Additional file 1: of Hpa1 is a type III translocator in Xanthomonas oryzae pv. oryzae

Table S1. Strains and plasmids used and created in this study. Table S2. Information on genes tested and primers used in this study. (DOCX 35 kb

miR-27a protects human mitral valve interstitial cell from TNF-α-induced inflammatory injury via up-regulation of NELL-1

<div><p>MicroRNAs (miRNAs) have been reported to be associated with heart valve disease, which can be caused by inflammation. This study aimed to investigate the functional impacts of miR-27a on TNF-α-induced inflammatory injury in human mitral valve interstitial cells (hMVICs). hMVICs were subjected to 40 ng/mL TNF-α for 48 h, before which the expressions of miR-27a and NELL-1 in hMVICs were altered by stable transfection. Trypan blue staining, BrdU incorporation assay, flow cytometry detection, ELISA, and western blot assay were performed to detect cell proliferation, apoptosis, and the release of proinflammatory cytokines. We found that miR-27a was lowly expressed in response to TNF-α exposure in hMVICs. Overexpression of miR-27a rescued hMVICs from TNF-α-induced inflammatory injury, as cell viability and BrdU incorporation were increased, apoptotic cell rate was decreased, Bcl-2 was up-regulated, Bax and cleaved caspase-3/9 were down-regulated, and the release of IL-1β, IL-6, and MMP-9 were reduced. NELL-1 was positively regulated by miR-27a, and NELL-1 up-regulation exhibited protective functions during TNF-α-induced cell damage. Furthermore, miR-27a blocked JNK and Wnt/β-catenin signaling pathways, and the blockage was abolished when NELL-1 was silenced. This study demonstrated that miR-27a overexpression protected hMVICs from TNF-α-induced cell damage, which might be via up-regulation of NELL-1 and thus modulation of JNK and Wnt/β-catenin signaling pathways.</p></div

Trend of OD₂₆₀/OD₂₈₀ in collaborative calibration study for <i>E. coli</i> DNA reference standard.

<p>Trend of OD₂₆₀/OD₂₈₀ in collaborative calibration study for <i>E. coli</i> DNA reference standard.</p

Results of real-time PCR for <i>E. coli</i> DNA reference standard.

<p>(A) Standard curve. Ct was plotted against each concentration (Log Co). Ct represents the cycle at which reporter signal was first detected. (B) Amplification curves. 1–6: 10², 10, 1, 1×10⁻¹, 1×10⁻², 1×10⁻³ pg/μL. (C) Melting curves. It showed primer pair was specific for <i>E. coli</i> DNA.</p

Preparation and quality control of <i>E. coli</i> DNA reference standard.

<p>(A) Analysis of <i>E. coli</i> genomic DNA with full wavelength scanning (240–350 nm). (B) Results of Agarose Gel electrophoresis for <i>E. coli</i> genomic DNA. M, λ-Hind III digest DNA marker. Lane 1, <i>E. coli</i> genomic DNA.</p

Comparison between 293GCAC3-based test and rabbit aortic strips test (RAST).

<p>The new 293GCAC3 assay and traditional RAST assay were conducted in five separate runs to compare sensitivity, precision and variability. The EC₅₀ values determined by two assays were found to be similar (unpaired t test, p = 0.8582). However, For the new assay, the RSD was just 13.1%, with the 95% CI being 5.387 ng/mL to 7.359 ng/mL, while for RAST, RSD was 65.9%, with the 95% CI falling between 1.098 ng/mL to 10.98 ng/mL.</p

The consistency analysis of <i>E. coli</i> DNA reference standard using PicoGreen fluorescence method.

<p>The consistency analysis of <i>E. coli</i> DNA reference standard using PicoGreen fluorescence method.</p

The calibration results of <i>E. coli</i> DNA content by UV absorption (μg/mL).

<p>Results, expressed in μg/mL, were obtained from six laboratories using UV absorption method. At least 15 independent tests were performed in each laboratory. Geometric means of each laboratory and overall geometric means with 95% confidence interval were calculated. Variability within laboratory and overall calibration were expressed as %CV.</p

Specifity of the assay.

<p><b>Panel A</b>. Effect of guanylin on 293GCAC3. Guanylin is also a little peptide which could stimulate the increase of cGMP by binding GC-C. The starting concentration of rhBNP and guanylin was 2 µg/mL, serially diluted by 4 times, with the effects of these two peptides on cGMP production tested simultaneously. The data showed the guanylin had no effect on 293GCAC3 compared with rhBNP. Each point illustrates the mean of 3 replicates. <b>Panel B</b>. The effects of neutralizing antibody on rhBNP-induced cGMP production. rhBNP (0.5 µg/mL) was pre-incubated with rhBNP anti-serum or negative serum of serial dilutions (1∶10⁷−1:10²) at 37°C for 60 minutes. Afterwards, the mixture was analyzed for its ability of stimulating cGMP production. The results indicated that rhBNP-induced cGMP production was inhibited by the neutralizing antibody in a dose-responsive fashion. Each point illustrates the mean of 3 replicates.</p

Development of a Reference Standard of <i>Escherichia coli</i> DNA for Residual DNA Determination in China

<div><p>This collaborative study developed the first national <i>Escherichia coli</i> (<i>E. coli</i>) DNA reference standard for standardizing quantitative residual DNA assay methods, fluorescence dye (PicoGreen) and quantitative PCR (q-PCR), which were widely employed to measure residual DNA contents of prokaryotic-derived recombinant products. High purity of <i>E. coli</i> strain <i>BL21</i> was extracted by the cetyl triethyl ammonium bromide (CTAB)/phenol chloroform method, analyzed by UV-visible spectrophotometry and electrophoresis, diluted with tris-EDTA (TE) buffer and manually dispensed. Then, with a cooperative calibration among six laboratories, including five manufacturers and one national control laboratory, the concentration of <i>E. coli</i> DNA standard solution was determined as 96.2 μg/mL (95% C.I: 95.5–96.9 μg/mL, CV 3.4%). The candidate showed excellent stability both from accelerated degradation study and real time stability study. The applicability study showed that the <i>E. coli</i> DNA reference could reach the sensitivity of 0.781 ng/mL and 1 fg/μL, respectively, in fluorescent dye and q-PCR assay, and also had good linearity and precision. The consistency of the reference could meet the requirements of the national reference standard. As a conclusion, the candidate material was suitable to serve as a China national standard for <i>E. coli</i> residual DNA determination. The successful establishment of the <i>E. coli</i> DNA standard will facilitate the standardization of quantitative methods for testing residual host cell DNA.</p></div