Additional file 2: of Genetic diversity and structure of core collection of winter mushroom (Flammulina velutipes) developed by genomic SSR markers

Allele frequence distributions between entire collection and core collection. (XLS 46 kb

Occurrence and Fate of Substituted <i>p</i>‑Phenylenediamine-Derived Quinones in Hong Kong Wastewater Treatment Plants

para-Phenylenediamine quinones (PPD-Qs) are a newly discovered class of transformation products derived from para-phenylenediamine (PPD) antioxidants. These compounds are prevalent in runoff, roadside soil, and particulate matter. One compound among these, N-1,3-dimethylbutyl-n′-phenyl-p-phenylenediamine quinone (6PPD-Q), was found to induce acute mortality of coho salmon, rainbow trout, and brook trout, with the median lethal concentrations even lower than its appearance in the surface and receiving water system. However, there was limited knowledge about the occurrence and fate of these emerging environmental contaminants in wastewater treatment plants (WWTPs), which is crucial for effective pollutant removal via municipal wastewater networks. In the current study, we performed a comprehensive investigation of a suite of PPD-Qs along with their parent compounds across the influent, effluent, and biosolids during each processing unit in four typical WWTPs in Hong Kong. The total concentrations of PPDs and PPD-Qs in the influent were determined to be 2.7–90 and 14–830 ng/L. In the effluent, their concentrations decreased to 0.59–40 and 2.8–140 ng/L, respectively. The median removal efficiency for PPD-Qs varied between 53.0 and 91.0% across the WWTPs, indicating that a considerable proportion of these contaminants may not be fully eliminated through the current processing technology. Mass flow analyses revealed that relatively higher levels of PPD-Qs were retained in the sewage sludge (20.0%) rather than in the wastewater (16.9%). In comparison to PPDs, PPD-Qs with higher half-lives exhibited higher release levels via effluent wastewater, which raises particular concerns about their environmental consequences to aquatic ecosystems

Ribozyme expression in cells assayed using Northern blot analysis.

<p>RNA fractions (40 μg) from the parental U251 cells (-) and cells expressing different ribozymes (F-M1-IE, C-M1-IE, F-R228-IE, and C-R228-IE) were hybridized to probes for detection of human H1 RNA (internal control) (lanes 6–10) and ribozymes (lanes 1–5).</p

Levels of human β-actin (internal control) and viral proteins assayed by Western blot analysis.

<p>Protein fractions (60 μg) from the parental U251 cells (-) and cells expressing different ribozymes (F-M1-IE, F-R228-IE, and C-R228-IE) were reacted with antibodies for detection of human actin, IE1 protein, IE2 protein, and UL99 protein. We observed no expression of viral proteins in mock-infected U251 cells [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186791#pone.0186791.ref030" target="_blank">30</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186791#pone.0186791.ref032" target="_blank">32</a>] (data not shown).</p

Overall cleavage rate [(k_cat/K_m)^s] and binding affinity (K_d) in reactions of substrates ie-39 with RNase P ribozymes.

<p>The values are derived from experiments that were performed in triplicate and repeated three times. “ND”: not determined.</p

Analysis of growth in LB broth of <i>Salmonella</i>.

<p><i>Salmonella</i> used in the study include the wild type strain ST14028s, mutant strain SL368, and its derivatives that carry the empty vector construct pVAX1 (Sal-vector), and constructs p5HA and p5NA (Sal-HA-NA). Experimental details can be found in Materials and Methods.</p

ELISPOT analysis of IFN-γ production by HA_533–541 specific T cells in vaccinated mice at 42 days after immunization.

<p>Mice were immunized intragastrically at day 0, 14, and 28 with PBS, the commercial H5N1 vaccine (cv-H5N1), and <i>Salmonella</i> SL368 carrying the empty vector pVAX1 (Sal-vector) and constructs p5HA and p5NA (Sal-HA-NA). Splenocytes (n = 5) were harvested from immunized mice at 42 days post immunization and stimulated with HA_533–541 peptide for 48 hours. The results were expressed as spot-forming cells (SFC) per million cells. The assays were performed in triplicate and the experiments were repeated three times. The values obtained were the average from these experiments. Statistical analysis was performed by two-way ANOVA. *<i>p</i> < <i>0</i>.<i>01</i>.</p

Toxicity and virulence of different <i>Salmonella</i> strains in BALB/c mice.

<p>Mice (5 animals per group) were either inoculated intramuscularly with the commercial H5N1 (cv-H5N1) and H1N1 vaccines (cv-H1N1) or intragastrically with PBS, the wild-type strain ST14028 (1 × 10³ CFU), and vaccine strain SL368 (1 × 10⁹ CFU) carrying the empty vector construct pVAX1 (Sal-vector) and constructs p5HA and p5NA (Sal-HA-NA). The mortality of the animals was monitored for 60 days. Humane (non-lethal) endpoints were used during the experiments and mice were euthanized with overdose inhalational CO₂ to limit suffering, when they exhibited weight loss of more than 30%, lethargy, ruffled hair coat, or hunched posture.</p

Levels of viral mRNAs assayed by Northern blot analysis.

<p>RNA fractions (45 μg) from the parental U251 cells (-) and cells expressing different ribozymes (F-M1-IE, F-R228-IE, and C-R228-IE) were hybridized to probes for detection of 5kb RNA, IE1 mRNA, IE2 mRNA, and US2 mRNA. We observed no expression of viral mRNAs in mock-infected U251 cells [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186791#pone.0186791.ref030" target="_blank">30</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186791#pone.0186791.ref032" target="_blank">32</a>].</p

Substrates for M1 RNA and RNase P.

<p>(A) pre-tRNA; (B) A target mRNA bound to an EGS; (3) a target mRNA bound to an M1GS ribozyme.</p