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Clinical studies have previously established the role of olive products in cardiovascular disease (CVD) prevention, whilst the identification of the responsible constituents for the beneficial effects is still pending. We sought to assess and compare the cardioprotective potential of oleuropein (OL), hydroxytyrosol (HT), oleocanthal (OC) and oleanolic Acid (OA), regarding Ischemia/Reperfusion Injury (IRI) and CVD risk factors alleviation. The scope of the study was to design a potent and safe combinatorial therapy for high-cardiovascular-risk patients on a bench-to-bedside approach. We evaluated the IRI-limiting potential of 6-weeks treatment with OL, HT, OC or OA at nutritional doses, in healthy and metabolic syndrome (MS)-burdened mice. Three combinatorial regimens were designed and the mixture with preponderant benefits (OL-HT-OC, Combo 2), including infarct sparing and antiglycemic potency, compared to the isolated compounds, was further investigated for its anti-atherosclerotic effects. In vivo experiments revealed that the combination regimen of Combo 2 presented the most favorable effects in limiting infarct size and hyperglycemia, which was selected to be further investigated in the clinical setting in Chronic Coronary Artery Syndrome (CCAS) patients. Cardiac function, inflammation markers and oxidative stress were assessed at baseline and after 4 weeks of treatment with the OL-HT-OC supplement in the clinical study. We found that OL, OC and OA significantly reduced infarct size in vivo compared to Controls. OL exhibited antihyperglycemic properties and OA attenuated hypercholesterolemia. OL-HT-OA, OL-HT-OC and OL-HT-OC-OA combination regimens were cardioprotective, whereas only OL-HT-OC mitigated hyperglycemia. Combo 2 cardioprotection was attributed to apoptosis suppression, enhanced antioxidant effects and upregulation of antioxidant enzymes. Additionally, it reduced atherosclerotic plaque extent in vivo. OL-HT-OC supplement ameliorated cardiac, vascular and endothelial function in the small-scale clinical study. Conclusively, OL-HT-OC combination therapy exerts potent cardioprotective, antihyperglycemic and anti-atherosclerotic properties in vivo, with remarkable and clinically translatable cardiovascular benefits in high-risk patients.

Abstract Single-cell open chromatin profiling via scATAC-seq has become a mainstream measurement of open chromatin in single-cells. Here we present epiAneufinder, an algorithm that exploits the read count information from scATAC-seq data to extract genome-wide copy number alterations (CNAs) for individual cells, allowing the study of CNA heterogeneity present in a sample at the single-cell level. Using different cancer scATAC-seq datasets, we show that epiAneufinder can identify intratumor clonal heterogeneity in populations of single cells based on their CNA profiles. We demonstrate that these profiles are concordant with the ones inferred from single-cell whole genome sequencing data for the same samples. EpiAneufinder allows the inference of single-cell CNA information from scATAC-seq data, without the need of additional experiments, unlocking a layer of genomic variation which is otherwise unexplored.

Background: Hypertension poses a significant global health burden and is associated with cardiovascular morbidity. Chios mastic gum (CMG), derived from Pistacia lentiscus var. Chia, shows potential as a phytotherapeutic agent, due to its multifaceted beneficial effects. However, its anti-hypertensive effects and vascular, circulatory, and renal-related dysfunction, have not been thoroughly investigated. Herein, we aimed to explore the antihypertensive potential of CMG, focusing on vascular and renal endothelium, in vivo. Methods: Two models of hypertension in male rats, induced by Angiotensin II and Deoxycorticosterone acetate (DOCA)–high-salt administration, were utilized. CMG was administered at 220 mg/kg daily for four weeks after hypertension onset and blood pressure was measured non-invasively. Whole blood RNA sequencing, metabolomics, real-time PCR, and Western blot analyses of kidney and aorta tissues were additionally performed. Results: CMG significantly lowered systolic, diastolic, and mean blood pressure in both models. RNA sequencing revealed that CMG modulated immunity in the Angiotensin II model and metabolism in the DOCA–HS model. CMG downregulated genes related to oxidative stress and endothelial dysfunction and upregulated endothelial markers such as Vegfa. Metabolomic analysis indicated improved endothelial homeostasis via lysophosphatidylinositol upregulation. Conclusions: CMG emerges as a potent natural antihypertensive therapy, demonstrating beneficial effects on blood pressure and renal endothelial function.

Circadian rhythms control organismal physiology throughout the day. At the cellular level, clock regulation is established by a self-sustained Bmal1-dependent transcriptional oscillator network. However, it is still unclear how different tissues achieve a synchronized rhythmic physiology. That is, do they respond independently to environmental signals, or require interactions with each other to do so? We show that unexpectedly, light synchronizes the Bmal1-dependent circadian machinery in single tissues in the absence of Bmal1 in all other tissues. Strikingly, light-driven tissue autonomous clocks occur without rhythmic feeding behavior and are lost in constant darkness. Importantly, tissue-autonomous Bmal1 partially sustains homeostasis in otherwise arrhythmic and prematurely aging animals. Our results therefore support a two-branched model for the daily synchronization of tissues: an autonomous response branch, whereby light entrains circadian clocks without any commitment of other Bmal1-dependent clocks, and a memory branch using other Bmal1-dependent clocks to "remember" time in the absence of external cues.

The draft genome of the moss model, Physcomitrella patens, comprised approximately 2000 unordered scaffolds. In order to enable analyses of genome structure and evolution we generated a chromosome-scale genome assembly using genetic linkage as well as (end) sequencing of long DNA fragments. We find that 57% of the genome comprises transposable elements (TEs), some of which may be actively transposing during the life cycle. Unlike in flowering plant genomes, gene- and TE-rich regions show an overall even distribution along the chromosomes. However, the chromosomes are mono-centric with peaks of a class of Copia elements potentially coinciding with centromeres. Gene body methylation is evident in 5.7% of the protein-coding genes, typically coinciding with low GC and low expression. Some giant virus insertions are transcriptionally active and might protect gametes from viral infection via siRNA mediated silencing. Structure-based detection methods show that the genome evolved via two rounds of whole genome duplications (WGDs), apparently common in mosses but not in liverworts and hornworts. Several hundred genes are present in colinear regions conserved since the last common ancestor of plants. These syntenic regions are enriched for functions related to plant-specific cell growth and tissue organization. The P. patens genome lacks the TE-rich pericentromeric and gene-rich distal regions typical for most flowering plant genomes. More non-seed plant genomes are needed to unravel how plant genomes evolve, and to understand whether the P. patens genome structure is typical for mosses or bryophytes.

Fatty acid uptake and altered metabolism constitute hallmarks of metastasis.sup.1,2, yet evidence of the underlying biology, as well as whether all dietary fatty acids are prometastatic, is lacking. Here we show that dietary palmitic acid (PA), but not oleic acid or linoleic acid, promotes metastasis in oral carcinomas and melanoma in mice. Tumours from mice that were fed a short-term palm-oil-rich diet (PA), or tumour cells that were briefly exposed to PA in vitro, remained highly metastatic even after being serially transplanted (without further exposure to high levels of PA). This PA-induced prometastatic memory requires the fatty acid transporter CD36 and is associated with the stable deposition of histone H3 lysine 4 trimethylation by the methyltransferase Set1A (as part of the COMPASS complex (Set1A/COMPASS)). Bulk, single-cell and positional RNA-sequencing analyses indicate that genes with this prometastatic memory predominantly relate to a neural signature that stimulates intratumoural Schwann cells and innervation, two parameters that are strongly correlated with metastasis but are aetiologically poorly understood.sup.3,4. Mechanistically, tumour-associated Schwann cells secrete a specialized proregenerative extracellular matrix, the ablation of which inhibits metastasis initiation. Both the PA-induced memory of this proneural signature and its long-term boost in metastasis require the transcription factor EGR2 and the glial-cell-stimulating peptide galanin. In summary, we provide evidence that a dietary metabolite induces stable transcriptional and chromatin changes that lead to a long-term stimulation of metastasis, and that this is related to a proregenerative state of tumour-activated Schwann cells. Palmitic acid induces stable transcriptional and chromatin changes that lead to long-term stimulation of metastasis in orthotopic models of cancer through the secretion by tumour-associated Schwann cells of a specialized proregenerative extracellular matrix, the ablation of which inhibits metastasis initiation., Author(s): Gloria Pascual [sup.1] , Diana Domínguez [sup.1] , Marc Elosúa-Bayes [sup.2] , Felipe Beckedorff [sup.3] , Carmelo Laudanna [sup.1] , Claudia Bigas [sup.1] , Delphine Douillet [sup.4] , Carolina [...]

The process of aging and circadian rhythms are intimately intertwined, but how peripheral clocks involved in metabolic homeostasis contribute to aging remains unknown. Importantly, caloric restriction (CR) extends lifespan in several organisms and rewires circadian metabolism. Using young versus old mice, fed ad libitum or under CR, we reveal reprogramming of the circadian transcriptome in the liver. These age-dependent changes occur in a highly tissue-specific manner, as demonstrated by comparing circadian gene expression in the liver versus epidermal and skeletal muscle stem cells. Moreover, de novo oscillating genes under CR show an enrichment in SIRT1 targets in the liver. This is accompanied by distinct circadian hepatic signatures in NAD+-related metabolites and cyclic global protein acetylation. Strikingly, this oscillation in acetylation is absent in old mice while CR robustly rescues global protein acetylation. Our findings indicate that the clock operates at the crossroad between protein acetylation, liver metabolism, and aging.

Background: Hypertension poses a significant global health burden and is associated with cardiovascular morbidity. Chios mastic gum (CMG), derived from Pistacia lentiscus var. Chia, shows potential as a phytotherapeutic agent, due to its multifaceted beneficial effects. However, its anti-hypertensive effects and vascular, circulatory, and renal-related dysfunction, have not been thoroughly investigated. Herein, we aimed to explore the antihypertensive potential of CMG, focusing on vascular and renal endothelium, in vivo. Methods: Two models of hypertension in male rats, induced by Angiotensin II and Deoxycorticosterone acetate (DOCA)–high-salt administration, were utilized. CMG was administered at 220 mg/kg daily for four weeks after hypertension onset and blood pressure was measured non-invasively. Whole blood RNA sequencing, metabolomics, real-time PCR, and Western blot analyses of kidney and aorta tissues were additionally performed. Results: CMG significantly lowered systolic, diastolic, and mean blood pressure in both models. RNA sequencing revealed that CMG modulated immunity in the Angiotensin II model and metabolism in the DOCA–HS model. CMG downregulated genes related to oxidative stress and endothelial dysfunction and upregulated endothelial markers such as Vegfa. Metabolomic analysis indicated improved endothelial homeostasis via lysophosphatidylinositol upregulation. Conclusions: CMG emerges as a potent natural antihypertensive therapy, demonstrating beneficial effects on blood pressure and renal endothelial function. [ABSTRACT FROM AUTHOR]

Abstract Stochastic changes in DNA methylation (i.e., spontaneous epimutations) contribute to methylome diversity in plants. Here, we describe AlphaBeta, a computational method for estimating the precise rate of such stochastic events using pedigree-based DNA methylation data as input. We demonstrate how AlphaBeta can be employed to study transgenerationally heritable epimutations in clonal or sexually derived mutation accumulation lines, as well as somatic epimutations in long-lived perennials. Application of our method to published and new data reveals that spontaneous epimutations accumulate neutrally at the genome-wide scale, originate mainly during somatic development and that they can be used as a molecular clock for age-dating trees.

Genome editing with the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR associated protein) system allows mutagenesis of a targeted region of the genome using a Cas endonuclease and an artificial guide RNA. Both because of variable efficiency with which such mutations arise and because the repair process produces a spectrum of mutations, one needs to ascertain the genome sequence at the targeted locus for many individuals that have been subjected to mutagenesis. We provide a complete protocol for the generation of amplicons up until the identification of the exact mutations in the targeted region. CRISPR-finder can be used to process thousands of individuals in a single sequencing run. We successfully identified an ISOCHORISMATE SYNTHASE 1 mutant line in which the production of salicylic acid was impaired compared to the wild type, as expected. These features establish CRISPR-finder as a high-throughput, cost-effective and efficient genotyping method of individuals whose genomes have been targeted using the CRISPR/Cas9 system.

Cryptophyte and chlorarachniophyte algae are transitional forms in the widespread secondary endosymbiotic acquisition of photosynthesis by engulfment of eukaryotic algae. Unlike most secondary plastid-bearing algae, miniaturized versions of the endosymbiont nuclei (nucleomorphs) persist in cryptophytes and chlorarachniophytes. To determine why, and to address other fundamental questions about eukaryote eukaryote endosymbiosis, we sequenced the nuclear genomes of the cryptophyte Guillardia theta and the chlorarachniophyte Bigelowiella natans. Both genomes have 21,000 protein genes and are intron rich, and B. natans exhibits unprecedented alternative splicing for a single-celled organism. Phylogenomic analyses and subcellular targeting predictions reveal extensive genetic and biochemical mosaicism, with both host- and endosymbiont-derived genes servicing the mitochondrion, the host cell cytosol, the plastid and the remnant endosymbiont cytosol of both algae. Mitochondrion-to-nucleus gene transfer still occurs in both organisms but plastid-to-nucleus and nucleomorph-to-nucleus transfers do not, which explains why a small residue of essential genes remains locked in each nucleomorph.

Abstract Background Our knowledge of natural genetic variation is increasing at an extremely rapid pace, affording an opportunity to come to a much richer understanding of how effects of specific genes are dependent on the genetic background. To achieve a systematic understanding of such GxG interactions, it is desirable to develop genome editing tools that can be rapidly deployed across many different genetic varieties. Results We present an efficient CRISPR/Cas9 toolbox of super module (SM) vectors. These vectors are based on a previously described fluorescence protein marker expressed in seeds allowing identification of transgene-free mutants. We have used this vector series to delete genomic regions ranging from 1.7 to 13 kb in different natural accessions of the wild plant Arabidopsis thaliana. Based on results from 53 pairs of sgRNAs targeting individual nucleotide binding site leucine-rich repeat (NLR) genes, we provide a comprehensive overview of obtaining heritable deletions. Conclusions The SM series of CRISPR/Cas9 vectors enables the rapid generation of transgene-free, genome edited plants for a diversity of functional studies.

Purpose: Pain is prevalent in cancer patients, appearing to be moderate to severe in more than one third of them. Despite the fact that fentanyl is widely used with effective analgesic results, some patients do not correspond to treatment, resulting in opioid change. Methods: This is a cohort study, performed in Greek patients with cancer. Its scope was to identify potential reasons responsible for opioid change, due to transdermal-fentanyl intolerance, resulting from inadequate analgesia (pain relief

Translation of the instrumental activities of daily living (IADL) was carried out and its psychometric properties were assessed in a Greek sample of patients with advanced cancer. The scale was translated with the forward-backward procedure into the Greek language. It was initially administered to 136 advanced cancer patients. To assess reliability, it was administered to 45 patients 3 days later. To assess the effect of treatment, 75 patients were studied. The patients also completed the Eastern Cooperative Oncology Group (ECOG) performance status and the linear analogue scale assessment (LASA) quality of life (QoL) scale. Confirmatory factor analysis of the IADL was carried out. Reliability was assessed in terms of internal consistency (Cronbach's alpha) and test-retest correlation (Pearson and ICC) of the IADL scale. Construct validity was assessed through correlation of IADL with ECOG and LASA QoL scores. Confirmatory factor analysis yielded a single-factor model. The homogeneity of the instrument proved to be satisfactory (alpha was 0.88 for men and 0.83 for women). Test-retest reliability was also satisfactory ("P" less than 0.0005). High correlation with ECOG (men, "r" = - 0.87; women, "r" = - 0.85) and LASA QoL (men, "r" = 0.55; women, "r" = 0.53) was observed. The Greek version of the IADL in cancer patients treated in a palliative care unit is a reliable and valid clinical instrument.

The moss Physcomitrella patens is used both as an evo-devo model and biotechnological production system for metabolites and pharmaceuticals. Strong in vivo expression of genes of interest is important for production of recombinant proteins, e.g., selectable markers, fluorescent proteins, or enzymes. In this regard, the choice of the promoter sequence as well as codon usage optimization are two important inside factors to consider in order to obtain optimum protein accumulation level. To reliably quantify fluorescence, we transfected protoplasts with promoter:GFP fusion constructs and measured fluorescence intensity of living protoplasts in a plate reader system. We used the red fluorescent protein mCherry under 2x 35S promoter control as second reporter to normalize for different transfection efficiencies. We derived a novel endogenous promoter and compared deletion variants with exogenous promoters. We used different codon-adapted green fluorescent protein (GFP) genes to evaluate the influence of promoter choice and codon optimization on protein accumulation in P. patens, and show that the promoter of the gene of P. patens chlorophyll a/b binding protein lhcsr1 drives expression of GFP in protoplasts significantly (more than twofold) better than the commonly used 2x 35S promoter or the rice actin1 promoter. We identified a shortened 677 bp version of the lhcsr1 promoter that retains full activity in protoplasts. The codon optimized GFP yields significantly (more than twofold) stronger fluorescence signals and thus demonstrates that adjusting codon usage in P. patens can increase expression strength. In combination, new promotor and codon optimized GFP conveyed sixfold increased fluorescence signal.