge STXM revealed the distributions of microbial cells, extracellular polymeric substances (EPS), and lipids using the protein, saccharide, and lipid signatures, respectively, together with highly localized Se 0 using the Se L III edge. Transmission electron microscopy showed the electron-dense particle diameter to be 50-700 nm, suggesting Se 0 nanoparticles. The intimate association of Se 0 particles with protein and polysaccharide biofilm components has implications for the bioavailability of selenium in the environment.

Sulfur K-edge X-ray absorption spectroscopy increasingly is used as a tool to provide speciation information about the sulfur chemical form in complex samples, with applications ranging from fossil fuels to soil science to health research. As part of an ongoing program of systematic investigations of the factors that affect the variability of sulfur K near-edge spectra, we have examined the X-ray absorption spectra of a series of organic symmetric disulfide compounds. We have used polarized sulfur K-edge spectra of single crystals of dibenzyl disulfide to confirm the assignments of the major transitions in the spectrum as 1s → (S-S)σ* and 1s → (S-C)σ*. We also have examined the solution spectra of an extended series of disulfides and show that the spectra change in a systematic and predictable manner with the nature of the external group.