Alterations in the DNA methylation status and BMP-6 mRNA expression in HCC cell lines treated with 5′-aza-CdR.

<p>The BMP-6 locus became hypomethylated following 5′-aza-CdR treatment. In SMMC-7721 cells, the rate of DNA methylation prior to treatment was 95.38%, and this decreased to 32.31% after 3 days of treatment (A); In Hep3B cells, the rate of DNA methylation prior to treatment was 96.92%, and this decreased to 33.85% after 3 days of treatment (C). The levels of BMP-6 expression were upregulated following treatment with 5′-aza-CdR in SMMC-7721 cells (B) and Hep3B cells (D). The status of DNA methylation was evaluated with the BSP-based assay, and the expression level of BMP-6 was assessed by qRT-PCR. These results were analysed using the Student's <i>t</i>-test.</p

The BMP-6 promoter is hypermethylated, and BMP-6 is expressed at a low level in HCC tissues.

<p>(A) An MSP-based assay was used to detect BMP-6 promoter methylation in 6 HCC cell lines and normal liver tissues. No methylation of the BMP-6 promoter was observed in 5 normal liver tissues. (B) An MSP-based assay was used to detect BMP-6 promoter methylation in 60 pairs of clinical HCC tissues. BMP-6 promoter methylation was demonstrated in 65% of HCC tissues, whereas this rate in the adjacent non-cancerous tissue was 50%. (C) The Taqman probe-based MethyLight assay was used to detect BMP-6 promoter methylation in 114 pairs of clinical HCC tissues, and the level of DNA methylation was upregulated by 23.44 fold in HCC tissues compared to adjacent non-cancerous tissues. (D) qRT-PCR was used to detect BMP-6 mRNA expression in normal liver tissue and HCC cells. The HCC cell lines used are shown, and the normal liver tissue was the NL1 sample shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087994#pone-0087994-g004" target="_blank">Figure 4A</a>. All BMP-6 expression levels in HCC cell lines were lower than those in normal liver tissue. (E) qRT-PCR was used to detect the BMP-6 mRNA expression in 60 pairs of HCC clinical samples, containing both DNA and RNA samples. We obtained similar results as those in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087994#pone-0087994-g001" target="_blank">Figure 1A</a>. (F) The expression level of BMP-6 was correlated with BMP-6 promoter methylation. The level of BMP-6 DNA methylation obtained according to the MethyLight assay in 60 pairs HCC tissues is listed from lowest to highest. The 60 tissues with the lowest DNA methylation levels were placed into the hypomethylation group, whereas the 60 tissues with the highest DNA methylation levels were placed into the hypermethylation group. The HCC patients with BMP-6 hypermethylation had low levels of BMP-6 mRNA. The results were analysed using the Student's <i>t</i>-test.</p

The BMP-6 IHC staining intensity is downregulated in HCC tissue.

<p>IHC was performed in 75 pairs of HCC tissue arrays. (A) The staining intensity of BMP-6 in HCC tissues was downregulated by 46% compared to adjacent non-cancerous tissues. Additionally, the rate of upregulation was 7%. The staining intensity was confirmed by histopathology. (B) The staining intensity score was lower in HCC tissue compared to adjacent non-cancerous tissue. Sixteen samples of HCC tissue scored 1 or 1–2, whereas these scores were not observed in the adjacent non-cancerous tissue samples; furthermore, scores of 2–3 emerged 37 times in HCC tissue samples as compared to 56 times in adjacent non-cancerous tissue samples. (C) The BMP-6 staining intensity was downregulated, as assessed using Image-Pro 6.0 software. The staining intensity was downregulated to 83.14% in HCC tissues compared to adjacent non-cancerous tissues. (D) The representative image shows that the BMP-6 staining intensity was downregulated in relation to the rate of cell staining or the direct staining intensity. (E) The BMP-6 staining intensity likely correlated with poor prognosis; however, in 75 pairs of HCC tissues, only 14 pairs of HCC clinical samples had outcome data available.</p

Chiral 1,2-Cyclohexane-Bridged Bis-NHC Palladium Catalysts for Asymmetric Suzuki–Miyaura Coupling: Synthesis, Characterization, and Steric Effects on Enantiocontrol

The series of chiral 1,2-cyclohexane-bridged bis-N-heterocyclic carbene ligand precursors H₂[(1<i>R</i>,2<i>R</i>)-(<b>1a</b>–<b>i</b>)]Br with different substituent groups and their neutral and cationic diaqua palladium complexes, namely {Pd­[(1<i>R</i>,2<i>R</i>)-(<b>1a</b>–<b>i</b>)]­Br₂} (<b>2a</b>–<b>i</b>), {Pd­[(1<i>R</i>,2<i>R</i>)-(<b>1a</b>)]­X₂} (X = Cl (<b>3</b>), OAc (<b>4-OAc</b>), OC­(O)­CF₃ (<b>4-OCH­(O)­CF</b>_<b>3</b>)), and {Pd­[(1<i>R</i>,2<i>R</i>)-(<b>L</b>^<b>OMe</b>)]­(OH₂)₂}­X₂ (X = OTf (<b>5-OTf</b>), SbF₆ (<b>5-SbF</b>_<b>6</b>)) have been prepared in moderate to good yields. These chiral palladium complexes were fully characterized by elemental analysis, high-resolution mass spectra, ¹H and ¹³C NMR, and optical rotation determinations. The crystal structures of the chiral complexes <b>2a</b> and <b>5-OTf</b> were further confirmed to adopt a distorted-square-planar coordination geometry around the palladium center. The obtained chiral NHC-Pd compounds were able to catalyze the asymmetric Suzuki–Miyaura couplings of aryl halides with arylboronic acids in good yields (up to 96%) and moderate enantioselectivities (up to 64% ee). The coligand and steric effects were studied carefully. The coligands, including Br^–, Cl^–, AcO^–, CF₃COO^–, and water molecules, have little influence on the catalytic results. However, a strong steric effect of the two aromatic substituents R on the enantiocontrol has been proved in the catalytic asymmetric Suzuki–Miyaura coupling reaction. The highest enantioselectivity of 64% ee could be achieved under the standard reaction conditions

Overexpression of BMP-6 in HCC cell lines inhibits colony formation.

<p>(A) We cotransfected the BMP-6 overexpression construct into SMMC-7721 and Hep-1 cells, and BMP-6 overexpression was confirmed by Western blotting using anti-BMP-6 and anti-FLAG antibodies. Overexpressed BMP-6 inhibited colony formation in SMMC-7721 (B) and Hep-1 cells (C); however, BMP-6 overexpression did not influence cell growth in these 2 cell lines (D and E).</p

MOESM1 of Extrahepatic cytochrome P450s play an insignificant role in triptolide-induced toxicity

Additional file 1. Minimum standards of reporting checklist

The low expression level of BMP-6 in HCC tissues is associated with a poor prognosis.

<p>(A) We analysed the expression levels of BMP-6 mRNA in 88 pairs of HCC tissues and found an 85.85% decrease in the expression level of BMP-6 in HCC tissues compared to adjacent non-cancerous tissues [<i>p</i> = 0.0005 (paired <i>t</i> test)]. BMP-6 expression was downregulated in 84.09% of these 88 pairs of tissues. (B) Downregulation of BMP-6 was associated with poor prognosis. BMP-6 expression in HCC tissues was grouped from lowest to highest; the 44 HCC tissues with the lowest BMP-6 expression levels were placed into the low-expression group, whereas the 44 HCC tissues with the highest BMP-6 expression levels were placed into the high-expression group. (C) Downregulation of BMP-6 was associated with a high AFP level.</p

The HCC cell mix showed hypermethylation of the BMP-6 promoter and low mRNA expression of BMP-6.

<p>(A) BMP-6 is located on chromosome 6, and there is a CpG island in the promoter of BMP-6 with a DNA methylation signal. The DNA methylation signal of the BMP-6 gene body was decreased in the HCC cell mix compared to normal liver tissue. (B) Enlarged region of the CpG island within the BMP-6 promoter. There was a significant DNA methylation signal in the HCC cell mix but not in normal liver tissue. (C) Hypermethylation of the BMP-6 promoter in HCC cells was detected according to the BSP-based assay. The levels of DNA methylation were 4.62% and 93.85% in normal liver tissue and the HCC cell mix, respectively. BMP-6 expression levels in normal liver tissue and the HCC cell mix were analysed by qRT-PCR. The BMP-6 expression level was analysed in each sample 3 times, and then the BMP-6 expression level in the HCC cell mix was analysed in 6 cell lines 3 times followed by calculation of the average value from the 6 cell lines. All mRNA expression levels were normalised to actin mRNA expression levels. These results were analysed using Student's <i>t</i>-test.</p

MOESM3 of Prognostic impact of HbA1c variability on long-term outcomes in patients with heart failure and type 2 diabetes mellitus

Additional file 3: Figure S2. Kaplan–Meier curves of freedom from all-cause mortality (A, B) and composite endpoints (C, D) for low and high HbA1c variability after 42-month follow-up in HFmrEF. The numbers at the bottom of the figure are “number at risk”

Exchange Processes in Shibasaki’s Rare Earth Alkali Metal BINOLate Frameworks and Their Relevance in Multifunctional Asymmetric Catalysis

Shibasaki’s rare earth alkali metal BINOLate (REMB) catalysts (REMB; RE = Sc, Y, La – Lu; M = Li, Na, K; B = 1,1-bi-2-naphtholate; RE/M/B = 1/3/3) are among the most successful enantioselective catalysts and have been employed in a broad range of mechanistically diverse reactions. Despite the phenomenal success of these catalysts, several fundamental questions central to their reactivity remain unresolved. Combined reactivity and spectroscopic studies were undertaken to probe the identity of the active catalyst(s) in Lewis-acid (LA) and Lewis-acid/Brønsted-base (LA/BB) catalyzed reactions. Exchange spectroscopy provided a method to obtain rates of ligand and alkali metal self-exchange in the RE/Li frameworks, demonstrating the utility of this technique for probing solution dynamics of REMB catalysts. Isolation of the first crystallographically characterized REMB complex with substrate bound enabled stoichiometric and catalytic reactivity studies, wherein we observed that substrate deprotonation by the catalyst framework was necessary to achieve selectivity. Our spectroscopic observations in LA/BB catalysis are inconsistent with previous mechanistic proposals, which considered only tris­(BINOLate) species as active catalysts. These findings significantly expand our understanding of the catalyst structure in these privileged multifunctional frameworks and identify new directions for development of new catalysts