Influence of CaO on structure and permeability of clayey soil

The aim of this study was to determine the effect of quicklime (1 - 8% CaO) and maturation time (1 - 540 days) on the structure of clayey soil compacted at optimum moisture content by Proctor standard energy and whether expected change in structure affects the long - term permeability. The change of pore space of compacted loess with 1-8% lime (CaO) was studied by mercury porosimetry (MIP) for a long period of maturation (from 1 to 540 days). Development of pozzolanic reactions were monitored by measuring the pH. The development of new mineral phases (calcium silicate hydrates, calcium aluminate hydrates and calcium aluminate carbonate hydrate) in the treated soil was investigated by using X-Ray diffraction. The MIP indicated that 2% of CaO were sufficient for long term pozzolanic reaction. The threshold value is below the initial consumption of lime determined from the pH measurements (Eades and Grim, 1966). The alteration of the voids of the lime treated soil is noticeable, but the pH value can not drop below 11.7. At 4% of CaO, at 8% of CaO respectively, the macroporosity kept decreasing due to increasing mesoporosity for 360 curing days, for 540 curing days respectively, due to the new mineral phases. At 2% of CaO, the decrease of the macroporosity stops after 120 days. Below 2% of lime, the..

Additional file 7: of Long non-coding RNAs display higher natural expression variation than protein-coding genes in healthy humans

A List of robust lncRNA transcripts in granulocytes (2,825 transcripts): columns are formatted as a BED12 file. B List of robust well expressed (RPKM >1) lncRNA transcripts in granulocytes (931 transcripts): columns are formatted as a BED12 file. (XLSX 250 kb

Tandem direct repeats regulate the length of <i>Airn</i>.

<p>(A) qPCR of total (spliced+unspliced) <i>Airn</i> in <i>S12/+</i> and four <i>S12/TDRΔ</i> cell lines (1A/1B/2A/2B), in undifferentiated (d0) and day 5 or 14 differentiated ES cells (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002540#pgen-1002540-g002" target="_blank">Figure 2</a> map for location of qPCR assays). Relative <i>Airn</i> levels were set to 100% in <i>S12/+</i> cells at d14. Bars and error bars: mean and standard deviation of three differentiation sets. <i>S12/+</i> and <i>S12/TDRΔ</i> were compared using an unpaired t-test (*P = 0.1–0.5, **P = 0.001–0.01, ***P<0.001). The data show that <i>Airn</i> steady-state levels are unchanged up to 53 kb but are greatly reduced and lost at the 3′ end. (B) qPCR of spliced <i>Airn</i> in <i>S12/+</i> and four <i>S12/TDRΔ</i> cell lines (1A/1B/2A/2B), in undifferentiated (d0) and day 5 or 14 differentiated ES cells. Details as in (A). These data show that the TDR deletion does not affect <i>Airn</i> splicing suppression but leads to a shortening at the 3′ end. (C) qPCR of unspliced <i>Airn</i> in 12.5–13.5 dpc mouse embryos confirms the significant loss of <i>Airn</i> steady-state levels at the 3′ end as seen in differentiated ES cells (A,B). Embryos from 3 litters were assayed carrying wildtype (<i>+/+</i>, <i>Thp/+</i>) or <i>TDRΔ</i> (<i>+</i>/<i>TDRΔ</i>, <i>Thp/TDRΔ</i>) paternal alleles. The <i>Thp</i> allele carries a deletion of the entire <i>Igf2r</i> cluster thus only the paternal allele is present. Samples of the same genotype were averaged and the horizontal lines and error bars show mean and standard deviation. Values for individual embryos are plotted as single data points. The number of samples is given below the genotype (n). Relative <i>Airn</i> levels were set to 100% for <i>+/+</i>, all others are displayed relative to it. Samples were compared to <i>+/+</i> using an unpaired t-test. Details as (A).</p

The <i>Airn</i> CGI plays a major role in <i>Airn</i> transcription and function.

<p>(A) <i>Airn</i> expression by genome-tiling array (left axis) and strand-specific expression analysis by RNA-Seq (right axis) for differentiated <i>S12/+</i> and <i>S12/CGIΔ</i>-1A cells. Dashed arrows: sharp drop of <i>Airn</i> hybridisation signals in the <i>Airn</i>-specific region (single) and absence after 73 kb (doublet). Below: qPCR assays relative to <i>Airn</i>-TSS with colour code as (B,C). Striped box: overlapping START+RP11 assays. (B) qPCR of total+unspliced <i>Airn</i> in d0/d5/d14 differentiated <i>S12/+</i> and four <i>S12/CGIΔ</i> clones shows unspliced <i>Airn</i> is reduced by ∼40% at the 5′ end (RP11/154 bp), but when assayed downstream (<i>Airn</i>-middle/53 kb, <i>Airn</i>-end/99 kb) or at positions which include splice variants (START), is reduced by >70% in <i>S12/CGIΔ</i> cells. Shown are mean and standard deviation of three differentiation sets (details as <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002540#pgen-1002540-g003" target="_blank">Figure 3A</a>). (C) <i>Airn</i> qPCR in <i>S12/+</i> and four <i>S12/CGIΔ</i> d14 clones shows that unspliced <i>Airn</i> is reduced by 79–83% at 0.57 kb and ∼85% at 7.3 kb, while spliced <i>Airn</i> reduced by >85%. Shown are mean and standard deviation of three differentiation sets (details as <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002540#pgen-1002540-g003" target="_blank">Figure 3A</a>). (D) ChIP for Ser5P/Ser2P RNAPII in <i>S12/+</i>, <i>S12/TDRΔ</i>-1A and <i>S12/CGIΔ</i>-1A d11 cells shows unaffected <i>Airn</i> initiation and elongation (except at <i>Airn</i>-end) in <i>TDRΔ</i> and a sharp RNAPII decrease in the <i>CGIΔ</i> allele. The mean and standard deviation of three technical replicates is shown. Assay <i>Airn</i>-132 controls for background from the overlapping <i>Igf2r</i> transcript, which is 2-fold higher in <i>CGIΔ</i> that fails to repress the paternal <i>Igf2r</i> promoter. Map for qPCR assays as <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002540#pgen-1002540-g002" target="_blank">Figure 2</a>. (E) DNA blot analysing methylation of the <i>Igf2r</i> promoter NotI site (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002540#pgen-1002540-g004" target="_blank">Figure 4A</a>). *methylated fragment in d0 cells originating from feeder-cells. This blot shows that cells carrying a paternal <i>CGIΔ</i> allele contrary to wildtype cells do not gain the methylated 5 kb band on the paternal <i>Igf2r</i> promoter. White lines: indicate the order of samples run on the same gel was changed electronically. (F) qPCR quantifying allelic expression shows absence of <i>Igf2r</i> imprinted expression (Mat∶Pat ratio is close to 1), in four <i>CGIΔ</i> (<i>S12/CGIΔ</i>) cell lines compared to wildtype (<i>S12/+</i>). Three differentiation sets are shown separately due to variability in Mat∶Pat ratios in wildtype controls for each set. Bars represent the mean, error bars the standard deviation of 3 technical replicates (details as <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002540#pgen-1002540-g004" target="_blank">Figure 4C</a>).</p

Tandem direct repeats play a role in <i>Airn</i> processivity.

<p><i>Airn</i> expression by genome tiling array in day 5 differentiated ES cells carrying a paternal wildtype (<i>S12/+</i>) or mutated (<i>S12/TDRΔ</i>-2A and <i>+/TDRΔ</i>) allele. Note the maternal allele is always written on the left side (Mat/Pat). x-axis: basepairs, y-axis: averaged relative signal intensities with standard deviation (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002540#s4" target="_blank">Materials and Methods</a>). Single and double dashed arrows: position after which consistent differences between wildtype and two <i>TDRΔ</i> cell lines are seen. Grey arrow: <i>Airn</i> hybridisation signals are lost after 90 kb in two <i>TDRΔ</i> cell lines. Below: <i>Airn</i> (wavy arrow) and <i>Airn</i> splice variants (black boxes: exons). Grey font: <i>Airn</i> qPCR assays with their distance from <i>Airn</i>-TSS. RP11, RP6, RP21, RP5, RP4 were combined with FP1+TQ-AS. This analysis shows that <i>Airn</i> in <i>TDRΔ</i> cells is reduced after 68 kb and lost after 90 kb.</p

Enhanced phagocytosis by <i>Trem-2</i>^−/− AM depends on C1q.

<p>(<b>A</b>) WT AM were adhered to C1q, control BSA or uncoated plates for 3 h prior to incubation with FITC labeled <i>S. pneumoniae</i> (MOI 100) and phagocytosis was assessed using FACS 1 h later (n = 3–4 per condition). (<b>B</b>) WT AM (n = 6–7 per condition) were pre-treated for 24 h with 10 µM PPAR-δ inhibitor GSK0660 or DMSO control after which, the cells were adhered to C1q or control plates for 3 h and phagocytosis of FITC labeled <i>S. pneumoniae</i> (MOI 100) was assessed 1 h later using FACS. (<b>C</b>) WT and <i>Trem-2</i>^−/− AM (n = 4 per genotype/condition) were pre-treated for 24 h with the indicated doses of the PPAR-δ inhibitor GSK0660 or DMSO control after which phagocytosis of FITC labeled <i>S. pneumoniae</i> (MOI 100) was assessed 1 h later using FACS. (<b>D</b>) WT and <i>Trem-2</i>^−/− AM were pre-treated for 1 h with 10 µg/ml C1q blocking antibody or isotype control and phagocytosis of FITC labeled <i>S. pneumoniae</i> (MOI 100) was assessed 1 h later using FACS (n = 4 per genotype/condition). (<b>E</b>) WT and <i>Trem-2</i>^−/− AM (n = 4–7 per condition) were adhered for 3 h to plates coated with the indicated conditions prior to incubation with FITC labeled <i>S. pneumoniae</i> (MOI 100) and phagocytosis was assessed using FACS. (<b>F</b>) WT and <i>Trem-2</i>^−/− AM (n = 4 per genotype) were incubated with FITC labeled <i>S. pneumoniae</i> (MOI 100) that were pre-opsonised with 10% WT or C1qa^−/− serum for 30 min after which phagocytosis was assessed 1 h later using FACS (<b>G</b>) WT and <i>Trem-2</i>^−/− mice (n = 17 per condition) were intranasally treated with 125 µg C1q blocking antibody or isotype control prior to infection with 6×10⁴ CFU <i>S. pneumoniae</i>. 48 h post infection lung bacterial CFUs were enumerated. Data represent mean ± SEM; ** p<0.005, *** p<0.001, **** p<0.0001. Data in (<b>A, C–F</b>) are representative of two independent experiments, (<b>B and G</b>) are pooled data from two independent experiments.</p

Pulmonary TREM-2 expression and function during <i>S. pneumoniae</i> induced inflammation.

<p>(<b>A</b>) TREM-1 and TREM-2 expression was evaluated in the indicated cell types using RT-PCR. (<b>B</b>) Western blot was used to evaluate TREM-2 expression on AM. (<b>C</b>) WT mice (n = 6 per time point) were intranasally inoculated with 10⁵ CFU <i>S. pneumoniae</i> and after indicated time points TREM-2 lung transcript levels were evaluated. (<b>D</b>) WT AM were treated with 2×10⁷ CFU/ml <i>S. pneumoniae</i> for indicated time points and TREM-2 RT-PCR was conducted. (<b>E–G</b>) WT and <i>Trem-2</i>^−/− mice (n = 7 per genotype) were intranasally infected with 10⁵ CFU <i>S. pneumoniae</i> for 6 h and levels of indicated cytokines were evaluated in the lung (<b>E</b>) or BALF (<b>F</b>) and neutrophil counts were determined in the BALF (<b>G</b>). Data represent mean ± SEM and are (<b>A–G</b>) representative of two independent experiments. Differences were calculated versus time point 0 (<b>C–D</b>) or versus WT (<b>E–G</b>) and are indicated as * p<0.05, ** p<0.005, **** p<0.0001.</p

TDRs are required for the regulation of ICE DNA methylation.

<p>(A) Genomic DNA digested with EcoRI (E) or EcoRI+methyl-sensitive MluI (E/M) and hybridised with probe <i>Airn</i>T. Cells carrying a paternal <i>TDRΔ</i> (<i>S12/TDRΔ</i>) or wildtype (<i>S12/+</i>) allele have a 6.2 kb band from the maternally-methylated plus 4.3 kb or 5.0 kb from the paternally-unmethylated allele. <i>S12/TDRΔ</i> cells additionally show at d0, a faint 5.5 kb band representing a paternal partially methylated allele (*) that is lost by d14 in differentiated cells. (B) Bisulfite sequencing of undifferentiated <i>S12/TDRΔ</i>-1A ES cells. Each row represents one allele, single columns: one CpG, double columns: two neighbouring CpGs. White/black circles: unmethylated/methylated CpGs. Arrows: position of MluI site and <i>TDRΔ</i> deletion. Primers DMR2-4F and -9R specifically amplify the paternal allele in <i>S12/TDRΔ</i> ES cells (details in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002540#pgen.1002540.s005" target="_blank">Figure S5B</a>). Bisulfite sequencing confirms a low level of DNA methylation on the paternal <i>TDRΔ</i> allele at d0 only. (C) Percent methylation levels for two <i>S12/TDRΔ</i> and control <i>R2Δ/+</i> ES cells. The <i>R2</i>Δ deletion removes the maternal ICE, thus only the wildtype paternal ICE is detected. Each point represents one sequenced clone and shows that low-level DNA methylation is a general feature of the paternal ICE in d0 ES cells and not a consequence of the TDR deletion. (D) DNA blot analysing the MluI methylation status as in (A) for 12.5–13.5 dpc embryos carrying a maternal <i>TDRΔ</i> (<i>TDRΔ/+, TDRΔ/Thp</i>) or wildtype (<i>+/+, +/Thp</i>) allele. The 5.0 kb methylated band is faint or absent in <i>TDRΔ/+</i> embryos, but present in <i>+/+</i> embryos (6.2 kb), showing that maternal inheritance of the <i>TDRΔ</i> allele leads to loss of DNA methylation. (E) Bisulfite sequencing of embryonic genomic DNA (details as in B) shows that maternal transmission of the TDR deletion leads to a major loss in DNA methylation. For <i>TDRΔ/Thp</i> only the maternal allele, for <i>+/+</i> both parental alleles were analysed. (F) Percent methylation level for <i>TDRΔ/Thp</i> and <i>+/+</i> embryos as in (C).</p

CpG islands lie upstream and downstream of the transcription start site.

<p>(A) ES cell imprinting model. In undifferentiated ES cells <i>Igf2r</i> is expressed from both chromosomes and <i>Airn</i> is not expressed <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002540#pgen.1002540-Latos1" target="_blank">[31]</a>. The ICE, which contains the <i>Airn</i> promoter, is methylated on the maternal allele (black hexagon) and unmethylated on the paternal allele (white hexagon). The <i>Igf2r</i> promoter is unmethylated on both parental alleles (white oval). During differentiation, <i>Airn</i> (wavy line) is expressed from the unmethylated paternal allele. <i>Igf2r</i> is upregulated up to 20-fold on the maternal allele, but its upregulation on the paternal allele is blocked by <i>Airn</i> expression <i>in cis</i>. The repressed paternal <i>Igf2r</i> promoter gains DNA methylation late in differentiation (black oval). (B) <i>Airn</i> 5′ region. T1,T2,T3: multiple closely-spaced <i>Airn</i> TSSs. 53 bp splice donor: shared by all <i>Airn</i> splice variants. MluI: MluI restriction site. Grey shading: CGI. Grey triangles labelled TDRs: tandem direct repeats. <i>CGIΔ</i>/<i>TDRΔ</i>: 1129/692 bp deletions generated here. <i>AirnPΔ</i>: 959 bp deletion defining <i>Airn</i> promoter <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002540#pgen.1002540-Stricker1" target="_blank">[32]</a> that lies upstream of the CGI. <i>R2Δ</i>: 3656 bp deletion defining the imprint control element or ICE <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002540#pgen.1002540-Wutz1" target="_blank">[10]</a>. Thicker grey line on <i>R2Δ</i>: maternally methylated region. Possible roles of the CGI and the TDRs on the two parental alleles are listed below. (C) Transcription start sites (TSSs) of mouse RefSeq genes are plotted relative to their CpG island (CGI) and 50% of the CGI length upstream and downstream (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002540#s4" target="_blank">Materials and Methods</a>). Dotted line: TSS of <i>Airn</i>, <i>Kcnq1ot1</i> and <i>Igf2r</i>. The <i>Airn</i> TSS lies upstream to the CGI in contrast to the majority of TSSs that lie inside of the CGI.</p

The methylation-free state of the paternal ICE depends on the CGI.

<p>(A) DNA blot assaying methylation of the <i>Airn</i> promoter MluI site as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002540#pgen-1002540-g005" target="_blank">Figure 5A</a>, in undifferentiated ES cells carrying a paternal <i>CGIΔ</i> or wildtype (<i>+</i>) allele. The 5.0 kb band identified by probe MEi (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002540#pgen-1002540-g005" target="_blank">Figure 5A</a> map) indicates a gain of methylation on the <i>CGIΔ</i> paternal allele. This band is weaker in cells with lower passage numbers that still retain the selection cassette (<i>S12/CGIΔ+cas</i>-1,-2) compared to cells that have been in culture for 8 more passages (<i>S12/CGIΔ</i>-1A,-1B,-2A,-2B) with a deleted selection cassette. The lower panel confirms this by showing a matching loss of the unmethylated 1.1 kb fragment specific to the paternal allele in cells with a higher passage number. Both panels were from the same blot and the intervening area lacking any hybridisation signal removed. (B) DNA blot as in (A) assaying <i>Airn</i> promoter MluI methylation during ES cell differentiation showing that the level of paternal methylation on the <i>CGIΔ</i> allele in undifferentiated ES (d0) cells (5 kb band) does not change in differentiated d5 and d14 cells. Probe MEi is a 1 kb EcoRI-MluI fragment shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002540#pgen-1002540-g005" target="_blank">Figure 5A</a> map. (C) Bisulfite sequencing of two undifferentiated <i>S12/CGIΔ</i> ES cell clones using primers spanning the deletion that specifically amplify the paternal <i>CGIΔ</i> allele, confirms the strong gain of DNA methylation, but also shows that some alleles are more methylated than others (details as <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002540#pgen-1002540-g005" target="_blank">Figure 5B</a>).</p