The redevelopment of Thomond Park, Limerick

Thomond Park, the historic home of Munster Rugby, has undergone major redevelopment (fig. 1) which will see its capacity rise to 25 756. This paper describes the design and construction of the new stand and terrace structures. Particular emphasis is given to the development of the engineering and architectural concepts , the wind tunnel testing and dynamic analysis undertaken during detailed design, and the fabrication and erection of the two 150m span 'rainbow' trusses

HIRA is enriched in the vicinity of <i>Tnni2</i>/<i>Tnnt3</i>, which is upregulated in conditionally deleted <i>Hira</i> mutants, and binds GAGA rich DNA.

<p><b>A.</b><i>In situ</i> hybridization <i>Tnni2</i> on transverse sections of E12.5 embryos with the indicated genotype, revealed an abnormally increased expression of <i>Tnni2</i> particularly in the ventricular free wall of the mutant hearts (arrows). <i>Scale bar</i>: <i>100μm</i> <b>B</b>. qChIP of the HIRA enrichment at the <i>TTe</i> locus. An intergenic region on chromosome 6 was used as a negative control. Data is normalized to the input (= 100) and is representative of 2 independent experiments. Errors bars represent the min/max from technical duplicates. Unpaired t-test: p<0.001 *** <b>C.</b> IGV profile showing the HIRA ChIP seq peak present at the <i>Tnni2/Lsp1/Tnnt3 (TTe</i>) site (situated 17Kb downstream of <i>Tnni2</i> and 34Kb upstream of <i>Tnnt3)</i>, co-localizing with NKX2.5 ChIPseq peaks (E11.5 heart [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161096#pone.0161096.ref011" target="_blank">11</a>]), NKX2.5 DamID peaks (HL-1 cell line [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161096#pone.0161096.ref012" target="_blank">12</a>]), and the active enhancer marks H3K4me1 and H3K27Ac ChIPseq peaks on WT E13.5 hearts (encode database ENCSR663VWL) and P300 (WT E12.5 heart [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0161096#pone.0161096.ref023" target="_blank">23</a>]). <b>D.</b> The Matrix-based nucleotide profiles display here the motifs that HIRA binds most frequently. 45% of HIRA enriched sites contained a GAGA/TCTC motif.</p

HIRA is required for the regulation of a subset of genes expressed during cardiac morphogenesis.

<p><b>A.</b> Venn diagram indicating the number of genes whose expression is up or downregulated in <i>Mesp1Cre</i>;<i>Hira</i>^<i>-/fl</i> compared to <i>Mesp1Cre</i>;<i>Hira</i>^<i>+/fl</i> hearts at E11.5 and E12.5. The table indicates the level of fold change observed at E11.5 and at E12.5 on a subset of genes. <b>B.</b> Heatmap displaying the differential gene expression in mutant <i>(Mesp1Cre</i>;<i>Hira</i>^<i>-/fl</i>) vs control <i>(Mesp1Cre</i>;<i>Hira</i>^<i>+/fl</i>) in triplicates at E12.5. <b>C.</b> Heatmap of the gene ontology analysis revealed a trend in upregulation of sarcomeric contractile fibre genes (aside from <i>Tnnt1 and Slc4a1</i> which were downregulated). <b>D.</b> qRT-PCR and RNASeq of the indicated genes within E12.5 hearts displayed as the fold induction in the mutants compared to their WT littermates (n = 3). Unpaired t-test: p<0.05 *, p<0.01 **, p<0.001 ***.</p

Embryonic phenotype resulting from the conditional ablation of <i>Hira</i>.

<p>Embryonic phenotype resulting from the conditional ablation of <i>Hira</i>.</p

HIRA interacts with WHSC1 and binds to the <i>TTe</i> in the cardiac lineage and heart, and is required for differentiation-associated H3.3 enrichment at this locus.

<p><b>A.</b> 20 E14.5 hearts from WT embryos were isolated, pooled and immunoprecipitated with HIRA Wc15 antibody and immunoblotted with WHSC1. Presence of HIRA in the IP was verified using Wc119 antibody. <b>B.</b> Mouse ESCs (control and <i>Hira</i>^<i>-/-</i> ES cells) were differentiated towards cardiomyocytes over 15 days in LIF free medium following the hanging drop method, and were subjected to RNA and protein isolation. qRT-PCR of <i>Brachyury (T)</i>, <i>Mesp1 and Nkx2</i>.<i>5</i> over the 15 days are presented and demonstrate sequential expression of the early mesoderm, cardiogenic mesoderm, and cardiac markers respectively. Data are shown as levels of mRNA normalized to <i>Gapdh</i> and are representative of five independent experiments. Errors bars represent the standard deviation of technical triplicates. <b>C.</b> Protein isolated from undifferentiated ESCs and 15 days differentiated control and <i>Hira</i>^<i>-/-</i> ESCs were immunoprecipitated with anti-HIRA Wc15 antibody and immunoblotted with anti-WHSC1 antibody. The results shown are all from the same western membrane but separated lanes are brought into proximity and separated by dashed bars. GAPDH is used as a control for input. <b>D.</b> HIRA and H3.3 qChIP on differentiating ESCs shows an enrichment of HIRA at the <i>TTe</i> at day 15. H3.3 enrichment at the <i>TTe</i> increases significantly in WT but not in <i>Hira</i> null ESCs following this differentiation. X6 is an intergenic negative control region. Unpaired t-test was used: p<0.05 *, p<0.01 **.</p

<i>Epha3</i> is downregulated when HIRA is conditionally deleted, and HIRA binds to the gene body of <i>Epha3</i>.

<p><b>A</b>. <i>In situ</i> hybridization of <i>Epha3</i> on transverse sections of E12.5 embryonic hearts with the indicated genotypes (n = 2). <i>Hira</i>-conditional mutant hearts displayed low expression of <i>Epha3</i> in both the membranous portion of the septum (upper panels) and the endocardial cushions (lower panels) compared to littermate controls as indicated by a black arrow. <i>Scale bar</i>: <i>100μm</i> <b>B.</b> IGV profile showing two HIRA enriched loci in intronic regions of <i>Epha3</i>.</p

HIRA is required in the developing heart.

<p><b>A.</b> Lateral view of littermate embryos with the indicated genotype. <i>Mesp1Cre</i>;<i>Hira</i>^<i>-/fl</i> embryos had a low penetrance external phenotype at E12.5: exencephaly (E) and light haemorrhage (H) are indicated in the mutants. At E15.5, all mutants showed severe oedema (O) as indicated. <b>B.</b> Transverse OPT reconstructions followed by virtual reslicing of E15.5 embryo trunks with the indicated genotype. VSD (V) and ASD (A) are indicated in the mutants. H&E of transverse sections of E14.5 embryos with the indicated genotype also showed a common atrioventricular junction (CJ) in the mutants as indicated. <b>C</b>. H&E staining of transverse sections from E12.5 embryos reveals a disruption of the endocardial cushion (EC) fusion (two controls and two littermate mutants shown). The muscular septum is deficient (<b>*</b>, top) and the relatively flat rather than crescentic cushion shape in the mutant are indicated (arrows). <i>Scale bars represent 2mm</i> <b>(A)</b>, <i>0</i>.<i>5mm</i> <b>(B-C)</b>. <b>D</b>. Transverse sections of E12.5 embryonic hearts of the indicated genotype immunostained with DAPI and Troponin C captured on confocal showing disrupted sarcomeric structure in the mutant ventricular free wall. <i>Scale bar</i>: <i>10μm</i>.</p

<i>Hira</i> requirements in distinct lineages contributing to the heart and vessels.

<p><b>A.</b> E15.5 <i>Nkx2</i>.<i>5Cre</i>;<i>Hira</i>^-<i>/fl</i> embryos and their littermate control embryos were examined as indicated. Lateral view of <i>Nkx2</i>.<i>5Cre</i>;<i>Hira</i>^<i>-/fl</i> mutants revealed a normal external appearance. OPT transverse section revealed a VSD (V) and an overriding of the aortic root of the muscular septum in as well as a constricted pulmonary trunk PT (cPT) in some mutants by 3D reconstruction. <b>B&C.</b> OPT transverse section of the heart of a 6 months old <i>Mef2cCre;Hira</i>^<i>-/fl</i> (right/left ventricle: RV/LV) <b>(B)</b> or of <i>Tie2Cre</i>;<i>Hira</i>^<i>-/fl</i> embryo and adult <b>(C)</b> with their respective littermate control. Mutant embryos did not show any defect and their adult heart only showed proportional size reduction to the body size. The Aorta (Ao), the PT, and the right and left atrium (RA/LA) are indicated. <b>D&E.</b> Great vessel structure <b>(D)</b> and OPT transverse section (<b>E</b>) on E18.5 <i>Wnt1Cre</i>;<i>Hira</i>^-/fl and their control littermate hearts. No heart defects were observed in these mutants. <i>Scale bars represent 2mm in whole embryo and 0</i>.<i>5mm in transverse sections</i>.</p

sj-docx-1-tam-10.1177_17588359231225028 – Supplemental material for Impact of SARS-CoV-2 vaccines and recent chemotherapy on COVID-19 morbidity and mortality in patients with soft tissue sarcoma: an analysis from the OnCovid registry

Supplemental material, sj-docx-1-tam-10.1177_17588359231225028 for Impact of SARS-CoV-2 vaccines and recent chemotherapy on COVID-19 morbidity and mortality in patients with soft tissue sarcoma: an analysis from the OnCovid registry by Bruno Vincenzi, Alessio Cortellini, Alessandro Mazzocca, Sarah Orlando, Davide Romandini, Juan Aguilar-Company, Isabel Ruiz-Camps, Claudia Valverde Morales, Simeon Eremiev-Eremiev, Carlo Tondini, Joan Brunet, Rossella Bertulli, Salvatore Provenzano, Mark Bower, Daniele Generali, Ramon Salazar, Anna Sureda, Aleix Prat, Michalarea Vasiliki, Mieke Van Hemelrijck, Ailsa Sita-Lumsden, Alexia Bertuzzi, Sabrina Rossi, Amanda Jackson, Federica Grosso, Alvin J. X. Lee, Cian Murphy, Katherine Belessiotis, Uma Mukherjee, Fanny Pommeret, Angela Loizidou, Gianluca Gaidano, Gino M. Dettorre, Salvatore Grisanti, Marco Tucci, Claudia A. M. Fulgenzi, Alessandra Gennari, Andrea Napolitano and David J. Pinato in Therapeutic Advances in Medical Oncology</p