Flow cytometric analysis of B cell populations resident in SMGs.

<p>Flow cytometry was performed on cells prepared from SMGs from male <i>Myd88</i>^+/+ mice and <i>Myd88</i>^-/- mice at 10 weeks old. A: Analysis of SMG cells obtained from four mice for expression CD45 and B220 (30,000 cells each). The percentage and cell number of the cells within the outlined area in dot plots (indicative of CD45⁺B220⁺ cells; others shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113333#pone.0113333.s002" target="_blank">Figure S2A</a>) were shown. The data of percentage were graphically shown as means ± SD (n = 4 per group) and are representative of three independent experiments. **, <i>P</i><0.01 (unpaired Student's <i>t</i>-test). B: Analysis of SMG cells obtained from four mice for expression CD138 and B220 (30,000 cells each). The percentage and cell number of the cells within the outlined area in dot plots (indicative of CD138⁺B220⁺ cells; others shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113333#pone.0113333.s002" target="_blank">Figure S2B</a>) were shown. The data of percentage were graphically shown as means ± SD (n = 4 per group) and are representative of three independent experiments. **, <i>P</i><0.01 (unpaired Student's <i>t</i>-test). **, <i>P</i><0.01 (unpaired Student's <i>t</i>-test). C: Analysis of SMG cells obtained from four mice for expression CD138 and IgA (30,000 cells each). The percentage and cell number of the cells within the outlined area in dot plots (indicative of CD138⁺IgA⁺ cells; others not shown) were shown. The data of percentage were graphically shown as means ± SD (n = 4 per group) and are representative of three independent experiments. *, <i>P</i><0.05 (unpaired Student's <i>t</i>-test). D: Analysis of B-1 cells and B-2 cells in SMG cells obtained from four mice (30,000 cells each). For discrimination of B-1a cells in the dot plots, B220-positive cells were separated into CD5⁺ cells (B-1a cells) and CD5^- cells (B-1b plus B-2 cells). For discrimination of B-2 cells, B220-positive cells were separated into CD23⁺ cells (B-2 cells) and CD23^- cells (B-1 cells). The percentage and cell number within the outlined area in dot plots were shown. The data of percentage of B220⁺CD23^- B-1 cells were graphically shown as means ± SD (n = 4 per group) and are representative of three independent experiments. *, <i>P</i><0.05 (unpaired Student's <i>t</i>-test).</p

Analysis of expression of the genes encoding defensins in SGs.

<p>A and B: Microarray analysis of the genes encoding α-defensins (A) and β-defensins (B). Total RNA was prepared from SMGs from <i>Myd88</i>^+/+ mice and <i>Myd88</i>^-/- mice at 10 weeks old (n = 3 each). The mean value of fold-change for each gene in SMGs from <i>Myd88</i>^-/- mice shown here was calculated relative to the mean value of expression of that gene in SMGs from <i>Myd88</i>^+/+ mice. Statistical analysis was performed by unpaired Student's <i>t</i>-test. C and D: qRT-PCR analysis for expression of <i>Defa1</i> (left) and <i>Defb1</i> (right) in SMGs (C) and SLGs (D). Total RNA was prepared separately from SMGs and SLGs collected from <i>Myd88</i>^+/+ mice and <i>Myd88</i>^-/- mice at 10 weeks old (n = 8 each). Expression levels of <i>Defa1 and Defb1</i> in SMGs and SLGs were calculated relative to expression of the <i>Hprt1</i> housekeeping gene. Means of each group were shown and <i>P</i> values were calculated by unpaired Student's <i>t</i>-test.</p

Analysis of expression of the genes encoding salivary AMPs in SGs.

<p>A: Microarray analysis of the expression of 45 genes encoding salivary AMPs. Total RNA was prepared from SMGs from <i>Myd88</i>^+/+ mice and <i>Myd88</i>^-/- mice at 10 weeks old (n = 3 each). The mean value of fold-change for each gene in <i>Myd88</i>^-/- mice shown here was calculated relative to the mean value of expression of that gene in <i>Myd88</i>^+/+ mice. Statistical analysis was performed by unpaired Student's <i>t</i>-test. The complete list of 45 genes is in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113333#pone.0113333.s008" target="_blank">Table S2</a>. *, <i>P</i><0.05, **, <i>P</i><0.01, N.D., not detectable. B: qRT-PCR analysis of <i>Slpi</i> expression. Total RNA was prepared separately from SMGs and SLGs from <i>Myd88</i>^+/+ mice and <i>Myd88</i>^-/- mice at 10 weeks old (n = 8 each). Each expression level of <i>Slpi</i> was calculated relative to expression of the <i>Hprt1</i> housekeeping gene. Means of each group were shown and <i>P</i> values were calculated by unpaired Student's <i>t</i>-test. *, <i>P</i><0.05. C: Fluorescent imaging of SLPI expression in SMGs. Sections of SMGs from male <i>Myd88</i>^+/+ mice (left) and <i>Myd88</i>^-/- mice (right) at 10 weeks old were deparaffinized and incubated with an anti-SLPI antibody/Alexa Fluor 488-conjugated secondary antibody (green) and propidium iodide to stain nuclei (red). Results are representative of three independent experiments. Scale, 50 µm.</p

Effect of MyD88 deficiency on histology of SGs.

<p>Paraffin sections of the major SGs from male <i>Myd88</i>^+/+ mice (left) and <i>Myd88</i>^-/- mice (right) at 10 weeks old were stained with hematoxylin and eosin. Results are representative of more than three independent experiments. A: Low magnification images of the whole tissues of major SGs. LN, lymph node, SLG, sublingual gland; SMG, submandibular gland. Scale bar, 1 mm. B: Middle magnification images of SMGs. Scale bar, 100 µm. C: High magnification images of SMGs. Scale bar, 20 µm.</p

Analysis of expression of the genes encoding S100 proteins in SGs.

<p>A: Microarray analysis of the genes encoding S100 proteins. Total RNA was prepared from SMGs from <i>Myd88</i>^+/+ mice and <i>Myd88</i>^-/- mice at 10 weeks old (n = 3 each). The mean value of fold-change for each gene in SMGs from <i>Myd88</i>^-/- mice shown here was calculated relative to expression of that gene in SMGs from <i>Myd88</i>^+/+ mice. Statistical analysis was performed by unpaired Student's <i>t</i>-test. *, <i>P</i><0.05. B and C: qRT-PCR analysis for expression of <i>S100a8</i> (left) and <i>S100a9</i> (right) in SMGs (B) and SLGs (C). Total RNA was prepared separately from SMGs and SLGs from <i>Myd88</i>^+/+ mice and <i>Myd88</i>^-/- mice at 10 weeks old (n = 8 each). Expression levels of <i>S100a8 and S100a9</i> in SMGs and SLGs were calculated relative to expression of the <i>Hprt1</i> housekeeping gene. Means of each group were shown and <i>P</i> values were calculated by unpaired Student's <i>t</i>-test. *, <i>P</i><0.05; **, <i>P</i><0.01.</p

Effect of MyD88 deficiency on salivary basal Ig production.

<p>A: Levels of salivary basal IgA (left), IgM (middle) and IgG3 (right). Saliva samples were collected from naive <i>Myd88</i>^+/+ mice and <i>Myd88</i>^-/- mice at 10 weeks old (n = 8 each) for determination of basal Ig levels by ELISA. Means of each group were shown and <i>P</i> values were calculated by unpaired Student's <i>t</i>-test. **, <i>P</i><0.01. B: Levels of salivary anti-PC IgA (left) and anti-PC IgM (right). Saliva samples were collected from naive <i>Myd88</i>^+/+ mice and <i>Myd88</i>^-/- mice at 10 weeks old for determination of the levels of anti-PC Igs by ELISA. Data are expressed as means ± SD (n = 4 each) and are representative of three independent experiments. *, <i>P</i><0.05 (unpaired Student's <i>t</i>-test).</p

Defensive effect of microRNA-200b/c against amyloid-beta peptide-induced toxicity in Alzheimer's disease models

<div><p>MiRNA molecules are important post-transcriptional regulators of gene expression in the brain function. Altered miRNA profiles could represent a defensive response against the pathogenesis of neurodegenerative disorders, such as Alzheimer's disease (AD). Endogenous miRNAs have lower toxic effects than other gene silencing methods, thus enhancing the expression of defensive miRNA could be an effective therapy. However, little is known about the potential of targeting miRNAs for the treatment of AD. Here, we examined the function of the miR-200 family (miR-200a, -141, -429, -200b, -200c), identified using miRNA microarray analysis of cortical tissue from Tg2576 transgenic mice. In murine primary neurons, we found that upregulation of miR-200b or -200c was induced by the addition of amyloid beta (Aβ). Neurons transfected with miR-200b or -200c reduced secretion of Aβ in conditioned medium. Moreover, mice infused with miR-200b/c into the brain were relieved of memory impairments induced by intracerebroventricular injection of oligomeric Aβ, and demonstrated proper spatial learning in the Barnes maze. To gain further understanding of the relationship between miR-200b/c and Aβ, we identified target mRNAs via an RNA-binding protein immunoprecipitation-microarray assay. Western blot analysis showed that expression of ribosomal protein S6 kinase B1 (S6K1), a candidate target, was inhibited by miR-200c. S6K1, a downstream effector of mammalian target of rapamycin (mTOR), serves as a negative feedback mediator that phosphorylates insulin receptor substrate 1 at serine residues (IRS-1pSer). S6K1-dependent IRS-1pSer suppresses insulin signaling leading to insulin resistance, which is frequently observed in AD brains. Notably, miR-200b/c transfection of SH-SY5Y cells reduced the levels of IRS-1pSer. This finding indicates that miR-200b/c has the potential to alleviate insulin resistance via modulation of S6K1. Taken together, miR-200b/c may contribute to reduce Aβ secretion and Aβ-induced cognitive impairment by promoting insulin signaling.</p></div

Additional file 2 of Plasma microRNA biomarker detection for mild cognitive impairment using differential correlation analysis

Supplement B. Details of t-test and the results. The results include boxplots and ROC curves based on each of 22 miRNAs selected by t-test between Normal and MCI. (PDF 55.1 kb

The members of the miR-200 family and related differentially expressed mRNA network are implicated in insulin signaling in the cortex of 10-month-old Tg2576 mice.

<p>(A) Volcano plot showing microarray data of significantly upregulated miRNAs in the cortices of 10-month-old (10 mo.) Tg2576 mice as compared to wild-type (WT) mice. The threshold values for fold change were set at > 2 or < 0.5 (outside the vertical lines) and P value < 0.05 (above the horizontal line); red points in the plots represent the significant differentially expressed miRNA. The miRNAs that were upregulated were members of the miR-200 and miR-183 families. (B) qRT-PCR was used to validate the upregulation of miR-200 family members using TaqMan assays. The upregulation of members of this miRNA family was confined to the 10 mo. period, a phase during which Aβ accumulation increases. (C) The top network of differentially expressed mRNA as identified by IPA analysis of the microarray data. Up- and down- regulated genes are colored in yellow and blue, respectively. Solid and dashed arrows indicate direct and indirect connections, respectively. Subscripted numbers indicate fold change. The network involves components of the insulin signaling pathway.</p

Additional file 1 of Plasma microRNA biomarker detection for mild cognitive impairment using differential correlation analysis

Supplement A. Scatterplots and ROC curves for each of top 20 pairs of miRNAs selected by differential correlation analysis between Normal and MCI. (PDF 63.2 kb