Chemical Biology in the Embryo: <i>In Situ</i> Imaging of Sulfur Biochemistry in Normal and Proteoglycan-Deficient Cartilage Matrix

Proteoglycans (PGs) are heavily glycosylated proteins that play major structural and biological roles in many tissues. Proteoglycans are abundant in cartilage extracellular matrix; their loss is a main feature of the joint disease osteoarthritis. Proteoglycan function is regulated by sulfation–sulfate ester formation with specific sugar residues. Visualization of sulfation within cartilage matrix would yield vital insights into its biological roles. We present synchrotron-based X-ray fluorescence imaging of developing zebrafish cartilage, providing the first <i>in situ</i> maps of sulfate ester distribution. Levels of both sulfur and sulfate esters decrease as cartilage develops through late phase differentiation (maturation or hypertrophy), suggesting a functional link between cartilage matrix sulfur content and chondrocyte differentiation. Genetic experiments confirm that sulfate ester levels were due to cartilage proteoglycans and support the hypothesis that sulfate ester levels regulate chondrocyte differentiation. Surprisingly, in the PG synthesis mutant, the total level of sulfur was not significantly reduced, suggesting sulfur is distributed in an alternative chemical form during lowered cartilage proteoglycan production. Fourier transform infrared imaging indicated increased levels of protein in the mutant fish, suggesting that this alternative sulfur form might be ascribed to an increased level of protein synthesis in the mutant fish, as part of a compensatory mechanism

X‑ray Absorption Spectroscopy of Aliphatic Organic Sulfides

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

Bioavailability, Toxicity and Biotransformation of Selenium in Midge (<i>Chironomus dilutus</i>) Larvae Exposed via Water or Diet to Elemental Selenium Particles, Selenite, or Selenized Algae

Elemental selenium (Se) is generally considered to be biologically inert due to its insolubility in water. It is a common form of Se in sediment near uranium mining and milling operations in northern Saskatchewan, Canada. Nanosized particles of many materials exhibit different properties compared with their bulk phases, in some cases posing health and ecological risks. Here we investigated the bioavailability and toxicity of Se nanoparticles (SeNPs) using 10-day waterborne and dietary exposures to larvae of <i>Chironomus dilutus</i>, a common benthic invertebrate. For comparison, larvae were also exposed to waterborne dissolved selenite and to dietary selenomethionine as selenized algae. Larval Se accumulation was evaluated using graphite furnace atomic absorption spectroscopy or inductively coupled plasma mass spectroscopy for total Se and X-ray absorption spectroscopy for Se chemical speciation. Exposure to nanoparticulate Se resulted in Se bioaccumulation, at high concentrations, inhibiting larval growth in both waterborne and dietary exposures; larvae predominantly accumulated selenomethionine-like species regardless of uptake route or form of Se tested. Despite the observed Se accumulation, our findings suggest there is little risk of direct SeNP toxicity to benthic invertebrates in Se-contaminated sediments in northern Saskatchewan. Nevertheless, elemental Se in sediments may be biologically available and may contribute directly or indirectly to the risk of Se toxicity to egg-laying vertebrates (fish and piscivorous birds) in Se-contaminated aquatic systems. It thus may be necessary to include elemental Se as a source of potential Se exposure in ecological risk assessments

Chemical Form Matters: Differential Accumulation of Mercury Following Inorganic and Organic Mercury Exposures in Zebrafish Larvae

Mercury, one of the most toxic elements, exists in various chemical forms each with different toxicities and health implications. Some methylated mercury forms, one of which exists in fish and other seafood products, pose a potential threat, especially during embryonic and early postnatal development. Despite global concerns, little is known about the mechanisms underlying transport and toxicity of different mercury species. To investigate the impact of different mercury chemical forms on vertebrate development, we have successfully combined the zebrafish, a well-established developmental biology model system, with synchrotron-based X-ray fluorescence imaging. Our work revealed substantial differences in tissue-specific accumulation patterns of mercury in zebrafish larvae exposed to four different mercury formulations in water. Methylmercury species not only resulted in overall higher mercury burdens but also targeted different cells and tissues than their inorganic counterparts, thus revealing a significant role of speciation in cellular and molecular targeting and mercury sequestration. For methylmercury species, the highest mercury concentrations were in the eye lens epithelial cells, independent of the formulation ligand (chloride <i>versus</i> l-cysteine). For inorganic mercury species, in absence of l-cysteine, the olfactory epithelium and kidney accumulated the greatest amounts of mercury. However, with l-cysteine present in the treatment solution, mercuric bis-l-cysteineate species dominated the treatment, significantly decreasing uptake. Our results clearly demonstrate that the common differentiation between organic and inorganic mercury is not sufficient to determine the toxicity of various mercury species

Insights into the Nature of the Chemical Bonding in Thiophene-2-thiol from X‑ray Absorption Spectroscopy

Thiophenes are the simplest aromatic sulfur-containing compounds; they are widespread in fossil fuels and a variety of natural products, and they have vital roles in determining characteristic aromas that are important in food chemistry. We used a combination of sulfur K-edge X-ray absorption spectroscopy and density functional theory to investigate the chemical bonding in the novel sulfur-containing heterocycle thiophene-2-thiol. We show that solutions of thiophene-2-thiol contain significant quantities of the thione tautomer, which may be the energetically preferred 5<i>H</i>-thiophene-2-thione or the more accessible 3<i>H</i>-thiophene-2-thione

Multispecies Biofilms Transform Selenium Oxyanions into Elemental Selenium Particles: Studies Using Combined Synchrotron X‑ray Fluorescence Imaging and Scanning Transmission X‑ray Microscopy

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 (Se⁰). 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 Se⁰ using the Se L_III edge. Transmission electron microscopy showed the electron-dense particle diameter to be 50–700 nm, suggesting Se⁰ nanoparticles. The intimate association of Se⁰ particles with protein and polysaccharide biofilm components has implications for the bioavailability of selenium in the environment

Radiochemical, Computational, and Spectroscopic Evaluation of High-Denticity Desferrioxamine Derivatives DFO2 and DFO2p toward an Ideal Zirconium-89 Chelate Platform

Desferrioxamine (DFO) has long been considered the gold standard chelator for incorporating [89Zr]Zr4+ 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-NO2-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

Quantification, Localization, and Speciation of Selenium in Seeds of Canola and Two Mustard Species Compared to Seed-Meals Produced by Hydraulic Press

<i>Brassica</i> plants accumulate selenium (Se) especially in seeds when grown in soils laden with Se. We report a chemical analysis of Se in <i>Brassica</i> seeds (canola, Indian mustard, and white mustard) and in their hydraulically pressed seed meals, which are used as a Se supplement in livestock animal feeds. Complementary techniques were used to measure total Se concentrations, to map the localization of Se, and to quantify different Se forms. Seeds and hydraulically pressed seed meals contained an average of 1.8 and 2.0 μg Se g^–1 DW, respectively. Selenium was primarily located in cotyledons and roots of seed embryos. Microfocused Se K-edge XANES and bulk XANES showed that seeds contained 90% of Se as C–Se–C forms. Hydraulically pressing seeds for oil caused changes in the forms of Se as follows: 40–55% C–Se–C forms, 33–42% selenocystine, 5–12% selenocysteine, and 11–14% trimethylselenonium ion. Aqueous extracts of seed and seed meals were also analyzed by SAX-HPLC/ICPMS and found to contain mainly the C–Se–C form SeMet, but also another C–Se–C form MeSeCys, which is of dietary pharmacological interest for cancer inhibition. In addition, SAX-HPLC/ICPMS also detected selenocystine and selenocysteine, further confirming the results obtained by XANES analyses

Subcellular Biochemical Investigation of Purkinje Neurons Using Synchrotron Radiation Fourier Transform Infrared Spectroscopic Imaging with a Focal Plane Array Detector

Coupling Fourier transform infrared spectroscopy with focal plane array detectors at synchrotron radiation sources (SR-FTIR-FPA) has provided a rapid method to simultaneously image numerous biochemical markers in situ at diffraction limited resolution. Since cells and nuclei are well resolved at this spatial resolution, a direct comparison can be made between FTIR functional group images and the histology of the same section. To allow histological analysis of the same section analyzed with infrared imaging, unfixed air-dried tissue sections are typically fixed (after infrared spectroscopic analysis is completed) via immersion fixation. This post fixation process is essential to allow histological staining of the tissue section. Although immersion fixation is a common practice in this filed, the initial rehydration of the dehydrated unfixed tissue can result in distortion of subcellular morphology and confound correlation between infrared images and histology. In this study, vapor fixation, a common choice in other research fields where postfixation of unfixed tissue sections is required, was employed in place of immersion fixation post spectroscopic analysis. This method provided more accurate histology with reduced distortions as the dehydrated tissue section is fixed in vapor rather than during rehydration in an aqueous fixation medium. With this approach, accurate correlation between infrared images and histology of the same section revealed that Purkinje neurons in the cerebellum are rich in cytosolic proteins and not depleted as once thought. In addition, we provide the first direct evidence of intracellular lactate within Purkinje neurons. This highlights the significant potential for future applications of SR-FTIR-FPA imaging to investigate cellular lactate under conditions of altered metabolic demand such as increased brain activity and hypoxia or ischemia

Selenium Biotransformations in an Engineered Aquatic Ecosystem for Bioremediation of Agricultural Wastewater via Brine Shrimp Production

An engineered aquatic ecosystem was specifically designed to bioremediate selenium (Se), occurring as oxidized inorganic selenate from hypersalinized agricultural drainage water while producing brine shrimp enriched in organic Se and omega-3 and omega-6 fatty acids for use in value added nutraceutical food supplements. Selenate was successfully bioremediated by microalgal metabolism into organic Se (seleno-amino acids) and partially removed via gaseous volatile Se formation. Furthermore, filter-feeding brine shrimp that accumulated this organic Se were removed by net harvest. Thriving in this engineered pond system, brine shrimp (Artemia franciscana Kellogg) and brine fly (Ephydridae sp.) have major ecological relevance as important food sources for large populations of waterfowl, breeding, and migratory shore birds. This aquatic ecosystem was an ideal model for study because it mimics trophic interactions in a Se polluted wetland. Inorganic selenate in drainage water was metabolized differently in microalgae, bacteria, and diatoms where it was accumulated and reduced into various inorganic forms (selenite, selenide, or elemental Se) or partially incorporated into organic Se mainly as selenomethionine. Brine shrimp and brine fly larva then bioaccumulated Se from ingesting aquatic microorganisms and further metabolized Se predominately into organic Se forms. Importantly, adult brine flies, which hatched from aquatic larva, bioaccumulated the highest Se concentrations of all organisms tested