Extra spuisluis in de Afsluitdijk: Effect op onderhoud havens

Datasets for constructing the miRNA-disease association prediction models. Four datasets such as three positive sample sets “positive_miR”, “positive_HMDD” and “positive_miRcancer” and the negative sample set “negative_expression” are stored. The three positive sample sets are retrieved from the three existing databases such as miR2Disease, HMDD v2 and miRCancer, while the negative sample set was obtained via analyzing the expression of the miRNAs.This file can also be downloaded from: https://drive.google.com/open?id=0B6lH3mKdA9CSTkg2OVBPS0ZfVnM . (XLS 947 kb

Generation of miR-132/212 knockout mice.

<p>miR-132/212 knockout mice were generated by insertion of LoxP sites in the 1^st intron and exon2 of the small non coding RNA gene that contains miR-132 and miR-212 (A). Targeted C57BL/6 ES cells were generated as described in the Methods section, and identified by Southern analysis of Kpn I digests using a probe 3′ to the sequence used in the targeting vector (B). Correctly targeted ES cells were used to generate a conditional miR-132/212 allele in mice using standard techniques. Mice were crossed to Flp transgenic mice to excise Neomycin resistance cassette and then Cre expressing mice to delete miR-132 and miR-212. Routine genotyping was carried out using 3 primers (p1, p2 and p3) which resulted in bands of 373 bp for the wild-type allele, 420 for the floxed allele and 550 for the deleted allele (C). Litter sizes (D) and pre weaning mortality (E) for matings of male and female knockout (n = 50 for litter size, n = 39 for pre weaning mortality), floxed (n = 20 or 17) or heterozygous (n = 53 or 41) mice are shown. Litters where pups were used for neuronal cultures were excluded from the pre weaning mortality values. Error bars represent standard deviation.</p

Deletion of miR212/132 influences hippocampal synaptic transmission and plasticity.

<p>A) Plot of fEPSP slope vs stimulus strength (mean ± SEM) showing that basal synaptic transmission is reduced by ∼30% in miR 212/132 KO slices (grey circles) compared to control (fl/fl) slices (black circles). Inset are representative fEPSPs at each of the stimulus strengths for fl/fl (left, black traces) and KO (right, grey traces) slices. Scale bar measures 5 ms and 1 mV. B) Paired-pulse facilitation (mean ± SEM) is no different between control (black circles) and miR KO slices (grey circles). Inset are representative fEPSPs at each of the inter-pulse intervals for control (left, black traces) and KO (right, grey traces) slices. Scale bar measures 100 ms and 1 mV. A and B represent data from 17 slices from 8 KO, and 11 slices from 6 control mice. C) LTP induced by tetanic stimulation (100 Hz for 1s) was not appreciably different between fl/fl (black circles) and KO mice (grey circles). Inset are fEPSPs taken before (smaller of the traces) and 60 mins after the induction of LTP (larger of the two traces) in fl/fl (left, black traces) and KO (right, grey traces) slices. Data from 11 slices from 8 KO mice and 6 slices from 4 control mice. Mean ± SEM. Scale bar measures 5 ms and 1 mV. D) In contrast, theta-burst stimulation (5 episodes at 10 s intervals of theta-burst stimulation (5 pulses at 100 Hz, repeated 10 times with 200 ms interval)) revealed greater LTP in KO slices (grey circles) compared to fl/fl (black circles) slices (p<0.05 at 55–65^th minute; unpaired t-test). Inset are fEPSPs taken before (smaller of the traces) and 60 mins after the induction of LTP (larger of the two traces) in fl/fl (left, black traces) and KO (right, grey traces) slices. The black and grey triangles hovering at 100% refer to the slope of fEPSPs from a simultaneously recorded control pathway for fl/fl and KO slices, respectively. Data from 13 slices from 5 KO mice and 6 slices from 2 fl/fl mice. Scale bar measures 5 ms and 1 mV. Data is presented as mean ± SD.</p

miR-132 and miR-212 are not required for cytokine induction in BMDMs.

<p>BMDMs were isolated from wild-type or miR-132/212 knockout mice. Cells were either left unstimulated or stimulated with 100 ng/ml LPS, 10 µg/ml poly I:C, 2 mM CpG, 100 ng/ml Pam3-CSK4, 100 ng/ml Pam3-CSK4 or 100 ng/ml CL097 for 6 h. Secreted levels of TNF, IL-12p40, IL-6 and IL-10 in the media were measured by a multiplex based assay as described in the methods. Error bars represent the standard deviation of independent cultures from 4 mice per genotype.</p

Primer sequences used for qPCR.

<p>Primer sequences used for qPCR.</p

miR-132 and miR-212 do not regulate LPS-induced cytokine production <i>in vivo</i>.

<p>Wild-type or miR-132/212 knockout mice were given an intraperitoneal injection with either PBS or LPS (2 mg/kg). The levels of TNF, IL-10, IL-12p70, IL-12p40, IL-13, KC, MCP1, Mip-1a and Mip-1b were then determined at 1, 4 or 8 h after the injection using a multiplex cytokine assay. Error bars represent the standard deviation of 4 to 6 mice per condition.</p

miR-132/212 knockout cortical cultures exhibit normal morphology.

<p>Cultures of cortical neurons were established on coverslips from P0 pups from either wild-type or miR-132/212 knockouts. After 24, 48, 72 or 96 h in culture, neurons were fixed with PFA and stained with MAP2 primary mouse monoclonal then fluorescein-labelled secondary anti-mouse antibody. Morphology was examined using confocal fluorescence microscopy and the images were analysed using the FilamentTracer Module of the Imaris software (Bitplane, Switzerland). Dendrite length (A) and branching (B) were quantified and representative images are shown with MAP2 staining in green and DAPI in blue (C). A p value (students t-test) between wild-type and knockout of <0.05 is indicated by * or <0.01 by **. Alternatively cultures were stimulated with 20 µM NMDA for the indicated times and total RNA isolated. The levels of pri-miR-132/212, miR-132, miR-212 (D), p250-Gap and MeCP2 (E) were determined. Error bars represent the standard deviation of 4 cultures per genotype. Similar experiments were also performed but the level of MeCP2 was determined by immunoblotting (E).</p

Deletion of miR132/212 influences synaptic transmission and plasticity in the neocortex.

<p>A) Representative examples of miniature spontaneous currents recorded in pyramidal neurons of the somatosensory cortex of a control (fl/fl mice, left panels) and miR-132/212 KO slice (right panels). Whole-cell recording were made at membrane potential of −80 mV in the presence of TTX (1 µM) and picrotoxin (100 µM). Scale bar measures 1 s and 10 pA. B) The superimposition of the average waveform (25 each) of mEPSCs recorded in the fl/fl neuron (black line) and miR-132/212 KO neuron (grey line) shown in A. Scale bar measures 20 ms and 5 pA. C) Superimposition of corresponding amplitude distributions of mEPSCs recorded in the fl/fl neuron (black) and miR-132/212 KO (grey) neuron. The leftward shift in the peak of amplitude distribution of mEPSCs in the KO neuron clearly indicates at decrease in the unitary size of the mEPSCs. D) Cumulative distributions of mEPSCs amplitudes pooled for 7 fl/fl (black line) and 8 miR-132/212 KO neurons (grey line) showing a consistent leftward shift towards reduced mEPSC amplitude in miR-132/212 KO neurons. E) Pooled data of average mEPSC and quantal, unitary size (mean ± SD for 7 fl/fl and 8 KO neurons). The difference in the average amplitude and quantal size between fl/fl and miR KO neurons was statistically significant with p<0.01 (ANOVA). F) Time course of changes in the slope of fEPSPs in layer 2/3 of somatosensory cortex after theta-burst stimulation (5 episodes at 10 s intervals of theta-burst stimulation (5 pulses at 100 Hz, repeated 10 times with 200 ms interval)). Each point represents the mean ± SD for 11 cortical slices of fl/fl mice (black circles) and 14 cortical slices of miR-132/212 KO mice (grey circles). The insets show the average fEPSP (20 each) waveforms recorded before (smaller of the two traces) and 60 min after theta-burst stimulation (larger of the two traces) in cortical slices of fl/fl (black lines) and miR-132/212 KO (grey lines) mice. The black and grey triangles hovering at 100% refer to the slope of fEPSPs from a simultaneously-recorded control pathway for fl/fl and KO slices, respectively. Long-term potentiation of excitatory synaptic transmission is impaired in the neocortex of miR-132/212 KO mice. Scale bar measures 5 ms and 1 mV.</p