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Background DNA methylation is one of the main epigenetic mechanisms for the regulation of gene expression in eukaryotes. In the standard model, methylation in gene promoters has received the most attention since it is generally associated with transcriptional silencing. Nevertheless, recent studies in human tissues reveal that methylation of the region downstream of the transcription start site is highly informative of gene expression. Also, in some cell types and specific genes it has been found that methylation of the first intron, a gene feature typically rich in enhancers, is linked with gene expression. However, a genome-wide, tissue-independent, systematic comparative analysis of the relationship between DNA methylation in the first intron and gene expression across vertebrates has not been explored yet. Results The most important findings of this study are: (1) using different tissues from a modern fish, we show a clear genome-wide, tissue-independent quasi-linear inverse relationship between DNA methylation of the first intron and gene expression. (2) This relationship is conserved across vertebrates, since it is also present in the genomes of a model pufferfish, a model frog and different human tissues. Among the gene features, tissues and species interrogated, the first intron’s negative correlation with the gene expression was most consistent. (3) We identified more tissue-specific differentially methylated regions (tDMRs) in the first intron than in any other gene feature. These tDMRs have positive or negative correlation with gene expression, indicative of distinct mechanisms of tissue-specific regulation. (4) Lastly, we identified CpGs in transcription factor binding motifs, enriched in the first intron, the methylation of which tended to increase with the distance from the first exon–first intron boundary, with a concomitant decrease in gene expression. Conclusions Our integrative analysis clearly reveals the important and conserved role of the methylation level of the first intron and its inverse association with gene expression regardless of tissue and species. These findings not only contribute to our basic understanding of the epigenetic regulation of gene expression but also identify the first intron as an informative gene feature regarding the relationship between DNA methylation and gene expression where future studies should be focused.

The molecular classification of glioblastoma (GBM) based on gene expression might better explain outcome and response to treatment than clinical factors. Whole transcriptome sequencing using next-generation sequencing platforms is rapidly becoming accepted as a tool for measuring gene expression for both research and clinical use. Fresh frozen (FF) tissue specimens of GBM are difficult to obtain since tumor tissue obtained at surgery is often scarce and necrotic and diagnosis is prioritized over freezing. After diagnosis, leftover tissue is usually stored as formalin-fixed paraffin-embedded (FFPE) tissue. However, RNA from FFPE tissues is usually degraded, which could hamper gene expression analysis. We compared RNA-Seq data obtained from matched pairs of FF and FFPE GBM specimens. Only three FFPE out of eleven FFPE-FF matched samples yielded informative results. Several quality-control measurements showed that RNA from FFPE samples was highly degraded but maintained transcriptomic similarities to RNA from FF samples. Certain issues regarding mutation analysis and subtype prediction were detected. Nevertheless, our results suggest that RNA-Seq of FFPE GBM specimens provides reliable gene expression data that can be used in molecular studies of GBM if the RNA is sufficiently preserved.

Low‐flow vascular malformations are congenital overgrowths composed of abnormal blood vessels potentially causing pain, bleeding and obstruction of different organs. These diseases are caused by oncogenic mutations in the endothelium, which result in overactivation of the PI3K/AKT pathway. Lack of robust in vivo preclinical data has prevented the development and translation into clinical trials of specific molecular therapies for these diseases. Here, we demonstrate that the Pik3ca H1047R activating mutation in endothelial cells triggers a transcriptome rewiring that leads to enhanced cell proliferation. We describe a new reproducible preclinical in vivo model of PI3K‐driven vascular malformations using the postnatal mouse retina. We show that active angiogenesis is required for the pathogenesis of vascular malformations caused by activating Pik3ca mutations. Using this model, we demonstrate that the AKT inhibitor miransertib both prevents and induces the regression of PI3K‐driven vascular malformations. We confirmed the efficacy of miransertib in isolated human endothelial cells with genotypes spanning most of human low‐flow vascular malformations. SYNOPSIS This work describes a robust preclinical model of PI3K‐driven vascular malformations using the postnatal mouse retina. We show that AKT inhibition by miransertib is an effective therapeutic strategy for these diseases. Pik3ca H1047R mutation in endothelial cells leads to enhanced cell cycle progression. Active angiogenesis is required for the formation of PI3K‐driven vascular malformations. PI3K‐driven vascular malformations are prevented and regressed upon miransertib treatment. Graphical Abstract This work describes a robust preclinical model of PI3K‐driven vascular malformations using the postnatal mouse retina. We show that AKT inhibition by miransertib is an effective therapeutic strategy for these diseases.

The advent of droplet-based single-cell RNA-sequencing (scRNA-seq) has dramatically increased data throughput, enabling the release of a diverse array of tissue cell atlases to the public. However, we will show that prominent initiatives such as the Human Cell Atlas [ 1 ], the Tabula Sapiens [ 2 ] and the Tabula Muris [ 3 ] contain a significant amount of contamination products (frequently affecting the whole organ) in their data portals due to suboptimal quality filtering. Our work addresses a critical gap by advocating for more stringent quality filtering, highlighting the imperative for a shift from existing standards, which currently lean towards greater permissiveness. We will show the importance of incorporating cell intronic fraction in quality control -or MALAT1 expression otherwise- showcasing its informative nature and potential to elevate cell atlas data reliability. In summary, here, we unveil the hidden intronic landscape of every tissue and highlight the importance of more rigorous single-cell RNA-sequencing quality assessment in cell atlases to enhance their applicability in diverse downstream analyses.

Sustainable tourism development is considered an essential challenge for improving resource management in coastal and maritime areas. In this context, various initiatives have been developed for facilitating the assessment and monitoring of tourism sustainability. Nevertheless, the perception of sustainability varies across different tourism stakeholders, since they approach tourism development under different perspectives while the issue of data availability has been a great barrier in measuring sustainability. The present paper examines the perceptions of sustainability observed over a Community of projects with the common aim of enhancing coastal and maritime tourism sustainability at the Mediterranean. Based on surveys, the Community of projects conceptualizes sustainability, reveals their own strategies in operationalizing sustainability assessment and evaluates the usefulness and the main gaps of various sustainability assessment toolkits. The findings of the study signify that tourism sustainability is a broad concept allowing for different interpretations. The assessment of sustainability seems to be affected by the perception and weight attributed to the economic, social, environmental, and governance pillar of sustainability by each project. Finally, the applicability of international assessment toolkits could be questioned as these do not reflect the objectives of the projects and tailored made approaches are considered as essential for operationalizing sustainability assessments.

Acute inflammation can either resolve through immunosuppression or persist, leading to chronic inflammation. These transitions are driven by distinct molecular and metabolic reprogramming of immune cells. The anti-diabetic drug Metformin inhibits acute and chronic inflammation through mechanisms still not fully understood. Here, we report that the anti-inflammatory and reactive-oxygen-species-inhibiting effects of Metformin depend on the expression of the plasticity factor ZEB1 in macrophages. Using mice lacking Zeb1 in their myeloid cells and human patient samples, we show that ZEB1 plays a dual role, being essential in both initiating and resolving inflammation by inducing macrophages to transition into an immunosuppressed state. ZEB1 mediates these diverging effects in inflammation and immunosuppression by modulating mitochondrial content through activation of autophagy and inhibition of mitochondrial protein translation. During the transition from inflammation to immunosuppression, Metformin mimics the metabolic reprogramming of myeloid cells induced by ZEB1. Mechanistically, in immunosuppression, ZEB1 inhibits amino acid uptake, leading to downregulation of mTORC1 signalling and a decrease in mitochondrial translation in macrophages. These results identify ZEB1 as a driver of myeloid cell metabolic plasticity, suggesting that targeting its expression and function could serve as a strategy to modulate dysregulated inflammation and immunosuppression. The immune suppression required for the resolution of acute inflammation is characterised by molecular and metabolic reprogramming of myeloid cells. Authors here show that the transcription factor ZEB1 is a key mediator of the pathway governing transition from inflammation to immunosuppression via regulating mitochondrial translation in macrophages.

Duchenne muscular dystrophy is a genetic disease produced by mutations in the dystrophin gene characterized by early onset muscle weakness leading to severe and irreversible disability. The cellular and molecular consequences of the lack of dystrophin in humans are only partially known, which is crucial for the development of new therapies aiming to slow or stop the progression of the disease. Here we have analyzed quadriceps muscle biopsies of seven DMD patients aged 2 to 4 years old and five age and gender matched controls using single nuclei RNA sequencing (snRNAseq) and correlated the results obtained with clinical data. SnRNAseq identified significant differences in the proportion of cell population present in the muscle samples, including an increase in the number of regenerative fibers, satellite cells, and fibro-adipogenic progenitor cells (FAPs) and a decrease in the number of slow fibers and smooth muscle cells. Muscle samples from the younger patients with stable mild weakness were characterized by an increase in regenerative fibers, while older patients with moderate and progressive weakness were characterized by loss of muscle fibers and an increase in FAPs. An analysis of the gene expression profile in muscle fibers identified a strong regenerative signature in DMD samples characterized by the upregulation of genes involved in myogenesis and muscle hypertrophy. In the case of FAPs, we observed upregulation of genes involved in the extracellular matrix regeneration but also several signaling pathways. Indeed, further analysis of the potential intercellular communication profile showed a dysregulation of the communication profile in DMD samples identifying FAPs as a key regulator of cell signaling in DMD muscle samples. In conclusion, our study has identified significant differences at the cellular and molecular levels in the different cell populations present in skeletal muscle samples of patients with DMD compared to controls.

Determining the effect of DNA methylation on chromatin structure and function in higher organisms is challenging due to the extreme complexity of epigenetic regulation. We studied a simpler model system, budding yeast, that lacks DNA methylation machinery making it a perfect model system to study the intrinsic role of DNA methylation in chromatin structure and function. We expressed the murine DNA methyltransferases in Saccharomyces cerevisiae and analyzed the correlation between DNA methylation, nucleosome positioning, gene expression and 3D genome organization. Despite lacking the machinery for positioning and reading methylation marks, induced DNA methylation follows a conserved pattern with low methylation levels at the 5’ end of the gene increasing gradually toward the 3’ end, with concentration of methylated DNA in linkers and nucleosome free regions, and with actively expressed genes showing low and high levels of methylation at transcription start and terminating sites respectively, mimicking the patterns seen in mammals. We also see that DNA methylation increases chromatin condensation in peri-centromeric regions, decreases overall DNA flexibility, and favors the heterochromatin state. Taken together, these results demonstrate that methylation intrinsically modulates chromatin structure and function even in the absence of cellular machinery evolved to recognize and process the methylation signal. Multi-layered epigenetic regulation in higher eukaryotes makes it challenging to disentangle the individual effects of modifications on chromatin structure and function. Here, the authors expressed mammalian DNA methyltransferases in yeast, which have no DNA methylation, to show that methylation has intrinsic effects on chromatin structure.

Phenotypic plasticity, the ability of an organism to express different phenotypes depending on the environment, provides an important mechanism by which an animal population can persist under rapid climate change. We experimentally tested both life‐history and transcriptional responses of an ecological model species, the three‐spined stickleback, to warm acclimation at the southern edge of its European range. We explored cross‐environment genetic correlations of key life‐history traits in male sticklebacks exposed to long‐term temperature changes to examine whether the plasticity pattern was variable among genotypes by using a character‐state approach. We also studied gene expression plasticity by analysing both whole‐transcriptome and candidate gene expression in brain and liver. Male sticklebacks that developed under warmer conditions during winter were smaller in size and invested less in nuptial coloration at the beginning of the breeding season, showing similar responses across different genotypes. The lack of genetic variation in life‐history responses may limit any future evolution of the thermal reaction norm in the study population. After long‐term exposure to increased winter temperatures, genes responsible for several metabolic and oxidation–reduction processes were upregulated, and some hormone genes involved in growth and reproduction were downregulated in the brain. In the liver, there was no significantly represented gene ontology by the differentially expressed genes. Since a higher temperature leads to a higher resting metabolic rate, living in warmer environments may incur higher energetic costs for ectotherms to maintain cellular homoeostasis, resulting in negative consequences for life‐history traits. The expression of genes related to metabolism, cellular homoeostasis and regulatory signalling may underlie temperature‐induced changes in life history.

The interpretation of clinical diagnostic results in suspected inborn errors of immunity, including Tregopathies, is hampered by the lack of age-stratified reference values for regulatory T cells (Treg) in the pediatric population and a consensus on which Treg immunophenotype to use. Regulatory B cells (Breg) are an important component of the regulatory system that have been poorly studied in the pediatric population. We analyzed (1) the correlation between the three immunophenotypic definitions of Treg (CD4 + CD25 hi CD127 low , CD4 + CD25 hi CD127 low FoxP3 + , CD4 + CD25 hi FoxP3 + ), and with CD4 + CD25 hi and (2) the changes in Treg and Breg frequencies and their maturation status with age. We performed peripheral blood immunophenotyping of Treg and Breg (CD19 + CD24 hi CD38 hi ) by flow cytometry in 55 healthy pediatric controls. We observed that Treg numbers varied depending on the definition used, and the frequency ranged between 3.3–9.7% for CD4 + CD25 hi CD127 low , 0.07-1.6% for CD4 + CD25 hi CD127 low FoxP3 + , and 0.24-2.83% for CD4 + CD25 hi FoxP3 + . The correlation between the three definitions of Treg was positive for most age ranges, especially between the two intracellular panels and with CD4 + CD25 hi vs CD4 + CD25 hi CD127 low . Treg and Breg frequencies tended to decline after 7 and 3 years onwards, respectively. Treg’s maturation status increased with age, with a decline of naïve Treg and an increase in memory/effector Treg from age 7 onwards. Memory Breg increased progressively from age 3 onwards. In conclusion, the number of Treg frequencies spans a wide range depending on the immunophenotypic definition used despite a good level of correlation exists between them. The decline in numbers and maturation process with age occurs earlier in Breg than in Treg.