The mitochondrial Cu+ transporter PiC2 (SLC25A3) is a target of MTF1 and contributes to the development of skeletal muscle in vitro.

The loading of copper (Cu) into cytochrome c oxidase (COX) in mitochondria is essential for energy production in cells. Extensive studies have been performed to characterize mitochondrial cuproenzymes that contribute to the metallation of COX, such as Sco1, Sco2, and Cox17. However, limited information is available on the upstream mechanism of Cu transport and delivery to mitochondria, especially through Cu-impermeable membranes, in mammalian cells. The mitochondrial phosphate transporter SLC25A3, also known as PiC2, binds Cu+ and transports the ion through these membranes in eukaryotic cells, ultimately aiding in the metallation of COX. We used the well-established differentiation model of primary myoblasts derived from mouse satellite cells, wherein Cu availability is necessary for growth and maturation, and showed that PiC2 is a target of MTF1, and its expression is both induced during myogenesis and favored by Cu supplementation. PiC2 deletion using CRISPR/Cas9 showed that the transporter is required for proliferation and differentiation of primary myoblasts, as both processes are delayed upon PiC2 knock-out. The effects of PiC2 deletion were rescued by the addition of Cu to the growth medium, implying the deleterious effects of PiC2 knockout in myoblasts may be in part due to a failure to deliver sufficient Cu to the mitochondria, which can be compensated by other mitochondrial cuproproteins. Co-localization and co-immunoprecipitation of PiC2 and COX also suggest that PiC2 may participate upstream in the copper delivery chain into COX, as verified by in vitro Cu+-transfer experiments. These data indicate an important role for PiC2 in both the delivery of Cu to the mitochondria and COX, favoring the differentiation of primary myoblasts.

Subretinal Pigment Epithelial Deposition of Drusen Components Including Hydroxyapatite in a Primary Cell Culture Model.

Purpose: Extracellular deposits containing hydroxyapatite, lipids, proteins, and trace metals that form between the basal lamina of the RPE and the inner collagenous layer of Bruch's membrane are hallmarks of early AMD. We examined whether cultured RPE cells could produce extracellular deposits containing all of these molecular components. Methods: Retinal pigment epithelium cells isolated from freshly enucleated porcine eyes were cultured on Transwell membranes for up to 6 months. Deposit composition and structure were characterized using light, fluorescence, and electron microscopy; synchrotron x-ray diffraction and x-ray fluorescence; secondary ion mass spectroscopy; and immunohistochemistry. Results: Apparently functional primary RPE cells, when cultured on 10-µm-thick inserts with 0.4-µm-diameter pores, can produce sub-RPE deposits that contain hydroxyapatite, lipids, proteins, and trace elements, without outer segment supplementation, by 12 weeks. Conclusions: The data suggest that sub-RPE deposit formation is initiated, and probably regulated, by the RPE, as well as the loss of permeability of the Bruch's membrane and choriocapillaris complex associated with age and early AMD. This cell culture model of early AMD lesions provides a novel system for testing new therapeutic interventions against sub-RPE deposit formation, an event occurring well in advance of the onset of vision loss.

Spectroscopic evidence of uranium immobilization in acidic wetlands by natural organic matter and plant roots.

Biogeochemistry of uranium in wetlands plays important roles in U immobilization in storage ponds of U mining and processing facilities but has not been well understood. The objective of this work was to study molecular mechanisms responsible for high U retention by Savannah River Site (SRS) wetland sediments under varying redox and acidic (pH = 2.6-5.8) conditions using U L3-edge X-ray absorption spectroscopy. Uranium in the SRS wetland sediments existed primarily as U(VI) bonded as a bidentate to carboxylic sites (U-C bond distance at ∼2.88 Å), rather than phenolic or other sites of natural organic matter (NOM). In microcosms simulating the SRS wetland processes, U immobilization on roots was 2 orders of magnitude higher than on the adjacent brown or more distant white sands in which U was U(VI). Uranium on the roots were both U(IV) and U(VI), which were bonded as a bidentate to carbon, but the U(VI) may also form a U phosphate mineral. After 140 days of air exposure, all U(IV) was reoxidized to U(VI) but remained as a bidentate bonding to carbon. This study demonstrated NOM and plant roots can highly immobilize U(VI) in the SRS acidic sediments, which has significant implication for the long-term stewardship of U-contaminated wetlands.

Retention and chemical speciation of uranium in an oxidized wetland sediment from the Savannah River Site.

Uranium speciation and retention mechanisms onto Savannah River Site (SRS) wetland sediments was studied using batch (ad)sorption experiments, sequential extraction, U L3-edge X-ray absorption near-edge structure (XANES) spectroscopy, fluorescence mapping and µ-XANES. Under oxidized conditions, U was highly retained by the SRS wetland sediments. In contrast to other similar but much lower natural organic matter (NOM) sediments, significant sorption of U onto the SRS sediments was observed at pH < 4 and pH > 8. Sequential extraction indicated that the U species were primarily associated with the acid soluble fraction (weak acetic acid extractable) and organic fraction (Na-pyrophosphate extractable). Uranium L3-edge XANES spectra of the U-bound sediments were nearly identical to that of uranyl acetate. Based on fluorescence mapping, U and Fe distributions in the sediment were poorly correlated, U was distributed throughout the sample and did not appear as isolated U mineral phases. The primary oxidation state of U in these oxidized sediments was U(VI), and there was little evidence that the high sorptive capacity of the sediments could be ascribed to abiotic or biotic reduction to the less soluble U(IV) species or to secondary mineral formation. Collectively, this study suggests that U may be strongly bound to wetland sediments, not only under reducing conditions by reductive precipitation, but also under oxidizing conditions through NOM-uranium bonding.

Abiotic reductive immobilization of U(VI) by biogenic mackinawite.

During subsurface bioremediation of uranium-contaminated sites, indigenous metal and sulfate-reducing bacteria may utilize a variety of electron acceptors, including ferric iron and sulfate that could lead to the formation of various biogenic minerals in situ. Sulfides, as well as structural and adsorbed Fe(II) associated with biogenic Fe(II)-sulfide phases, can potentially catalyze abiotic U(VI) reduction via direct electron transfer processes. In the present work, the propensity of biogenic mackinawite (Fe 1+x S, x = 0 to 0.11) to reduce U(VI) abiotically was investigated. The biogenic mackinawite produced by Shewanella putrefaciens strain CN32 was characterized by employing a suite of analytical techniques including TEM, SEM, XAS, and Mössbauer analyses. Nanoscale and bulk analyses (microscopic and spectroscopic techniques, respectively) of biogenic mackinawite after exposure to U(VI) indicate the formation of nanoparticulate UO2. This study suggests the relevance of sulfide-bearing biogenic minerals in mediating abiotic U(VI) reduction, an alternative pathway in addition to direct enzymatic U(VI) reduction.

Real-time X-ray absorption spectroscopy of uranium, iron, and manganese in contaminated sediments during bioreduction.

The oxidation status of uranium in sediments is important because the solubility of this toxic and radioactive element is much greater for U(VI) than for U(IV) species. Thus, redox manipulation to promote precipitation of UO2 is receiving interest as a method to remediate U-contaminated sediments. Presence of Fe and Mn oxides in sediments at much higher concentrations than U requires an understanding of their redox status as well. This study was conducted to determine changes in oxidation states of U, Fe, and Mn in U-contaminated sediments from Oak Ridge National Laboratory. Oxidation states of these elements were measured in real-time and nondestructively using X-ray absorption spectroscopy on sediment columns supplied with synthetic groundwater containing organic carbon (OC, 0, 3, 10, 30, and 100 mM OC as lactate) for over 400 days. In sediments supplied with OC > or = 30 mM, 80% of the U was reduced to U(IV), with transient reoxidation at about 150 days. Mn(III,IV) oxides were completely reduced to Mn(II) in sediments infused with OC > or = 3 mM. However, Fe remained largely unreduced in all sediment columns, showing that Fe(III) can persist as an electron acceptor in reducing sediments over long times. This result in combination with the complete reduction of all other potential electron acceptors supports the hypothesis that the reactive Fe(III) fraction was responsible for reoxidizing U(IV).

Effect of saline waste solution infiltration rates on uranium retention and spatial distribution in Hanford sediments.

The accidental overfilling of waste liquid from tank BX-102 at the Hanford Site in 1951 put about 10 t of U(VI) into the vadose zone. In order to understand the dominant geochemical reactions and transport processes that occurred during the initial infiltration and to help understand current spatial distribution, we simulated the waste liquid spilling event in laboratory sediment columns using synthesized metal waste solution. We found that, as the plume propagated through sediments, pH decreased greatly (as much as 4 units) at the moving plume front. Infiltration flow rates strongly affect U behavior. Slower flow rates resulted in higher sediment-associated U concentrations, and higher flow rates (> or =5 cm/day) permitted practically unretarded U transport. Therefore, given the very high Ksat of most of Hanford formation, the low permeability zones within the sediment could have been most important in retaining high concentrations of U during initial release into the vadose zone. Massive amount of colloids, including U-colloids, formed at the plume fronts. Total U concentrations (aqueous and colloid) within plume fronts exceeded the source concentration by up to 5-fold. Uranium colloid formation and accumulation at the neutralized plume front could be one mechanism responsible for highly heterogeneous U distribution observed in the contaminated Hanford vadose zone.

Zinc coordination to multiple ligand atoms in organic-rich surface soils.

We report on the solid-phase speciation of naturally occurring Zn in metalliferous organic-matter-rich surface soils. Synchrotron-based studies were used to probe elemental distribution and associations in soil particles (micro-XRF) together with the mineralogy (micro-XRD) and Zn bonding environment (Zn-micro-XANES) at the micrometer-scale level. The average bonding environment of Zn was also probed for bulk soils using XANES. We found the distribution of elements within soil particles to be heterogeneous; however, some elements are consistently co-located. While conventional XRD analyses of whole soils did not identify any Zn mineral phase, synchrotron-based-micro-XRD analyses indicated that sphalerite (ZnS) is present in a particle from a wetland soil (soil labeled G3). Linear combination fit (LCF) analyses of XANES spectra collected for bulk soils (Zn-XANES) and microm-regions (Zn-micro-XANES) within soil particles suggest Zn bonds to oxygen-, nitrogen-, and sulfur-functional groups in these sulfur-, nitrogen-, and zinc-rich organic surface soils. The XANES spectra of all bulk soils and of all microm-regions except for the wetland soil (G3), where ZnS was the most significant constituent, were best fitted by the Zn-arginine reference compound and therefore seems to indicate Zn bonding to nitrogen. Thus, these results provide compelling evidence of the formation of highly covalent Zn-organic bonds in the organic-rich surface soils that were studied. This may explain in part why metal partition coefficients (Kd) are generally higher in organic soils, and why the toxic thresholds for total metal concentrations are higher in organic than in mineral soils.

Uranium reduction in sediments under diffusion-limited transport of organic carbon.

Costly disposal of uranium (U) contaminated sediments is motivating research on in situ U(VI) reduction to insoluble U(IV) via directly or indirectly microbially mediated pathways. Delivery of organic carbon (OC) into sediments for stimulating U bioreduction is diffusion-limited in less permeable regions of the subsurface. To study OC-based U reduction in diffusion-limited regions, one slightly acidic and another calcareous sediment were treated with uranyl nitrate, packed into columns, then hydrostatically contacted with tryptic soy broth solutions. Redox potentials, U oxidation state, and microbial communities were well correlated. At average supply rates of 0.9 micromol OC (g sediment)(-1) day(-1), the U reduction zone extended to only about35-45 mm into sediments. The underlying unreduced U(VI) zone persisted over 600 days because the supply of OC was diffusion-limited and metabolized within a short distance. These results also suggestthat low U concentrations in groundwater samples from OC-treated sediments are not necessarily indicative of pervasive U reduction because interior and exterior regions of such sediment blocks can contain primarily U(VI) and U(IV), respectively.

Geochemical signature of contaminated sediment remobilization revealed by spatially resolved X-ray microanalysis of annual rings of Salix nigra.

An X-ray microprobe was used to determine the concentration and distribution of Ni, U, and other metals within annual rings of willows (Salix nigra L.) from a former de facto radiological settling basin (Steed Pond; SP) and a depositional environment downstream (Tims Branch; TB) on the Savannah River Site (SRS). Geochemical and historical information about both areas are well documented. Following spillway breaches at SP in 1984 and the early 1990s, TB is inundated with contaminated sediments during storms. Bulk elemental composition of tree cores was determined using ICP-OES. Synchrotron X-rayfluorescence (SXRF) analysis showed that the metal contents of SP and TB cores were an order of magnitude higher than those from a reference site. TB cores were enriched with Ni in 1984 and 1991, corresponding with SP spillway breaches (containing 790 mg kg(-1) Ni in 1991). Cores from SP exhibited an extremely high Ni peak in 1996, approximately 5000 mg kg(-1), even though contaminant levels at SP did not change. The geochemical signature of contaminants recorded in TB annual rings reflected the significant sediment remobilization events consistent with the detailed history of the site, and at concentrations relative to their proximity to the source term. However, physiological processes occurring within impacted trees strongly influence the chronological accuracy of dendroanalysis and must be investigated further.