Simple Solution Plasma Synthesis of Hierarchical Nanoporous MnO₂ for Organic Dye Removal

We have demonstrated a simple and green approach to synthesize hierarchical nanoporous MnO₂ by applying plasma in a liquid precursor; the approach is named the “solution plasma process (SPP).” Three types of sugar, i.e., glucose, fructose, and sucrose, were used as inducers for the nanoporous MnO₂ formation (hereafter called G-MnO₂, F-MnO₂, and S-MnO₂). These were successfully synthesized within a few minutes (7–19 min) under ambient conditions. It was confirmed that the generated numerous reactive species (e.g., electrons, radicals, and ions) accelerated the reduction of MnO₄^–. The reaction rate as well as the physical and chemical features of resulting products were found to be related to the type of sugars. Their high surface areas (F-MnO₂ (169.1 m²·g^–1) > G-MnO₂ (141.0 m²·g^–1) > S-MnO₂ (85.5 m²·g^–1)) provided efficient capability for the adsorption of cationic dye molecules, i.e., methylene blue. The dye removal efficiencies of all samples were >99% for an initial dye concentration (<i>C</i>₀) of 10 mg·L^–1 within 2 min and >82% for <i>C</i>₀ = 50 mg·L^–1 within 30 min. We expect that the synthesis route presented in this study can be extended to the large-scale production of effective adsorbents and to find practical applications for the industrial and green infrastructure

The Impact of Polymerization Chemistry on the Mechanical Properties of Poly(dimethylsiloxane) Bottlebrush Elastomers

We compare the low-strain mechanical properties of bottlebrush elastomers (BBEs) synthesized using ring-opening metathesis and free radical polymerization. Through comparison of experimentally measured elastic moduli and those predicted by an ideal, affine model, we evaluate the efficiency of our networks in forming stress-supporting strands. This comparison allowed us to develop a structural efficiency ratio that facilitates the prediction of mechanical properties relative to polymerization chemistry (e.g., softer BBEs when polymerizing under dilute conditions). This work highlights the impact that polymerization chemistry has on the structural efficiency ratio and the resultant mechanical properties of BBEs with identical side chains, providing another “knob” by which to control polymer network properties

Differences in the Epigenetic Regulation of Cytochrome P450 Genes between Human Embryonic Stem Cell-Derived Hepatocytes and Primary Hepatocytes

<div><p>Human pluripotent stem cell-derived hepatocytes have the potential to provide <i>in vitro</i> model systems for drug discovery and hepatotoxicity testing. However, these cells are currently unsuitable for drug toxicity and efficacy testing because of their limited expression of genes encoding drug-metabolizing enzymes, especially cytochrome P450 (CYP) enzymes. Transcript levels of major <i>CYP</i> genes were much lower in human embryonic stem cell-derived hepatocytes (hESC-Hep) than in human primary hepatocytes (hPH). To verify the mechanism underlying this reduced expression of <i>CYP</i> genes, including <i>CYP1A1</i>, <i>CYP1A2</i>, <i>CYP1B1</i>, <i>CYP2D6</i>, and <i>CYP2E1</i>, we investigated their epigenetic regulation in terms of DNA methylation and histone modifications in hESC-Hep and hPH. CpG islands of <i>CYP</i> genes were hypermethylated in hESC-Hep, whereas they had an open chromatin structure, as represented by hypomethylation of CpG sites and permissive histone modifications, in hPH. Inhibition of DNA methyltransferases (DNMTs) during hepatic maturation induced demethylation of the CpG sites of <i>CYP1A1</i> and <i>CYP1A2</i>, leading to the up-regulation of their transcription. Combinatorial inhibition of DNMTs and histone deacetylases (HDACs) increased the transcript levels of <i>CYP1A1</i>, <i>CYP1A2</i>, <i>CYP1B1</i>, and <i>CYP2D6</i>. Our findings suggest that limited expression of <i>CYP</i> genes in hESC-Hep is modulated by epigenetic regulatory factors such as DNMTs and HDACs.</p></div

Bioimaging of Hyaluronate–Interferon α Conjugates Using a Non-Interfering Zwitterionic Fluorophore

We conducted real-time bioimaging of the hyaluronate–interferon α (HA–IFNα) conjugate using a biologically inert zwitterionic fluorophore of ZW800-1 for the treatment of hepatitis C virus (HCV) infection. ZW800-1 was labeled on the IFNα molecule of the HA–IFNα conjugate to investigate its biodistribution and clearance without altering its physicochemical and targeting characteristics. Confocal microscopy clearly visualized the effective <i>in vitro</i> cellular uptake of the HA–IFNα conjugate to HepG2 cells. After verifying the biological activity in Daudi cells, we conducted the pharmacokinetic analysis of the HA–IFNα conjugate, which confirmed its target-specific delivery to the liver with a prolonged residence time longer than that of PEGylated IFNα. <i>In vivo</i> and <i>ex vivo</i> bioimaging of the ZW800-1-labeled HA–IFNα conjugate directly showed real-time biodistribution and clearance of the conjugate that are consistent with the biological behaviors analyzed by an enzyme-linked immunosorbent assay. Furthermore, the elevated level of OAS1 mRNA in the liver confirmed <i>in vivo</i> antiviral activity of HA–IFNα conjugates. With the data taken together, we could confirm the feasibility of ZW800-1 as a biologically inert fluorophore and target-specific HA–IFNα conjugate for the treatment of HCV infection

DNA methylation and histone modifications in regulatory regions of <i>CYP</i> genes.

<p>Each diagram shows the locations of the sites of <i>CYP1A1</i> (A), <i>CYP1A2</i> (B), <i>CYP1B1</i> (C), <i>CYP2D6</i> (D), and <i>CYP2E1</i> (E) within gene promoters, which were examined by bisulfite sequencing and chromatin immunoprecipitation (ChIP). The methylation status of CpG dinucleotides in target regions was examined in hPH and hESC-Hep (day 20) by bisulfite sequencing (upper panel). Each row represents the methylation status of each CpG in one bacterial clone. A series of 9–10 clones is shown. Black circles represent metyhylated CpG sites while white circles represent unmethylated CpG sites. Numbers indicate nucleotide positions in relation to the transcription start site (TSS, +1). ChIP analysis of histone modifications including two active histone marks H3Ac and H3K4me3 and one repressive histone mark H3K27me3 in hPH and hESC-Hep is shown (lower graph). Data validated by real-time PCR are presented as fold enrichment of precipitated DNA associated with a given histone modification relative to a 100-fold dilution of input chromatin. Data represent mean ± SD from two independent experiments. * p<0.05, ** p<0.01, *** p<0.001, significant values in comparison with hPH (t-test followed by Wilcoxon matched pairs test).</p

Gene expression levels of drug-metabolizing enzymes.

<p>Expression of genes encoding nuclear receptors (A), phase I enzymes (B), phase II enzymes (C), and phase III transporters (D) was examined by real-time RT-PCR in human primary hepatocytes (hPH) and human embryonic stem cell-derived hepatocytes (hESC-Hep, day 20). Data represent mean ± SD from three independent experiments. * p<0.05, ** p<0.01, significant values in comparison with hPH (t-test followed by Wilcoxon matched pairs test).</p

Transcriptional regulation of <i>CYP</i> genes by inhibition of DNA methyltransferases (DNMTs) and histone deacetylases (HDACs) during hepatic differentiation.

<p>(A) Methylation frequencies in the promoter and gene body regions of <i>CYP</i> genes were examined by bisulfite sequencing in hESC-Hep treated with DMSO or a DNMT inhibitor (DAC or RG108). Data represent methylation frequencies from two independent experiments. (B) Expression levels of <i>CYP</i> genes were examined by real-time RT-PCR in hESC-Hep treated with DMSO, DAC, or RG108. Data represent mean ± SD from three independent experiments. * p<0.05, ** p<0.01, significant values in comparison with DMSO (ANOVA followed by Dunn’s multiple comparison test). (C) Expression levels of <i>CYP</i> genes were examined by real-time RT-PCR in hESC-Hep treated with DMSO or a HDAC inhibitor (1 mM sodium butyrate (SB)) with or without a DNMT inhibitor (DAC or RG108). Data represent mean ± SD from three independent experiments. * p<0.05, ** p<0.01, *** p<0.001, significant values in comparison with DMSO (ANOVA followed by Bonferroni’s multiple comparison test).</p

IL-17 production via direct interaction of CD40 and CD40L increases STAT3 activation and the proliferation of MDA-MD231 cells.

<p>(A) MDA-MB231 cells and activated T cells were directly co-cultured at the ratio of 1:5 for 24 hrs in the presence of anti-IL-17 neutralizing antibody or anti-CD40 neutralizing antibody (2 μg/ml/each) on 96-well plate, and then cells were cultured for 24 hrs. After the addition of 1 μCi/mL of [³H]-thymidine, cells were culture for another 18 hrs. And the proliferation of cells was measured as described in <i>Materials and Methods</i>. Data represents mean ± S.D. The assay was performed in quadruplicate and result is the representative of three independent experiments. (B) MDA-MB231 cells were cultured in the presence of recombinant IL-17 (rIL-17, 50 ng/ml) for 15, 30 and 60 min. In addition, cells were cultured with direct co-culture supernatant of MDA-MB231 cells and activated T cells in the presence or absence of anti-IL-17 neutralizing antibody (nAb). And then, the activation of STAT3 was examined by western blot as described in <i>Materials and Methods</i>. Lane 1: Control, Lane 2: rIL-17 (50 ng/ml), Lane 3: Direct co-culture supernatant of MDA-MB231 cells and activated T cells, Lane 4: Direct co-culture supernatant of MDA-MB231 cells and activated T cells with IL-17 nAb, Lane 5: Indirect co-culture supernatant of MDA-MB231 cells and activated T cells. β-actin was used as a loading control. Result is the representative of three independent experiments. (C) MDA-MB231 cells and activated T cells were directly co-cultured at the ratio of 1:5 for 24 hrs in the presence of 20 μM of AG490 (STAT3 inhibitor) or anti-IL-17 nAb (2 μg/ml) on 96-well plate, and then cells were cultured for 24 hrs. After the addition of 1 μCi/mL of [³H]-thymidine, cells were culture for another 18 hrs. Direct co-culture supernatant of CD40-non expressing breast cancer cell line, Hs578T and activated T cells was used as a negative control. The proliferation of cells was measured as described in <i>Materials and Methods</i>. Data represents mean ± S.D. The assay was performed in quadruplicate and result is the representative of three independent experiments.</p

CD40 ligand stimulation on the activated T cells induces Th17 differentiation.

<p>(A) Human T cells (2.5x10⁶/ml) purified from human PBMCs were activated as described in <i>Materials and Methods</i>. After increase of CD40L expression on activated T cells was confirmed by flow cytometry analysis, they were co-cultured with MDA-MB231 cells in the same well on 6-well plate at the ratio of 5:1 for 24 hrs. Th17 differentiation was confirmed by staining of intracellular IL-17 in CD4⁺ T cells with Alexa Fluor 647-conjugated anti-human IL-17A antibody (1 μg/ml) and FITC-conjugated anti-human CD4 antibody (1 μg/ml). To confirm the role of CD40 on Th17 differentiation, the interaction between CD40 and CD40L was interfered with the addition of anti-CD40 neutralizing antibody (2 μg/ml) for 1 hr. Result is the representative of three independent experiments. (B-D) Culture supernatant from the experiment described in Fig 3A were collected and the amount of IL-1β (B), IL-6 (C) and IL-21 (D) were measured by ELISA, according to manufacturer’s instruction. Data represents mean ± S.D. Result is the representative of three independent experiments and each experiment was performed in triplicate. *p < 0.05. (E) Human T cells (2.5x10⁶/ml) purified from human PBMCs were activated as described in <i>Materials and Methods</i>. After increase of CD40L expression on activated T cells was confirmed by flow cytometry analysis, activated T cells were stimulated by anti-CD40L agonistic antibody (1 μg/ml) or isotype (1 μg/ml) for 24 and 36 hrs. The expression of ROR gamma t (RORγt), the orphan nuclear receptor for IL-17 transcription and Th17 differentiation was determined by intracellular flow cytometry analysis, after staining with PE-conjugated anti- RORγt antibody (1 μg/ml) and PE-conjugated Rat IgG (1 μg/ml) as described in <i>Materials and Methods</i>. Result is the representative of three independent experiments.</p

TGF-β production is up-regulated by CD40 stimulation in a breast cancer cell line, MDA-MB231.

<p>(A) The breast cancer cell lines, MDA-MB231 and Hs578T were collected at continuous log phase of growth. The expression of CD40 was examined by staining with PE-conjugated anti-human CD40 antibody (1 μg/ml) as described in <i>Materials and Methods</i>. Result is the representative of three independent experiments. (B) MDA-MB231 cells (2.5 x 10³/well) were stimulated with the various concentrations of anti-CD40 agonistic antibody (2, 4, 8, and 16 μg/ml) and the same amount of isotype antibody for 1 hr on 96-well plate, and then cells were cultured for 24 hrs. After the addition of 1 μCi/mL of [³H]-thymidine, cells were culture for another 18 hrs. And then, the proliferation of cells by CD40 stimulation was measured as described in <i>Materials and Methods</i>. Data represents mean ± S.D. The assay was performed in quadruplicate and result is the representative of three independent experiments. There was no statistical significance among groups. (C and D) The expression of TGF-β mRNA transcript in MDA-MB231 cells by CD40 stimulation was investigated with semi-quantitative RT-PCR (C) and quantitative real-time PCR (D). Cells (1.5 x 10⁵/well) were stimulated 2 μg/ml of anti-CD40 agonistic antibody for 3, 6, 9 and 12 hrs on 6-well plate. Cells were harvested and total RNA was extracted with Trizol, according to manufacturer’s instruction. After quantification, 1μg of RNA was converted to cDNA by reverse transcriptase. And then RT-PCR and real-time PCR were performed with specific primers for TGF-β as described in <i>Materials and Methods</i>. β-actin or GAPDH was used as a loading control. ***p < 0.001. (E) CD40 on MDA-MB231 cells (1.5 x 10⁵/well) were stimulated with anti-CD40 agonistic antibody (2 and 4 μg/ml) and soluble CD40L (3 μg/ml) for 1 hr, and then cells were cultured for 24 hrs on 6-well plate. The amount of TGF-β in the culture supernatant was measured by ELISA, according to manufacturer’s instruction. Data represents mean ± S.D. Result is the representative of three independent experiments and each experiment was performed in triplicate. **p < 0.01 vs. untreated control. (F) Cells (1.5 x 10⁵/well) were seeded on 6-well plate and then transfected with 20 nM of CD40 siRNA and control siRNA in the mixture of serum free media and oligofectamine as described in <i>Materials and Methods</i>. After 72 hrs, the down-regulation of CD40 expression on MDA-MB231 cells by CD40 siRNA transfection was confirmed by flow cytometry analysis. And then, cells (1.5 x 10⁵/well) were stimulated with anti-CD40 agonistic antibody (2 μg/ml) and soluble CD40L (3 μg/ml) for 1 hr, and then cells were cultured for 24 hrs on 6-well plate. And then, the amount of TGF-β in the culture supernatant was measured by ELISA. Data represents mean ± S.D. Result is the representative of three independent experiments and each experiment was performed in triplicate. ***p < 0.001 vs. untreated control.</p