Effects of SMG on NADPH oxidase2 (Nox2) and NADPH oxidase4 (Nox4) expression in <i>Rad9</i>^<i>+/+</i> and <i>Rad9</i>^<i>-/-</i> MES cells.

<p>(A) Quantitative real-time PCR analysis of Nox2 mRNA expression in <i>Rad9</i>^{<b><i>+/+</i></b>} and <i>Rad9</i>^{<b><i>-/-</i></b>} MES cells exposed to 1G or SMG condition for 1 or 5 days. The expression levels of Nox2 were normalized to the endogenous control GAPDH expression. (B) Quantitative real-time PCR analysis of Nox4 mRNA expression in <i>Rad9</i>^{<b><i>+/+</i></b>} and <i>Rad9</i>^{<b><i>-/-</i></b>} MES cells exposed to 1G or SMG condition for 1 or 5 days. The expression levels of Nox4 were normalized to the endogenous control GAPDH expression. (C) Western blot analysis of Nox2 protein expression in <i>Rad9</i>^{<b><i>+/+</i></b>} and <i>Rad9</i>^{<b><i>-/-</i></b>} MES cells exposed to 1G or SMG condition for 1 or 5 days. GAPDH was used as an internal control. The representative results of three independent experiments were shown. (D) Quantitative comparison of Nox2 expression. Data were derived from three independent experiments. The expression levels of Nox2 protein were normalized to the endogenous control GAPDH protein expression. The data represent mean ± SD of three independent experiments. Student’s t test, *P<0.05.</p

Effects of SMG on DNA damage and apoptosis in <i>Rad9</i>^<i>+/+</i> and <i>Rad9</i>^<i>-/-</i> MES cells.

<p>(A) Evaluation of DNA double strand break by neutral comet assay in <i>Rad9</i>^{<b><i>+/+</i></b>} and <i>Rad9</i>^{<b><i>-/-</i></b>} MES cells cultured under 1G or SMG condition. Time points were 1, 2, 3, 4 and 5 days. At least 50 cells for each condition were scored for comet tail moment. (B) Flow cytometric analysis of γ-H2AX formation in <i>Rad9</i>^{<b><i>+/+</i></b>} and <i>Rad9</i>^{<b><i>-/-</i></b>} mMES cells cultured under 1G or SMG condition for 1 or 5 days. (C) Evaluation of DNA damage by alkaline comet assay in <i>Rad9</i>^{<b><i>+/+</i></b>} and <i>Rad9</i>^{<b><i>-/-</i></b>} MES cells cultured under 1G or SMG condition for 1 or 5 days. At least 50 cells for each condition were scored for comet tail moment. (D) Evaluation of DNA damage by alkaline comet assay in <i>Rad9</i>^{<b><i>-/-</i></b>} MES cells with ectopic expression of <i>Rad9</i> (<i>Rad9</i>^{<b><i>-/-</i></b>}<i>+Rad9</i> MES cells) cultured under 1G or SMG condition for 1 or 5 days. At least 50 cells for each condition were scored for comet tail moment. (E) Flow cytometric analysis of <i>Rad9</i>^{<b><i>+/+</i></b>} and <i>Rad9</i>^{<b><i>-/-</i></b>} MES cells cultured under 1G or SMG condition for 1 day to assess apoptosis using Annexin V labeling. Experiments were performed three times and representative analyses are shown (upper). The lower part is the quantitative comparison of apoptosis between the 1G Group and the SMG Group. The data represent mean ± SD of at least three independent experiments. Student’s t test, *P<0.05.</p

Effects of SMG on the generation of endogenous reactive oxygen species(ROS)in <i>Rad9</i>^<i>+/+</i> and <i>Rad9</i>^<i>-/-</i> MES cells.

<p>(A) Flow cytometric analysis of ROS activity in <i>Rad9</i>^{<b><i>+/+</i></b>} and <i>Rad9</i>^{<b><i>-/-</i></b>} MES cells exposed to 1G or SMG condition for 1 or 5 days. (B) Flow cytometric analysis of ROS activity in <i>Rad9</i>^{<b><i>-/-</i></b>}<i>+Rad9</i> MES cells exposed to 1G or SMG condition for 1 or 5 days. (C) N-acetylcysteine inhibited SMG-induced increase of ROS formation in <i>Rad9</i>^{<b><i>-/-</i></b>} MES cells. <i>Rad9</i>^{<b><i>-/-</i></b>} MES cells were mock-treated or treated with 0.05, 0.1 or 0.5 mM N-acetylcysteine under SMG for 1 day. (D) Evaluation of DNA damage by alkaline comet assay in <i>Rad9</i>^{<b><i>-/-</i></b>} MES cells mock-treated or treated with 0.5 mM N-acetylcysteine under 1G of SMG condition for 1 day. (E) Evaluation of DNA damage by neutral comet assay in <i>Rad9</i>^{<b><i>-/-</i></b>} MES cells mock-treated or treated with 0.5 mM N-acetylcysteine under 1G of SMG condition for 1 day. The data represent mean ± SD of three independent experiments. Student’s t test *P<0.05, **P<0.01.</p

Effects of SMG on antioxidant enzyme activity in MES cells.

<p><i>Rad9</i>^{<b><i>+/+</i></b>} MES cells and <i>Rad9</i>^{<b><i>-/-</i></b>} MES cells were cultured under 1G and SMG for 1 and 5 days, respectively. The activities of the antioxidant enzymes in MES cell lysates were determined. (A) Histograms of superoxide dismutase enzyme activity. (B) Histograms of catalase enzyme activity. (C) Histogram of Glutahione peroxidase. The data represent mean ± SD of three independent experiments.</p

Effects of SMG on DNA damage in <i>Mdc1</i>^<i>+/+</i> and <i>Mdc1</i>^<i>-/-</i> MEF cells.

<p>Evaluation of DNA double strand break by neutral comet assay in <i>Mdc1</i>^{<b><i>+/+</i></b>} and <i>Mdc1</i>^{<b><i>-/-</i></b>} MEF cells cultured under 1G or SMG condition for 1 or 5 days. At least 50 cells for each condition were scored for comet tail moment. The data represent mean ± SD of three independent experiments. Student’s t test, *P<0.05.</p

Cell Number Expansion analysis of MES and MEF cells after five days incubation under 1G or SMG.

<p>(A) The initial MES cells seeding number was 3×10⁴. The cell doubling curve was generated by dividing the cell number by10⁴ and then transforming the values to logarithm base2. (B) The initial MEF cells seeding number was 10⁵. The cell doubling curve was generated by dividing the cell number by10⁴ and then transforming the values to logarithm base2. The data represent mean ±SD of three independent experiments.</p

Effects of SMG on NADPH oxidase2 (Nox2) expression in <i>Mdc1</i>^<i>+/+</i> and <i>Mdc1</i>^<i>-/-</i> MEF cells.

<p>(A) Western blot analysis of Nox2 protein expression in <i>Mdc1</i>^{<b><i>+/+</i></b>} and <i>Mdc1</i>^{<b><i>-/-</i></b>} MEF cells exposed to 1G or SMG condition for 1 or 5days. GAPDH was used as an internal control. The representative results of three independent experiments were shown. (B) Quantitative comparison of Nox2 expression. Data were derived from three independent experiments as in A). The expression levels of Nox2 were normalized to the endogenous control GAPDH expression. The data represent mean ± SD of three independent experiments. Student’s t test, *P<0.05.</p

Evolved Bacterial Biosensor for Arsenite Detection in Environmental Water

Arsenic, a ubiquitous presence in the biosphere, often occurs from both natural and anthropogenic sources. Bacterial biosensors based on genetically engineered bacteria have promising applications in detecting the chemical compound and its toxicity. However, most of the bacteria biosensors take advantage of the existing wild-type substrate-induced promoters, which are often low in specificity, affinity and sensitivity, and thus limiting their applications in commercial or field use. In this study, we developed an in vivo evolution procedure with a bidirectional selection scheme for improving the sensitivity of an arsenite-responsive bacterial biosensor through optimization of the inducible operon. As a proof of concept, we evolved the arsenite-induced arsR operon for both low background and high expression through three successive rounds of fluorescence activated cell sorting (FACS) with bidirectional strategy. An arsR operon variant with 12-fold higher activity over the control was isolated, confirming multiple rounds of construction and screening of mutation library, as described here, can be efficiently applied to bacterial biosensor optimization. The evolved arsenite-responsive biosensor demonstrated an excellent performance in the detection of low trace arsenite in environmental water. These results indicate that the technologies of directed evolution could be used to improve the performance of bacterial biosensors, which will be helpful in promoting the practical application of bacterial biosensors