Telomere length as biomarker of nutritional therapy for prevention of type 2 diabetes mellitus development in patients with coronary heart disease: CORDIOPREV randomised controlled trial.

BACKGROUND: Telomere Length (TL), a marker of cellular aging, holds promise as a biomarker to elucidate the molecular mechanism of diabetes. This study aimed to investigate whether shorter telomeres are associated with a higher risk of type 2 diabetes mellitus (T2DM) incidence in patients with coronary heart disease; and to determine whether the most suitable dietary patterns, particularly a Mediterranean diet or a low-fat diet, can mitigate the development of diabetes in these patients after a follow-up period of five years. METHODS: The CORonary Diet Intervention with Olive oil and cardiovascular PREVention study (CORDIOPREV study) was a single-centre, randomised clinical trial done at the Reina Sofia University Hospital in Córdoba, Spain. Patients with established coronary heart disease (aged 20-75 years) were randomly assigned in a 1:1 ratio by the Andalusian School of Public Health to receive two healthy diets. Clinical investigators were masked to treatment assignment; participants were not. Quantitative-PCR was used to assess TL measurements. FINDINGS: 1002 patients (59.5 ± 8.7 years and 82.5% men) were enrolled into Mediterranean diet (n = 502) or a low-fat diet (n = 500) groups. In this analysis, we included all 462 patients who did not have T2DM at baseline. Among them, 107 patients developed T2DM after a median of 60 months. Cox regression analyses showed that patients at risk of short telomeres (TL < percentile 20th) are more likely to experience T2DM than those at no risk of short telomeres (HR 1.65, p-value 0.023). In terms of diet, patients at high risk of short telomeres had a higher risk of T2DM incidence after consuming a low-fat diet compared to patients at no risk of short telomeres (HR 2.43, 95CI% 1.26 to 4.69, p-value 0.008), while no differences were observed in the Mediterranean diet group. CONCLUSION: Patients with shorter TL presented a higher risk of developing T2DM. This association could be mitigated with a specific dietary pattern, in our case a Mediterranean diet, to prevent T2DM in patients with coronary heart disease. TRIAL REGISTRATION: Clinicaltrials.gov number NCT00924937.

Safety of inhaled nitric oxide withdrawal in severe chronic pulmonary hypertension.

Inhaled nitric oxide (iNO) is a potent and selective pulmonary vasodilator with a safety concern due to rebound pulmonary hypertension (PH) associated with its withdrawal. We report short-term pulsed iNO in patients with severe pulmonary arterial hypertension (PAH) and nonoperable chronic thromboembolic PH (nCTEPH). This is a retrospective analysis of 33 patients: 22 with PAH and 11 with nCTEPH. We assessed hemodynamic, echocardiographic, and other noninvasive variables to evaluate safety and efficacy of iNO. We performed an iNO withdrawal test during right heart catheterization and after 3 days of iNO treatment. iNO significantly improved all variables examined in 22 patients with PAH and 11 with nCTEPH. Two patterns of response were observed after sudden iNO withdrawal. Twenty-nine patients (88%) showed minimal hemodynamic, oxygenation and clinical changes. Four patients (12%) had a reduction in cardiac index ≥20% and PaO2 ≥ 5%, three patients did not show clinical deterioration, and one patient developed hemodynamic collapse that needed iNO administration. This retrospective study suggests that short-term iNO improves hemodynamics and clinical conditions in some patients with PAH an nCTPEH. However, pulsed iNO withdrawal PH rebound could be a serious concern in these patients. Given the lack of evidence, we do not recommend the use of pulsed iNO in the treatment of patients with chronic PH.

Sex-specific differences in intestinal microbiota associated with cardiovascular diseases.

BACKGROUND: Cardiovascular diseases (CVD), including coronary heart disease (CHD), display a higher prevalence in men than women. This study aims to evaluate the variations in the intestinal microbiota between men and women afflicted with CHD and delineate these against a non-CVD control group for each sex. METHODS: Our research was conducted in the framework of the CORDIOPREV study, a clinical trial which involved 837 men and 165 women with CHD. We contrasted our findings with a reference group of 375 individuals (270 men, 105 women) without CVD. The intestinal microbiota was examined through 16S metagenomics on the Illumina MiSeq platform and the data processed with Quiime2 software. RESULTS: Our results showed a sex-specific variation (beta diversity) in the intestinal microbiota, while alpha-biodiversity remained consistent across both sexes. Linear discriminant analysis effect size (LEfSe) analysis revealed sex-centric alterations in the intestinal microbiota linked to CVD. Moreover, using random forest (RF) methodology, we identified seven bacterial taxa-g_UBA1819 (Ruminococcaceae), g_Bilophila, g_Subdoligranulum, g_Phascolarctobacterium, f_Barnesiellaceae, g_Ruminococcus, and an unknown genus from the Ruminococcaceae family (Ruminococcaceae incertae sedis)-as key discriminators between men and women diagnosed with CHD. The same taxa also emerged as critical discriminators between CHD-afflicted and non-CVD individuals, when analyzed separately by sex. CONCLUSION: Our findings suggest a sex-specific dysbiosis in the intestinal microbiota linked to CHD, potentially contributing to the sex disparity observed in CVD incidence. Trial registration Clinical Trials.gov.Identifier NCT00924937.

The frequency with which cardiovascular diseases occur differs in men and women as it appears with greater frequency in men. Moreover, it has been known for years that the community of bacteria living in our intestine, also known as the gut microbiota, influences the development of these diseases. Indeed, nowadays it known the influence of the intestinal microbiota in the development of atherosclerosis, the pathological process which is responsible for the three main causes of cardiovascular diseases: coronary heart disease, cerebrovascular disease and peripheral arterial disease. This study shows the differences in the community of bacteria living in the gut of men and those living in the gut of women, so that these differences could explain, at least in part, the differences in the frequency with which cardiovascular diseases appear between men and women. Our results suggest that the dysbiosis of the intestinal microbiota associated with CHD seems to be partially sex-specific, which may influence the sexual dimorphism in its incidence. Moreover, the identification of the mechanisms responsible for sexual dimorphism in the incidence of metabolic and cardiovascular disease is of particular importance when developing effective strategies and therapies aimed at reducing their incidence and recurrence. Indeed, the strategies and therapies used to treat the dysbiosis of the gut microbiota should be sex-specific.

Proteome and Interactome Linked to Metabolism, Genetic Information Processing, and Abiotic Stress in Gametophytes of Two Woodferns.

Ferns and lycophytes have received scant molecular attention in comparison to angiosperms. The advent of high-throughput technologies allowed an advance towards a greater knowledge of their elusive genomes. In this work, proteomic analyses of heart-shaped gametophytes of two ferns were performed: the apomictic Dryopteris affinis ssp. affinis and its sexual relative Dryopteris oreades. In total, a set of 218 proteins shared by these two gametophytes were analyzed using the STRING database, and their proteome associated with metabolism, genetic information processing, and responses to abiotic stress is discussed. Specifically, we report proteins involved in the metabolism of carbohydrates, lipids, and nucleotides, the biosynthesis of amino acids and secondary compounds, energy, oxide-reduction, transcription, translation, protein folding, sorting and degradation, and responses to abiotic stresses. The interactome of this set of proteins represents a total network composed of 218 nodes and 1792 interactions, obtained mostly from databases and text mining. The interactions among the identified proteins of the ferns D. affinis and D. oreades, together with the description of their biological functions, might contribute to a better understanding of the function and development of ferns as well as fill knowledge gaps in plant evolution.

The Shared Proteome of the Apomictic Fern Dryopteris affinis ssp. affinis and Its Sexual Relative Dryopteris oreades.

Ferns are a diverse evolutionary lineage, sister to the seed plants, which is of great ecological importance and has a high biotechnological potential. Fern gametophytes represent one of the simplest autotrophic, multicellular plant forms and show several experimental advantages, including a simple and space-efficient in vitro culture system. However, the molecular basis of fern growth and development has hardly been studied. Here, we report on a proteomic study that identified 417 proteins shared by gametophytes of the apogamous fern Dryopteris affinis ssp. affinis and its sexual relative Dryopteris oreades. Most proteins are predicted to localize to the cytoplasm, the chloroplast, or the nucleus, and are linked to enzymatic, binding, and structural activities. A subset of 145 proteins are involved in growth, reproduction, phytohormone signaling and biosynthesis, and gene expression, including homologs of SHEPHERD (SHD), HEAT SHOCK PROTEIN 90-5 (CR88), TRP4, BOBBER 1 (BOB1), FLAVONE 3'-O-METHYLTRANSFERASE 1 (OMT1), ZEAXANTHIN EPOXIDASE (ABA1), GLUTAMATE DESCARBOXYLASE 1 (GAD), and dsRNA-BINDING DOMAIN-LIKE SUPERFAMILY PROTEIN (HLY1). Nearly 25% of the annotated proteins are associated with responses to biotic and abiotic stimuli. As for biotic stress, the proteins PROTEIN SGT1 HOMOLOG B (SGT1B), SUPPRESSOR OF SA INSENSITIVE2 (SSI2), PHOSPHOLIPASE D ALPHA 1 (PLDALPHA1), SERINE/THREONINE-PROTEIN KINASE SRK2E (OST1), ACYL CARRIER PROTEIN 4 (ACP4), and NONHOST RESISTANCE TO P. S. PHASEOLICOLA1 (GLPK) are worth mentioning. Regarding abiotic stimuli, we found proteins associated with oxidative stress: SUPEROXIDE DISMUTASE[CU-ZN] 1 (CSD1), and GLUTATHIONE S-TRANSFERASE U19 (GSTU19), light intensity SERINE HYDROXYMETHYLTRANSFERASE 1 (SHM1) and UBIQUITIN-CONJUGATING ENZYME E2 35 (UBC35), salt and heavy metal stress included MITOCHONDRIAL PHOSPHATE CARRIER PROTEIN 3 (PHT3;1), as well as drought and thermotolerance: LEA7, DEAD-BOX ATP-DEPENDENT RNA HELICASE 38 (LOS4), and abundant heat-shock proteins and other chaperones. In addition, we identified interactomes using the STRING platform, revealing protein-protein associations obtained from co-expression, co-occurrence, text mining, homology, databases, and experimental datasets. By focusing on ferns, this proteomic study increases our knowledge on plant development and evolution, and may inspire future applications in crop species.

Mapping the spatial variability of rainfall from a physiographic-based multilinear regression: model development and application to the Southwestern Iberian Peninsula.

A physiographic-based multilinear regression model supported by GIS was developed to estimate spatial rainfall variability in the Southwest Iberian Peninsula. The area study includes a wide diversity of landscape features and comprises four Portuguese regions and one Spanish province (totalizing 28,860 km2). The region suffers a very strong Mediterranean influence, with a major cleavage between winter and summer seasons. Thus, the analysis was carried out separately for the wet (October to March) and dry (April to September) semesters. From an initial set of 10 explanatory physiographic variables, five were selected to be used in the multilinear regression, as they allowed generating models by map algebra that fitted well with the last 40 years of monthly rainfall data records. These records were obtained from 163 weather stations, filtered from an initial set of 230 (142 stations in Portugal and 88 in Spain). The correlation between the physiographic-based multilinear regression model and a model obtained by interpolation from rainfall historical data showed to be good or very good in approximately 75% of the area under study. Results show that physiographic-based models can be effectively used to estimate rainfall where there is a lack of rain gauges, or to densify spatial resolution of rainfall between rain gauges.

Sexual and Apogamous Species of Woodferns Show Different Protein and Phytohormone Profiles.

The gametophyte of ferns reproduces either by sexual or asexual means. In the latter, apogamy represents a peculiar case of apomixis, in which an embryo is formed from somatic cells. A proteomic and physiological approach was applied to the apogamous fern Dryopteris affinis ssp. affinis and its sexual relative D. oreades. The proteomic analysis compared apogamous vs. female gametophytes, whereas the phytohormone study included, in addition to females, three apogamous stages (filamentous, spatulate, and cordate). The proteomic profiles revealed a total of 879 proteins and, after annotation, different regulation was found in 206 proteins of D. affinis and 166 of its sexual counterpart. The proteins upregulated in D. affinis are mostly associated to protein metabolism (including folding, transport, and proteolysis), ribosome biogenesis, gene expression and translation, while in the sexual counterpart, they account largely for starch and sucrose metabolism, generation of energy and photosynthesis. Likewise, ultra-performance liquid chromatography-tandem spectrometry (UHPLC-MS/MS) was used to assess the levels of indol-3-acetic acid (IAA); the cytokinins: 6-benzylaminopurine (BA), trans-Zeatine (Z), trans-Zeatin riboside (ZR), dyhidrozeatine (DHZ), dyhidrozeatin riboside (DHZR), isopentenyl adenine (iP), isopentenyl adenosine (iPR), abscisic acid (ABA), the gibberellins GA3 and GA4, salicylic acid (SA), and the brassinosteroids: brassinolide (BL) and castasterone (CS). IAA, the cytokinins Z, ZR, iPR, the gibberellin GA4, the brassinosteoids castasterone, and ABA accumulated more in the sexual gametophyte than in the apogamous one. When comparing the three apogamous stages, BA and SA peaked in filamentous, GA3 and BL in spatulate and DHRZ in cordate gametophytes. The results point to the existence of large metabolic differences between apogamous and sexual gametophytes, and invite to consider the fern gametophyte as a good experimental system to deepen our understanding of plant reproduction.

Splicing factor SF3B1 is overexpressed and implicated in the aggressiveness and survival of hepatocellular carcinoma.

Splicing alterations represent an actionable cancer hallmark. Splicing factor 3B subunit 1 (SF3B1) is a crucial splicing factor that can be targeted pharmacologically (e.g. pladienolide-B). Here, we show that SF3B1 is overexpressed (RNA/protein) in hepatocellular carcinoma (HCC) in two retrospective (n = 154 and n = 172 samples) and in five in silico cohorts (n > 900 samples, including TCGA) and that its expression is associated with tumor aggressiveness, oncogenic splicing variants expression (KLF6-SV1, BCL-XL) and decreased overall survival. In vitro, SF3B1 silencing reduced cell viability, proliferation and migration and its pharmacological blockade with pladienolide-B inhibited proliferation, migration, and formation of tumorspheres and colonies in liver cancer cell lines (HepG2, Hep3B, SNU-387), whereas its effects on normal-like hepatocyte-derived THLE-2 proliferation were negligible. Pladienolide-B also reduced the in vivo growth and the expression of tumor-markers in Hep3B-induced xenograft tumors. Moreover, SF3B1 silencing and/or blockade markedly modulated the activation of key signaling pathways (PDK1, GSK3b, ERK, JNK, AMPK) and the expression of cancer-associated genes (CDK4, CD24) and oncogenic SVs (KLF6-SV1). Therefore, the genetic and/or pharmacological inhibition of SF3B1 may represent a promising novel therapeutic strategy worth to be explored through randomized controlled trials.

Differential gene expression profiling of one- and two-dimensional apogamous gametophytes of the fern Dryopteris affinis ssp. affinis.

Apomixis was originally defined as the replacement of sexual reproduction by an asexual process that does not involve fertilization but, in angiosperms, it is often used in the more restricted sense of asexual reproduction through seeds. In ferns, apomixis combines the production of unreduced spores (diplospory) and the formation of sporophytes from somatic cells of the prothallium (apogamy). The genes that control the onset of apogamy in ferns are largely unknown. In this study, we describe the gametophyte transcriptome of the apogamous fern Dryopteris affinis ssp. affinis using an RNA-Seq approach to compare the gene expression profiles of one- and two-dimensional gametophytes, the latter containing apogamic centers. After collapsing highly similar de novo transcripts, we obtained 166,191 unigenes, of which 30% could be annotated using public databases. Multiple quality metrics indicate a good quality of the de novo transcriptome with a low level of fragmentation. Our data show a total of 10,679 genes (6% of all genes) to be differentially expressed between gametophytes of filamentous (one-dimensional) and prothallial (two-dimensional) architecture. 6,110 genes were up-regulated in two-dimensional relative to one-dimensional gametophytes, some of which are implicated in the regulation of meristem growth, auxin signaling, reproduction, and sucrose metabolism. 4,570 genes were down-regulated in two-dimensional versus one-dimensional gametophytes, which are enriched in stimulus and defense genes, as well as genes involved in epigenetic gene regulation and ubiquitin degradation. Our results provide insights into free-living gametophyte development, focusing on the filamentous-to-prothallus growth transition, and provide a useful resource for further investigations of asexual reproduction.

PattRec: An easy-to-use CNV detection tool optimized for targeted NGS assays with diagnostic purposes.

Genetic laboratories use custom-commercial targeted next-generation sequencing (tg-NGS) assays to identify disease-causing variants. Although the high coverage achieved with these tests allows for the detection of copy number variants (CNVs), which account for an important proportion of the genetic burden in human diseases, an easy-to-use tool for automatic CNV detection is still lacking. This article presents a new CNV detection tool optimized for tg-NGS data: PattRec. PattRec was evaluated using a wide range of data, and its performance compared with those of other CNV detection tools. The software includes features for selecting optimal controls, discarding polymorphic CNVs prior to analysis, and filtering out deletions based on SNV zygosity, and automatically creates an in-house CNV database. There is no need for high level bioinformatic expertise and users can choose color-coded xlsx output that helps to prioritize potentially pathogenic CNVs. PattRec is presented as a Java based GUI, freely available online: https://github.com/irotero/PattRec.

Free-access copy-number variant detection tools for targeted next-generation sequencing data.

Copy number variants (CNVs) are intermediate-scale structural variants containing copy number changes involving DNA fragments of between 1 kb and 5 Mb. Although known to account for a significant proportion of the genetic burden in human disease, the role of CNVs (especially small CNVs) is often underestimated, as they are undetectable by traditional Sanger sequencing. Since the development of next-generation sequencing (NGS) technologies, several research groups have compared depth of coverage (DoC) patterns between samples, an approach that may facilitate effective CNV detection. Most CNV detection tools based on DoC comparisons are designed to work with whole-genome sequencing (WGS) or whole-exome sequencing (WES) data. However, few methods developed to date are designed for custom/commercial targeted NGS (tg-NGS) panels, the assays most commonly used for diagnostic purposes. Moreover, the development and evaluation of these tools is hindered by (i) the scarcity of thoroughly annotated data containing CNVs and (ii) a dearth of simulation tools for WES and tg-NGS that mimic the errors and biases encountered in these data. Here, we review DoC-based CNV detection methods described in the current literature, assess their performance with simulated tg-NGS data, and discuss their strengths and weaknesses when integrated into the daily laboratory workflow. Our findings suggest that the best methods for CNV detection in tg-NGS panels are DECoN, ExomeDepth, and ExomeCNV. Regardless of the method used, there is a need to make these programs more user-friendly to enable their use by diagnostic laboratory staff who lack bioinformatics training.

Proteogenomic Analysis Greatly Expands the Identification of Proteins Related to Reproduction in the Apogamous Fern Dryopteris affinis ssp. affinis.

Performing proteomic studies on non-model organisms with little or no genomic information is still difficult. However, many specific processes and biochemical pathways occur only in species that are poorly characterized at the genomic level. For example, many plants can reproduce both sexually and asexually, the first one allowing the generation of new genotypes and the latter their fixation. Thus, both modes of reproduction are of great agronomic value. However, the molecular basis of asexual reproduction is not well understood in any plant. In ferns, it combines the production of unreduced spores (diplospory) and the formation of sporophytes from somatic cells (apogamy). To set the basis to study these processes, we performed transcriptomics by next-generation sequencing (NGS) and shotgun proteomics by tandem mass spectrometry in the apogamous fern D. affinis ssp. affinis. For protein identification we used the public viridiplantae database (VPDB) to identify orthologous proteins from other plant species and new transcriptomics data to generate a "species-specific transcriptome database" (SSTDB). In total 1,397 protein clusters with 5,865 unique peptide sequences were identified (13 decoy proteins out of 1,410, protFDR 0.93% on protein cluster level). We show that using the SSTDB for protein identification increases the number of identified peptides almost four times compared to using only the publically available VPDB. We identified homologs of proteins involved in reproduction of higher plants, including proteins with a potential role in apogamy. With the increasing availability of genomic data from non-model species, similar proteogenomics approaches will improve the sensitivity in protein identification for species only distantly related to models.

Proteomic approaches to sexual development mediated by antheridiogen in the fern Blechnum spicant L.

This report is intended to compare proteomic profiles between female and male gametophytes of Blechnum spicant L. For this purpose, female gametophytes were obtained from spores germinated on Murashige and Skoog medium (1962), and the male ones by culturing 30-day-old gametophytes on the same medium supplemented with extract derived from mature gametophytes, which exhibits anteridiogen effect. Proteins were extracted using the phenolic method, and after two-dimensional gel electrophoresis, 581 spots were detected in Coomassie stained gels within the 3-11 pH range and 10-100 kDa Mr ranges. Quantitative and qualitative differences in spot intensity between both types of gametophytes were found. The induction of male gametophytes by antheridiogens was correlated to a global increase in stress and defence-related pathways, which affect flavonoid signaling and cell division, while reducing protein biosynthesis, photosynthesis, and other energy-related pathways, reflecting how stressed are the gametophytes during the induction process. This is the first proteomic study that examines proteins associated to sexual development in the gametophyte of the fern B. spìcant.

A dig into the past mitochondrial diversity of Corsican goats reveals the influence of secular herding practices.

The goat (Capra hircus) is one of the earliest domesticated species ca. 10,500 years ago in the Middle-East where its wild ancestor, the bezoar (Capra aegagrus), still occurs. During the Neolithic dispersal, the domestic goat was then introduced in Europe, including the main Mediterranean islands. Islands are interesting models as they maintain traces of ancient colonization, historical exchanges or of peculiar systems of husbandry. Here, we compare the mitochondrial genetic diversity of both medieval and extant goats in the Island of Corsica that presents an original and ancient model of breeding with free-ranging animals. We amplified a fragment of the Control Region for 21 medieval and 28 current goats. Most of them belonged to the A haplogroup, the most worldwide spread and frequent today, but the C haplogroup is also detected at low frequency in the current population. Present Corsican goats appeared more similar to medieval goats than to other European goat populations. Moreover, 16 out of the 26 haplotypes observed were endemic to Corsica and the inferred demographic history suggests that the population has remained constant since the Middle Ages. Implications of these results on management and conservation of endangered Corsican goats currently decimated by a disease are addressed.

Divergent mtDNA lineages of goats in an Early Neolithic site, far from the initial domestication areas.

Goats were among the first farm animals domesticated, approximately 10,500 years ago, contributing to the rise of the "Neolithic revolution." Previous genetic studies have revealed that contemporary domestic goats (Capra hircus) show far weaker intercontinental population structuring than other livestock species, suggesting that goats have been transported more extensively. However, the timing of these extensive movements in goats remains unknown. To address this question, we analyzed mtDNA sequences from 19 ancient goat bones (7,300-6,900 years old) from one of the earliest Neolithic sites in southwestern Europe. Phylogenetic analysis revealed that two highly divergent goat lineages coexisted in each of the two Early Neolithic layers of this site. This finding indicates that high mtDNA diversity was already present >7,000 years ago in European goats, far from their areas of initial domestication in the Near East. These results argue for substantial gene flow among goat populations dating back to the early neolithisation of Europe and for a dual domestication scenario in the Near East, with two independent but essentially contemporary origins (of both A and C domestic lineages) and several more remote and/or later origins.

Gemmation in cultured gametophytes ofOsmunda regalis.

Gametophytes ofOsmunda regalis were culturedin vitro to determine the optimal conditions for growth and development. Culture media with low ammonium concentration (Knop, Knudson or 1/4 Murashige and Skoog dilution) were the most effective for growth of this organism. Addition of sucrose and mannitol to the culture medium at the same osmoticum, inhibits or enhances, respectively, growth and development of the gametophytes, which show a strong autotrophyin vitro. In darkness, both two-dimensional growth and sexual organ formation took place. Gemmae formation is described for the first time in this species and this process does not occur without sucrose in the culture medium or in darkness.