9 research outputs found

    Detection of Cyanobacteria in Eutrophic Water Using a Portable Electrocoagulator and NanoGene Assay

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    We have demonstrated the detection of cyanobacteria in eutrophic water samples using a portable electrocoagulator and NanoGene assay. The electrocoagulator is designed to preconcentrate cyanobacteria from water samples prior to analysis via NanoGene assay. Using <i>Microcystis aeruginosa</i> laboratory culture and environmental samples (cell densities ranging from 1.7 × 10<sup>5</sup> to 4.1 × 10<sup>6</sup> and 6.5 × 10<sup>3</sup> to 6.6 × 10<sup>7</sup> cells·mL<sup>–1</sup>, respectively), the electrocoagulator was evaluated and compared with a conventional centrifuge. Varying the operation duration from 0 to 300 s with different cell densities was first investigated. Preconcentration efficiencies (obtained via absorbance measurement) and dry cell weight of preconcentrated cyanobacteria were then obtained and compared. For laboratory samples at cell densities from 3.2 × 10<sup>5</sup> to 4.1 × 10<sup>6</sup> cells·mL<sup>–1</sup>, the preconcentration efficiencies of electrocoagulator appeared to be stable at ∼60%. At lower cell densities (1.7 and 2.2 × 10<sup>5</sup> cells·mL<sup>–1</sup>), the preconcentration efficiencies decreased to 33.9 ± 0.2 and 40.4 ± 5.4%, respectively. For environmental samples at cell densities of 2.7 × 10<sup>5</sup> and 6.6 × 10<sup>7</sup> cells·mL<sup>–1</sup>, the electrocoagulator maintained its preconcentration efficiency at ∼60%. On the other hand, the centrifuge’s preconcentration efficiencies decreased to nondetectable and below 40%, respectively. This shows that the electrocoagulator outperformed the centrifuge when using eutrophic water samples. Finally, the compatibility of the electrocoagulator with the NanoGene assay was verified via the successful detection of the microcystin synthetase D (<i>mcyD</i>) gene in environmental samples. The viability of the electrocoagulator as an in situ compatible alternative to the centrifuge is also discussed

    Noninvasive Measurement of Membrane Potential Modulation in Microorganisms: Photosynthesis in Green Algae

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    Cell membrane potential (CMP) modulation is a physical measurement to quantitatively probe cell physiology in real time at high specificity. Electrochemical field effect transistors (eFETs) made from graphene and Si nanowire provide strong mechanical and electrical coupling with neurons and muscle cells to noninvasively measure CMP at high sensitivity. To date, there are no noninvasive methods to study electrophysiology of microorganisms because of stiff cell walls and significantly smaller membrane polarizations. An eFET made from the smallest possible nanostructure, a nanoparticle, with sensitivity to a single-electron charge is developed to noninvasively measure CMP modulation in algae. The applicability of the device is demonstrated by measuring CMP modulation due to a light-induced proton gradient inside the chloroplast during photosynthesis. The ∼9 mV modulation in CMP in algae is consistent with the absorbance spectrum of chlorophyll, photosynthetic pathway, and inorganic carbon source concentration in the environment. The method can potentially become a routine method to noninvasively study electrophysiology of cells, such as microorganisms for biofuels

    Noninvasive Measurement of Electrical Events Associated with a Single Chlorovirus Infection of a Microalgal Cell

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    Chlorovirus Paramecium bursaria chlorella virus 1 (PBCV-1) contains a viral-encoded K<sup>+</sup> channel imbedded in its internal membrane, which triggers host plasma membrane depolarization during virus infection. This early stage of infection was monitored at high resolution by recording the cell membrane depolarization of a single Chlorella cell during infection by a single PBCV-1 particle. The measurement was achieved by depositing the cells onto a network of one-dimensional necklaces of Au nanoparticles, which spanned two electrodes 70 μm apart. The nanoparticle necklace array has been shown to behave as a single-electron device at room temperature. The resulting electrochemical field-effect transistor (eFET) was gated by the cell membrane potential, which allowed a quantitative measurement of the electrophysiological changes across the rigid cell wall of the microalgae due to a single viral attack at high sensitivity. The single viral infection signature was quantitatively confirmed by coupling the eFET measurement with a method in which a single viral particle was delivered for infection by a scanning probe microscope cantilever

    Dex-IR induced H1650 cell cycle arrest.

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    <p>(A) Cell cycle analysis following drug treatment for 72 h. The bar graph of the results of the cell cycle analysis of drug-treated H1650 cells shows the percentages of cells in different phases of the cell cycle (G<sub>0</sub>/G<sub>1</sub>, S, and G<sub>2</sub>/M). The bar graph shows the mean ± SEM of three independent experiments (*<i>P</i> < 0.05 <i>vs</i>. G<sub>0</sub>/G<sub>1</sub> phase in the medium; <sup>#</sup><i>P</i> < 0.05 <i>vs</i>. S phase in the medium; and <sup>§</sup><i>P</i> < 0.05 <i>vs</i>. G2/M phase in the medium). (B) Cell lysates were analyzed by Immunoblot analysis using specific antibodies against the cyclins A2, D1, B1 and Phospho-Rb. Protein loading was normalized based on GAPDH. (C) Cells were treated with 400 nM nocodazole for 24 h, and then further treated with Dex or Dex-IR for 24 h. They were then harvested, and subjected to cell cycle analysis. The bar graph shows the mean ± SEM of three independent experiments (*P < 0.05 vs. G<sub>0</sub>/G<sub>1</sub> phase in the Nocodazole).</p

    Dex-IR inhibits the invasiveness of H1650 cells.

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    <p>(A) Invasion assay of H1650 cells treated with the drugs as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194341#pone.0194341.g001" target="_blank">Fig 1</a>. The Transwell invasion assay showed that Dex-IR suppressed the invasion of H1650 cells. Images were captured at a magnification of 400× (A, left panel). Scale bars, 100 μm. Graphical representation of the number of invasive H1650 cells per microscopic field. Each column and bar shows the SEM from three independent experiments (*<i>P</i> < 0.05 <i>vs</i>. vehicle) (A, right panel). (B) Inhibition of MMP9 activity in conditioned medium from H1650 cells treated with Dex-IR at the indicated concentration and incubated for 18 h was evaluated using gelatin zymography. Representative data from a single experiment are shown. The left lanes are standard markers. (C) qRT-PCR analysis of the MMP2, MMP9, integrin α2, and integrin α5 gene expression in cells 6 h after treatment with drugs. Each experiment was repeated three times and the results shown are representative of the three independent experiments. The bar graph shows the mean ± SEM of three independent experiments (*P < 0.05 vs. MMP2 expression in vehicle; <sup>#</sup>P < 0.05 vs. MMP9 expression in vehicle; <sup>§</sup>P < 0.05 vs. integrin α2 expression in vehicle).</p

    Ionizing-radiation-irradiated Dex (Dex-IR) inhibits the proliferation of non-small cell lung cancer (NSCLC) cells.

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    <p>(A) The chromatograms of Dex (top) and the fraction of crude extracts with Dex-IR (bottom). The chromatography conditions are given in the Materials and Methods. The arrows indicate the retention time of each peak. (B) Lung cancer cell lines were treated with increasing concentrations of Dex and Dex-IR for 24 h. The effects of Dex-IR at the indicated concentration on the viability of lung cancer cells were determined using the MTT assay and were compared with those of Dex-treated cells. Data are presented as the mean ± standard error of the mean (SEM) of three independent experiments (*<i>P</i> < 0.05). (C) H1650 cells were treated with vehicle (1% DMSO), Dex (100 ug/mL), Dex-IR (100 ug/mL), or doxorubicin (DOXO; 1 μM) for 72 h. DOXO was used as a positive control. Cells were observed using phase-contrast microscopy (40× magnification). The scale bar is 200 μm.</p

    Increased apoptotic cell death induced by Dex-IR in H1650 lung cancer cells.

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    <p>(A) Annexin V/propidium iodide double staining analysis of apoptosis in H1650 cells. H1650 cells were treated with Dex, Dex-IR, or DOXO as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194341#pone.0194341.g001" target="_blank">Fig 1</a> for 72 h. The bar graph shows the percentages of dead, living, early-apoptotic, and late-apoptotic cells according to treatment. Data are presented as the mean ± SEM of three independent experiments (*<i>P</i> < 0.05 <i>vs</i>. live cells in the medium; <sup>#</sup><i>P</i> < 0.05 <i>vs</i>. early apoptotic cells in the medium; and <sup>§</sup><i>P</i> < 0.05 <i>vs</i>. late apoptotic cells in the medium). (B) After treating H1650 cells with the same doses of the indicated drugs as described above for 24 h, the proteins were analyzed by Immunoblotting with antibodies against pro-Casp-3, cleaved Casp-3, PARP, and cleaved PARP. GAPDH was used to normalize the protein contents. (C) Dex-IR-induced apoptotic cells were detected by TUNEL assay (400× magnification). H1650 cells were treated with the drugs as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194341#pone.0194341.g001" target="_blank">Fig 1</a> for 12 h.</p

    Terpenylated Coumarins As SIRT1 Activators Isolated from <i>Ailanthus altissima</i>

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    Four new terpenylated coumarins (<b>1</b>–<b>4</b>) were isolated from the stem bark of <i>Ailanthus altissima</i> by bioactivity-guided fractionation using an in vitro SIRT1 deacetylation assay. Their structures were identified as (2′<i>R</i>,3′<i>R</i>)-7-(2′,3′-dihydroxy-3′,7′-dimethylocta-6′-enyloxy)-6,8-dimethoxycoumarin (<b>1</b>), 6,8-dimethoxy-7-(3′,7′-dimethylocta-2′,6′-dienyloxy)­coumarin (<b>2</b>), (2′<i>R</i>,3′<i>R</i>,6′<i>R</i>)-7-(2′,3′-dihydroxy-6′,7′-epoxy-3′,7′-dimethyloctaoxy)-6,8-dimethoxycoumarin (<b>3</b>), and (2′<i>R</i>,3′<i>R</i>,4′<i>S</i>,5′<i>S</i>)-6,8-dimethoxy-7-(3′,7′-dimethyl-4′,5′-epoxy-2′-hydroxyocta-6′-enyloxy)­coumarin (<b>4</b>). Compounds <b>1</b>–<b>4</b> strongly enhanced SIRT1 activity in an in vitro SIRT1-NAD/NADH assay and an in vivo SIRT1-p53 luciferase assay. These compounds also increased the NAD-to-NADH ratio in HEK293 cells. The present results suggest that terpenylated coumarins from <i>A. altissima</i> have a direct stimulatory effect on SIRT1 deacetylation activity and may serve as lead molecules for the treatment of some age-related disorders