Additional file 1 of Relationship between autism and brain cortex surface area: genetic correlation and a two-sample Mendelian randomization study

Supplementary Material 1: Supplementary Figure 1. Local genetic covariance estimates of Heritability Estimation from Summary Statistics. Supplementary Figure 2. Forest plot of significant estimates identified with IVW. Supplementary Figure 3. Funnel plot from genetically predicted ASD on S

PrA conditioning reduced PBMC IP-10 secretion <i>in vitro</i>.

<p>PBMCs isolated from control recipient rats on day four were cultured with different doses of PrA for 72(n = 3 per group). (A) PBMC viability was measured by MTT assay. The highest dose induced a significant decrease in viable cell count, therefore, only 5 nM and 20 nM concentrations were used in subsequent experiments. (B) Culture supernatants of PBMC with or without PrA condition were harvested and analyzed for the production of IP-10 by ELISA. * indicates <i>p</i><0.05 when comparing treatment condition to control.</p

PrA reduced T cell migration to PBMC supernatant through inhibition of IP-10-CXCR3 receptor interaction.

<p>IP-10 (50 ng/ml), Mig (100 ng/ml), or SDF-1 (100 ng/ml) was added to the PBMC supernatant. T cells were incubated with the isotype control Ab (20 µg/ml) or anti-CXCR3 Ab (20 µg/ml) prior to chemotactic migration assays (n = 3 in each group). (A) IP-10 addition to the supernatant of PrA-exposed PBMCs improved the migration of PrA-treated recipients T cells. Furthermore, addition of anti-CXCR3 Ab restored PrA inhibition of IP-10-induced T cell migration, but isotype Ab did not have the same effect. (B) The addition of Mig and SDF-1 did not significantly increase the migration of PrA-treated recipients T cells. However the addition of Mig, but not SDF-1 addition increased the migration of non PrA-treated recipients T cells. *indicates <i>p</i><0.05, and bars indicate comparators.</p

PrA inhibited IP-10 expression in infiltrating monocytes of cardiac allografts.

<p>Immunofluorescence staining of heart allograft sections stained with Abs to CD68 (FITC, green) and IP-10 (TMRITC, red). Nuclei were counterstained with DAPI (blue). The IP-10 expression in CD68 positive infiltrating monocytes was reduced after PrA treatment. The results are representative of three independent experiments (n = 3).</p

The migrating capacity of naïve T cells was slightly affected by PrA.

<p>The naïve T cells isolated from rat spleen were cultured with or without PrA (20 nM) for 72 h. A chemotactic migration assay was used to assess the migration of naïve T cells in the presence or absence of PrA towards different doses of either recombinant IP-10 or Mig. (A) PrA-conditioned T cells exhibited decreased migration in presence of 25 ng/mL of IP-10, but not at 50 ng/mL or 100 ng/mL, compared with those of non-condition T cells. (B) The migration of PrA-conditioned T cells was decreased at 50 ng/mL and 100 ng/mL of Mig, but not at 200 ng/mL, as compared with those of non-conditioned T cells. The result suggests that the capacity of T cell migration was partially affected by PrA <i>in vitro</i>. * indicates <i>p</i><0.05, and bars indicate comparators.</p

PrA prevented CXCR3⁺T cell infiltration into allografts.

<p>(A) The heart allograft was harvested on day seven post heart transplantation, sectioned, and stained for TCR (TMRITC, red) and CXCR3 (FITC, green). The extent of colocalization (yellow) of TCR (T cell marker) and CXCR3 indicating CXCR3⁺ T cell infiltration into allografts was significantly reduced after PrA administration. The results are representative of three independent experiments. (B) The relative quantitative analysis to determine the percentage of CXCR3⁺TCR⁺ cells within infiltrating cells of allografts further showed that PrA treatment inhibited the CXCR3⁺ T cell infiltration into allografts. *indicates <i>p</i><0.05 when comparing treatment to control.</p

PrA inhibited recipient T cell migration towards PBMC supernatant.

<p>PBMC and spleen T cells were isolated from recipient control and PrA-treated rats on day seven posttransplantation. The migration of T cells toward PBMC supernatant was tested using a chemotactic migration assay. In the presence of PBMC supernatant from control recipients, the migration of T cells from PrA-treated recipients was not impaired compared with those from control recipients. However, in the presence of PBMC supernatant from PrA-treated recipients, the migration of T cells was significantly impaired when cells were harvested from either PrA-treated or control recipients. * indicates <i>p</i><0.05, and bars indicate comparators.</p

PrA depressed IP-10 mRNA level in allografts.

<p>(A) The effect of PrA administration on IP-10 mRNA expression in heart allografts of recipient rats was assessed by relative qRT-PCR on day 0, 3, 7, and 9 posttransplantation. The IP-10 mRNA level decreased with PrA addition (n = 8) as compared to controls (n = 8) on day seven and nine. (B) The gene expression of Mig, ITAC and RANTES was assessed in cardiac allografts on day 7 posttransplantation. However, no significant difference between control (n = 8) and PrA treated group (n = 8) was detected. * indicates <i>p</i><0.05 when comparing treatment to control.</p

PrA treatment reduced IP-10 secretion from PBMCs in recipient rats.

<p>Control and PrA-treated PBMCs from recipient rats (n = 8) were separately isolated on day seven posttransplantation and cultured at a concentration of 2×10⁶ cells/ml for 4 h. Culture supernatant was harvested and analyzed for the IP-10 secretion by ELISA. * indicates <i>p</i><0.05 when comparing treatment to control.</p

Quantitative Profiling of Polar Metabolites in Herbal Medicine Injections for Multivariate Statistical Evaluation Based on Independence Principal Component Analysis

<div><p>Botanical primary metabolites extensively exist in herbal medicine injections (HMIs), but often were ignored to control. With the limitation of bias towards hydrophilic substances, the primary metabolites with strong polarity, such as saccharides, amino acids and organic acids, are usually difficult to detect by the routinely applied reversed-phase chromatographic fingerprint technology. In this study, a proton nuclear magnetic resonance (¹H NMR) profiling method was developed for efficient identification and quantification of small polar molecules, mostly primary metabolites in HMIs. A commonly used medicine, Danhong injection (DHI), was employed as a model. With the developed method, 23 primary metabolites together with 7 polyphenolic acids were simultaneously identified, of which 13 metabolites with fully separated proton signals were quantified and employed for further multivariate quality control assay. The quantitative ¹H NMR method was validated with good linearity, precision, repeatability, stability and accuracy. Based on independence principal component analysis (IPCA), the contents of 13 metabolites were characterized and dimensionally reduced into the first two independence principal components (IPCs). IPC1 and IPC2 were then used to calculate the upper control limits (with 99% confidence ellipsoids) of χ² and Hotelling T² control charts. Through the constructed upper control limits, the proposed method was successfully applied to 36 batches of DHI to examine the out-of control sample with the perturbed levels of succinate, malonate, glucose, fructose, salvianic acid and protocatechuic aldehyde. The integrated strategy has provided a reliable approach to identify and quantify multiple polar metabolites of DHI in one fingerprinting spectrum, and it has also assisted in the establishment of IPCA models for the multivariate statistical evaluation of HMIs.</p></div