Additional file 2: of Elevation of brain-enriched miRNAs in cerebrospinal fluid of patients with acute ischemic stroke

Comparison between study populations in our previous and present study. (DOCX 17 kb

Neuronal differentiation in NT2-D1 cells.

<p>NT2-D1 cells were treated with Retinoic Acid (RA) for 1 and 3 days or left untreated (day 0). Protein-, RNA- and ChIP-samples were harvested. (A) Western blot analysis of protein samples taken at day 0, 1 and 3. (B) Relative mRNA levels at day 0 and 3 of <i>REST</i>, <i>OCT4</i> and <i>NANOG</i> and <i>HOXA1</i>. (C) Group of genes, which loose binding of both REST and PcG proteins during differentiation. ChIP analysis was performed for day 0 and 3 of RA treatment. (D) Relative mRNA levels of the genes displayed in (C), for untreated cells and cells treated with RA for 3 days. (E) Group of genes, which maintain REST binding but loose PcG proteins during differentiation. ChIP analysis at day 0 and 3 after RA treatment. (F) Relative mRNA levels of the genes displayed in (E) after 0 and 3 days of RA treatment. Error bars represent standard deviations calculated from triplicate qPCR reactions.</p

Occupancy of PRC1 and PRC2 at Rest peaks in <i>Wt</i> and <i>Rest</i>−/− mES cells.

<p>(A) Average ChIP-seq read counts in <i>Wt</i> control and <i>Rest</i>−/− mES cells, normalized to IgG controls for Rnf2, Suz12 and Jarid2 within 3 Kb of the 3,378 peaks identified for Rest in the <i>Wt</i> control cells. Black arrow indicates the direction of the nearest TSS. (B) Heat-maps showing Rnf2, Suz12 and Jarid2 occupancy in a 2 Kb window centered around individual Rest peaks identified in <i>Wt</i> control mES cells. The ratiometric heat-maps depict the part of the profiles that were increased, unaffected, or decreased in <i>Rest−/−</i> mES cells as red, green and blue, respectively. Out of the 3,378 Rest peak positions identified in the <i>Wt</i> control mES cells, positions with very low levels or no PcG (Rnf2, Suz12 and Jarid2) ChIP-seq signal in either <i>Wt</i> control or <i>Rest−/−</i> mES cells were filtered out (for thresholds and genomic peak positions see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002494#pgen.1002494.s011" target="_blank">Table S3</a>). The regions were sorted according to the fold change in PcG (Rnf2, Suz12 and Jarid2) signal between the <i>Wt</i> control and <i>Rest−/−</i> mES cells, within the Rest peaks. P-values were calculated by comparing the number of Rest binding sites with 1.5 fold increase or decrease to those expected from a population with similar distribution as the matched control using chi²-tests (C) ChIP-seq profiles for the positions marked 1 to 6 in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002494#pgen-1002494-g004" target="_blank">Figure 4B</a> as examples. Data from <i>Wt</i> and <i>Rest−/−</i> mES cells are shown in black and grey, respectively (K27 = H3K27Me3). (D) Diagram showing the number of Rest peaks and matched control regions (no Rest peaks) scored positive for Rnf2, Suz12 or Jarid2 signal. The filtering process described in the legend for <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002494#pgen-1002494-g004" target="_blank">Figure 4B</a>. P-values compares binding sites scoring positive for PcG binding to those that scored positive by chance in the matched control regions using chi²-tests (E) Heat-maps of the matched control regions shown in 4D.</p

Rest regulates recruitment of PRC1 and PRC2 at specific genomic loci.

<p>(A) Western blot analysis of <i>Wt</i> and <i>Rest</i>−/− mES cell lysates. Histones were extracted using LSB. (B) ChIP-seq profiles for Rnf2, Suz12 and H3K27Me3 levels in <i>Wt</i> and <i>Rest−/−</i> mES cells for three Rest target genes (<i>Brunol6</i>, <i>Best2</i> and <i>Prrxl1</i>) and as control, one Polycomb target gene with no Rest binding site (<i>Gjb2</i>). Direct ChIP for the three Rest target genes shown in (B) comparing <i>Wt</i> and <i>Rest−/−</i> mES cells (primer positions are indicated by arrow heads in B and K27 = H3K27Me3) and mRNA levels (C). Error bars represent standard deviations calculated from triplicate qPCR reactions.</p

The recruitment of PRC1 at a number of Rest binding sites is independent of PRC2 activity in mES cells.

<p>(A) Histograms showing the number of Rnf2 peaks in <i>Wt</i> and <i>Eed</i>−/− mES cells at positions relative to CpG islands. Upper panel: Rnf2 peaks that overlap with CpG-islands relative to the distance between the center of the peak and the center of the CpG-island. Lower panel: Rnf2 peaks, not overlapping with annotated CpG-islands, plotted relative to the distance between the borders of the Rnf2 peak and the border of the CpG-island. (B) Average ChIP-seq reads in E14 <i>Wt</i> and <i>Eed</i>−/− for Rnf2 (upper panel) and Rest (lower panel) within 3 Kb of the 5,097 Rest peaks identified in E14 <i>Wt</i> mES cells (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002494#pgen.1002494.s013" target="_blank">Table S5</a>). Read counts were normalized to the IgG control. The black arrow indicates the direction of the nearest TSS. (C) Heat-maps of Rest and Rnf2 in <i>Wt</i> and <i>Eed</i>−/− mES cells at 1,907 Rest peaks within 10 Kb of a TSS. (D) Direct ChIP in E14 <i>Wt</i> and <i>Eed</i>−/− mES cells for <i>Calb1</i>, <i>Nudt9</i>, <i>Gpc2/Stag3</i> and <i>Gjb2</i> (positions of primers used are indicated by arrow heads in E). Error bars represent standard deviations calculated from triplicate qPCR reactions. (E) ChIP-seq profiles for the <i>Nudt9, Gpc2/Stag3 and Gjb2</i> loci using antibodies for Rnf2, Rest and control IgG. (F) Co-occurrence matrix of Rest and Rnf2 peaks from <i>Wt</i> and <i>Eed−/−</i> mES cells. The maps are based on scans, with a 2,500 bp window, along different positions near the transcription start site (TSS) or transcription end points (TE) annotated in the mouse genome (Mouse genome: mm9). For each scan the number of observed peaks were calculated and normalized to the number of expected peaks based on the frequency of Rest and Rnf2 peaks in that particular position, relative to the TSS or TE. Scans with a higher or lower frequency than expected from random localization, were colored blue or red, respectively. Scans with a P-value below 0.05 (Bonferroni corrected for multiple sampling), are overlaid with transparent green color.</p

REST and Polycomb Repressor Complex 1 (PRC1) and PRC2 interact <i>in vivo</i>.

<p>(A) Nuclear extracts from NT2-D1 cells were processed for size-exclusion chromatography followed by Western blotting to reveal the profiles of Polycomb proteins, the transcription factors REST and E2F6. Pooled fractions (1: Fractions F7–9; 2: F11–13; 3: F21–22) were used for immunoprecipitation (IP) with anti-REST or control IgG and processed for Western blotting with antibodies as indicated (total lysate: 12 µg of protein). (B) IPs using REST or control IgG on total nuclear extract (NT2-D1 cells; 500 µg per IP). After IPs the samples were either treated with a combination of RNase V1 and RNase A or left without RNase followed by repeated washes. Eluted proteins were processed for Western blotting using antibodies as indicated. Lower panel: Control experiment for the efficiency of RNase treatment using either 2 µg (left part) or 4 µg (right part) of RNA. Samples were incubated under the conditions used for REST IPs. (C–D) Nuclear protein extracts from mouse embryonic stem cells (mES) of different genetic background (<i>Wt</i>, <i>Eed−/−</i> or <i>Rnf2−/−</i>) were separated by size-exclusion chromatography (C–D: upper panels) and pooled fractions (1: F8–10; 2: F11–13; 3: F22) were processed for IPs (C–D: lower panels) using antibodies for Rest or control IgG. Western blots were processed with antibodies as indicated. Input corresponds to 3% of the material used for each IP. (C) Represents IPs comparing <i>Wt</i> and <i>Eed−/−</i> mES cells and (D) represents IPs in the <i>Rnf2−/−</i> mES cells. The samples were processed for Western blotting with antibodies as indicated. Lanes marked “M” represents loading of a pre-stained molecular weight marker.</p

REST is required for PRC1 binding and the maintenance of H3K27Me3.

<p>(A) shRNA mediated knockdown of <i>REST</i> and <i>RNF2</i> in NT2-D1 cells. Samples were processed for Western blot analysis (upper panel) and quantitative real-time RT-PCR (lower panel). (B) Expression array analysis of RNA isolated from NT2-D1 cells treated with a control shRNA or a shRNA against either <i>REST</i> or <i>RNF2</i>. Left panel: Cluster analysis of regulated genes displayed as a heat-map. Right panel: Venn diagram for genes up-regulated more than 2 fold and a P-value<0.05. In C and D relative mRNA levels in NT2-D1 cells treated with shRNA against <i>REST</i>, <i>RNF2</i>, or <i>RCOR1</i> are shown. Vertical punctured line separate two independent experiments (knockdown of <i>REST</i> and <i>RNF2</i> were done in parallel and knockdown of <i>RCOR1</i> was done separately). (C) Group of genes repressed by REST and RNF2. (D) Group of genes repressed by REST, RNF2 and/or CoREST. (E) ChIP analysis on NT2-D1 cells treated with shRNA against <i>REST</i> and <i>RNF2</i> using antibodies as indicated (K27 = H3K27Me3). Error bars represent standard deviations calculated from triplicate qPCR reactions.</p

Chemical composition and antimicrobial activity of the essential oils of some species of Anthemis sect. Anthemis (Asteraceae) from Sicily

The chemical composition of the essential oils isolated from the aerial parts of Anthemis arvensis L. subsp. arvensis, Anthemis cretica subsp. messanensis (Brullo) Giardina & Raimondo and from flowers and leaves of Anthemis cretica subsp. columnae (Ten.) Frezén were determinated by GC–FID and GC–MS analyses. Torreyol (85.4%) was recognised as the main constituent of the Anthemis arvensis subsp. arvensis essential oil, while in the essential oils of Anthemis cretica subsp. messanensis, collected on the rock and cultivated in Hortus Botanicus Panormitanus, (E)-chrysanthenyl acetate (28.8 and 24.2% resp.), 14-hydroxy-α-humulene (8.1 and 5.3% resp.), santolina triene (8 and 5.8% resp.) and α-pinene (6.7 and 5.4% resp.) prevailed. 18-cineole (13.3 and 12.2% resp.), was the main component of both flower and leaf oils of Anthemis cretica subsp. columnae together with δ-cadinene (9.0 and 8.2% resp.) and (E)-caryophyllene (8.3 and 5.6% resp.)

Osservatorio della Corte costituzionale (n. 3/2017)

Corte cost. n. 18 del 2017 (in tema di incompatibilit\ue0 endoprocessuale, udienza preliminare e invito al pubblico ministero a modificare l\u2019imputazione per fatto diverso); Corte cost. n. 17 del 2017 (in tema di custodia in carcere, tutela della maternit\ue0 e figli minori che abbiano gi\ue0 compiuto i sei anni); Corte cost. n. 24 del 2017 (in tema di prescrizioni, frodi comunitarie e giurisprudenza della Corte di Giustizia: un nuovo rinvio pregiudiziale a Lussemburgo per il caso Taricco); Corte cost. n. 21 del 2917 (in tema di testimonianza assistita e dichiarante assolto \u201cperch\ue9 il fatto non sussiste\u201d e di incostituzionalit\ue0 dell\u2019art. 197 bis, commi 3 e 6, c.p.p.)

Additional file 3: Table S3. of The segregation of different submicroscopic imbalances underlying the clinical variability associated with a familial karyotypically balanced translocation

Primers used in qRT-PCR analysis of duplicated (LMNB1, MARCH3 and FBN2) and reference (ACTB) genes. (XLSX 11 kb