霍乱毒素B亚单位

G-richsequencesubiquitouslyexistedinmRNA3′-UTRmicroRNAbindingsites.A,SchematicrepresentationofthevalidatedmicroRNAtargetswithpotentialG-quadruplexbinding sites.BluecirclesrepresentG-richsequences,greencirclesrepresentmicroRNAtargetgenes,purplecirclesrepresentsexperimentallysupportedgenesandredshapesindicatemicroRNA targetswithpotentialG-quadruplexbindingsites.B,GOenrichmentanalysisofbiologicalprocessesoftargetgenes.C,GOenrichmentanalysisofthecellularcomponentoftargetgenes.D, GOenrichmentanalysis of molecular functionsoftarget genes.

CandidateG-richsequencesformedG-quadruplexesinpotassiumsolution.A–D.CirculardichroismspectrumofSCN1B,KCNA7,CLIC5,andKCNJ11G-richsequences(2μMRNAin 30mM Tris-HCl) inthepresenceand absence of 100 mM potassiumsolution

KCNJ11 mRNA G-quadruplexes affected luciferase expression. A, Schematic of luciferase reporter plasmidstructure. B, Luciferase reporter assay with KCNJ11 wild type and two mutation plasmidswith miR-331-3p overexpression.***P b 0.001, n = 6.

G-quadruplex ligand TMPyP4 destabilized KCNJ11 G-quadruplexes and suppressed luciferase expression. A, The chemical structural formula of TMPyP4. B, Circular dichroism spectrumofKCNJ11 G-richsequencewith TMPyP4 (2 μM RNAwith/without 10μM TMPyP4 in150 mM KCl and 30 mM Tris-HCl). C,Luciferase reporter assay of KCNJ11 wildtype and mutated G-rich sequence plasmids with 10 μM TMPyP4. Luciferase reporter assay of KCNJ11 wild type and mutated G-rich sequence plasmids with 10 μM TMPyP4 and 10 nM agomir (D)/antagomir-331-3p (E). *P b 0.05,**P b 0.01 n = 6.

TMPyP4 togetherwith miR-331-3p inhibitedKCNJ11expression incardiomyocytes. A,KCNJ11mRNA expression incardiomyocytesafter 10nMmiR-331-3p and10 μM TMPyP4 coincubation.B,KCNJ11 protein expressionincardiomyocytes after10nMmiR-331-3pand 10 μM TMPyP4 coincubation.*P b 0.05, n = 6.

Increased expression of succinate receptor GPR91 in the biopsies of MU patients A: IHC staining showed the elevated expression of GPR91 in the cornea and conjunctiva (magnification ×400)

Effects of SCE on the expression of the immunoreactivity of BDNF in the mouse hippocampus. (A) Hippocampus were stained with specific antibodies against BDNF (red). Nuclei were stained with DAPI (blue). The scale bar shows 100 μm. (B) The average fluorescence intensities of BDNF were quantified after SCE treatment. The data represented the values of mean ± S.E.M. from 10 mice/group. *p < 0.05, **p < 0.01 vs. CUMS group. ##p < 0.01 vs. Control group.

Combination of PEI and H9N2 WIV. Native gel electrophoresis of H9N2 WIV-PEI complexes in different weight ratios (PEI/H9N2 WIV, wt/wt): H9N2 WIV alone (lane 1) and H9N2 WIV-PEI complex ratios 0.01 (lane 2), 0.02 (lane 3), 0.04 (lane 4), 0.08 (lane 5), and 0.12 (lane 6). Results are representative of three independent experiments.

PEI assists H9N2 WIV in enhancing the local immune responses. Antigen-specific mucosa IgA titers in nasal wash (A), tracheal wash (B), and lung wash (C) of immunized mice (n = 12) 2 weeks after the last vaccination. Data are shown as the means ± SD. *, P < 0.05; **, P < 0.01.

PEI assists H9N2 WIV in enhancing the level of serum antibody. Antigen-specific serum IgG titers (A), IgG1 titers (B), IgG2a titers (C), and HI titers (D) of immunized mice (n = 12) 2 weeks after the last vaccination. Data are shown as the means ± SD. *, P < 0.05; **, P < 0.01.

PEI assists H9N2 WIV in enhancing the percentages of CD3+ CD4+ and CD3+ CD8+ T cells. At 28 days after the primary immunization, the percentages of CD3+ CD4+ (A and C) and CD3+ CD8+ (B and D) spleen T cells from the immunized mice were analyzed by FACS. Data are shown as the means ± SD. *, P < 0.05; **, P < 0.01.

PEI assists H9N2 WIV in enhancing the CD69 expression and proliferative index of lymphocytes isolated from the spleen. At 28 days after the primary immunization, splenic lymphocytes of immunized mice were isolated and restimulated with H9N2 WIV (10 μg/ml) in vitro. (A and B) FACS analysis of splenocyte activation was assessed as CD69 expression; (C) proliferative index of spleen was analyzed using the WST-8 assay. Data are shown as the means ± SD. *, P < 0.05; **, P < 0.01.

PEI improves the viral adhesion to Calu-3 cells and the cellular uptake of H9N2 WIV by DCs. The Calu-3 cells (A to C) or DCs (D and E) were incubated with H9N2 WIV or H9N2 WIV-PEI complexes (PEI/H9N2 WIV weight ratios of 0.04, 0.08, and 0.12) for the indicated times: 0.5 h (A to C) or 10 min or 0.5 h (D, E). H9N2 WIV samples were labeled with the fluorescent probe DyLight 405 (A) or DyLight 633 (B to D) NHS Ester. Calu-3 cells and DCs were collected and were washed 3 times with PBS and then were detected by FACS. MFI, mean fluorescence intensity. Data are shown as the means ± SD. *, P < 0.05; **, P < 0.01. All results are representative of three independent experiments. Bars in panel A, 20 μm.

PEI enhances the cellular endosomal escape of H9N2 WIV in DCs. (A) Intracellular trafficking of internalized H9N2 WIV or H9N2 WIV-PEI complexes (PEI/H9N2 WIV weight ratio of 0.08) was observed by CLSM. Lysosomes (red), H9N2 WIV (green). Bars, 5 μm. (B) Overlay analysis shows the detailed colocalization information: empty lysosome (1), escaped antigen (2), and colocalized antigen (3). Data are representative of more than three independent experiments.

PEI assists H9N2 WIV in enhancing the phenotypic maturation of DCs. DCs were stimulated with medium, PEI, H9N2 WIV (H9N2 WIV concentration, 14.3 μg/ml), or H9N2 WIV-PEI complexes (PEI/H9N2 WIV weight ratios of 0.04, 0.08, and 0.12) for 24 h. The expressions of MHC-II (A and C) and CD40 (B and D) on DCs were analyzed by FACS. All of the data are shown as the means ± SD of three replicates and are representative of three independent experiments. *, P < 0.05; **, P < 0.01.

Exosome treatment inhibits M1 phenotype polarization and promotes M2 polarization of microglia in rat spinal cords both in protein level and mRNA level. (B)Immunofluorescent double staining of CD68 and CD206, and the quantificational analysis of the percentage of CD206 or CD68 positive area comparing to the entire section is presented as means ± standard deviation. N =4 per group, P < 0.05, compared to the control group; **P < 0.01, ***P < 0.001 compared to the LPSgroup.

Antigen-specific (B) serum IgG, (C) nasal lavage IgA, alveolar (D) IgA, (E) intestine lavage IgA titers of mice vaccinated with PBS, FreeF-CpG, NCF-CpG, NHCF-CpG, NHCF-CTB, and NHCF-GAMP at 6 weeks.