Drug resistance analysis before and after second-line treatment.

<p><b>*</b>A score >60 get from Stanford University drug resistance database was regarded as high drug resistance.</p><p>Drug resistance analysis before and after second-line treatment.</p

CD4 count, viral load and viral inhibition rates before, 6 months after, and 12 months after drug switch.

<p>CD4 count, viral load and viral inhibition rates before, 6 months after, and 12 months after drug switch.</p

General information of subjects before drug switch.

<p>General information of subjects before drug switch.</p

Resistance to 3TC and TDF before and after drug switch.

<p>Resistance to 3TC and TDF before and after drug switch.</p

Practical Asymmetric Hydrogenation-Based Synthesis of a Class-Selective Histone Deacetylase Inhibitor

Two syntheses of the class-selective histone deacetylase inhibitor <b>1</b> are reported. In the first, eight-step entailing synthesis, the key transformations were a highly efficient [3 + 2] dipolar cycloaddition affording <i>trans</i>-<i>rac</i>-<b>5</b> and its resolution. In the second, asymmetric approach, the key steps were a highly selective asymmetric hydrogenation to produce the <i>cis</i>-(<i>S,S</i>)-3,4-disubstituted pyrrolidine <b>18</b> followed by an amide formation with simultaneous chiral inversion of the carboxy stereocenter to generate the key intermediate <i>trans</i>-(<i>R,S</i>)-3,4-disubstituted pyrrolidine <b>19</b>. The overall yield increased from ∼6% for the resolution approach to ∼26% for the enantioselective approach

Efficacy of <i>S-</i>(-)equol in protecting against oxidative stress-induced toxicity through Nrf2.

<p>EA.hy926 cells that were pretreated with 100<i>S-</i>(-)equol or sham treated for 24 h were exposed to H₂O₂ (100–800 µM) (A) or tBHP (20–80 µM) (B) in the presence or absence of 100 nM <i>S-</i>(-)equol for 24 h. HUVECs were pretreated with 100 nM <i>S-</i>(-)equol or sham treated for 24 h were exposed to H₂O₂ (100 µM) (C). Cell survival after oxidative stress was measured using a CCK-8 assay. The data are reported as the mean ± SD, n = 6. (D) Cells untreated or pretreated with 250 nM <i>S-</i>(-)equol for 24 h and then treated with H₂O₂ for another 24 h. Apoptotic cell death was detected using Annexin V-FITC and flow cytometry. The mean ± SD was calculated from three independent experiments. (E) Apoptotic cells were visualized by TUNEL. Immunoblot analysis showing Nrf2 protein in EA.hy926 cells (F, right) or HUVECs (G, right) transfected with a control or Nrf2-siRNA. Nrf2 protein was measured with immunoblot analysis using an anti-Nrf2 antibody to confirm knockdown of Nrf2 expression. (F and G) Untransfected cells and cells transfected with indicated siRNAs were incubated with 100 nM <i>S-</i>(-)equol for 24 h in the presence of 650 µM H₂O₂ or with H₂O₂ alone. The apoptotic index for each sample was determined using a cell death ELISA kit. The data are reported as the mean ± SD, n = 3 (*<i>p</i><0.05, **<i>p</i><0.01, ***<i>p</i><0.001 compared with control group, ^#<i>p</i><0.05 compared with <i>S-</i>(-)equol treated group).</p

Identification of <i>S-</i>(-)equol as an Nrf2 activator.

<p>Where indicated, EA.hy926 cells or HUVECs were transfected with plasmids containing an ARE-dependent firefly luciferase reporter gene (<i>F</i>) and the renilla firefly luciferase gene (<i>R</i>), the latter of which was included in each transfection reaction as a control for transfection efficiency. Medium was changed after 6 hours of incubation. (A) Cells were treated with <i>S-</i>(-)equol (10 nM, 50 nM, and 250 nM) or tBHQ (50 µM) for 16 h. (B) Cells were treated with <i>S-</i>(-)equol (250 nM) and tBHQ (50 µM) for the time periods indicated. The potency of induction is expressed as the relative luminescence (R/F) of the treated samples over the untreated transfected controls (mean ± SD, n = 3). *<i>p</i><0.05.</p

The effects of PI3K/Akt and ER inhibitors on <i>S-</i>(-)equol-induced Nrf2 activation in endothelial cells.

<p>(A) EA.hy926 cells or HUVECs were transfected with ARE-dependent firefly luciferase reporter gene (<i>F</i>) and the renilla firefly luciferase gene (<i>R</i>), and treated with 250 nM <i>S-</i>(-)equol in the presence or absence of 10 µM LY294002 or 100 nM ICI182,780 for 16 h. The potency of induction is expressed as the relative luminescence (R/F) measured using the dual luciferase reporter assay system (mean ± SD, n = 3). *<i>p</i><0.05 versus with control group, ^#<i>p</i><0.05 versus with <i>S-</i>(-)equol treated group. (B) EA.hy926 cells or HUVECs were incubated with or without 10 µM LY294002 or 100 nM ICI182,780 for 30 min and then with or without 250 nM <i>S-</i>(-)equol or 500 nM daidzein for 24 h. Whole cell lysates were then immunoblotted with antibodies against Nrf2, HO-1, NQO1, and β-tubulin. Values are means of three independent experiments with standard deviations represented by vertical bars. Mean values were significantly different compared with controls (*<i>p</i><0.05). (C) EA.hy926 cells or HUVECs cotransfected with the HA-Nrf2 and renilla firefly luciferase expression plasmids were treated with 250 nM <i>S-</i>(-)equol in the presence or absence of 10 µM LY294002 or 100 nM ICI182,780 for 16 h, and HA-Nrf2 localization was analyzed by confocal microscopy. (D) EA.hy926 cells were incubated with 250 nM <i>S-</i>(-)equol, 500 nM daidzein with or without 10 µM LY294002 or 100 nM ICI182,780 for 16 h and then immunostained with an antibody against Nrf2, and Nrf2 localization was analyzed by confocal microscopy. Values (intensity of nuclear versus cytoplasmic) are means of counting 100 cells with standard deviations represented by vertical bars. *<i>p</i><0.05 versus with control group, ^#<i>p</i><0.05 versus with <i>S-</i>(-)equol treated group.</p

<i>S-</i>(-)equol primarily up-regulated Nrf2 protein expression activating ARE-dependent response.

<p>(A) Whole EA.hy926 cells lysates of cells treated with 250 nM <i>S-</i>(-)equol, 500 nM daidzein, or 50 µM tBHQ for 24 h were subjected to immunoblot analysis with anti-Nrf2, anti-HO-1, anti-NQO1, and anti-β-tubulin antibodies. (B) Total HUVECs lysates of cell treated with 250 nM <i>S-</i>(-)equol for 24 h were immunoblotted with anti-Nrf2 and anti-β-tubulin antibodies. (C) EA.hy926 cells were transfected with plasmids containing an HA-tagged Nrf2 open reading frame and the renilla firefly luciferase gene. Medium was changed after 6 hours of incubation. Where indicated, extracts from HA-Nrf2 EA hy.92 cells were additionally probed with anti-HA antibody, anti-Nrf2, anti-HO-1, anti-NQO1 and anti-β-tubulin antibodies. Values are means of three independent experiments with standard deviations represented by vertical bars. Mean values were significantly different compared to controls (*<i>p</i><0.05). Total RNA was extracted from EA.hy926 cells or HUVECs treated as indicated, and Nrf2 (D), HO-1 (E), and NQO1 (F) mRNA was measured by real-time RT-PCR analysis. The values shown represent the mean ± SD obtained for three independent experiments (*<i>p</i><0.05).</p

ERβ plays a role in activation of Nrf2 by <i>S-</i>(-)equol in endothelial cells.

<p>(A) EA.hy926 cells were transfected with ARE-dependent firefly luciferase reporter gene (<i>F</i>) and the renilla firefly luciferase gene (<i>R</i>), and treated with 100 nM <i>S-</i>(-)equol in the presence or absence of 10 nM DPN, 10 nM PPT, 100 nM MPP or 100 nM PHTPP for 2 h following further incubation with or without 100 nM <i>S-</i>(-)equol. The potency of induction is expressed as the relative luminescence (R/F) measured using the dual luciferase reporter assay system (mean ± SD, n = 6). *<i>p</i><0.05 versus with control group, ^#<i>p</i><0.05 versus with <i>S-</i>(-)equol treated group. (B) EA.hy926 cells were incubated with 0.1% DMSO (1), 10 nM PPT (3), or 100 nM <i>S-</i>(-)equol (5) for 16 h and then immunostained with an antibody against ERα; and cells also were incubated with 0.1% DMSO (2), 10 nM PPT (4), or 100 nM <i>S-</i>(-)equol (6) for 16 h and then immunostained with an antibody against ERβ, then analyzed by confocal microscopy. (C) Cells were treated with 100 nM <i>S-</i>(-)equol in the presence or absence of 10 nM DPN, 10 nM PPT, 100 nM MPP or 100 nM PHTPP for 2 h following further incubation with or without 100 nM <i>S-</i>(-)equol, and Nrf2 localization was analyzed by confocal microscopy. Values (intensity of nuclear versus cytoplasmic) are means of counting 100 cells with standard deviations represented by vertical bars. *<i>p</i><0.05 versus with control group, ^#<i>p</i><0.05 versus with <i>S-</i>(-)equol treated group.</p