Regulation of Myc transcription by an enhancer cluster dedicated to pluripotency and early embryonic expression.

MYC plays various roles in pluripotent stem cells, including the promotion of somatic cell reprogramming to pluripotency, the regulation of cell competition and the control of embryonic diapause. However, how Myc expression is regulated in this context remains unknown. The Myc gene lies within a ~ 3-megabase gene desert with multiple cis-regulatory elements. Here we use genomic rearrangements, transgenesis and targeted mutation to analyse Myc regulation in early mouse embryos and pluripotent stem cells. We identify a topologically-associated region that homes enhancers dedicated to Myc transcriptional regulation in stem cells of the pre-implantation and early post-implantation embryo. Within this region, we identify elements exclusively dedicated to Myc regulation in pluripotent cells, with distinct enhancers that sequentially activate during naive and formative pluripotency. Deletion of pluripotency-specific enhancers dampens embryonic stem cell competitive ability. These results identify a topologically defined enhancer cluster dedicated to early embryonic expression and uncover a modular mechanism for the regulation of Myc expression in different states of pluripotency.

P53 and BCL-2 family proteins PUMA and NOXA define competitive fitness in pluripotent cell competition.

Cell Competition is a process by which neighboring cells compare their fitness. As a result, viable but suboptimal cells are selectively eliminated in the presence of fitter cells. In the early mammalian embryo, epiblast pluripotent cells undergo extensive Cell Competition, which prevents suboptimal cells from contributing to the newly forming organism. While competitive ability is regulated by MYC in the epiblast, the mechanisms that contribute to competitive fitness in this context are largely unknown. Here, we report that P53 and its pro-apoptotic targets PUMA and NOXA regulate apoptosis susceptibility and competitive fitness in pluripotent cells. PUMA is widely expressed specifically in pluripotent cells in vitro and in vivo. We found that P53 regulates MYC levels in pluripotent cells, which connects these two Cell Competition pathways, however, MYC and PUMA/NOXA levels are independently regulated by P53. We propose a model that integrates a bifurcated P53 pathway regulating both MYC and PUMA/NOXA levels and determines competitive fitness.

mTert induction in p21-positive cells counteracts capillary rarefaction and pulmonary emphysema.

Lung diseases develop when telomeres shorten beyond a critical point. We constructed a mouse model in which the catalytic subunit of telomerase (mTert), or its catalytically inactive form (mTertCI), is expressed from the p21Cdkn1a locus. Expression of either TERT or TERTCI reduces global p21 levels in the lungs of aged mice, highlighting TERT non-canonical function. However, only TERT reduces accumulation of very short telomeres, oxidative damage, endothelial cell (ECs) senescence and senile emphysema in aged mice. Single-cell analysis of the lung reveals that p21 (and hence TERT) is expressed mainly in the capillary ECs. We report that a fraction of capillary ECs marked by CD34 and endowed with proliferative capacity declines drastically with age, and this is counteracted by TERT but not TERTCI. Consistently, only TERT counteracts decline of capillary density. Natural aging effects are confirmed using the experimental model of emphysema induced by VEGFR2 inhibition and chronic hypoxia. We conclude that catalytically active TERT prevents exhaustion of the putative CD34 + EC progenitors with age, thus protecting against capillary vessel loss and pulmonary emphysema.

Whole Grape Pomace Flour as Nutritive Ingredient for Enriched Durum Wheat Pasta with Bioactive Potential.

In this study, grape pomace is used as an ingredient to fortify pasta. The grape pomace phenolic component is highly accessible and available for metabolization in the human gut. Hence, grape pomace can be exploited as a source of polyphenols and fiber for sustainable and dietary beneficial food production. Analyses of soluble and bound phenols and volatile compounds in raw and cooked pasta were performed. In the uncooked pasta fortified with pomace, the content of soluble and bound phenolic molecules increased significantly. During the cooking process, the bound phenols were lost, while the soluble phenols doubled. The whole grape pomace flour as a pasta ingredient increased the fiber component by at least double, increased the soluble polyphenol component by at least 10 times, and doubled the isoprenoids (toco-chromanols and carotenoids) while maintaining the unaltered fatty acid content after cooking. In accordance with the polyphenol content, antioxidant activity resulted higher than that of the control pasta. Analysis of volatile compounds in fortified pasta, both uncooked and cooked, indicated an improvement in aromatic profile when compared to the control pasta. Our results show that durum wheat pasta fortified with whole pomace flour has bioactive potential for the reuse of food industry byproducts.

A Human Hereditary Cardiomyopathy Shares a Genetic Substrate With Bicuspid Aortic Valve.

BACKGROUND: The complex genetics underlying human cardiac disease is evidenced by its heterogenous manifestation, multigenic basis, and sporadic occurrence. These features have hampered disease modeling and mechanistic understanding. Here, we show that 2 structural cardiac diseases, left ventricular noncompaction (LVNC) and bicuspid aortic valve, can be caused by a set of inherited heterozygous gene mutations affecting the NOTCH ligand regulator MIB1 (MINDBOMB1) and cosegregating genes. METHODS: We used CRISPR-Cas9 gene editing to generate mice harboring a nonsense or a missense MIB1 mutation that are both found in LVNC families. We also generated mice separately carrying these MIB1 mutations plus 5 additional cosegregating variants in the ASXL3, APCDD1, TMX3, CEP192, and BCL7A genes identified in these LVNC families by whole exome sequencing. Histological, developmental, and functional analyses of these mouse models were carried out by echocardiography and cardiac magnetic resonance imaging, together with gene expression profiling by RNA sequencing of both selected engineered mouse models and human induced pluripotent stem cell-derived cardiomyocytes. Potential biochemical interactions were assayed in vitro by coimmunoprecipitation and Western blot. RESULTS: Mice homozygous for the MIB1 nonsense mutation did not survive, and the mutation caused LVNC only in heteroallelic combination with a conditional allele inactivated in the myocardium. The heterozygous MIB1 missense allele leads to bicuspid aortic valve in a NOTCH-sensitized genetic background. These data suggest that development of LVNC is influenced by genetic modifiers present in affected families, whereas valve defects are highly sensitive to NOTCH haploinsufficiency. Whole exome sequencing of LVNC families revealed single-nucleotide gene variants of ASXL3, APCDD1, TMX3, CEP192, and BCL7A cosegregating with the MIB1 mutations and LVNC. In experiments with mice harboring the orthologous variants on the corresponding Mib1 backgrounds, triple heterozygous Mib1 Apcdd1 Asxl3 mice showed LVNC, whereas quadruple heterozygous Mib1 Cep192 Tmx3;Bcl7a mice developed bicuspid aortic valve and other valve-associated defects. Biochemical analysis suggested interactions between CEP192, BCL7A, and NOTCH. Gene expression profiling of mutant mouse hearts and human induced pluripotent stem cell-derived cardiomyocytes revealed increased cardiomyocyte proliferation and defective morphological and metabolic maturation. CONCLUSIONS: These findings reveal a shared genetic substrate underlying LVNC and bicuspid aortic valve in which MIB1-NOTCH variants plays a crucial role in heterozygous combination with cosegregating genetic modifiers.

Encapsulation of Grape (Vitis vinifera L.) Pomace Polyphenols in Soybean Extract-Based Hydrogel Beads as Carriers of Polyphenols and pH-Monitoring Devices.

In this work, novel bio-based hydrogel beads were fabricated by using soybean extract as raw waste material loaded with Lambrusco extract, an Italian grape cultivar. The phenolic profile and the total amount of anthocyanins from the Lambrusco extract were evaluated before encapsulating it in soybean extract-based hydrogels produced through an ionotropic gelation technique. The physical properties of the produced hydrogel beads were then studied in terms of their morphological and spectroscopic properties. Swelling degree was evaluated in media with different pH levels. The release kinetics of Lambrusco extract were then studied over time as a function of pH of the release medium, corroborating that the acidity/basicity could affect the release rate of encapsulated molecules, as well as their counter-diffusion. The pH-sensitive properties of wine extract were studied through UV-Vis spectroscopy while the colorimetric responses of loaded hydrogel beads were investigated in acidic and basic solutions. Finally, in the framework of circular economy and sustainability, the obtained data open routes to the design and fabrication of active materials as pH-indicator devices from food industry by-products.

Pluripotency factors regulate the onset of Hox cluster activation in the early embryo.

Pluripotent cells are a transient population of the mammalian embryo dependent on transcription factors, such as OCT4 and NANOG, which maintain pluripotency while suppressing lineage specification. However, these factors are also expressed during early phases of differentiation, and their role in the transition from pluripotency to lineage specification is largely unknown. We found that pluripotency factors play a dual role in regulating key lineage specifiers, initially repressing their expression and later being required for their proper activation. We show that Oct4 is necessary for activation of HoxB genes during differentiation of embryonic stem cells and in the embryo. In addition, we show that the HoxB cluster is coordinately regulated by OCT4 binding sites located at the 3' end of the cluster. Our results show that core pluripotency factors are not limited to maintaining the precommitted epiblast but are also necessary for the proper deployment of subsequent developmental programs.

Grape Pomace Extract Attenuates Inflammatory Response in Intestinal Epithelial and Endothelial Cells: Potential Health-Promoting Properties in Bowel Inflammation.

Inflammatory bowel disease (IBD) implies the chronic inflammation of the gastrointestinal tract, combined with systemic vascular manifestations. In IBD, the incidence of cardiovascular disease appears to be related to an increase of oxidative stress and endothelial dysfunction. Grape pomace contains high levels of anti-oxidant polyphenols that are able to counteract chronic inflammatory symptoms. The aim of this study was to determine whether grape pomace polyphenolic extract (GPE) was able to mitigate the overwhelming inflammatory response in enterocyte-like cells and to improve vascular function. Intestinal epithelial Caco-2 cells, grown in monolayers or in co-culture with endothelial cells (Caco-2/HMEC-1), were treated with different concentrations of GPE (1, 5, 10 µg/mL gallic acid equivalents) for 2 h and then stimulated with lipopolysaccharide (LPS) and tumor necrosis factor (TNF)-α for 16 h. Through multiple assays, the expression of intestinal and endothelial inflammatory mediators, intracellular reactive oxygen species (ROS) levels and NF-κB activation, as well as endothelial-leukocyte adhesion, were evaluated. The results showed that GPE supplementation prevented, in a concentration-dependent manner, the intestinal expression and release of interleukin (IL)-6, monocyte chemoattractant protein (MCP)-1, and matrix metalloproteinases (MMP)-9 and MMP-2. In Caco-2 cells, GPE also suppressed the gene expression of several pro-inflammatory markers, such as IL-1ß, TNF-α, macrophage colony-stimulating factor (M-CSF), C-X-C motif ligand (CXCL)-10, intercellular adhesion molecule (ICAM)-1, vascular cell adhesion molecule (VCAM)-1, and cyclooxygenase (COX)-2. The GPE anti-inflammatory effect was mediated by the inhibition of NF-κB activity and reduced intracellular ROS levels. Furthermore, transepithelial GPE suppressed the endothelial expression of IL-6, MCP-1, VCAM-1, and ICAM-1 and the subsequent adhesion of leukocytes to the endothelial cells under pro-inflammatory conditions. In conclusion, our findings suggest grape pomace as a natural source of polyphenols with multiple health-promoting properties that could contribute to the mitigation of gut chronic inflammatory diseases and improve vascular endothelial function.

Comparison of Antibacterial and Antioxidant Properties of Red (cv. Negramaro) and White (cv. Fiano) Skin Pomace Extracts.

Wine pomace has attracted the attention of the food industry, due to its high content in bioactive compounds, and its multiple healthy activities. In this work, whole and separated skin pomaces from fermented (red) and un-fermented (white) grape by-products were characterized for their antioxidant and antimicrobial activities in order to exploit them as functional food ingredient. Antioxidant activity, measured by both ORAC and TEAC assays, was higher in whole than in skin pomace extracts. The characterization of phenolic composition in whole and skin pomace extracts confirmed the peculiarity of some compounds such as anthocyanins (107.84 + 10.3 mg/g TP) in red skin pomace and a great amount of flavanols (80.73 + 4.04 mg/g TP) in white skin pomace. Whole and skin pomace extracts displayed the same antibacterial activity at 250 µg gallic acid equivalents (GAE)/mL. Red and white skin pomace extracts showed a Minimum Inhibitory Concentration (MIC) of 31.25-62.5 GAE/mL against Staphylococcus aureus and Enterococcus faecalis. Pseudomonas spp. were more sensitive to red skin pomace extracts rather than white skin pomace extracts. Given these results, both red and white pomace extracts could be exploited for future application in food, pharmaceutical and cosmetic industry.

Meis2 Is Required for Inner Ear Formation and Proper Morphogenesis of the Cochlea.

Meis genes have been shown to control essential processes during development of the central and peripheral nervous system. Here we have explored the roles of the Meis2 gene during vertebrate inner ear induction and the formation of the cochlea. Meis2 is expressed in several tissues required for inner ear induction and in non-sensory tissue of the cochlear duct. Global inactivation of Meis2 in the mouse leads to a severely reduced size of the otic vesicle. Tissue-specific knock outs of Meis2 reveal that its expression in the hindbrain is essential for otic vesicle formation. Inactivation of Meis2 in the inner ear itself leads to an aberrant coiling of the cochlear duct. By analyzing transcriptomes obtained from Meis2 mutants and ChIPseq analysis of an otic cell line, we define candidate target genes for Meis2 which may be directly or indirectly involved in cochlear morphogenesis. Taken together, these data show that Meis2 is essential for inner ear formation and provide an entry point to unveil the network underlying proper coiling of the cochlear duct.

Control of mouse limb initiation and antero-posterior patterning by Meis transcription factors.

Meis1 and Meis2 are homeodomain transcription factors that regulate organogenesis through cooperation with Hox proteins. Elimination of Meis genes after limb induction has shown their role in limb proximo-distal patterning; however, limb development in the complete absence of Meis function has not been studied. Here, we report that Meis1/2 inactivation in the lateral plate mesoderm of mouse embryos leads to limb agenesis. Meis and Tbx factors converge in this function, extensively co-binding with Tbx to genomic sites and co-regulating enhancers of Fgf10, a critical factor in limb initiation. Limbs with three deleted Meis alleles show proximal-specific skeletal hypoplasia and agenesis of posterior skeletal elements. This failure in posterior specification results from an early role of Meis factors in establishing the limb antero-posterior prepattern required for Shh activation. Our results demonstrate roles for Meis transcription factors in early limb development and identify their involvement in previously undescribed interaction networks that regulate organogenesis.

Strategies to Modulate Specialized Metabolism in Mediterranean Crops: From Molecular Aspects to Field.

Plant specialized metabolites (SMs) play an important role in the interaction with the environment and are part of the plant defense response. These natural products are volatile, semi-volatile and non-volatile compounds produced from common building blocks deriving from primary metabolic pathways and rapidly evolved to allow a better adaptation of plants to environmental cues. Specialized metabolites include terpenes, flavonoids, alkaloids, glucosinolates, tannins, resins, etc. that can be used as phytochemicals, food additives, flavoring agents and pharmaceutical compounds. This review will be focused on Mediterranean crop plants as a source of SMs, with a special attention on the strategies that can be used to modulate their production, including abiotic stresses, interaction with beneficial soil microorganisms and novel genetic approaches.

Resveratrol and other Stilbenes: Effects on Dysregulated Gene Expression in Cancers and Novel Delivery Systems.

Trans-resveratrol (RESV), pterostilbene, trans-piceid and trans-viniferins are bioactive stilbenes present in grapes and other plants. Several groups applied biotechnology to introduce their synthesis in plant crops. Biochemical interaction with enzymes, regulation of non-coding RNAs, and activation of signaling pathways and transcription factors are among the main effects described in literature. However, solubility in ethanol, short half-life, metabolism by gut bacteria, make the concentration responsible for the effects observed in cultured cells difficult to achieve. Derivatives obtained by synthesis, trans-resveratrol analogs and methoxylated stilbenes show to be more stable and allow the synthesis of bioactive compounds with higher bioavailability. However, changes in chemical structure may require testing for toxicity. Thus, the delivery of RESV and its natural analogs incorporated into liposomes or nanoparticles, is the best choice to ensure stability during administration and appropriate absorption. The application of RESV and its derivatives with anti-inflammatory and anticancer activity is presented with description of novel clinical trials.

Can Natural Polyphenols Help in Reducing Cytokine Storm in COVID-19 Patients?

SARS-CoV-2 first emerged in China during late 2019 and rapidly spread all over the world. Alterations in the inflammatory cytokines pathway represent a strong signature during SARS-COV-2 infection and correlate with poor prognosis and severity of the illness. The hyper-activation of the immune system results in an acute severe systemic inflammatory response named cytokine release syndrome (CRS). No effective prophylactic or post-exposure treatments are available, although some anti-inflammatory compounds are currently in clinical trials. Studies of plant extracts and natural compounds show that polyphenols can play a beneficial role in the prevention and the progress of chronic diseases related to inflammation. The aim of this manuscript is to review the published background on the possible effectiveness of polyphenols to fight SARS-COV-2 infection, contributing to the reduction of inflammation. Here, some of the anti-inflammatory therapies are discussed and although great progress has been made though this year, there is no proven cytokine blocking agents for COVID currently used in clinical practice. In this regard, bioactive phytochemicals such as polyphenols may become promising tools to be used as adjuvants in the treatment of SARS-CoV-2 infection. Such nutrients, with anti-inflammatory and antioxidant properties, associated to classical anti-inflammatory drugs, could help in reducing the inflammation in patients with COVID-19.

Strategies for Reuse of Skins Separated From Grape Pomace as Ingredient of Functional Beverages.

Wine grape pomace, the by-product of wine making, is a source of polyphenols, metals, and organic acids, and may be exploited for the production of functional beverages. Among red wines, Primitivo and Negramaro varieties possess an interesting amount of polyphenolic compounds and other chemicals. Consequently, study of the biological activity of Primitivo and Negramaro vinification by-products is of great interest as well as optimizing the extraction of its bioactive components. In order to stabilize the grape pomace, different methods of drying grape pomace were tested. After stabilization of the pomace, the grape skins were manually separated from the seeds and any woody parts. The chemical characterizations of acidified alcoholic (methanol/ethanol) and water extracts and either microwave-assisted or ultrasound-assisted extractions of separated grape skins were compared. Besides that, the in vitro antioxidant activity of wine pomace skin extracts was also investigated as Trolox equivalents antioxidant capacity (TEAC) and oxygen radical absorbance capacity (ORAC). Overall, the alcoholic extractions were found to be the most effective for recovering phenolic compounds, when compared with those in water. Ultrasound- and microwave-assisted extraction of pomace skin using acidified water allowed the highest TEAC value. Taking into account the water extraction result, in order to reuse grape pomace skins to produce a functional beverage, we utilized them in combination with black tea, karkadè (Hibiscus sabdariffa L.), or rooibos (Aspalathus linearis Burm.) to produce an infusion. The combination of grape skins and black tea showed the highest ratio of total phenol content to antioxidant activity. Moreover, skin isolated from pomace, with or without black tea infusions, were shown to have anti-inflammatory capacity in human cell culture. Our results raise the value of grape skin pomace as a rich source of bioactive compounds with antioxidant and anti-inflammatory activity and suggest its exploitation as an ingredient for functional beverages.

Proximo-distal positional information encoded by an Fgf-regulated gradient of homeodomain transcription factors in the vertebrate limb.

The positional information theory proposes that a coordinate system provides information to embryonic cells about their position and orientation along a patterning axis. Cells interpret this information to produce the appropriate pattern. During development, morphogens and interpreter transcription factors provide this information. We report a gradient of Meis homeodomain transcription factors along the mouse limb bud proximo-distal (PD) axis antiparallel to and shaped by the inhibitory action of distal fibroblast growth factor (FGF). Elimination of Meis results in premature limb distalization and HoxA expression, proximalization of PD segmental borders, and phocomelia. Our results show that Meis transcription factors interpret FGF signaling to convey positional information along the limb bud PD axis. These findings establish a new model for the generation of PD identities in the vertebrate limb and provide a molecular basis for the interpretation of FGF signal gradients during axial patterning.

Severe Cardiac Dysfunction and Death Caused by Arrhythmogenic Right Ventricular Cardiomyopathy Type 5 Are Improved by Inhibition of Glycogen Synthase Kinase-3ß.

BACKGROUND: Arrhythmogenic cardiomyopathy/arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited cardiac disease characterized by fibrofatty replacement of the myocardium, resulting in heart failure and sudden cardiac death. The most aggressive arrhythmogenic cardiomyopathy/ARVC subtype is ARVC type 5 (ARVC5), caused by a p.S358L mutation in TMEM43 (transmembrane protein 43). The function and localization of TMEM43 are unknown, as is the mechanism by which the p.S358L mutation causes the disease. Here, we report the characterization of the first transgenic mouse model of ARVC5. METHODS: We generated transgenic mice overexpressing TMEM43 in either its wild-type or p.S358L mutant (TMEM43-S358L) form in postnatal cardiomyocytes under the control of the α-myosin heavy chain promoter. RESULTS: We found that mice expressing TMEM43-S358L recapitulate the human disease and die at a young age. Mutant TMEM43 causes cardiomyocyte death and severe fibrofatty replacement. We also demonstrate that TMEM43 localizes at the nuclear membrane and interacts with emerin and ß-actin. TMEM43-S358L shows partial delocalization to the cytoplasm, reduced interaction with emerin and ß-actin, and activation of glycogen synthase kinase-3ß (GSK3ß). Furthermore, we show that targeting cardiac fibrosis has no beneficial effect, whereas overexpression of the calcineurin splice variant calcineurin Aß1 results in GSK3ß inhibition and improved cardiac function and survival. Similarly, treatment of TMEM43 mutant mice with a GSK3ß inhibitor improves cardiac function. Finally, human induced pluripotent stem cells bearing the p.S358L mutation also showed contractile dysfunction that was partially restored after GSK3ß inhibition. CONCLUSIONS: Our data provide evidence that TMEM43-S358L leads to sustained cardiomyocyte death and fibrofatty replacement. Overexpression of calcineurin Aß1 in TMEM43 mutant mice or chemical GSK3ß inhibition improves cardiac function and increases mice life span. Our results pave the way toward new therapeutic approaches for ARVC5.

Autochthonous Saccharomyces cerevisiae Starter Cultures Enhance Polyphenols Content, Antioxidant Activity, and Anti-Inflammatory Response of Apulian Red Wines.

Several biotic and abiotic factors can influence the amount of polyphenols in grape tissues. During vinification, the temperature, presence of grape seeds and peel, and use of enzymes, can influence the extraction of polyphenols. However, little information is available on the effects of yeast strains used in the polyphenolic composition of wine. With this aim, two selected Saccharomyces cerevisiae strains, ITEM 14093 and ITEM 14077 were used to produce wine from two Italian grape cultivars, Primitivo and Negroamaro. At the end of the alcoholic fermentation, the content of total polyphenols and of particular classes of polyphenols (stilbenes, phenolic acids, flavonols, and flavanols) and the antioxidant activity were evaluated and compared in the obtained wines. We also examined the effects of red wine extracts in a culture model of vascular inflammation. The results obtained comparing wine extracts obtained by utilizing commercial or autochthonous yeast showed that wines obtained with selected yeast significantly inhibited vascular inflammation. The results are positively directed towards the healthy properties of wine drinking.

Dual ifgMosaic: A Versatile Method for Multispectral and Combinatorial Mosaic Gene-Function Analysis.

Improved methods for manipulating and analyzing gene function have provided a better understanding of how genes work during organ development and disease. Inducible functional genetic mosaics can be extraordinarily useful in the study of biological systems; however, this experimental approach is still rarely used in vertebrates. This is mainly due to technical difficulties in the assembly of large DNA constructs carrying multiple genes and regulatory elements and their targeting to the genome. In addition, mosaic phenotypic analysis, unlike classical single gene-function analysis, requires clear labeling and detection of multiple cell clones in the same tissue. Here, we describe several methods for the rapid generation of transgenic or gene-targeted mice and embryonic stem (ES) cell lines containing all the necessary elements for inducible, fluorescent, and functional genetic mosaic (ifgMosaic) analysis. This technology enables the interrogation of multiple and combinatorial gene function with high temporal and cellular resolution.