Induction of apoptosis in tumor cells.

<p>A total of 2x10^5 cells of Hela cells and lent-clones in triplicates were co-cultured with human NK cells for 24 hours. After the removal of suspension NK92 cells, the caspase 3 activities in the remaining tumor cells were detected using a Caspase 3 Assay Kit (abcam). The statistical analyses were conducted between the corresponding un-induced and induced groups using one-way analysis of variance (ANOVA) with the Tukey’s post test (***p<0.001).</p

Activation of human NK92 cells.

<p>(a) IFN-γ ELISA. Cells of Lent-clones and Hela cells in triplicates were co-cultured with human NK92 cells and the supernatants were collected. IFN-γ activity in the supernatants was detected using Human IFN-γ ELISA Kit. The statistical analyses were conducted between the control (Hela+NK) and each Lent-clone (as the bars represented in the figure) using one-way analysis of variance (ANOVA) with Tukey’s post test. (*p<0.05; **p<0.01) (b) Cytotoxicity of NK92 cells. Cells of each lent-clone and Hela in triplicates were co-cultured with human NK92 cells. After co-culture, NK92 cells were removed and remaining tumor cells’ proliferation was measured with Promega’s CellTiter 96ueous nonRadioactive cell proliferation assay kit. The statistical analyses were conducted between the control (Hela+NK) and each virally transduced lent-clone using one-way analysis of variance (ANOVA) with the Tukey’s post test (*p<0.05; **p<0.01).</p

RT-PCR Confirmation of gene expression in Lent-clones.

<p>Total RNAs were isolated from Lent-clones and un-transduced Hela cells and a two-step RT-PCR was performed using Phusion RT-PCR kit (Thermo Scientific). Corresponding pairs of primers were used to amply different fragments. House-keeping gene β-actin expression was also included as controls.</p

IHC staining of Lent-clones.

<p>Cells from Lent-clones and Hela cells were harvested and cytospun onto glass slides. After fixation, the cells were stained with anti-mouse IL-12 antibody or isotype control antibody followed by anti-mouse IgG secondary antibody conjugated with HRP. The cells were then treated with substrate DAB and observed under microscope.</p

Lentiviral titer determination.

<p>Each virally transduced 293-Lent clones (Lent-IF/Lent-IL12/Lent-Fas/Lent-LacZ) were collected for EmGFP-based cytometry analysis. Viral titer of each lent-clone was calculated based on cytometry reading and represented.</p

hnRNP A2/B1 is a choroid plexus and CSF vesicle protein.

<p>A. Nanoparticle tracking analysis of serum free media from human CPE cultures and size frequency distributions. Media contained 559.1 (x10⁹) EVs per mL (N = 3). B. The average size of an EV was 160.3 nm (N = 3). C. Transmission electron micrographs of CPE EVs. Scale bar: 100 nm. D. Immunoblot of the exosome protein ALIX from vesicles isolated from CPEs cultured in the presence or absence of serum. E. The consensus sequence contains features of a recently described exosome miRNA motif recognized by hnRNPA2/B1. F. Multiple sequence alignment of vesicle proteins indicates conservation of miRNA sequence. G. Immunoblot of CPE derived media in the absence of serum or media alone containing serum for ALIX H. Immunoblot of hnRNPA2/B1 from CPE derived media in the absence of serum or media alone containing serum for. I. Immunoblot of hnRNPA2/B1 from CSF EVs demonstrating an age dependent decrease. J. Quantification of immunoblots for hnRNPA2/B1. *: p = 0.0138 Error Bars: SEM.</p

Temporal dynamics of CSF vesicle miRNAs.

<p>A. miRNA microarray heat-map of CSF vesicle content from patients less than 2 years or greater than 70 years where red is higher expression and green is lower expression. B. Rank expression of miRNAs based on microarray probe intensity. Red is microarray data from patients less than 2 years and black is from patients greater than 70 years. C. Log(₂) signal of the most abundant miRNAs. D. Hierarchical clustering of miRNA microarrays at different ages. E. Venn diagram of high abundance or (F.) low abundance miRNAs at different ages.</p

CSF EVs undergo temporal declines.

<p>A. CSF from patients ranging from 1 month to 85 years was collected and subjected to nanoparticle tracking analysis. The average size of EVs was unchanged. B. The average number of EVs was highest in patients less than 2 years and decreased by 10–15 years and 70 years. C. Overlay of size distribution of vesicles from patients less than 2 years, 10-15 years, and greater than 70 years. D–F. Size distribution averages with SEM depicted. G–I. Size distribution averages with area under the curve depicted. *: p<.01 Error Bars: SEM.</p

Next generation sequencing of CSF vesicle RNA content from patients less than 2 years old.

<p>A. Reads aligned to the human genome as a distribution across chromosomes. B. Rank sum distribution of mapped reads as a function of relative abundance (FPKM). C. Frequency distribution of reads in relation to read length. D. Alignment of a novel short RNA, CUFF .1222 to Hg19 chromosome 17 as visualized by integrated genomics viewer. The sequence of Hg19 is seen on the X axis. The chromosomal alignment is shown on the Y axis with red delineating the boundaries of the alignment.</p

Anti-inflammatory and Quinone Reductase Inducing Compounds from Fermented Noni (<i>Morinda citrifolia</i>) Juice Exudates

A new fatty acid ester disaccharide, 2-<i>O</i>-(β-d-glucopyranosyl)-1-<i>O</i>-(2<i>E</i>,4<i>Z</i>,7<i>Z</i>)-deca-2,4,7-trienoyl-β-d-glucopyranose (<b>1</b>), a new ascorbic acid derivative, 2-caffeoyl-3-ketohexulofuranosonic acid γ-lactone (<b>2</b>), and a new iridoid glycoside, 10-dimethoxyfermiloside (<b>3</b>), were isolated along with 13 known compounds (<b>4</b>–<b>16</b>) from fermented noni fruit juice (<i>Morinda citrifolia</i>). The structures of the new compounds, together with <b>4</b> and <b>5</b>, were determined by 1D and 2D NMR experiments, as well as comparison with published values. Compounds <b>2</b> and <b>7</b> showed moderate inhibitory activities in a TNF-α-induced NF-κB assay, and compounds <b>4</b> and <b>6</b> exhibited considerable quinone reductase-1 (QR1) inducing effects