Table_1_Physiological responses and antibiotic-degradation capacity of duckweed (Lemna aequinoctialis) exposed to streptomycin.xlsx

Aquatic plants are constantly exposed to various water environmental pollutants. Few data on how antibiotics affect duckweed health and its removal ability. The aim of this study was to investigate the impact of streptomycin on the physiological change and uptake capability in duckweed (Lemna aequinoctialis) after exposure at different time points (0, 5, 10, 15 and 20 days). Duckweeds were exposed to streptomycin at a range of concentrations (0.1-10 mM). Results indicated that the high streptomycin concentrations (≥1 mM) resulted in a lower duckweed biomass (21.5-41.5%), RGR (0.258-0.336 g d−1), decrease in total Chl and increase in carotenoids. Antioxidative enzymes, including CAT (18-42.88 U mg protein-1), APX (0.41-0.76 U mg protein-1), and SOD (0.52-0.71 U mg protein-1) were found to accumulate in the streptomycin groups in comparison to the control group. The significant reduction (72-82%) in streptomycin content at 20 d compared to the control (40-55%) suggested that duckweed has a high ability in removing streptomycin. Transcriptome analysis showed that the secondary metabolic pathways including phenylpropanoid biosynthesis and flavonoid biosynthesis were significantly upregulated in the streptomycin setup compared to the control. Therefore, our findings suggested that duckweed can contribute to the streptomycin degradation, which should be highly recommended to the treatment of aquaculture wastewater and domestic sewage.</p

Table_2_Physiological responses and antibiotic-degradation capacity of duckweed (Lemna aequinoctialis) exposed to streptomycin.docx

Aquatic plants are constantly exposed to various water environmental pollutants. Few data on how antibiotics affect duckweed health and its removal ability. The aim of this study was to investigate the impact of streptomycin on the physiological change and uptake capability in duckweed (Lemna aequinoctialis) after exposure at different time points (0, 5, 10, 15 and 20 days). Duckweeds were exposed to streptomycin at a range of concentrations (0.1-10 mM). Results indicated that the high streptomycin concentrations (≥1 mM) resulted in a lower duckweed biomass (21.5-41.5%), RGR (0.258-0.336 g d−1), decrease in total Chl and increase in carotenoids. Antioxidative enzymes, including CAT (18-42.88 U mg protein-1), APX (0.41-0.76 U mg protein-1), and SOD (0.52-0.71 U mg protein-1) were found to accumulate in the streptomycin groups in comparison to the control group. The significant reduction (72-82%) in streptomycin content at 20 d compared to the control (40-55%) suggested that duckweed has a high ability in removing streptomycin. Transcriptome analysis showed that the secondary metabolic pathways including phenylpropanoid biosynthesis and flavonoid biosynthesis were significantly upregulated in the streptomycin setup compared to the control. Therefore, our findings suggested that duckweed can contribute to the streptomycin degradation, which should be highly recommended to the treatment of aquaculture wastewater and domestic sewage.</p

DHA-induced apoptosis is caused by ROS.

<p>(A) Apoptosis of pancreatic cancer cells. BxPC-3 and PANC-1 cells were treated with DHA (50 µmol/L) and Apo2L/TRAIL (100 ng/ml) as indicated. Flow cytometry was performed to measure apoptosis rates (%). A significant increase in the apoptosis rate compared with the control is denoted by “*” (<i>P</i><0.05), a significant increase compared with DHA- or Apo2L/TRAIL-treated cells is denoted by “†” (<i>P</i><0.01), and a significant decrease compared with DHA+Apo2L/TRAIL-treated cells is denoted by “‡” (<i>P</i><0.01). Representative histograms from cytometrically analyzed BxPC-3 and PANC-1 cells treated with control, DHA, Apo2L/TRAIL, DHA+Apo2L/TRAIL or NAC (10 mM). (B) Laser scanning confocal microscopy of cells. Representative photographs were taken of the control BxPC-3 and PANC-1 cells and of BxPC-3 and PANC-1 cells treated with DHA+Apo2L/TRAIL and DHA+Apo2L/TRAIL+NAC. (C) Levels of intracellular ROS measured in vitro. BxPC-3 and PANC-1 cells were treated with DHA (50 µmol/L), Apo2L/TRAIL (100 ng/ml), DHA+Apo2L/TRAIL, or pretreated with NAC (10 mM) and then treated with DHA+Apo2L/TRAIL for 6 h. Untreated cells served as the control. The cells were incubated with DCFHDA and then subjected to flow cytometry to measure the levels of intracellular ROS, as represented by DCF fluorescence. A significant increase in DCF fluorescence compared with the control is denoted by “*” (<i>P</i><0.05), a highly significant difference compared with the control is denoted by “**” (<i>P</i><0.01), and a significant reduction compared with the DHA+Apo2L/TRAIL treatment is denoted by “†” (<i>P</i><0.05). (D) Representative photographs are shown for DCFHDA-stained cells observed using laser scanning confocal microscopy. The green fluorescence represents intracellular ROS. (E) The mean fluorescence intensity was measured for the DCFHDA-stained cells, and the respective 3-dimensional horizontal plane images were produced by laser scanning confocal microscopy. A significant difference from the control is denoted by “*” (<i>P</i><0.01), and a significant difference from the DHA+Apo2L/TRAIL treatment is denoted by “†” (<i>P</i><0.05).</p

The expression of apoptosis-related genes.

<p>BxPC-3 and PANC-1 cells were treated with various concentrations of DHA (0, 25, 50, 100 µmol/L) and pretreated with NAC followed by DHA (100 µmol/L) for 72 h. Whole cell extracts were prepared and analyzed by western blotting using antibodies against Bcl-2, Bax, surviving, caspase-3, caspase-8, and caspase-9. DHA significantly up-regulated the expression of Bax, caspase-3, caspase-8 and caspase-9, and down-regulated the expression of Bcl-2. However, DHA had little influence on the expression of survivin. DHA with NAC (10 mM) pretreatment did not up-regulate the expression of caspase-8, Bax, caspase-9, and caspase-3. β-actin served as an internal control.</p

Up-regulation of DR5 by DHA was mediated by ROS.

<p>(A) BxPC-3 and PANC-1 cells were treated with the indicated doses of DHA for 48 h. Whole cell extracts were prepared and analyzed for DR5 expression using western blotting. DHA had dose-dependent effects on the expression of DR5. The DHA-induced increase in DR5 levels was significantly blocked by pretreatment with 10 mM NAC. β-actin served as an internal control. (B) The cells were collected and analyzed using flow cytometry. A gradual increase in fluorescence was observed in cells treated with 25, 50 and 100 µmol/L DHA, respectively, indicating a dose-dependent increase in the production of ROS in response to DHA treatment in the two cell lines. The production of ROS was markedly inhibited by pretreating the cells with NAC (10 mM). (C) The cells were treated with DHA (50 µmol/L) alone, Apo2L/TRAIL (100 ng/ml) alone or a combination of the two agents. The cells were also treated with NAC (10 mM) alone or pretreated with NAC and incubated at 37°C for 72 h. The viability of the cells was assessed using the MTT method and the viability index (%) was calculated. Significant differences are denoted by “*” (<i>P</i><0.01).</p

DHA synergistically enhances Apo2L/TRAIL-induced cell death in BxPC-3 cells.

<p>(A) Cells were treated with DHA alone, Apo2L/TRAIL alone or a combination of the two agents and incubated at 37°C for 72 h. The viability of the cells was assessed using the MTT method. Combination index (CI) versus fraction affected (Fa) plots obtained from median-effect analysis of Chou-Talalay. A CI>1 indicates antagonism,  = 1 indicates additivity, and <1 indicates synergy. (B) The clonogenic assay was performed as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0037222#s4" target="_blank">Materials and Methods</a> section. The cells were treated with DHA (50 µmol/L) alone, Apo2L/TRAIL (100 ng/ml) alone or the combination of the two drugs for 24 h and washed with PBS. The cells were then incubated for an additional 7 d and stained with crystal violet. (C) Clonogenic survival is presented as the percentage of surviving colonies formed in drug-treated cells with respect to untreated cells.</p

Effects of knockdown of DR5 expression on DHA-induced cytotoxicity and cell apoptosis of Apo2L/TRAIL.

<p>BxPC-3 and PANC-1 cells were transfected with DR5 siRNA and control siRNA, either alone or in combination. After 48 h, the cells were treated with 50 µmol/L DHA for 24 h, and whole cell extracts were subjected to western blotting to test for the expression of DR5. Transfection of cells with siRNA targeting DR5 specifically silenced the expression of DR5. The cells were seeded on a chamber slide and transfected with siRNAs. After 48 h, the cells were treated with 50 µmol/L DHA, 100 ng/mL Apo2L/TRAIL, either alone or in combination, and incubated at 37°C for 72 h. The viability of the cells was assessed using the MTT method, and the viability index (%) was calculated. Silencing of DR5 by siRNA reduced the cytotoxic effect of the combination of DHA and Apo2L/TRAIL but not of DHA alone. Significant differences are denoted by “*” (<i>P</i><0.01).</p

Tumor growth, tumor gene expression, tumor proliferation and apoptosis <i>in vivo</i>.

<p>(A) BxPC-3 tumors were established subcutaneously in mice. When the tumors reached approximately 120 mm³ in volume, the mice were randomly assigned to control, DHA, Apo2L/TRAIL, or DHA+Apo2L/TRAIL groups and treated as described in the methods section. The sizes (measured in mm³) of the tumors were monitored and recorded. A significant difference in tumor volume from the control is denoted by “*” (<i>P</i><0.05), and a significant reduction compared to the DHA or Apo2L/TRAIL-treated tumors is denoted by “**” (<i>P</i><0.01). (B) Representative animals and tumors are shown for each group. (C) Tumors from control mice and from mice treated with DHA, Apo2L/TRAIL, and DHA+Apo2L/TRAIL were homogenized and subjected to western blot analysis to detect the expression of caspase-3 and caspase-8. β-actin served as an internal control. (D) Analysis of proliferation marker PCNA by immunohistochemistry and apoptotic status of tumor cells by in situ TUNEL assay. PCNA positive (E) and TUNEL-positive (F) cells were also counted under microscope to calculate the proliferation index and apoptotic index, respectively. “*”: <i>P</i><0.05, compared with control. “**”: <i>P</i><0.01, compared with single agent.</p

A collection of upregulated miRNAs detected by deep sequencing in cancerous penile tissues compared with the matched adjacent normal penile tissues.

<p>^arpm: reads count per million reads count</p><p>A collection of upregulated miRNAs detected by deep sequencing in cancerous penile tissues compared with the matched adjacent normal penile tissues.</p

The list of top 10 most abundant novel miRNAs expressed in adjacent normal penile tissues and cancerous penile tissues.

<p>The list of top 10 most abundant novel miRNAs expressed in adjacent normal penile tissues and cancerous penile tissues.</p