Indirect immunofluorescence shows that LMKB localizes to P-bodies.

<p>GFP-LMKB (green, i) localized to discrete, dot-like structures in the cytoplasm of transfected HEp-2 cells and co-localized with co-expressed FLAG-Ge-1 (red, ii). To determine the cellular location of <i>endogenous</i> LMKB, rabbit anti-LMKB antiserum was used to stain Hut78 cells. LMKB (green, iv) co-localized with Ge-1 (red, v), identified using human serum 0121. After exposure to arsenite for 1 hour, TIA (a marker of stress granules) was detected in cytoplasmic granules (red, viii). LMKB (green, vii) did not co-localize with TIA in stress granules, but was instead detected in adjacent P-bodies. Merge of fluorescence in i and ii, iv and v, vii and viii is shown in iii, vi and ix. DAPI staining in iii and vi (blue) indicates the location of nuclei. White arrows in vii and ix indicate representative LMKB-containing P-bodies adjacent to stress granules. White bar in ix indicates 5.0 µm.</p

A. A modified two-hybrid assay was used to test for interaction between LMKB and Ge-1 and to identify the portion of LMKB that mediates interaction with Ge-1.

<p>Expression of a plasmid encoding Ge-1 fused to a nuclear localization sequence (NLS) shifted some of the protein from the cytoplasm to the nucleus of HEp-2 cells (red, i, iv and vii), where it localized to dot-like structures. Co-transfection of plasmids encoding GFP fused to full-length LMKB (green, ii) and NLS-Ge-1 resulted in co-localization of the two proteins in nuclear dots. GFP-LMKB fragment fusion proteins encoding amino acids 1622–1742 (green, v) also co-localized with co-expressed NLS-Ge-1 (red, iv) in nuclear dots. GFP-LMKB fragments encoding 1–687 and 389–1199 (both not shown) and 1–1622 (green, viii) did not co-localize with NLS-Ge-1 in nuclear dots. Merge of fluorescence in i and ii, iv and v, vii and viii is shown in iii, vi and ix. DAPI staining (blue) in iii, vi and ix indicates the location of nuclei. Human serum 0121 containing anti-Ge-1 antibodies and mouse monoclonal anti-GFP antibodies were used to detect Ge-1 and GFP, respectively. White arrows in vii and ix indicate nuclear dots containing NLS-Ge-1. White bar in ix indicates 5.0 µm. <b>B</b>. Schematic representation of the structure of LMKB and summary of results. LMKB contains an N-terminal globular domain (“LK”, amino acids 351–493) that may serve as a cation binding domain. Two RRM-type RNA binding domains are located between amino acids 510–600 and 792–867. The C-terminus of LMKB contains eight helix-turn-helix folds (“OST domains”) that are predicted to bind to dsRNA (amino acids 879–937, 1004–1074, 1100–1170, 1176–1247, 1259–1330, 1337–1406, 1412–1484, 1487–1557; numbering system is as indicated in GenBank #NM_014647). + indicates an interaction between a LMKB fragment and Ge-1; - indicates a fragment of LMKB that does not interact with Ge-1. The black rectangle indicates the smallest tested portion of LMKB that interacts with Ge-1.</p

Bmpr2 expression in PaSMCs obtained from WT or <i>Bmpr2</i>^{<i>Δtd/+</i>} mice.

<p>(A) Levels of <i>Bmpr2</i> mRNA were measured in WT (Bmpr2^+/+) or <i>Bmpr2</i>^{<i>Δtd/+</i>} PaSMCs by qPCR using hydrolysis probes for <i>Bmpr2</i> exon junctions 6–7 and 12–13. <i>Bmpr2</i> mRNA levels were normalized to <i>Gapdh</i> and expressed as the fold-change relative to <i>Bmpr2</i>^<i>+/+</i> PaSMCs. *P < 0.01 compared to <i>Bmpr2</i>^<i>+/+</i> PaSMCs. (B) Immunoblots prepared from lysates of <i>Bmpr2</i>^<i>+/+</i> and <i>Bmpr2</i>^{<i>Δtd/+</i>} PaSMCs were incubated with an antibody directed against the tail domain of Bmpr2 to detect Bmpr2‑WT or with an anti-GFP antibody to detect Bmpr2‑ΔTD. Immunoblots were subsequently incubated with an antibody directed against Gapdh as a control for protein loading. (C) Confocal microscopy image of a PaSMC transiently transfected with a plasmid directing expression of <i>Bmpr2</i>^<i>Δtd</i> and reacted with an anti-GFP antibody showing localization of Bmpr2‑ΔTD at the cell membrane.</p

The Analysis of a Novel Computational Thinking Test in a First Year Undergraduate Computer Science Course

In Ireland, Computer Science is not yet a state examination subject. In recent years, steps to include it have been taken - it was introduced as a Leaving Certificate subject in the academic year of 2018-19 on a pilot basis and will be examined for the first time in 2020 (O’Brien, 2017). Prior to this, the only Computer Science course offered at second level was a Junior Certificate Coding short course (NCCA, 2017). Research shows that an early introduction to computing is an advantage for students. It can build confidence in dealing with complexity and with open-ended problems (Yevseyeva &Towhidnejad, 2012). Problem-solving skills can be extended and transferred as reported by Koh et al. (2013) and students’ analytical skills can be improved according to Lishinski et al. (2016) and Van Dyne and Braun (2014). It has been shown by Webb and Rosson (2013) that students’ self-efficacy for computational problem solving, abstraction, debugging and terminology can be in-creased. It has also been found that teaching Computational Thinking can provide a better understanding of how programming is about solving a problem (not just coding) and that it can improve female students’ attitudes and confidence towards programming (Davies, 2008). One especially interesting finding is that exposure to Computational Thinking can be used as an early indicator and predictor of academic success since Computational Thinking scores have been found to correlate strongly with general academic achievement by Haddad and Kalaani (2015). This paper examines first year undergraduate Computer Science students who took a novel test to assess their Computational Thinking skills and in addition a survey gathering their views on Computer Science and Computational Thinking. This survey was administered twice within the academic year and comparisons are drawn on the changes between these survey results

Bmpr2‑TD attenuates Alk2‑mediated BMP7 signaling in PaSMCs.

<p>Alk3‑deficient <i>Bmpr2</i>^{<i>Δtd/+</i>} PaSMCs were transfected with specific siRNA to silence <i>Bmpr2</i>^<i>+</i> (si<i>Bmpr2</i>‑ex12) or <i>Bmpr2</i>^<i>Δtd</i> (si<i>Egfp</i>) transcripts. After 48 h, the ability of BMP7 to induce <i>Id1</i> and <i>Smad6</i> gene expression was measured by qPCR, normalized to <i>Gapdh</i> and expressed as fold-change relative to <i>Bmpr2</i>^{<i>Δtd/+</i>}; <i>Alk3</i>^{<i>del/del</i>} PaSMCs treated with siNC. *P < 0.01 compared to control cells (siNC) treated with BMP7. Silencing efficiency was quantified by qPCR.</p

Bmpr2‑ΔTD contributes to BMP7 signaling in <i>Bmpr2</i>^{<i>Δtd/+</i>} PaSMCs.

<p>(A) <i>Bmpr2</i>^{<i>Δtd/+</i>} PaSMCs were transfected with negative control siRNA (siNC), si<i>Bmpr2</i>‑ex12, or si<i>Egfp</i> (30 nM). After 48 h, the ability of BMP7 (10 ng/ml for 1.5 h) to induce <i>Id1</i> and <i>Smad6</i> mRNA expression was measured by qPCR, normalized to <i>Gapdh</i> and expressed as fold-change relative to <i>Bmpr2</i>^{<i>Δtd/+</i>} PaSMCs transfected with siNC. *P < 0.01 compared to siNC group treated with BMP7, ^† P<0.01 compared to si<i>Bmpr2</i>‑ex12 group treated with BMP7. Efficiency of silencing <i>Bmpr2</i>^<i>+</i> (si<i>Bmpr2</i>‑ex12) and <i>Bmpr2</i>^<i>Δtd</i> (si<i>Egfp</i>) transcripts was measured by qPCR. (B) <i>Bmpr2</i>^{<i>Δtd/flox</i>} and <i>Bmpr2</i>^{<i>Δtd/del</i>} PaSMCs were treated with BMP4 or BMP7 (10 ng/ml) for 30 and 60 minutes, upon which the activation of Smad1/5/8 was evaluated by immunoblotting. Quantification of the Smad1/5/8 activation is plotted as the ratio of pSmad1/5/8 to total Smad1/5/8. (C) The ability of BMP4 or BMP7 to induce <i>Id1</i> and <i>Smad6</i> gene expression in <i>Bmpr2</i>^{<i>Δtd/flox</i>} and <i>Bmpr2</i>^{<i>Δtd/del</i>} PaSMCs was measured by qPCR, normalized to <i>Gapdh</i> and expressed as fold-change relative to untreated <i>Bmpr2</i>^{<i>Δtd/flox</i>} PaSMCs. *P < 0.01 compared to <i>Bmpr2</i>^{<i>Δtd/flox</i>} PaSMC group treated with BMP7.</p

List of genes differentially expressed in BJAB cells after LMKB knockdown compared with control BJAB cells.

<p>List of genes differentially expressed in BJAB cells after LMKB knockdown compared with control BJAB cells.</p

The effects of LMKB depletion on gene expression in BJAB and Hut78 cells

<p>. SiRNA-mediated knock-down of LMKB in BJAB and Hut78 cells resulted in a greater than 80% decrease in the level of <i>LMKB</i> mRNA (i). LMKB depletion had no effect on the level of <i>PPP2cb</i> mRNA in either cell line (ii). The level of <i>IFI44L</i> mRNA was increased in both BJAB and Hut78 cell lines after LMKB knock-down (iii).</p

Loss of LMKB immunoreactivity after LMKB knockdown in BJAB and Hut78 cells.

<p>Treatment of BJAB cells with siRNA directed against LMKB resulted in loss of P-body staining as determined by indirect immunofluorescence (i). LMKB was detected in a cytoplasmic dot staining pattern in cells treated with control siRNA (green, iv). DAPI staining (blue) in ii and v indicate the location of nuclei. Merge of the preceding panels is shown in iii and vi. Treatment of Hut78 cells with siRNA directed against LMKB also resulted in loss of cytoplasmic dot staining (vii), compared with cells treated with control siRNA (green, x). Loss of LMKB did not alter the cellular location of Ge-1-containing P-bodies (red, viii and xi) demonstrating that LMKB is not required for P-body formation. Merge of vii and viii, and x and xi, is shown in ix and xii. DAPI staining (blue) in the merged panels indicates the location of nuclei. White bar in xii indicates 5.0 µm.</p

A. Delineation of the smallest portion of Ge-1 that mediates interaction with LMKB.

<p>GFP-NLS-Ge-1 fragment (amino acids 630–1437) localized to nuclear dots (green, ii) in HEp-2 cells, but RFP-LMKB remained in P-bodies and was not detected in the nucleus (red, i), suggesting that N-terminal amino acids in Ge-1 are required for interaction with LMKB. To identify the N-terminal portion of Ge-1 that interacts with LMKB, RFP-LMKB was tested for the ability to recruit N-terminal fragments of Ge-1 to P-bodies. In the presence of RFP-LMKB (red, iv), GFP-Ge-1(1–1094) localized to cytoplasmic dots resembling P-bodies (green, v). In cells expressing RFP-LMKB (red, vii) and GFP-Ge-1(1–1094) (green, viii), both proteins co-localized with endogenous Ge-1 (blue, ix), confirming that these structures are P-bodies. In the absence of LMKB (RFP alone, red, x), GFP-Ge-1(1–1094) did not localize to P-bodies (green, xi), but was instead distributed throughout the cytoplasm. Smaller fragments of Ge-1, including amino acids 1–935 (green, xiv) and 104–1094 (green, xvii) did not co-localize with co-expressed LMKB in P-bodies. Merge of fluorescence in i and ii, iv and v, x and xi, xiii and xiv, and xvi and xvii is shown in iii, vi, xii, xv and xviii. DAPI staining (blue in the merged panels) indicates the location of nuclei. White bar in xviii indicates 5.0 µm. <b>B.</b> Schematic representation of Ge-1 and summary of results. The N-terminus of Ge-1 contains a WD40 repeat domain. The C-terminus contains four regions that have repeating hydrophobic residue periodicity (ψ(X_2-3)-repeat domains). + indicates a fragment of Ge-1 that interacts with LMKB; - indicates a fragment of Ge-1 that does not interact with LMKB. The black rectangle indicates the smallest tested portion of Ge-1 that interacts with LMKB.</p