Elemental Mercury in Natural Waters: Occurrence and Determination of Particulate Hg(0)

Elemental mercury, Hg(0), is ubiquitous in water and involved in key Hg biogeochemical processes. It is extensively studied as a purgeable dissolved species, termed dissolved gaseous mercury (DGM). Little information is available regarding nonpurgeable particulate Hg(0) in water, Hg(0) bound to suspended particulate matter (SPM), which is presumably present due to high affinity of Hg(0) adsorption on solids. By employing stable isotope tracer and isotope dilution (ID) techniques, we investigated the occurrence and quantification of particulate Hg­(0) after Hg(0) being spiked into natural waters, aiming to provide firsthand information on particulate Hg(0) in water. A considerable fraction of ²⁰¹Hg­(0) spiked in water (about 70% after 4 h equilibration) was bound to SPM and nonpurgeable, suggesting the occurrence of particulate Hg(0) in natural waters. A scheme, involving isotope dilution, purge and trap, and inductively coupled plasma mass spectrometry detection, was proposed to quantify particulate Hg(0) by the difference between DGM and total Hg(0), determined immediately and at equilibration after spiking ID Hg isotope, respectively. The application of this newly established method revealed the presence of particulate Hg(0) in Florida Everglades water, as the determined DGM levels (0.14 to 0.22 ng L^–1) were remarkably lower than total Hg(0) (0.41 to 0.75 ng L^–1)

Human Metapneumovirus M2-2 Protein Inhibits Innate Immune Response in Monocyte-Derived Dendritic Cells

<div><p>Human metapneumovirus (hMPV) is a leading cause of lower respiratory infection in young children, the elderly and immunocompromised patients. Repeated hMPV infections occur throughout life. However, immune evasion mechanisms of hMPV infection are largely unknown. Recently, our group has demonstrated that hMPV M2-2 protein, an important virulence factor, contributes to immune evasion in airway epithelial cells by targeting the mitochondrial antiviral-signaling protein (MAVS). Whether M2-2 regulates the innate immunity in human dendritic cells (DC), an important family of immune cells controlling antigen presenting, is currently unknown. We found that human DC infected with a virus lacking M2-2 protein expression (rhMPV-ΔM2-2) produced higher levels of cytokines, chemokines and IFNs, compared to cells infected with wild-type virus (rhMPV-WT), suggesting that M2-2 protein inhibits innate immunity in human DC. In parallel, we found that myeloid differentiation primary response gene 88 (MyD88), an essential adaptor for Toll-like receptors (TLRs), plays a critical role in inducing immune response of human DC, as downregulation of MyD88 by siRNA blocked the induction of immune regulatory molecules by hMPV. Since M2-2 is a cytoplasmic protein, we investigated whether M2-2 interferes with MyD88-mediated antiviral signaling. We found that indeed M2-2 protein associated with MyD88 and inhibited MyD88-dependent gene transcription. In this study, we also identified the domains of M2-2 responsible for its immune inhibitory function in human DC. In summary, our results demonstrate that M2-2 contributes to hMPV immune evasion by inhibiting MyD88-dependent cellular responses in human DC.</p></div

M2-2 interacts with MyD88.

<p>(<b>A–B</b>) M2-2 forms a complex with MyD88 in the overexpression system. 293 cells were transfected with plasmids encoding HA-tagged MyD88 and V5-tagged M2-2 or their control vectors as indicated. Total cell lysates were immunoprecipitated with an anti-V5 antibody followed by Western blotting using an anti-Flag antibody to detect MyD88 (<b>A</b>). Reverse immunoprecipitation was also done, where MyD88 was immunoprecipitated using an anti-HA antibody and M2-2 protein was then detected using an anti-V5 antibody (<b>B</b>). Membranes were stripped and reprobed to check for proper IP of M2-2 and MyD88. A small aliquot was also prepared before the IP for a Western blot for equal input of MyD88 and proper expression of M2-2 in 293 cells. (<b>C</b>) Viral M2-2 binds to endogenous MyD88 in the context of hMPV infection. THP1 cells were mock infected or infected with rhMPV-WT or -ΔM2-2, at an MOI of 5, and harvested at 24 h p.i. to prepare total cell lysates. Samples were subjected to immunoprecipitation using an anti-MyD88 antibody or control isotype. The immunoprecipitated complexes were then subjected to SDS-PAGE followed by Western blotting using an anti-hMPV antibody. The membrane was then stripped and reprobed with an anti-MyD88 antibody to determine levels of immunoprecipitated MyD88. Data are representative of two independent experiments.</p

Evaluation of the Possible Sources and Controlling Factors of Toxic Metals/Metalloids in the Florida Everglades and Their Potential Risk of Exposure

The Florida Everglades is an environmentally sensitive wetland ecosystem with a number of threatened and endangered fauna species susceptible to the deterioration of water quality. Several potential toxic metal sources exist in the Everglades, including farming, atmospheric deposition, and human activities in urban areas, causing concerns of potential metal exposure risks. However, little is known about the pollution status of toxic metals/metalloids of potential concern, except for Hg. In this study, eight toxic metals/metalloids (Cd, Cr, Pb, Ni, Cu, Zn, As, and Hg) in Everglades soils were investigated in both dry and wet seasons. Pb, Cr, As, Cu, Cd, and Ni were identified to be above Florida SQGs (sediment quality guidelines) at a number of sampling sites, particularly Pb, which had a level of potential risk to organisms similar to that of Hg. In addition, a method was developed for quantitative source identification and controlling factor elucidation of toxic metals/metalloids by introducing an index, enrichment factor (EF), in the conventional multiple regression analysis. EFs represent the effects of anthropogenic sources on metals/metalloids in soils. Multiple regression analysis showed that Cr and Ni were mainly controlled by anthropogenic loading, whereas soil characteristics, in particular natural organic matter (NOM), played a more important role for Hg, As, Cd, and Zn. NOM may control the distribution of these toxic metals/metalloids by affecting their mobility in soils. For Cu and Pb, the effects of EFs and environmental factors are comparable, suggesting combined effects of loading and soil characteristics. This study is the first comprehensive research with a vast amount of sampling sites on the distribution and potential risks of toxic metals/metalloids in the Everglades. The finding suggests that in addition to Hg other metals/metalloids could also potentially be an environmental problem in this wetland ecosystem

Characterization of WT and ΔM2-2 in moDC.

<p>moDC were infected with WT or ΔM2-2 at MOI of 2. Mock infected was used as a control. At 24 h p.i., cells were harvested to prepare total cell lysates or total cell RNA or nuclear extracts. Viral protein expression in total cell lysates was determined by Western blot assay (<b>A</b>), viral gene transcription in total RNA samples was assayed by qRT-PCR (<b>B</b>), and the nuclear translocation of p64, IRF-7 and IRF-3 was determined by Western blot (<b>C</b>). Data are representative of two-three independent experiments.</p

Primers used for qRT-PCR and RT-PCR.

<p>Primers used for qRT-PCR and RT-PCR.</p

Effect of M2-2 protein deletion on cytokine, chemokine, and IFN secretion.

<p>moDCs were infected with hMPV, either WT or ΔM-2, and harvested at different time points p.i. to measure secretion of cytokines, chemokines, (<b>A</b>), as well as IFN (<b>B</b>), by Bio-Plex or ELISA. Results shown are representative of three separate experiments.</p

M2-2 inhibits MyD88-mediated signaling.

<p>(<b>A</b>) M2-2 in suppresses TLR-4 signaling. 293 cells constitutively expressing TLR-4 in triplicates were transfected with a luciferase reporter plasmid under the control of NF-κB binding site NF-κB-Luc (0.05 µg/well), a plasmid encoding M2-2 (0.5 µg/ml) or its control vectors. At 36 h post transfection, cells were either untreated or treated with agonist LPS (100 ng/ml). At 15 h post treatment, cells were harvested for luciferase acitivity measurement. For each plate, luciferase was normalized to the β-galactosidase reporter activity. Data are expressed as means ± SE from 2 experiments. **, <i>P</i><0.01, relative to CN+LPS. (<b>B</b>) Inhibition of MyD88-induced IFN-β transcription by M2-2. 293 cells in triplicates (24-well plate) were transfected with a luciferase reporter plasmid (IFN-β-Luc), plasmids encoding either MyD88 (0.2 µg/ml) or their control vectors (CV), or a plasmid expressing hMPV M2-2 or control proteins or the empty vector (0.2 µg/well). Cells were harvested 30 h posttransfection to measure luciferase activity. (<b>C</b>) M2-2 domains responsible for MyD88-mediated signaling. 293 cells in triplicates were transfected with IFN-β-Luc (0.2 µg/well), a plasmid encoding MyD88 (0.1 µg/ml) or its control vectors, and a plasmid expressing WT M2-2 or indicated M2-2 mutants or the empty vector. Cells were harvested 30 h posttransfection to measure luciferase activity. For each plate, luciferase was normalized to the β-galactosidase reporter activity. Data are expressed as means ± SE. In panel B **, <i>P</i><0.01, relative to MyD88+ pCAGGS. In Panel C, **, <i>P</i><0.01, column 3 and 4 relative to column 2, and column 5 and 6 relative to column 3.</p

Administration of curcumin protects CBA/J mice from the development of ALI/ARDS in response to reovirus 1/L.

<p>CBA/J mice were inoculated i.n. with 10⁷pfu reovirus 1/L and were either untreated (−) or treated (+) with 50 mg/kg curcumin by i.p. injection beginning 5 days prior to infection and daily, thereafter. Whole lungs were removed at the indicated time points, fixed in 2% paraformaldehyde and embedded in paraffin. Staining was performed on 4-micron sections. (A) H&E Staining on days 9 and 14; (B) Mason's Trichrome and Sirius Red Staining on day 14. Objective magnification: 10X (A) and 20X (B). Representative of at least four independent experiments evaluating 3 mice per time point.</p

Administration of curcumin modulates TGFß RII expression during reovirus 1/L-ALI/ARDS.

<p>CBA/J mice were inoculated i.n. with 10⁷pfu reovirus 1/L and were either untreated (−, solid bars) or treated (+, open bars) with 50 mg/kg curcumin by i.p. injection beginning 5 days prior to infection and daily, thereafter. At the indicated time points, RNA was prepared from whole lung tissue and the relative expression of (A) TGFß1 was assessed by RT-PCR; (B) TGFß1 and TGFß RII mRNA expression was also assessed by qRT-PCR. Saline inoculated mice were used as controls (S, stippled bars). Histograms are the mean +/−S.D. of two experiments with three mice per time point. *p<0.05 versus saline (control), **p<0.05 versus reovirus 1/L-ALI/ARDS.</p