Table1_Population pharmacokinetic modeling of ilaprazole in healthy subjects and patients with duodenal ulcer in China.DOCX

Aims: This study aimed to develop a population pharmacokinetic (PopPK) model of ilaprazole in healthy subjects and patients with duodenal ulcer in Chinese and investigate the effect of potential covariates on pharmacokinetic (PK) parameters.Methods: Pharmacokinetic data from 4 phase I clinical trials and 1 phase IIa clinical trial of ilaprazole were included in PopPK analysis. Phoenix NLME 8.3 was used to establish a PopPK model and quantify the effects of covariate, such as demographic data, biochemical indicators and disease state on the PK parameters of ilaprazole. The final model was evaluated by goodness-of-fit plots, bootstrap analysis, and visual predictive check.Results: A two-compartment model with first-order elimination successfully described the pharmacokinetic properties of ilaprazole. In the final PopPK model, body weight and sex were identified as statistically significant covariates for volume of peripheral compartment (Vp) and clearance of central compartment (CL), respectively, and disease status was also screened as a significant covariate affecting both CL and Vp. The validation results demonstrated the good predictability of the model, which was accurate and reliable.Conclusion: This is the first population pharmacokinetics study of ilaprazole in the Chinese, and the PopPK model developed in this study is expected to be helpful in providing relevant PK parameters and covariates information for further studies of ilaprazole.</p

SDS-PAGE electrophoresis and immunoblotting of recombinant MPT64 protein.

<p>(A) Proteins were transferred to nitrocellulose and probed with antibody specific for MPT64, and a final detection was made using DAB substrate. (B) Proteins were stained with Coomassie Briliant Blue. Lane 1: crude lysate of <i>k802</i> transformed by pGEX5T after induction. Lane 2: the expression product of <i>k802</i> transduced with pGEX5T-MPT64. Lane 3: purified MPT64 protein. Lane 4: crude lysate of <i>k802</i> transformed by pGEX5T after induction. Lanes 5/6: the expression product of <i>k802</i> transduced with pGEX5T-MPT64, before or after induction. Lane 7: purified MPT64. Lane 8: protein markers.</p

The control effect of miRNA21 on bcl-2 after MPT64 treatment RAW264.7.

<p>Macrophages were treated as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100949#pone-0100949-g002" target="_blank">Fig. 2</a>. The miRNA21 level was analyzed by real time-PCR (5A). **<i>P</i><0.01, the PPD group vs the PPD-MPT64 (15 µg/ml) group. The control effect of miRNA21 on bcl-2 was also analyzed by relative luciferase activity in RAW264.7 cells (5B). The wild-type or mutated type of bcl-2-utr was cloned into a pMIR-reportTM luciferase vector. Then, the recombinant plasmids and miR21 were transfected into RAW264.7 cells. The relative luciferase activity was detected. miR-mock and miR335 were used as control miRNAs. *<i>P</i><0.01, the miR21 group vs the miR-mock group after the transfection by wild-type bcl-2-utr. Data shown are representative of three independent experiments.</p

Apoptosis inhibition of macrophages by MPT64 protein.

<p>PMA-differentiated RAW264.7 cells were put into 24-well flat bottom tissue culture plates at a density of 1×10⁵ cells per well. Then, RAW264.7 macrophages were incubated with PBS, BCG-PPD (10 µg/ml) or a mixture of PPD-MPT64 at a different concentrations for 16 h. (A) Apoptosis was detected by measuring the membrane exposure of PS using annexin V by flow cytometry, and the results were analyzed. <b>Fig. 2A</b> Significant differences of the apoptosis percentages. *<i>P</i><0.05, the PPD group vs the MPT64 (10 µg/ml) group; ** <i>P</i><0.01, the PBS group vs the PPD group, and the PPD group vs the MPT64 (15 µg/ml or 20 µg/ml) group. The apoptosis percentage is not significantly different for the GST group or heat-treated MPT64 protein compared with the PPD group. <b>Fig. 2A</b> Ne, the PBS group; HAMPT64, heat treated MPT64; M10, MPT64 at 10 µg/ml of concentration; M15, MPT64 at 15 µg/ml of concentration; M20, MPT64 at 20 µg/ml of concentration. <b>Fig. 2B</b> A: The PBS group. B: The PPD group. C: The MPT64 (15 µg/ml) group. Data shown are representative of three independent experiments.</p

Apoptosis-related gene expression after MPT64 treatment.

<p>PMA-differentiated RAW264.7 macrophages were incubated with PBS, BCG-PPD (10 µg/ml), or a PPD-MPT64 (15 µg/ml) mixture for 16 h. Then, the bcl-2 mRNA (3A) and bax mRNA levels were detected by real-time PCR (3B) or Western-blot (3C) for the expression of bcl-2. For the Western-blot, reactive bands were detected by an ECL chemiluminescence system.*<i>P</i><0.05, the PPD group vs the PPD-MPT64 (15 µg/ml) group. <b>Fig. 3C</b> A: The PBS group. B: The MPT64 (15 µg/ml) group. C: The PPD group. Data shown are representative of three independent experiments.</p

Primers for NF-kB in ChIP assay.

<p>Primers for NF-kB in ChIP assay.</p

miR21 is directly regulated by NF-kB.

<p>Real-time PCR results for miR-21 levels were performed with siRNA against NF-κB. After the transfection of siRNA against NF-κB, the miR-21 level was detected by real-time PCR (6A). **<i>P</i><0.01, the siRNA treatment group vs the non- siRNA treatment group. Then, the apoptosis level was analyzed by flow cytometry (6D). **<i>P</i><0.01, the siRNA treatment group vs the non- siRNA treatment group. Five primers were designed according to a software analysis, and ChIP assays were performed in RAW264.7 cells to explore possible binding sites. A specific band was observed for primer for site 3 (6B). No specific band was observed for the primers for sites 1, 2, 4 and 5 (6C). Negative controls were incubated without primary antibody. The experiment was repeated three times.</p

MicroRNA-572 Improves Early Post-Operative Cognitive Dysfunction by Down-Regulating Neural Cell Adhesion Molecule 1

<div><p>Post-operative cognitive dysfunction (POCD) is a commonly-seen postoperative complication in elderly patients. However, the underlying mechanisms of POCD remain unclear. miRNAs, which are reported to be involved in the pathogenesis of the nervous system diseases, may also affect POCD. In this study, miRNA microarray technology was used to analyze the circulating miRNA expression profile of POCD patients. Among the altered miRNAs, miR-572 had the greatest decrease, which was also verified <i>in vivo</i> in rat POCD model. Further analysis found that miR-572 could regulate the expression of NCAM1 in the hippocampal neurons and interfering miR-572 expression could facilitate the restoration of cognitive function <i>in vivo.</i> Moreover, clinical correlation analysis found that the miR-572 expression was associated with the incidence of POCD. Collectively, miR-572 is involved in the development and restoration of POCD and it may serve as a biological marker for early diagnosis of POCD.</p></div

Primers Sequence.

<p>Primers Sequence.</p

Targeted regulation of the expression of NCAM by miR-572.

<p>A. Schematics of miR-572 binding to the 3'UTR region of the NCAM1 mRNA (wildtype and mutant) in the dual-luciferase experiment. B. The dual-luciferase assay showed that miR-572 significantly reduced the luciferase activity of plasmids containing the wildtype 3'UTR region of mouse NCAM1 mRNA. C. Overexpression of miR-572 in mouse HT22 cells could significantly reduce the NCAM1 expression at the mRNA and protein levels. D. Inhibition of miR-572 in mouse HT22 cells could significantly promote NCAM1 expression at the mRNA and protein levels. E. Immunohistochemical detection showed that after inhibiting miR-572 expression in the POCD rat brain, the NCAM1 expression was elevated. WT, wildtype; MUT, mutant; NC, negative control.</p