43 research outputs found

    Table_1.XLSX

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    <p>Tuberculosis (TB) is a major comorbidity in HIV patients as well as a serious co-epidemic. Traditional detection methods are not effective or sensitive for the detection of Mycobacterium tuberculosis at the early stage. TB has become a major cause of lethal on HIV patients. We employed isobaric tags for relative and absolute quantitation (iTRAQ) technology to identify the different host responses in HIV-noTB and HIV-TB patients’ sera. Given the diversity of HIV subtypes, which results in a variety of host responses in different human populations, we focused on the Chinese patients. Of the 25 proteins identified, 7 were increased and 18 were decreased in HIV-TB co-infected patients. These proteins were found to be involved in host immune response processes. We identified a candidate protein, endoglin (ENG), which showed an 4.9 times increase by iTRAQ and 11.5 times increase by ELISA. ENG demonstrated the diagnostic efficacy and presented a novel molecular biomarker for TB in HIV-infected Chinese patients. This study provides new insight into the challenges in the diagnosis and effective management of patients with HIV-TB.</p

    Scheme of diethyl flavon7-yl phosphate (FP) synthesized by a simplified Atheron-Todd reaction.

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    <p>Scheme of diethyl flavon7-yl phosphate (FP) synthesized by a simplified Atheron-Todd reaction.</p

    Calculation of binding constants.

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    <p>A) Stern-Volmer plots of FP and CaM-Ca<sup>2+</sup>-PDE system at different temperature (25°C and 37°C). B) Line weaver-Burk plots of FP versus CaM-Ca<sup>2+</sup>-PDE at 25°C and 37°C; <i>k</i><sub>25°C</sub> = 6.24×10<sup>4</sup> M<sup>−1</sup> and <i>k</i><sub>37°C</sub> = 4.8×10<sup>4</sup> M<sup>−1</sup>, <i>K</i> represents binding constant.</p

    Effects of HF and FP on p21, caspase-3, and PARP in Hela cells by western blotting analyses.

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    <p>A) Histogram representing p21. B) Representative western blots of p21/Waf1 in FP, HF and control groups after 24 and 48 h. C) Histogram representing cleaved caspase-3 (19 kD) and cleaved PARP-1 (90 kD). D) Representative western blots of cleaved caspase-3 and cleaved PARP-I in the FP, HF and control groups after 24, 48 and 72 h. Total extracts obtained from HeLa cells without HF or FP treatment (Con), with 20 µM HF treatment (HF) and with 20 µM FP treatment (FP) were subjected to western blot analysis as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036652#s4" target="_blank">Materials and Methods</a>. The densitometry measurements represent the amount of p21, caspase 3, and PARP1 relative to β-actin and are given as means from triplicate experiments expressed as % of β-actin ± SE. β-actin was used as a loading control. *<i>p</i><0.001 versus control cells; #<i>p</i><0.01 versus control group; <sup>$</sup><i>p</i><0.01 versus FP group; <sup>&</sup><i>p</i><0.01 versus HF group.</p

    PCNA-immunoreactivity (IR) was detected by brownish staining in HeLa cells after treatment for 48 h, 1,000×.

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    <p>A) PCNA-IR integration values (IV) of each group after treatment for 24, 48 and 72 h were analyzed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036652#s4" target="_blank">Materials and Methods</a>. B) PCNA-IR was localized in the nuclei in the control group. C) PCNA-IR was attenuated in the FP group. D) PCNA-IR was attenuated and inactive PCNA-IR was also located in the cytoplasm in the HF group. Values are given as means ± SE. *<i>p</i><0.001 versus control cells; #<i>p</i><0.01 versus HF group; <sup>$</sup><i>p</i><0.01 versus 24 h FP group.</p

    Effects of FP/HF on cell cycle distribution.

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    <p>HeLa cells were treated with different concentrations of FP/HF for 24, 48, 72 and 96 h. Cells were harvested and stained with propidium iodide. Cell cycle distribution was analyzed using a BD FACSCanto II flow cytometer. The distributions of cells in G0/G1, S, and G2/M phases were determined using Modfit software. A) Representative cell cycle distributions after treatment with FP at 10, 20, 40 and 80 µM for 24 h. B) Representative cell cycle distributions after treatment with HF at 10, 20, 40 and 80 µM for 24 h. C) Representative cell cycle distributions after treatment with 20 µM FP/HF at 72 h and control at 24 and 72 h. D) G0/G1, S and G2/M population percentages in each group following treatment with 10, 20, 40 and 80 µM HF or FP for 24 h. E) G0/G1, S and G2/M population percentages in each group following treatment with 20 µM HF or FP and control group for 24, 48, 72 and 96 h.</p

    Effects of FP/HF on cell apoptosis.

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    <p>A) TUNEL staining for apoptosis in HeLa cells after treatment for 48 h in control, FP and HF groups; methyl green counter-stained, 1,000×. Control group, few apoptotic cells were found; FP group, more apoptotic cells were found. B) Quantitative analysis of apoptotic cells using Annexin V-FITC/PI double staining in exponentially growing HeLa cells. C) Apoptosis percentages in the different groups detected using the TUNEL method at 24, 48 and 72 h. D) Apoptosis percentages (upper right (UR)+lower right (LR)) in each group following treatment with 10, 20, 40 and 80 µM HF or FP detected by flow cytometry at 24 h. E) Apoptosis percentages (UR+LR) in each group analyzed by flow cytometry at 24, 48, 72 and 96 h. Flow cytometry was performed on 5×10<sup>5</sup> cells and the percentages of apoptotic, live, and dead cells were measured. Upper left (UL; Annexin V-FITC<sup>−</sup>/PI<sup>+</sup>) represents dead cells, upper right (UR; Annexin V-FITC<sup>+</sup>/PI<sup>+</sup>) represents late apoptotic plus necrotic cells, lower left (LL; Annexin V-FITC<sup>−</sup>/PI<sup>−</sup>) represents live cells, and lower right (LR; Annexin V-FITC<sup>+</sup>/PI<sup>−</sup>) represents early apoptotic cells. Data are given as means ± SD and are representative of three separate experiments. *<i>p</i><0.01 versus control cells; #<i>p</i><0.01 versus HF group; <sup>$</sup><i>p</i><0.01 versus 24 h FP group.</p

    cAMP levels in Hela cells in each group and PDE inhibition activities <i>in vitro</i>.

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    <p>A) cAMP concentrations in Hela cells in each group at 12 and 24 h. CON, control; HF, 20 µM HF group; FP, 20 µM FP group. B) Histogram representing inhibitory effects of FP and HP on PDE and CaM-activated PDE1 activity. C) Inhibitory effects of FP and HP on CaM-activated PDE1 activity. D) Inhibitory effects of FP and HP on PDE activity from bovine brain. Values are given as mean ± SE; RLU (relative light units) on the y axes of panels C and D is positively related with CaM-activated PDE1 or PDE activities. The IC<sub>50</sub> was calculated by using a sigmoidal dose-response (variable slope) equation analysis. <sup>&</sup><i>p</i><0.01 versus control group; *<i>p</i><0.001 versus HF group; <sup>#</sup><i>p</i><0.01 versus control group; <sup>$</sup><i>p</i><0.01 versus 24 h FP group.</p

    Electrospray ionization mass spectrum of CaM-Ca<sup>2+</sup> with FP.

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    <p>The solution was prepared by mixing equal volumes of a 0.4 mM methanol solution of FP and 0.04 mM CaM-Ca<sup>2+</sup>. •Multiply-charged ion peaks of CaM-Ca<sup>2+</sup>;•○ multiply-charged ion peaks of CaM-Ca<sup>2+</sup>-FP complex.</p

    Effect of PE on fluorescence spectra in different protein solutions.

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    <p>A) Effect of FP on fluorescence spectra of CaM. B) Effect of FP on fluorescence spectra of PDE; C) Enhanced fluorescence spectra of CaM and PDE in presence of Ca<sup>2+</sup>. D) Effect of FP on fluorescence spectra of CaM-Ca<sup>2+</sup>-PDE at 37°C. E) Effect of FP on fluorescence spectra of CaM-Ca<sup>2+</sup>-PDE at 25°C.</p
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