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Since the 2000s, transcatheter closure has been the primary treatment for ostium secundum atrial septal defect (osASD) in children and adults. This study aims to identify factors associated with short-term adverse outcomes following this procedure in a large cohort. A prospective, single-center cohort study included 2,253 consecutive patients (median age 28 years; children: n = 865, 38.4%) who underwent transcatheter ASD closure with the Amplatzer Septal Occluder (ASO; Amplatzer™ Atrial Septal Occluder Device, Abbott, Chicago, USA) from May 1998 to December 2021. Peri-procedural data associated with major adverse events were investigated retrospectively. The mean ASD diameter, as measured by transthoracic echocardiography, was 18 mm. About 8.9% of patients had an ASD size-to-body surface area (BSA) ratio of ≥ 20 mm/m2. Deficient rims (< 5 mm) were identified in 27.9% of patients, with retroaortic rim deficiency in 22.7% and inferior rim deficiency in 0.9%. The median ASO diameter was 24 mm, with a procedural success rate of 98.2%. ASD/BSA ≥ 20 mm/m2 was associated with procedural failure, while age and weight were not. Major peri-procedural adverse events occurred in 31 patients (1.4%), with 19 device embolizations and 2 cardiac erosions. No peri-procedural deaths were reported. Multivariate analysis showed that deficiency of the inferior vena cava rim and an ASD size/BS ratio ≥ 20 mm/m2 were significantly associated with major adverse events (P = 0.002 and P = 0.035, respectively) (Fig. 1). Transcatheter osASD closure using ASO is safe and effective in a large spectrum population with low rate of peri-procedural adverse events and favorable short-term outcomes. ASD size-to-body surface area ratio (≥ 20 mm/m2) and inferior rim deficiency are key morphological risk markers for major adverse events following this procedure. [ABSTRACT FROM AUTHOR]

Rationale: Pulmonary arterial hypertension (PAH) is a progressive and fatal disease characterized by pulmonary arterial muscularization due to excessive pulmonary vascular cell proliferation and migration, a phenotype dependent upon growth factors and activation of receptor tyrosine kinases (RTKs). p130(Cas) is an adaptor protein involved in several cellular signaling pathways that control cell migration, proliferation, and survival.Objectives: We hypothesized that in experimental and human PAH p130(Cas) signaling is overactivated, thereby facilitating the intracellular transmission of signal induced by fibroblast growth factor (FGF)2, epidermal growth factor (EGF), and platelet-derived growth factor (PDGF).Measurements and Main Results: In patients with PAH, levels of p130(Cas) protein and/or activity are higher in the serum, in the walls of distal pulmonary arteries, in cultured smooth muscle cells (PA-SMCs), and in pulmonary endothelial cells (P-ECs) than in control subjects. These abnormalities in the p130(Cas) signaling were also found in the chronically hypoxic mice and monocrotaline-injected rats as models of human PAH. We obtained evidence for the convergence and amplification of the growth-stimulating effect of the EGF-, FGF2-, and PDGF-signaling pathways via the p130(Cas) signaling pathway. We found that daily treatment with the EGF-R inhibitor gefitinib, the FGF-R inhibitor dovitinib, and the PDGF-R inhibitor imatinib started 2 weeks after a subcutaneous monocrotaline injection substantially attenuated the abnormal increase in p130(Cas) and ERK1/2 activation and regressed established pulmonary hypertension.Conclusions: Our findings demonstrate that p130(Cas) signaling plays a critical role in experimental and idiopathic PAH by modulating pulmonary vascular cell migration and proliferation and by acting as an amplifier of RTK downstream signals. [ABSTRACT FROM AUTHOR]

Abstract: In this work we analyzed the transcriptome profiles of chicken hepatoma cells (LMH) in response to T0901317, a pharmacological agonist of the liver X receptor (LXR). Through an in silico search for LXRE (LXR response element) consensus sequences in the promoter of genes whose expression was shown to be sensitive to TO901317, we identified a LXRE in the promoter of the LPCAT3 (lysophosphatidylcholine acyltransferase 3). This motif is highly conserved between species. We further investigated the regulation of this gene and showed that the expression of LPCAT3 was induced both in chicken and human hepatoma cells (LMH and HuH-7, respectively) in response to T0901317. Transactivation and electrophoretic mobility shift assays allowed us to locate a functional LXRE in the chicken LPCAT3 promoter. Altogether these data evidence for the first time that the chicken LPCAT3 gene is a direct target of LXR and therefore suggest a new role for LXR in phospholipid homeostasis. [ABSTRACT FROM AUTHOR]

Abstract Inheritance of the ovulation rate (OR) in the Lacaune meat breed was studied through records from a small nucleus of 36 hyper-prolific ewes screened on farms on the basis of their natural litter size, and from progeny data of three selected Lacaune sires. These sires were chosen at the AI centre according to their breeding values estimated for the mean and the variability of their daughters' litter size. Non-carrier Lacaune dairy ewes were inseminated to produce 121 F1 daughters and 27 F1 sons. Twelve sons (four from each sire) were used in turn to inseminate non-carrier Lacaune dairy ewes providing 260 BC progeny ewes. F1 and BC progeny were brought from private farms and gathered after weaning on an experimental farm where ovulation rates were recorded in the first and second breeding seasons. With an average of 6.5 records each, the mean OR of hyper-prolific ewes was very high (5.34), and 38.4% of records showed a rate of 6 or more. F1 data showed high repeatability of OR (r = 0.54) within ewe, with significant variability among ewes. High OR (≥ 4) were observed in each family. A segregation analysis provided a significant likelihood ratio and classified the three founders as heterozygous. BC ewes also displayed high repeatability of OR (r = 0.47) and the mean OR varied considerably between families (from 1.24 to 1.78). Seven of the 12 BC families presented high-ovulating ewes (at least one record ≥ 4) and segregation analysis yielded a highly significant likelihood ratio as compared to an empirical test distribution. The high variability of the mean ovulation rate shown by a small group of daughters of BC ewes inseminated by putative carrier F1 rams, and the very high ovulation rate observed for some of these ewe lambs, confirmed the segregation of a major gene with two co-dominant alleles borne by an autosome. The difference between homozygous non-carriers and heterozygous ewes was about one ovulation on the observed scale and 2.2 standard deviations on the underlying scale.

Abstract Background Starvation triggers a complex array of adaptative metabolic responses including energy-metabolic responses, a process which must imply tissue specific alterations in gene expression and in which the liver plays a central role. The present study aimed to describe the evolution of global gene expression profiles in liver of 4-week-old male chickens during a 48 h fasting period using a chicken 20 K oligoarray. Results A large number of genes were modulated by fasting (3532 genes with a pvalue corrected by Benjamini-Hochberg < 0.01); 2062 showed an amplitude of variation higher than +/- 40% among those, 1162 presented an human ortholog, allowing to collect functional information. Notably more genes were down-regulated than up-regulated, whatever the duration of fasting (16 h or 48 h). The number of genes differentially expressed after 48 h of fasting was 3.5-fold higher than after 16 h of fasting. Four clusters of co-expressed genes were identified by a hierarchical cluster analysis. Gene Ontology, KEGG and Ingenuity databases were then used to identify the metabolic processes associated to each cluster. After 16 h of fasting, genes involved in ketogenesis, gluconeogenesis and mitochondrial or peroxisomal fatty acid beta-oxidation, were up-regulated (cluster-1) whereas genes involved in fatty acid and cholesterol synthesis were down-regulated (cluster-2). For all genes tested, the microarray data was confirmed by quantitative RT-PCR. Most genes were altered by fasting as already reported in mammals. A notable exception was the HMG-CoA synthase 1 gene, which was up-regulated following 16 and 48 h of fasting while the other genes involved in cholesterol metabolism were down-regulated as reported in mammalian studies. We further focused on genes not represented on the microarray and candidates for the regulation of the target genes belonging to cluster-1 and -2 and involved in lipid metabolism. Data are provided concerning PPARa, SREBP1, SREBP2, NR1H3 transcription factors and two desaturases (FADS1, FADS2). Conclusion This study evidences numerous genes altered by starvation in chickens and suggests a global repression of cellular activity in response to this stressor. The central role of lipid and acetyl-CoA metabolisms and its regulation at transcriptional level are confirmed in chicken liver in response to short-term fasting. Interesting expression modulations were observed for NR1H3, FADS1 and FADS2 genes. Further studies are needed to precise their role in the complex regulatory network controlling lipid metabolism.

Abstract Background The recent sequencing of full genomes has led to the availability of many SNP markers which are very useful for the mapping of complex traits. In livestock production, there are still no commercial arrays and many studies use home-made sets of SNPs. Thus, the current methodologies for SNP genotyping are still expensive and it is a crucial step to select the SNPs to use. Indeed, the main factors affecting the power of the linkage analyses are the density of the genetic map and the heterozygosity of markers in tested animal parents. Findings This is why we have developed a PERL program selecting a defined number of markers based on their locations on the genome and their informativity in specific experimental designs. As an option, different experimental designs can be combined in order to select the best possible common marker set. The program has been tested using different conditions of marker informativity and density with both real and simulated datasets. The results show the efficiency of our program to select the most informative markers even if there is a wide range of informativity for whole genome scan mapping analyses. In case of combination of different experimental crosses, the multidesign mode can optimize the SNP markers selection. Conclusion Written in PERL, it assures a maximum portability to other operating systems (OS) and the source code availability for user modifications. Except for the simulation mode which could be time consuming, MarkerSet can compute results in a very short time.

Abstract: Several institutions provide genomic annotation data, and therefore these data show a significant segmentation and redundancy. Public databases allow access, through their own methods, to genomic and proteomic sequences and related annotation. Although some cross-reference tables are available, they don't cover the complete datasets provided by these databases. The Genomic Annotation Gathering project intends to unify annotation data provided by GenBank and Ensembl. We introduce an intra-species, cross-bank method. Generated results provide an enriched set of cross- references. This method allows for identifying an average of 30% of new cross-references that can be integrated to other utilities dedicated to analyzing related annotation data. By using only sequence comparison, we are able to unify two datasets that previously didn't share any stable cross-bank accession method. The whole process is hosted by the GenOuest platform to provide public access to newly generated cross-references and to allow for regular updates (http://gag.genouest.org). [Copyright &y& Elsevier]

Whole Genome Sequencing (WGS) remains a costly or unsuitable method for routine genotyping of laying hens. Until now, breeding companies have been using or developing SNP chips. Nevertheless, alternatives methods based on sequencing have been developed. Among these, reduced representation sequencing approaches can offer sequencing quality and cost-effectiveness by reducing the genomic regions covered by sequencing. The aim of this study was to evaluate the ability of double digested Restriction site Associated DNA sequencing (ddRAD-seq) to identify and genotype SNPs in laying hens, by comparison with a presumed reliable WGS approach. Firstly, the sensitivity and precision of variant calling and the genotyping reliability of ddRADseq were determined. Next, the SNP Call Rate (CRSNP) and mean depth of sequencing per SNP (DPSNP) were compared between both methods. Finally, the effect of multiple combinations of thresholds for these parameters on genotyping reliability and amount of remaining SNPs in ddRAD-seq was studied. In raw form, the ddRAD-seq identified 349,497 SNPs evenly distributed on the genome with a CRSNP of 0.55, a DPSNP of 11X and a mean genotyping reliability rate per SNP of 80%. Considering genomic regions covered by expected enzymatic fragments (EFs), the sensitivity of the ddRAD-seq was estimated at 32.4% and its precision at 96.4%. The low CRSNP and DPSNP values were explained by the detection of SNPs outside the EFs theoretically generated by the ddRAD-seq protocol. Indeed, SNPs outside the EFs had significantly lower CRSNP (0.25) and DPSNP (1X) values than SNPs within the EFs (0.7 and 17X, resp.). The study demonstrated the relationship between CRSNP, DPSNP, genotyping reliability and the number of SNPs retained, to provide a decision-support tool for defining filtration thresholds. Severe quality control over ddRAD-seq data allowed to retain a minimum of 40% of the SNPs with a CcR of 98%. Then, ddRAD-seq was defined as a suitable method for variant calling and genotyping in layers., Competing Interests: The authors declare that they have no competing interests., (Copyright: © 2024 Doublet et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Purpose: The purpose of this study was to evaluate the accuracy of four-dimensional flow cardiac magnetic resonance imaging (4D flow MRI) compared to right heart catheterization in measuring pulmonary flow (Qp), systemic flow (Qs) and pulmonary-to-systemic flow ratio (Qp/Qs) in patients with pulmonary arterial hypertension associated with congenital heart disease (PAH-CHD)., Materials and Methods: The study was registered on Clinical-trial.gov (NCT03928002). Sixty-four patients with PAH-CHD who underwent 4D flow MRI were included. There were 16 men and 48 women with a mean age of 45.3 ± 13.7 (standard deviation [SD]) years (age range: 21-77 years). Fifty patients (50/64; 78%) presented with pre-tricuspid shunt. Qp (L/min), Qs (L/min) and Qp/Qs were measured invasively using direct Fick method during right heart catheterization and compared with measurements assessed by 4D flow MRI within a 24-48-hour window., Results: The average mean pulmonary artery pressure was 51 ± 17 (SD) mm Hg with median pulmonary vascular resistance of 8.8 Wood units (Q1, Q3: 5.3, 11.7). A strong linear correlation was found between Qp measurements obtained with 4D flow MRI and those obtained with the Fick method (r = 0.96; P < 0.001). Bland Altman analysis indicated a mean difference of 0.15 ± 0.48 (SD) L/min between Qp estimated by 4D flow MRI and by right heart catheterization. A strong correlation was found between Qs and Qp/Qs measured by 4D flow MRI and those obtained with the direct Fick method (r = 0.85 and r = 0.92; P < 0.001 for both)., Conclusion: Qp as measured by 4D flow MRI shows a strong correlation with measurements derived from the direct Fick method. Further investigation is needed to develop less complex and standardized methods for measuring essential PAH parameters, such as pulmonary arterial pressures and pulmonary vascular resistance., Competing Interests: Declaration of competing interest The authors declare no actual or potential conflict of interest related to this study., (Copyright © 2024 Société française de radiologie. Published by Elsevier Masson SAS. All rights reserved.)

Gene atlases for livestock are steadily improving thanks to new genome assemblies and new expression data improving the gene annotation. However, gene content varies across databases due to differences in RNA sequencing data and bioinformatics pipelines, especially for long non-coding RNAs (lncRNAs) which have higher tissue and developmental specificity and are harder to consistently identify compared to protein coding genes (PCGs). As done previously in 2020 for chicken assemblies galgal5 and GRCg6a, we provide a new gene atlas, lncRNA-enriched, for the latest GRCg7b chicken assembly, integrating "NCBI RefSeq", "EMBL-EBI Ensembl/GENCODE" reference annotations and other resources such as FAANG and NONCODE. As a result, the number of PCGs increases from 18,022 (RefSeq) and 17,007 (Ensembl) to 24,102, and that of lncRNAs from 5789 (RefSeq) and 11,944 (Ensembl) to 44,428. Using 1400 public RNA-seq transcriptome representing 47 tissues, we provided expression evidence for 35,257 (79%) lncRNAs and 22,468 (93%) PCGs, supporting the relevance of this atlas. Further characterization including tissue-specificity, sex-differential expression and gene configurations are provided. We also identified conserved miRNA-hosting genes with human counterparts, suggesting common function. The annotated atlas is available at gega.sigenae.org., (© 2024. The Author(s).)

Introduction: Percutaneous pulmonary valve implantation (PPVI) with a SAPIEN 3 valve is effective for treating treat right ventricle outflow (RVOT) dysfunction. A modified technique was developed without prestenting using a protective valve delivery method. We aimed to compare the procedural results of the modified technique group (MTG) to those of patients in a conventional technique group (CTG)., Methods: We designed a matched before-after study. All consecutive PPVI with SAPIEN 3 performed in the MTG over 9 months were matched, based on the RVOT type and size, to consecutive procedures performed previously with SAPIEN 3., Results: A total of 54 patients were included, equally distributed in the two groups. The sizes of the SAPIEN 3 valves were 23 mm (n = 9), 26 mm (n = 9), 29 mm (n = 36). The two groups were similar regarding demographic data, RVOT type, and pre-procedure hemodynamics. PPVI was performed in a single procedure in all patients of the MTG, whereas six (22.2%) patients of the CTG group underwent prestenting as a first step and valve implantation later ( p = 0.02). The procedures were successful in all cases. Stent embolization was reported in two patients (7.4%) in the CTG, which were impacted in pulmonary arteries. In one case (3.7%), in the MTG, an unstable 29 mm SAPIEN 3 valve was stabilized with two stents and additional valve-in-valve implantation. The hemodynamics results were good in all cases, without significant differences between the two groups. The procedures' durations and fluoroscopy times were significantly reduced in the MTG (48.1 versus 82.6 min, p < 0.0001; 15.2 versus 29.8 min, p = 0.0002). During follow-up, neither stent fracture nor valve dysfunction was noticed in either group., Conclusion: PPVI without prestenting and with a protective delivery method of the SAPIEN 3 valve significantly reduces the procedure's complexity, the duration, and the irradiation while maintaining excellent hemodynamics results in selected cases.

Background: To reduce the cost of genomic selection, a low-density (LD) single nucleotide polymorphism (SNP) chip can be used in combination with imputation for genotyping selection candidates instead of using a high-density (HD) SNP chip. Next-generation sequencing (NGS) techniques have been increasingly used in livestock species but remain expensive for routine use for genomic selection. An alternative and cost-efficient solution is to use restriction site-associated DNA sequencing (RADseq) techniques to sequence only a fraction of the genome using restriction enzymes. From this perspective, use of RADseq techniques followed by an imputation step on HD chip as alternatives to LD chips for genomic selection was studied in a pure layer line., Results: Genome reduction and sequencing fragments were identified on reference genome using four restriction enzymes (EcoRI, TaqI, AvaII and PstI) and a double-digest RADseq (ddRADseq) method (TaqI-PstI). The SNPs contained in these fragments were detected from the 20X sequence data of the individuals in our population. Imputation accuracy on HD chip with these genotypes was assessed as the mean correlation between true and imputed genotypes. Several production traits were evaluated using single-step GBLUP methodology. The impact of imputation errors on the ranking of the selection candidates was assessed by comparing a genomic evaluation based on ancestry using true HD or imputed HD genotyping. The relative accuracy of genomic estimated breeding values (GEBVs) was investigated by considering the GEBVs estimated on offspring as a reference. With AvaII or PstI and ddRADseq with TaqI and PstI, more than 10 K SNPs were detected in common with the HD SNP chip, resulting in an imputation accuracy greater than 0.97. The impact of imputation errors on genomic evaluation of the breeders was reduced, with a Spearman correlation greater than 0.99. Finally, the relative accuracy of GEBVs was equivalent., Conclusions: RADseq approaches can be interesting alternatives to low-density SNP chips for genomic selection. With more than 10 K SNPs in common with the SNPs of the HD SNP chip, good imputation and genomic evaluation results can be obtained. However, with real data, heterogeneity between individuals with missing data must be considered., (© 2023. The Author(s).)

Background: Anxiety before an invasive intervention is associated in children with persistent psychological disorders. We studied the effect of the transfer to the catheterisation room by an electric toy car on the anxiety of children and their parents before a cardiac catheterisation., Methods: Forty-eight children with a median age of 5.6 years [4.2-7.0] were randomised to either riding on an electric car to go to the catheterisation laboratory or being transported lying supine on a gurney. Anxiety assessments were performed by a physician blinded to group allocation on the day before the procedure (T0) and at anaesthesia induction (T1). The modified Yale Preoperative Anxiety Scale Short Form (mYPAS-SF) and visual analogue scale for anxiety (VAS-A) were used in the children and the VAS-A in the parents., Results: The mYPAS-SF, VAS-A-child, and the VAS-A-parent scores were significantly higher at T1 than at T0 (p < 0.001, p < 0.001, and p = 0.005, respectively). The primary outcome (the median mYPAS-SF score at T1) was not significantly different in the two groups when males and females were combined. At T1, the VAS-A-child score, however, was significantly lower in the intervention than the control group (22 versus 55, p < 0.001). In the boys, the median mYPAS-SF score at T1 was significantly lower in the intervention group (25.0 versus 51.0, p = 0.024). No difference was observed in girls. The VAS-A parent score was lower at T1 in the intervention group (60 versus 87, p = 0.05)., Conclusion: Riding to the catheterisation laboratory on an electric toy car decreased anxiety in boys and decreased parental anxiety.

Background: Transcatheter pulmonary valve implantation (TPVI) is effective for treating right ventricle outflow tract (RVOT) dysfunction. Factors associated with long-term valve durability remain to be investigated., Methods: Consecutive patients successfully treated by TPVI with Melody valves (n = 32) and SAPIEN valves (n = 182) between 2008 and 2020 at a single tertiary centre were included prospectively and monitored., Results: The 214 patients had a median age of 28 years (range, 10-81). The RVOT was a patched native pulmonary artery in 96 (44.8%) patients. Median follow-up was 2.8 years (range, 3 months-11.4 years). Secondary pulmonary valve replacement (sPVR) was performed in 23 cases (10.7%), due to stenosis (n = 22, 95.7%) or severe regurgitation (n = 1, 4.3%), yielding an incidence of 7.6/100 patient-years with melody valves and 1.3/100 patient-years with SAPIEN valves (P = 0.06). The 5- and 10-year sPVR-freedom rates were 78.1% and 50.4% with Melody vs. 94.3% and 82.2% with SAPIEN, respectively (P = 0.06). The incidence of infective endocarditis (IE) was 5.5/100 patient-years with Melody and 0.2/100 patient-years with SAPIEN (P < 0.0001). Factors associated with sPVR by univariate analysis were RV obstruction before TPVI (P = 0.04), transpulmonary maximal velocity > 2.7 m/s after TPVI (p = 0.0005), valve diameter ≤ 22 mm (P < 0.003), IE (P < 0.0001), and age < 25 years at TPVI (P = 0.04). By multivariate analysis adjusted for IE occurrence, transpulmonary maximal velocity remained associated with sPVR., Conclusions: TPVI is effective for treating RVOT dysfunction. Incidence of sPVR is higher in patients with residual RV obstruction or IE. IE add a substantial risk of TPVI graft failure and is mainly linked to the Melody valve., Social Media Abstract: Transcatheter pulmonary valve implantation is effective for treating right ventricular outflow tract dysfunction in patients with congenital heart diseases. Incidence of secondary valve replacement is higher in patients with residual obstruction or infective endocarditis., Competing Interests: Declaration of Competing Interest Sebastien Hascoët has received proctoring and consultant fees from Abbott. None of the other authors has any conflicts of interest to disclose., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Most single-nucleotide polymorphisms (SNPs) are located in non-coding regions, but the fraction usually studied is harbored in protein-coding regions because potential impacts on proteins are relatively easy to predict by popular tools such as the Variant Effect Predictor. These tools annotate variants independently without considering the potential effect of grouped or haplotypic variations, often called "multi-nucleotide variants" (MNVs). Here, we used a large RNA-seq dataset to survey MNVs, comprising 382 chicken samples originating from 11 populations analyzed in the companion paper in which 9.5M SNPs- including 3.3M SNPs with reliable genotypes-were detected. We focused our study on in-codon MNVs and evaluate their potential mis-annotation. Using GATK HaplotypeCaller read-based phasing results, we identified 2,965 MNVs observed in at least five individuals located in 1,792 genes. We found 41.1% of them showing a novel impact when compared to the effect of their constituent SNPs analyzed separately. The biggest impact variation flux concerns the originally annotated stop-gained consequences, for which around 95% were rescued; this flux is followed by the missense consequences for which 37% were reannotated with a different amino acid. We then present in more depth the rescued stop-gained MNVs and give an illustration in the SLC27A4 gene. As previously shown in human datasets, our results in chicken demonstrate the value of haplotype-aware variant annotation, and the interest to consider MNVs in the coding region, particularly when searching for severe functional consequence such as stop-gained variants., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Degalez, Jehl, Muret, Bernard, Lecerf, Lagoutte, Désert, Pitel, Klopp and Lagarrigue.)

In addition to their common usages to study gene expression, RNA-seq data accumulated over the last 10 years are a yet-unexploited resource of SNPs in numerous individuals from different populations. SNP detection by RNA-seq is particularly interesting for livestock species since whole genome sequencing is expensive and exome sequencing tools are unavailable. These SNPs detected in expressed regions can be used to characterize variants affecting protein functions, and to study cis -regulated genes by analyzing allele-specific expression (ASE) in the tissue of interest. However, gene expression can be highly variable, and filters for SNP detection using the popular GATK toolkit are not yet standardized, making SNP detection and genotype calling by RNA-seq a challenging endeavor. We compared SNP calling results using GATK suggested filters, on two chicken populations for which both RNA-seq and DNA-seq data were available for the same samples of the same tissue. We showed, in expressed regions, a RNA-seq precision of 91% (SNPs detected by RNA-seq and shared by DNA-seq) and we characterized the remaining 9% of SNPs. We then studied the genotype (GT) obtained by RNA-seq and the impact of two factors (GT call-rate and read number per GT) on the concordance of GT with DNA-seq; we proposed thresholds for them leading to a 95% concordance. Applying these thresholds to 767 multi-tissue RNA-seq of 382 birds of 11 chicken populations, we found 9.5 M SNPs in total, of which ∼550,000 SNPs per tissue and population with a reliable GT (call rate ≥ 50%) and among them, ∼340,000 with a MAF ≥ 10%. We showed that such RNA-seq data from one tissue can be used to ( i ) detect SNPs with a strong predicted impact on proteins, despite their scarcity in each population (16,307 SIFT deleterious missenses and 590 stop-gained), ( ii ) study, on a large scale, cis -regulations of gene expression, with ∼81% of protein-coding and 68% of long non-coding genes (TPM ≥ 1) that can be analyzed for ASE, and with ∼29% of them that were cis -regulated, and ( iii ) analyze population genetic using such SNPs located in expressed regions. This work shows that RNA-seq data can be used with good confidence to detect SNPs and associated GT within various populations and used them for different analyses as GTEx studies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Jehl, Degalez, Bernard, Lecerf, Lagoutte, Désert, Coulée, Bouchez, Leroux, Abasht, Tixier-Boichard, Bed’hom, Burlot, Gourichon, Bardou, Acloque, Foissac, Djebali, Giuffra, Zerjal, Pitel, Klopp and Lagarrigue.)

No prior proteomic screening study has centred on the right ventricle (RV) in pulmonary arterial hypertension (PAH). This study investigates the circulating proteomic profile associated with right heart maladaptive phenotype (RHMP) in PAH.Plasma proteomic profiling was performed using multiplex immunoassay in 121 (discovery cohort) and 76 (validation cohort) PAH patients. The association between proteomic markers and RHMP, defined by the Mayo right heart score (combining RV strain, New York Heart Association (NYHA) class and N-terminal pro-brain natriuretic peptide (NT-proBNP)) and Stanford score (RV end-systolic remodelling index, NYHA class and NT-proBNP), was assessed by partial least squares regression. Biomarker expression was measured in RV samples from PAH patients and controls, and pulmonary artery banding (PAB) mice.High levels of hepatocyte growth factor (HGF), stem cell growth factor-β, nerve growth factor and stromal derived factor-1 were associated with worse Mayo and Stanford scores independently from pulmonary resistance or pressure in both cohorts (the validation cohort had more severe disease features: lower cardiac index and higher NT-proBNP). In both cohorts, HGF added value to the REVEAL score in the prediction of death, transplant or hospitalisation at 3 years. RV expression levels of HGF and its receptor c-Met were higher in end-stage PAH patients than controls, and in PAB mice than shams.High plasma HGF levels are associated with RHMP and predictive of 3-year clinical worsening. Both HGF and c-Met RV expression levels are increased in PAH. Assessing plasma HGF levels might identify patients at risk of heart failure who warrant closer follow-up and intensified therapy., Competing Interests: Conflict of interest: M. Amsallem has received a 2016 Young Investigator Seed Grant from the Vera Moulton Wall Center at Stanford, a 2019–2020 Stanford Maternal and Child Health Research Institute research seed grant, a research grant from Actelion-Janssen and speaker fees from Bayer. Conflict of interest: A.J. Sweatt has nothing to disclose. Conflict of interest: J. Arthur Ataam has nothing to disclose. Conflict of interest: J. Guihaire has nothing to disclose. Conflict of interest: F. Lecerf has nothing to disclose. Conflict of interest: M. Lambert has nothing to disclose. Conflict of interest: M.R. Ghigna has nothing to disclose. Conflict of interest: M.K. Ali has nothing to disclose. Conflict of interest: Y. Mao has nothing to disclose. Conflict of interest: E. Fadel has nothing to disclose. Conflict of interest: M. Rabinovitch has nothing to disclose. Conflict of interest: V. de Jesus Perez has nothing to disclose. Conflict of interest: E. Spiekerkoetter is funded by Stanford Cardiovascular Institute, National Heart Lung Blood Institute (NHLBI) at the National Institute of Health (NIH) grant R01 HL128734 and Department of Defense grant PR161256. Conflict of interest: O. Mercier has been supported by a public grant overseen by the French National Research Agency as part of the second Investissement d'Avenir program (ANR-15-RHUS-0002). Conflict of interest: F. Haddad has received research grants from Actelion-Janssen and Philips. Conflict of interest: R.T. Zamanian has nothing to disclose., (Copyright ©ERS 2021.)

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted at the request of the Authors. This request follows an examination by The Editors of the uncut gels provided by the authors, which led the Editors to conclude that data were compromised in the following western blot images: Figure 3C, Figure 5B and Figure 6B. Duplicated data for the beta actin images were found in Figures 5 and 6. Examination of the raw data used for the western blot quantification also revealed frequent duplicated data. The microscopy data in Figure 5A also has features compatible with compromised data although the raw data were not available to the Editors due to the regrettable death of Dr. Saadia Eddahibi. All of the remaining authors agree with the retraction and apologize to the Editors and the readers of The Journal for difficulties this issue has caused., (Copyright © 2021 International Society for Heart and Lung Transplantation. Published by Elsevier Inc. All rights reserved.)

Trichloroethylene exposure is a major risk factor for pulmonary veno-occlusive disease. We demonstrated that trichloroethylene alters the endothelial barrier integrity, at least in part, through vascular endothelial (VE)-Cadherin internalisation, and suggested that this mechanism may play a role in the development of pulmonary veno-occlusive disease., Competing Interests: J.C. and M.R. report grants from Fondation du Souffle, during the conduct of the study. M.H. reports personal fees from Actelion, grants and personal fees from Bayer, grants and personal fees from GSK, personal fees from Merck, personal fees from United Therapeutics, outside the submitted work. G.M., M.K.N., M.Q., C.R.-M., P.D., F.L, M.C.V, S.C.-K. and F.P. have nothing to disclose., (© The Author(s) 2020.)

Pulmonary veno-occlusive disease (PVOD) occurs in humans either as a heritable form (hPVOD) due to biallelic inactivating mutations of EIF2AK4 ( encoding GCN2) or as a sporadic form in older age (sPVOD). The chemotherapeutic agent mitomycin C (MMC) is a potent inducer of PVOD in humans and in rats (MMC-PVOD). Here, we compared human hPVOD and sPVOD, and MMC-PVOD pathophysiology at the histological, cellular, and molecular levels to unravel common altered pathomechanisms. MMC exposure in rats was associated primarily with arterial and microvessel remodeling, and secondarily by venous remodeling, when PVOD became symptomatic. In all forms of PVOD tested, there was convergent GCN2-dependent but eIF2α-independent pulmonary protein overexpression of HO-1 (heme oxygenase 1) and CHOP (CCAAT-enhancer-binding protein [C/EBP] homologous protein), two downstream effectors of GCN2 signaling and endoplasmic reticulum stress. In human PVOD samples, CHOP immunohistochemical staining mainly labeled endothelial cells in remodeled veins and arteries. Strong HO-1 staining was observed only within capillary hemangiomatosis foci, where intense microvascular proliferation occurs. HO-1 and CHOP stainings were not observed in control and pulmonary arterial hypertension lung tissues, supporting the specificity for CHOP and HO-1 involvement in PVOD pathobiology. In vivo loss of GCN2 ( EIF2AK4 mutations carriers and Eif2ak4 -/- rats) or in vitro GCN2 inhibition in cultured pulmonary artery endothelial cells using pharmacological and siRNA approaches demonstrated that GCN2 loss of function negatively regulates BMP (bone morphogenetic protein)-dependent SMAD1/5/9 signaling. Exogenous BMP9 was still able to reverse GCN2 inhibition-induced proliferation of pulmonary artery endothelial cells. In conclusion, we identified CHOP and HO-1 inhibition, and BMP9, as potential therapeutic options for PVOD.

Background: Pulmonary endothelial cells play a key role in the pathogenesis of Chronic Thromboembolic Pulmonary Hypertension (CTEPH). Increased synthesis and/or the release of intercellular adhesion molecule-1 (ICAM-1) by pulmonary endothelial cells of patients with CTEPH has been recently reported, suggesting a potential role for ICAM-1 in CTEPH., Methods: We studied pulmonary endarterectomy specimens from 172 patients with CTEPH and pulmonary artery specimens from 97 controls undergoing lobectomy for low-stage cancer without metastasis., Results: ICAM-1 was overexpressed in vitro in isolated and cultured endothelial cells from endarterectomy specimens. Endothelial cell growth and apoptosis resistance were significantly higher in CTEPH specimens than in the controls (p < 0.001). Both abnormalities were abolished by pharmacological inhibition of ICAM-1 synthesis or activity. The overexpression of ICAM-1 contributed to the acquisition and maintenance of abnormal EC growth and apoptosis resistance via the phosphorylation of SRC, p38 and ERK1/2 and the overproduction of survivin. Regarding the ICAM-1 E469K polymorphism, the KE heterozygote genotype was significantly more frequent in CTEPH than in the controls, but it was not associated with disease severity among patients with CTEPH., Conclusions: ICAM-1 contributes to maintaining the abnormal endothelial cell phenotype in CTEPH., (Copyright © 2019 International Society for Heart and Lung Transplantation. Published by Elsevier Inc. All rights reserved.)

Idiopathic pulmonary arterial hypertension (IPAH) is a complex disease associated with vascular remodeling and a proliferative disorder in pulmonary artery smooth muscle cells (PASMCs) that has been variably described as having neoplastic features. To decode the phenotype of PASMCs in IPAH, PASMCs from explanted lungs of patients with IPAH (IPAH-PASMCs) and from controls (C-PASMCs) were cultured. The IPAH-PASMCs grew faster than the controls; however, both growth curves plateaued, suggesting contact inhibition in IPAH cells. No proliferation was seen without stimulation with exogenous growth factors, suggesting that IPAH cells are incapable of self-sufficient growth. IPAH-PASMCs were more resistant to apoptosis than C-PASMCs, consistent with the increase in the Bcl2/Bax ratio. As cell replication is governed by telomere length, these parameters were assessed jointly. Compared to C-PASMCs, IPAH-PASMCs had longer telomeres, but a limited replicative capacity. Additionally, it was noted that IPAH-PASMCs had a shift in energy production from mitochondrial oxidative phosphorylation to aerobic glycolysis. As DNA damage and genomic instability are strongly implicated in IPAH development a comparative genomic hybridization was performed on genomic DNA from PASMCs which showed multiple break-points unaffected by IPAH severity. Activation of DNA damage/repair factors (γH2AX, p53, and GADD45) in response to cisplatin was measured. All proteins showed lower phosphorylation in IPAH samples than in controls, suggesting that the cells were resistant to DNA damage. Despite the cancer-like processes that are associated with end-stage IPAH-PASMCs, we identified no evidence of self-sufficient proliferation in these cells-the defining feature of neoplasia.

Background: Monoallelic mutations in the gene encoding bone morphogenetic protein receptor 2 ( Bmpr2) are the main genetic risk factor for heritable pulmonary arterial hypertension (PAH) with incomplete penetrance. Several Bmpr2 transgenic mice have been reported to develop mild spontaneous PAH. In this study, we examined whether rats with the Bmpr2 mutation were susceptible to developing more severe PAH., Methods: The zinc finger nuclease method was used to establish rat lines with mutations in the Bmpr2 gene. These rats were then characterized at the hemodynamic, histological, electrophysiological, and molecular levels., Results: Rats with a monoallelic deletion of 71 bp in exon 1 (Δ 71 rats) showed decreased BMPRII expression and phosphorylated SMAD1/5/9 levels. Δ 71 Rats develop age-dependent spontaneous PAH with a low penetrance (16%-27%), similar to that in humans. Δ 71 Rats were more susceptible to hypoxia-induced pulmonary hypertension than wild-type rats. Δ 71 Rats exhibited progressive pulmonary vascular remodeling associated with a proproliferative phenotype and showed lower pulmonary microvascular density than wild-type rats. Organ bath studies revealed severe alteration of pulmonary artery contraction and relaxation associated with potassium channel subfamily K member 3 (KCNK3) dysfunction. High levels of perivascular fibrillar collagen and pulmonary interleukin-6 overexpression discriminated rats that developed spontaneous PAH and rats that did not develop spontaneous PAH. Finally, detailed assessments of cardiomyocytes demonstrated alterations in morphology, calcium (Ca 2+ ), and cell contractility specific to the right ventricle; these changes could explain the lower cardiac output of Δ 71 rats. Indeed, adult right ventricular cardiomyocytes from Δ 71 rats exhibited a smaller diameter, decreased sensitivity of sarcomeres to Ca 2+ , decreased [Ca 2+ ] transient amplitude, reduced sarcoplasmic reticulum Ca 2+ content, and short action potential duration compared with right ventricular cardiomyocytes from wild-type rats., Conclusions: We characterized the first Bmpr2 mutant rats and showed some of the critical cellular and molecular dysfunctions described in human PAH. We also identified the heart as an unexpected but potential target organ of Bmpr2 mutations. Thus, this new genetic rat model represents a promising tool to study the pathogenesis of PAH.

Aims: Multiple surgical revisions are often necessary in individuals with congenital heart defects affecting the RVOT or pulmonary valve. There are no multicentre data on the feasibility and safety of percutaneous pulmonary valve implantation (PPVI) using the SAPIEN 3 (S3) transcatheter heart valve. The aim of this study was to explore the short-term safety, feasibility, and haemodynamic outcomes of PPVI using the S3 transcatheter heart valve., Methods and Results: Pulmonic S3 is an observational registry of patients undergoing PPVI with the S3 valve across centres in Europe and Canada. Data for 82 patients (mean age 27.3 years) were obtained. The most common underlying diagnosis was tetralogy of Fallot (ToF) (58.5%), with 16.0% of patients having native RVOT anatomy; 90.2% received pre-stenting. Prosthesis dislodgement occurred in one patient and conduit perforation in another. Both were successfully resolved without the need for open surgery. Peak systolic gradient over the RVOT fell from 46.3 mmHg to 17.2 mmHg, moderate/severe pulmonary regurgitation from 86.3% to 0.0%, and NYHA ≥II from 86.0% to 15.2%. During follow-up, valve thrombosis was observed in two patients which resolved with adequate anticoagulation. No other procedural complications, endocarditis, stent fracture or death were reported within two years., Conclusions: PPVI with the S3 valve appears feasible and safe in a wide range of patients with congenital heart defects, with good short-term haemodynamic and functional outcomes.

Idiopathic pulmonary arterial hypertension (iPAH) is characterized by obstructive hyperproliferation and apoptosis resistance of distal pulmonary artery smooth muscle cells (PASMCs). T-type Ca 2+ channel blockers have been shown to reduce experimental pulmonary hypertension, although the impact of T-type channel inhibition remains unexplored in PASMCs from iPAH patients. Here we show that T-type channels Cav3.1 and Cav3.2 are present in the lung and PASMCs from iPAH patients and control subjects. The blockade of T-type channels by the specific blocker, TTA-A2, prevents cell cycle progression and PASMCs growth. In iPAH cells, T-type channel signaling fails to activate phosphatase PP2A, leading to an increase in ERK1/2, P38 activation. Moreover, T-type channel signaling is redirected towards the activation of the kinase Akt1, leading to increased expression of the anti-apoptotic protein survivin, and a decrease in the pro-apoptotic mediator FoxO3A. Finally, in iPAH cells, Akt1 is no longer able to regulate caspase 9 activation, whereas T-type channel overexpression reverses PP2A defect in iPAH cells but reinforces the deleterious effects of Akt1 activation. Altogether, these data highlight T-type channel signaling as a strong trigger of the pathological phenotype of PASMCs from iPAH patients (hyper-proliferation/cells survival and apoptosis resistance), suggesting that both T-type channels and PP2A may be promising therapeutic targets for pulmonary hypertension., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: Chronic thromboembolic pulmonary hypertension results from chronic mechanical obstruction of the pulmonary arteries after acute venous thromboembolism. However, the mechanisms that result in the progression from unresolved thrombus to fibrotic vascular remodeling are unknown. We hypothesized that pulmonary artery endothelial cells contribute to this phenomenon via paracrine growth factor and cytokine signaling., Methods: Using enzyme-linked immunosorbent assay and cell migration assays, we investigated the circulating growth factors and cytokines of chronic thromboembolic pulmonary hypertension patients as well as the cross talk between pulmonary endothelial cells and pulmonary artery smooth muscle cells and monocytes from patients with chronic thromboembolic pulmonary hypertension in vitro., Results: Culture medium from the pulmonary endothelial cells of chronic thromboembolic pulmonary hypertension patients contained higher levels of growth factors (fibroblast growth factor 2), inflammatory cytokines (interleukin 1β, interleukin 6, monocyte chemoattractant protein 1), and cell adhesion molecules (vascular cell adhesion molecule 1 and intercellular adhesion molecule 1). Furthermore, exposure to the culture medium of pulmonary endothelial cells from patients with chronic thromboembolic pulmonary hypertension elicited marked pulmonary artery smooth muscle cell growth and monocyte migration., Conclusions: These findings implicate pulmonary endothelial cells as key regulators of pulmonary artery smooth muscle cell and monocyte behavior in chronic thromboembolic pulmonary hypertension and suggest a potential mechanism for the progression from unresolved thrombus to fibrotic vascular remodeling., (Copyright © 2017 International Society for Heart and Lung Transplantation. Published by Elsevier Inc. All rights reserved.)

Background: Improving functional annotation of the chicken genome is a key challenge in bridging the gap between genotype and phenotype. Among all transcribed regions, long noncoding RNAs (lncRNAs) are a major component of the transcriptome and its regulation, and whole-transcriptome sequencing (RNA-Seq) has greatly improved their identification and characterization. We performed an extensive profiling of the lncRNA transcriptome in the chicken liver and adipose tissue by RNA-Seq. We focused on these two tissues because of their importance in various economical traits for which energy storage and mobilization play key roles and also because of their high cell homogeneity. To predict lncRNAs, we used a recently developed tool called FEELnc, which also classifies them with respect to their distance and strand orientation to the closest protein-coding genes. Moreover, to confidently identify the genes/transcripts expressed in each tissue (a complex task for weakly expressed molecules such as lncRNAs), we probed a particularly large number of biological replicates (16 per tissue) compared to common multi-tissue studies with a larger set of tissues but less sampling., Results: We predicted 2193 lncRNA genes, among which 1670 were robustly expressed across replicates in the liver and/or adipose tissue and which were classified into 1493 intergenic and 177 intragenic lncRNAs located between and within protein-coding genes, respectively. We observed similar structural features between chickens and mammals, with strong synteny conservation but without sequence conservation. As previously reported, we confirm that lncRNAs have a lower and more tissue-specific expression than mRNAs. Finally, we showed that adjacent lncRNA-mRNA genes in divergent orientation have a higher co-expression level when separated by less than 1 kb compared to more distant divergent pairs. Among these, we highlighted for the first time a novel lncRNA candidate involved in lipid metabolism, lnc&#95;DHCR24, which is highly correlated with the DHCR24 gene that encodes a key enzyme of cholesterol biosynthesis., Conclusions: We provide a comprehensive lncRNA repertoire in the chicken liver and adipose tissue, which shows interesting patterns of co-expression between mRNAs and lncRNAs. It contributes to improving the structural and functional annotation of the chicken genome and provides a basis for further studies on energy storage and mobilization traits in the chicken.

Background: The vascular remodeling responsible for pulmonary arterial hypertension (PAH) involves predominantly the accumulation of α-smooth muscle actin-expressing mesenchymal-like cells in obstructive pulmonary vascular lesions. Endothelial-to-mesenchymal transition (EndoMT) may be a source of those α-smooth muscle actin-expressing cells., Methods and Results: In situ evidence of EndoMT in human PAH was obtained by using confocal microscopy of multiple fluorescent stainings at the arterial level, and by using transmission electron microscopy and correlative light and electron microscopy at the ultrastructural level. Findings were confirmed by in vitro analyses of human PAH and control cultured pulmonary artery endothelial cells. In addition, the mRNA and protein signature of EndoMT was recognized at the arterial and lung level by quantitative real-time polymerase chain reaction and Western blot analyses. We confirmed our human observations in established animal models of pulmonary hypertension (monocrotaline and SuHx). After establishing the first genetically modified rat model linked to BMPR2 mutations (BMPR2(Δ140Ex1/+) rats), we demonstrated that EndoMT is linked to alterations in signaling of BMPR2, a gene that is mutated in 70% of cases of familial PAH and in 10% to 40% of cases of idiopathic PAH. We identified molecular actors of this pathological transition, including twist overexpression and vimentin phosphorylation. We demonstrated that rapamycin partially reversed the protein expression patterns of EndoMT, improved experimental PAH, and decreased the migration of human pulmonary artery endothelial cells, providing the proof of concept that EndoMT is druggable., Conclusions: EndoMT is linked to alterations in BPMR2 signaling and is involved in the occlusive vas cular remodeling of PAH, findings that may have therapeutic implications., (© 2015 American Heart Association, Inc.)