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

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

Ge-1 co-precipitates with C-terminal fragments of LMKB.

<p>COS-7 cells were transfected with plasmids encoding FLAG-Ge-1, GFP-LMKBc (containing amino acids 1457–1742), GFP-LMKBc’ (amino acids 1622–1742), FLAG and GFP as indicated. Extracts were prepared and incubated with mouse anti-GFP antibody and protein G coupled to Sepharose beads. Precipitates (top two panels) are compared with 5% of the total COS-7 cell extract input (bottom two panels). FLAG-Ge-1 co-precipitated with GFP-LMKBc(1457–1742) and with GFP-LMKBc’(1622–1742), but not with GFP alone.</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

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

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

Antibodies in the serum of patient 0081 react with LMKB.

<p>The human serum contained antibodies directed against GST-LMKB amino acids 1457–1742, but did not react with GST alone. Black arrow indicates location of the GST-LMKB fusion protein.</p

Aortic expression of VSMC markers in wild-type and MGP^-/- mice.

<p>RNA was isolated from aortas of WT and MGP^-/- mice and from LDN-193189-treated MGP^-/- mice at 7 and 14 days of age (n = 4–8 in each group). Levels of mRNAs encoding myocardin, α smooth muscle actin (SMA), transgelin, and calponin are depicted. The aortas of 14-day-old MGP^-/- mice have decreased expression of VSMC markers compared to WT mice. Treatment with LDN-193189 did not restore the expression of VSMC markers to WT levels. # P<0.05 compared to 7-day-old MGP^-/- mice.</p

Vascular calcification associated with MGP deficiency occurs in the absence of vascular inflammation.

<p>(<b>A</b>) At 27 days of age, OsteoSense-680 and Prosense-750 were injected via the tail vein of wild-type (WT) and MGP^-/- mice. Aortas were harvested 24 hours later and imaged. Although aortas from MGP^-/- mice exhibited extensive vascular calcification, this calcification was not associated with increased macrophage activity. (<b>B</b>) Aortas were harvested from WT and MGP^-/- mice at 28 days of age, sectioned, and stained for macrophages with an antibody directed towards MAC-2. Aortas from LDLR^-/- mice on a high fat diet were used as a positive control. Nuclei were stained with DAPI. Similar to WT mice, macrophages were not detected by immunohistochemistry in the aortas of MGP^-/- mice.</p

BMP signaling is required for the increased aortic expression of osteogenic markers associated with MGP deficiency.

<p>RNA was isolated from aortas of WT and MGP^-/- mice at 1, 7, 14, and 28 days of age and from LDN-193189-treated MGP^-/- mice at 7, 14, and 28 days of age (n = 4–11 in each group, as indicated). Expression of genes encoding Runx2 and osteopontin (OPN) was measured. MGP^-/- mice had increased levels of aortic Runx2 and OPN mRNA compared to WT mice. Treatment of MGP^-/- mice with LDN-193189 reduced aortic Runx2 and OPN mRNA levels. * P<0.001 compared to WT mice of same age. # P<0.05 compared to age-matched MGP^-/- mice treated with vehicle.</p