Derivation and characterisation of endothelial cells from patients with chronic thromboembolic pulmonary hypertension.

Pulmonary endarterectomy (PEA) resected material offers a unique opportunity to develop an in vitro endothelial cell model of chronic thromboembolic pulmonary hypertension (CTEPH). We aimed to comprehensively analyze the endothelial function, molecular signature, and mitochondrial profile of CTEPH-derived endothelial cells to better understand the pathophysiological mechanisms of endothelial dysfunction behind CTEPH, and to identify potential novel targets for the prevention and treatment of the disease. Isolated cells from specimens obtained at PEA (CTEPH-EC), were characterized based on morphology, phenotype, and functional analyses (in vitro and in vivo tubule formation, proliferation, apoptosis, and migration). Mitochondrial content, morphology, and dynamics, as well as high-resolution respirometry and oxidative stress, were also studied. CTEPH-EC displayed a hyperproliferative phenotype with an increase expression of adhesion molecules and a decreased apoptosis, eNOS activity, migration capacity and reduced angiogenic capacity in vitro and in vivo compared to healthy endothelial cells. CTEPH-EC presented altered mitochondrial dynamics, increased mitochondrial respiration and an unbalanced production of reactive oxygen species and antioxidants. Our study is the foremost comprehensive investigation of CTEPH-EC. Modulation of redox, mitochondrial homeostasis and adhesion molecule overexpression arise as novel targets and biomarkers in CTEPH.

Interspecific and intraspecific gene variability in a 1-Mb region containing the highest density of NBS-LRR genes found in the melon genome.

BACKGROUND: Plant NBS-LRR -resistance genes tend to be found in clusters, which have been shown to be hot spots of genome variability. In melon, half of the 81 predicted NBS-LRR genes group in nine clusters, and a 1 Mb region on linkage group V contains the highest density of R-genes and presence/absence gene polymorphisms found in the melon genome. This region is known to contain the locus of Vat, an agronomically important gene that confers resistance to aphids. However, the presence of duplications makes the sequencing and annotation of R-gene clusters difficult, usually resulting in multi-gapped sequences with higher than average errors. RESULTS: A 1-Mb sequence that contains the largest NBS-LRR gene cluster found in melon was improved using a strategy that combines Illumina paired-end mapping and PCR-based gap closing. Unknown sequence was decreased by 70% while about 3,000 SNPs and small indels were corrected. As a result, the annotations of 18 of a total of 23 NBS-LRR genes found in this region were modified, including additional coding sequences, amino acid changes, correction of splicing boundaries, or fussion of ORFs in common transcription units. A phylogeny analysis of the R-genes and their comparison with syntenic sequences in other cucurbits point to a pattern of local gene amplifications since the diversification of cucurbits from other families, and through speciation within the family. A candidate Vat gene is proposed based on the sequence similarity between a reported Vat gene from a Korean melon cultivar and a sequence fragment previously absent in the unrefined sequence. CONCLUSIONS: A sequence refinement strategy allowed substantial improvement of a 1 Mb fragment of the melon genome and the re-annotation of the largest cluster of NBS-LRR gene homologues found in melon. Analysis of the cluster revealed that resistance genes have been produced by sequence duplication in adjacent genome locations since the divergence of cucurbits from other close families, and through the process of speciation within the family a candidate Vat gene was also identified using sequence previously unavailable, which demonstrates the advantages of genome assembly refinements when analyzing complex regions such as those containing clusters of highly similar genes.

High presence/absence gene variability in defense-related gene clusters of Cucumis melo.

BACKGROUND: Changes in the copy number of DNA sequences are one of the main mechanisms generating genome variability in eukaryotes. These changes are often related to phenotypic effects such as genetic disorders or novel pathogen resistance. The increasing availability of genome sequences through the application of next-generation massive sequencing technologies has allowed the study of genomic polymorphisms at both the interspecific and intraspecific levels, thus helping to understand how species adapt to changing environments through genome variability. RESULTS: Data on gene presence/absence variation (PAV) in melon was obtained by resequencing a cultivated accession and an old-relative melon variety, and using previously obtained resequencing data from three other melon cultivars, among them DHL92, on which the current draft melon genome sequence is based. A total of 1,697 PAV events were detected, involving 4.4% of the predicted melon gene complement. In all, an average 1.5% of genes were absent from each analyzed cultivar as compared to the DHL92 reference genome. The most populated functional category among the 304 PAV genes of known function was that of stress response proteins (30% of all classified PAVs). Our results suggest that genes from multi-copy families are five times more likely to be affected by PAV than singleton genes. Also, the chance of genes present in the genome in tandem arrays being affected by PAV is double that of isolated genes, with PAV genes tending to be in longer clusters. The highest concentration of PAV events detected in the melon genome was found in a 1.1 Mb region of linkage group V, which also shows the highest density of melon stress-response genes. In particular, this region contains the longest continuous gene-containing PAV sequence so far identified in melon. CONCLUSIONS: The first genome-wide report of PAV variation among several melon cultivars is presented here. Multi-copy and clustered genes, especially those with putative stress-response functions, were found to be particularly affected by PAV polymorphisms. As cucurbits are known to possess a significantly lower number of defense-related genes compared to other plant species, PAV variation may play an important role in generating new pathogen resistances at the subspecies level. In addition, these results show the limitations of single reference genome sequences as the only basis for characterization and cloning of resistance genes.