La membrana vitelina de embriones bovinos, un modelo experimental de interés para estudios proteómicos.Análisis del perfil proteico mediante electroforesis bidimensional

Comunicaciones a congreso

Deletion of the Sequence Encoding the Tail Domain of the Bone Morphogenetic Protein type 2 Receptor Reveals a Bone Morphogenetic Protein 7-Specific Gain of Function

The bone morphogenetic protein (BMP) type II receptor (BMPR2) has a long cytoplasmic tail domain whose function is incompletely elucidated. Mutations in the tail domain of BMPR2 are found in familial cases of pulmonary arterial hypertension. To investigate the role of the tail domain of BMPR2 in BMP signaling, we generated a mouse carrying a Bmpr2 allele encoding a non-sense mediated decay-resistant mutant receptor lacking the tail domain of Bmpr2. We found that homozygous mutant mice died during gastrulation, whereas heterozygous mice grew normally without developing pulmonary arterial hypertension. Using pulmonary artery smooth muscle cells (PaSMC) from heterozygous mice, we determined that the mutant receptor was expressed and retained its ability to transduce BMP signaling. Heterozygous PaSMCs exhibited a BMP7‑specific gain of function, which was transduced via the mutant receptor. Using siRNA knockdown and cells from conditional knockout mice to selectively deplete BMP receptors, we observed that the tail domain of Bmpr2 inhibits Alk2‑mediated BMP7 signaling. These findings suggest that the tail domain of Bmpr2 is essential for normal embryogenesis and inhibits Alk2‑mediated BMP7 signaling in PaSMCs

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

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

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

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. 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

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

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