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.

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.

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.

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.

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.

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.

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.

Myc-driven endogenous cell competition in the early mammalian embryo.

The epiblast is the mammalian embryonic tissue that contains the pluripotent stem cells that generate the whole embryo. We have established a method for inducing functional genetic mosaics in the mouse. Using this system, here we show that induction of a mosaic imbalance of Myc expression in the epiblast provokes the expansion of cells with higher Myc levels through the apoptotic elimination of cells with lower levels, without disrupting development. In contrast, homogeneous shifts in Myc levels did not affect epiblast cell viability, indicating that the observed competition results from comparison of relative Myc levels between epiblast cells. During normal development we found that Myc levels are intrinsically heterogeneous among epiblast cells, and that endogenous cell competition refines the epiblast cell population through the elimination of cells with low relative Myc levels. These results show that natural cell competition in the early mammalian embryo contributes to the selection of the epiblast cell pool.

Telomere Length Defines the Cardiomyocyte Differentiation Potency of Mouse Induced Pluripotent Stem Cells.

Induced pluripotent stem cells (iPSCs) can be differentiated in vitro and in vivo to all cardiovascular lineages and are therefore a promising cell source for cardiac regenerative therapy. However, iPSC lines do not all differentiate into cardiomyocytes (CMs) with the same efficiency. Here, we show that telomerase-competent iPSCs with relatively long telomeres and high expression of the shelterin-complex protein TRF1 (iPSChighT ) differentiate sooner and more efficiently into CMs than those with relatively short telomeres and low TRF1 expression (iPSClowT ). Ascorbic acid, an enhancer of cardiomyocyte differentiation, further increases the cardiomyocyte yield from iPSChighT but does not rescue the cardiomyogenic potential of iPSClowT . Interestingly, although iPSCslowT differentiate very poorly to the mesoderm and endoderm lineages, they differentiate very efficiently to the ectoderm lineage, indicating that cell fate can be determined by in vitro selection of iPSCs with different telomere content. Our findings highlight the importance of selecting iPSCs with ample telomere reserves in order to generate high numbers of CMs in a fast, reliable, and efficient way. Stem Cells 2017;35:362-373.

Diffusible signals and epigenetic timing cooperate in late proximo-distal limb patterning.

Developing vertebrate limbs initiate proximo-distal patterning by interpreting opposing gradients of diffusible signaling molecules. We report two thresholds of proximo-distal signals in the limb bud: a higher threshold that establishes the upper-arm to forearm transition; and a lower one that positions a later transition from forearm to hand. For this last transition to happen, however, the signal environment seems to be insufficient, and we show that a timing mechanism dependent on histone acetylation status is also necessary. Therefore, as a consequence of the time dependence, the lower signaling threshold remains cryptic until the timing mechanism reveals it. We propose that this timing mechanism prevents the distal transition from happening too early, so that the prospective forearm has enough time to expand and form a properly sized segment. Importantly, the gene expression changes provoked by the first transition further regulate proximo-distal signal distribution, thereby coordinating the positioning of the two thresholds, which ensures robustness. This model is compatible with the most recent genetic analyses and underscores the importance of growth during the time-dependent patterning phase, providing a new mechanistic framework for understanding congenital limb defects.

Multiple anti-inflammatory and anti-atherosclerotic properties of red wine polyphenolic extracts: differential role of hydroxycinnamic acids, flavonols and stilbenes on endothelial inflammatory gene expression.

PURPOSE: The aim of the study was to evaluate the vascular anti-inflammatory effects of polyphenolic extracts from two typical South Italy red wines, the specific contribution of individual polyphenols and the underlying mechanisms of action. METHODS: Human endothelial cells were incubated with increasing concentrations (1-50 µg/mL) of Primitivo and Negroamaro polyphenolic extracts (PWPE and NWPE, respectively) or pure polyphenols (1-25 µmol/L), including hydroxycinnamic acids (p-coumaric, caffeic and caftaric acids), flavonols (kaempferol, quercetin, myricetin) or stilbenes (trans-resveratrol, trans-piceid) before stimulation with lipopolysaccharide. Through multiple assays, we analyzed the endothelial-monocyte adhesion, the endothelial expression of adhesion molecules (ICAM-1, VCAM-1 and E-Selectin), monocyte chemoattractant protein-1 (MCP-1) and macrophage colony-stimulating factor (M-CSF), as well as ROS intracellular levels and the activation of NF-κB and AP-1. RESULTS: Both PWPE and NWPE, already at 1 µg/mL, inhibited monocyte adhesion to stimulated endothelial cells, a key event in triggering vascular inflammation. They down-regulated the expression of adhesion molecules, ICAM-1, VCAM-1, E-Selectin, as well as MCP-1 and M-CSF, at mRNA and protein levels. All polyphenols reduced intracellular ROS, and everything, except caftaric acid, inhibited the endothelial expression of adhesion molecules and MCP-1, although with different potency. Flavonols and resveratrol significantly reduced also the endothelial expression and release of M-CSF. The decrease in endothelial inflammatory gene expression was related to the inhibition of NF-κB and AP-1 activation but not to intracellular oxidative stress. CONCLUSIONS: This study showed multiple anti-inflammatory and anti-atherosclerotic properties of red wine polyphenolic extracts and indentified specific bioactive polyphenols which could counteract inflammatory diseases including atherosclerosis.

Red Grape Skin Polyphenols Blunt Matrix Metalloproteinase-2 and -9 Activity and Expression in Cell Models of Vascular Inflammation: Protective Role in Degenerative and Inflammatory Diseases.

Matrix metalloproteinases (MMPs) are endopeptidases responsible for the hydrolysis of various components of extracellular matrix. MMPs, namely gelatinases MMP-2 and MMP-9, contribute to the progression of chronic and degenerative diseases. Since gelatinases' activity and expression are regulated by oxidative stress, we sought to evaluate whether supplementation with polyphenol-rich red grape skin extracts modulated the matrix-degrading capacity in cell models of vascular inflammation. Human endothelial and monocytic cells were incubated with increasing concentrations (0.5-25 µg/mL) of Negroamaro and Primitivo red grape skin polyphenolic extracts (NSPE and PSPE, respectively) or their specific components (0.5-25 µmol/L), before stimulation with inflammatory challenge. NSPE and PSPE inhibited, in a concentration-dependent manner, endothelial invasion as well as the MMP-9 and MMP-2 release in stimulated endothelial cells, and MMP-9 production in inflamed monocytes, without affecting tissue inhibitor of metalloproteinases (TIMP)-1 and TIMP-2. The matrix degrading inhibitory capacity was the same for both NSPE and PSPE, despite their different polyphenolic profiles. Among the main polyphenols of grape skin extracts, trans-resveratrol, trans-piceid, kaempferol and quercetin exhibited the most significant inhibitory effects on matrix-degrading enzyme activities. Our findings appreciate the grape skins as rich source of polyphenols able to prevent the dysregulation of vascular remodelling affecting degenerative and inflammatory diseases.

Functional Properties of Grape and Wine Polyphenols.

Grape berries polyphenols are mainly synthesized in the skin tissues and seeds and they are extracted during the winemaking process. These substances have a potentially positive effect, on human health, thus giving to grape and red wine "functional properties" that can contribute to prevent a number of human illness. Nevertheless, the research community is showing that the real effect is a result of a combination of different factors, notably daily intake, bioavailability, or in vivo antioxidant activity that are yet to be resolved. Viticulture and winemaking practices, determine the concentration of polyphenols in grape and wine. To date, reduced knowledge is existing on the effects of different yeast strains on the final concentration of polyphenols in red wine. We summarize the recent findings concerning the effects of polyphenols on human chronic disease and the future directions for research to increase the amount of these compounds in wine.

Two new targeted alleles for the comprehensive analysis of Meis1 functions in the mouse.

Meis1 is a highly conserved transcription factor that is activated in a regionally restricted manner from early stages of development. Meis1 belongs to the three amino acid loop extension (TALE) homeodomain family. Together with Pbx1, Meis1 plays a major role as a Hox cofactor, and therefore, plays an essential role in the development of several embryonic organs and systems, including limbs, heart, blood, and vasculature. In addition, Meis1 is required for the development of Hox-free embryonic regions and interacts with non-Hox homeodomain and non-homeodomain transcription factors. During post-natal life Meis1 is involved in adult cardiomyocyte homeostasis and has been associated with pre-disposition to human neural and cardiac pathologies. Given the relevance of this transcription factor, we have developed two new Meis1 gene knockin models; a direct ECFP knockin insertion that allows the direct identification of Meis1-expressing cells in living tissues, and a CreERT2 insertion that allows the inducible genetic tracing of Meis1-expressing cells in a time-controlled manner. Importantly, these two alleles represent the first Meis1 mutations in which Meis1 protein production is completely eliminated. These newly targeted Meis1 alleles will be valuable tools to further our understanding of the role of this critical transcription factor during development and disease.

Apical ectodermal ridge morphogenesis in limb development is controlled by Arid3b-mediated regulation of cell movements.

The apical ectodermal ridge (AER) is a specialized epithelium located at the distal edge of the limb bud that directs outgrowth along the proximodistal axis. Although the molecular basis for its function is well known, the cellular mechanisms that lead to its maturation are not fully understood. Here, we show that Arid3b, a member of the ARID family of transcriptional regulators, is expressed in the AER in mouse and chick embryos, and that interference with its activity leads to aberrant AER development, in which normal structure is not achieved. This happens without alterations in cell numbers or gene expression in main signalling pathways. Cells that are defective in Arid3b show an abnormal distribution of the actin cytoskeleton and decreased motility in vitro. Moreover, movements of pre-AER cells and their contribution to the AER were defective in vivo in embryos with reduced Arid3b function. Our results show that Arid3b is involved in the regulation of cell motility and rearrangements that lead to AER maturation.

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.

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.

Resveratrol biosynthesis: plant metabolic engineering for nutritional improvement of food.

The plant polyphenol trans-resveratrol (3, 5, 4'-trihydroxystilbene) mainly found in grape, peanut and other few plants, displays a wide range of biological effects. Numerous in vitro studies have described various biological effects of resveratrol. In order to provide more information regarding absorption, metabolism, and bioavailability of resveratrol, various research approaches have been performed, including in vitro, ex vivo, and in vivo models. In recent years, the induction of resveratrol synthesis in plants which normally do not accumulate such polyphenol, has been successfully achieved by molecular engineering. In this context, the ectopic production of resveratrol has been reported to have positive effects both on plant resistance to biotic stress and the enhancement of the nutritional value of several widely consumed fruits and vegetables. The metabolic engineering of plants offers the opportunity to change the content of specific phytonutrients in plant - derived foods. This review focuses on the latest findings regarding on resveratrol bioproduction and its effects on the prevention of the major pathological conditions in man.