CARATTERIZZAZIONE MOLECOLARE ATTRAVERSO ANALISI DI IMMUNOFLUORESCENZA DI CARDIOMIOCITI PACEMAKER DERIVATI DA CELLULE STAMINALI EMBRIONALI MURINE INGEGNERIZZATE

Introduction and Aim. Mouse embryonic stem cells (mESCs) are able to differentiate in vitro into many cell types derived from the three germ layers, including pacemaker cardiomyocytes. ESCs thus represent a potential cell source for generation of biological pacemakers repair. The applicability of ESCs in regenerative therapies requires the isolation of a pure population of differentiated cells in order to avoid the contamination with teratogenic undifferentiated ESCs. To identify ESC-derived pacemaker cardiomyocytes, we established a mESC line stably expressing the enhanced green fluorescent protein (EGFP) under the transcriptional control of the promoter of the HCN4 gene (pHCN4-EGFP). Although EGFP-positive spontaneously beating cells can be detected, non-beating EGFP-positive cells can be detected as well. The transmembrane protein CD166 is specifically expressed in the developing murine heart tube at embryonic day 8.5. Previous works have demonstrated that CD166 can be used as surface antigen to select a differentiated cell population enriched in cardiomyocytes. The aim of this work is to characterize the cardiac nature of CD166-positive and negative cell populations isolated from differentiating mESC cultures at day 7+1. Materials and Methods. pHCN4-EGFP mESCs-D3 were differentiated into embryoid bodies (EBs) by the \u201changing drops\u201d method. EBs at differentiation day 7+1 were dissociated into single cell-suspensions and sorted by flow cytometry. CD166+ and CD166\uf02d cells were plated in very low adhesion dish for 24 hours to facilitate their re-aggregation and then plated on cell culture dishes. Immunofluorescence experiments were carried out both on CD166+ and CD166\uf02d cell cultures. Samples were fixed in paraformaldehyde and labelled with antibodies against sarcomeric \u3b1-actinin to identify cardiomyocytes and against HCN4 and caveolin 3 to specifically detect cells with a pacemaker-like phenotype. Results. While CD166+ cells form spontaneously and synchronously beating aggregate, CD166\uf02d cells form EBs-like aggregates in which only very few cells show signs of automaticity. Immunofluorescence analysis of these aggregates demonstrate that 60,13 % of CD166+ cells expresses a contractile apparatus organized in sarcomeric-like structures, as shown by \u3b1-actinin staining. In contrast CD166\uf02d aggregates present a much lower degree of \u3b1-actinin staining (3,60 % of cells). To identify cardiac cells with pacemaker-like phenotype, double staining with anti-HCN4 and anti-\u3b1-actinin antibodies was carried out. In CD166+ cell population 42,77 % of \u3b1-actinin positive cell expresses HCN4 channels whereas in CD166\uf02d cell population the percentage is only 5,12 %. Moreover CD166+ cells also stained positive for caveolin 3, a structural protein of muscular caveolae, known to interact with pacemaker channels in both native SAN myocytes and ESC-derived pacemaker cells. Interestingly, most CD166+ cells were also EGFP-positive confirming a persistent activation of the HCN4 promoter in these cells and thus their pacemaker phenotype. Conclusions. Our data demonstrate that CD166 can be used, in a specific stage of differentiation, as a surface antigen to select a cell population enriched in cardiomyocytes presenting some peculiar features of sinoatrial pacemaker cells. For this reason, CD166+ cell population has the characteristics that made them a suitable cellular substrate for the creation of biological pacemakers

Safety and Liveness of Quantitative Automata

The safety-liveness dichotomy is a fundamental concept in formal languages which plays a key role in verification. Recently, this dichotomy has been lifted to quantitative properties, which are arbitrary functions from infinite words to partially-ordered domains. We look into harnessing the dichotomy for the specific classes of quantitative properties expressed by quantitative automata. These automata contain finitely many states and rational-valued transition weights, and their common value functions Inf, Sup, LimInf, LimSup, LimInfAvg, LimSupAvg, and DSum map infinite words into the totally-ordered domain of real numbers. In this automata-theoretic setting, we establish a connection between quantitative safety and topological continuity and provide an alternative characterization of quantitative safety and liveness in terms of their boolean counterparts. For all common value functions, we show how the safety closure of a quantitative automaton can be constructed in PTime, and we provide PSpace-complete checks of whether a given quantitative automaton is safe or live, with the exception of LimInfAvg and LimSupAvg automata, for which the safety check is in ExpSpace. Moreover, for deterministic Sup, LimInf, and LimSup automata, we give PTime decompositions into safe and live automata. These decompositions enable the separation of techniques for safety and liveness verification for quantitative specifications.Comment: Full version of the paper to appear in CONCUR 202

A caveolin-binding domain in the HCN4 channels mediates functional interaction with caveolin proteins

Pacemaker (HCN) channels have a key role in the generation and modulation of spontaneous activity of sinoatrial node myocytes. Previous work has shown that compartmentation of HCN4 pacemaker channels within caveolae regulates important functions, but the molecular mechanism responsible is still unknown. HCN channels have a conserved caveolin-binding domain (CBD) composed of three aromatic amino acids at the N-terminus; we sought to evaluate the role of this CBD in channel-protein interaction by mutational analysis. We generated two HCN4 mutants with a disrupted CBD (Y259S, F262V) and two with conservative mutations (Y259F, F262Y). In CHO cells expressing endogenous caveolin-1 (cav-1), alteration of the CBD shifted channels activation to more positive potentials, slowed deactivation and made Y259S and F262V mutants insensitive to cholesterol depletion-induced caveolar disorganization. CBD alteration also caused a significant decrease of current density, due to a weaker HCN4-cav-1 interaction and accumulation of cytoplasmic channels. These effects were absent in mutants with a preserved CBD. In caveolin-1-free fibroblasts, HCN4 trafficking was impaired and current density reduced with all constructs; the activation curve of F262V was not altered relative to wt, and that of Y259S displayed only half the shift than in CHO cells. The conserved CBD present in all HCN isoforms mediates their functional interaction with caveolins. The elucidation of the molecular details of HCN4-cav-1 interaction can provide novel information to understand the basis of cardiac phenotypes associated with some forms of caveolinopathies

Folding model analysis of alpha radioactivity

Radioactive decay of nuclei via emission of $\alpha$ particles has been studied theoretically in the framework of a superasymmetric fission model using the double folding (DF) procedure for obtaining the $\alpha$-nucleus interaction potential. The DF nuclear potential has been obtained by folding in the density distribution functions of the $\alpha$ nucleus and the daughter nucleus with a realistic effective interaction. The M3Y effective interaction has been used for calculating the nuclear interaction potential which has been supplemented by a zero-range pseudo-potential for exchange along with the density dependence. The nuclear microscopic $\alpha$-nucleus potential thus obtained has been used along with the Coulomb interaction potential to calculate the action integral within the WKB approximation. This subsequently yields microscopic calculations for the half lives of $\alpha$ decays of nuclei. The density dependence and the exchange effects have not been found to be very significant. These calculations provide reasonable estimates for the lifetimes of $\alpha$ radioactivity of nuclei.Comment: 7 pages including 1 figur

Spontaneous heavy cluster emission rates using microscopic potentials

The nuclear cluster radioactivities have been studied theoretically in the framework of a microscopic superasymmetric fission model (MSAFM). The nuclear interaction potentials required for binary cold fission processes are calculated by folding in the density distribution functions of the two fragments with a realistic effective interaction. The microscopic nuclear potential thus obtained has been used to calculate the action integral within the WKB approximation. The calculated half lives of the present MSAFM calculations are found to be in good agreement over a wide range of observed experimental data.Comment: 4 pages, 4 figure

Beta decay of 71,73Co; probing single particle states approaching doubly magic 78Ni

Low-energy excited states in 71,73Ni populated via the {\beta} decay of 71,73Co were investigated in an experiment performed at the National Superconducting Cyclotron Laboratory (NSCL) at Michigan State University (MSU). Detailed analysis led to the construction of level schemes of 71,73Ni, which are interpreted using systematics and analyzed using shell model calculations. The 5/2- states attributed to the the f5/2 orbital and positive parity 5/2+ and 7/2+ states from the g9/2 orbital have been identified in both 71,73Ni. In 71Ni the location of a 1/2- {\beta}-decaying isomer is proposed and limits are suggested as to the location of the isomer in 73Ni. The location of positive parity cluster states are also identified in 71,73Ni. Beta-delayed neutron branching ratios obtained from this data are given for both 71,73Co.Comment: Accepted for publication in PR

Resveratrol promotes myogenesis and hypertrophy in murine myoblasts

Background: Nutrigenomics elucidate the ability of bioactive food components to influence gene expression, protein synthesis, degradation and post-translational modifications.Resveratrol (RSV), natural polyphenol found in grapes and in other fruits, has a plethora of health benefits in a variety of human diseases: cardio- and neuroprotection, immune regulation, cancer chemoprevention, DNA repair, prevention of mitochondrial disorder, avoidance of obesity-related diseases. In skeletal muscle, RSV acts on protein catabolism and muscle function, conferring resistance against oxidative stress, injury and cell death, but its action mechanisms and protein targets in myogenesis process are not completely known. Myogenesis is a dynamic multistep process regulated by Myogenic Regulator Factors (MRFs), responsible of the commitment of myogenic cell into skeletal muscle: mononucleated undifferentiated myoblasts break free from cell cycle, elongate and fuse to form multinucleated myotubes. Skeletal muscle hypertrophy can be defined as a result of an increase in the size of pre-existing skeletal muscle fibers accompanied by increased protein synthesis, mainly regulated by Insulin Like Growth Factor 1 (IGF-1), PI3-K/AKT signaling pathways.Aim of this work was the study of RSV effects on proliferation, differentiation process and hypertrophy in C2C12 murine cells.Methods: To study proliferative phase, cells were incubated in growth medium with/without RSV (0.1 or 25 \u3bcM) until reaching sub confluence condition (24, 48, 72 h). To examine differentiation, at 70% confluence, cells were transferred in differentiation medium both with/without RSV (0.1 or 25 \u3bcM) for 24, 48, 72, 96 hours. After 72 hours of differentiation, the genesis of hypertrophy in neo-formed myotubes was analyzed.Results: Data showed that RSV regulates cell cycle exit and induces C2C12 muscle differentiation. Furthermore, RSV might control MRFs and muscle-specific proteins synthesis. In late differentiation, RSV has positive effects on hypertrophy: RSV stimulates IGF-1 signaling pathway, in particular AKT and ERK 1/2 protein activation, AMPK protein level and induces hypertrophic morphological changes in neo-formed myotubes modulating cytoskeletal proteins expression.Conclusions: RSV might control cell cycle promoting myogenesis and hypertrophy in vitro, opening a novel field of application of RSV in clinical conditions characterized by chronic functional and morphological muscle impairment

Gene Co-expression Analysis Identifies Histone Deacetylase 5 and 9 Expression in Midbrain Dopamine Neurons and as Regulators of Neurite Growth via Bone Morphogenetic Protein Signaling

Parkinson’s disease is characterized by the intracellular accumulation of α-synuclein which has been linked to early dopaminergic axonal degeneration. Identifying druggable targets that can promote axonal growth in cells overexpressing α-synuclein is important in order to develop strategies for early intervention. Class-IIa histone deacetylases (HDACs) have previously emerged as druggable targets, however, it is not known which specific class-IIa HDACs should be targeted to promote neurite growth in dopaminergic neurons. To provide insight into this, we used gene co-expression analysis to identify which, if any, of the class-IIa HDACs had a positive correlation with markers of dopaminergic neurons in the human substantia nigra. This revealed that two histone deacetylases, HDAC5 and HDAC9, are co-expressed with TH, GIRK2 and ALDH1A1 in the human SN. We further found that HDAC5 and HDAC9 are expressed in dopaminergic neurons in the adult mouse substantia nigra. We show that siRNAs targeting HDAC5 or HDAC9 can promote neurite growth in SH-SY5Y cells, and that their pharmacological inhibition, using the drug MC1568, promoted neurite growth in cultured rat dopaminergic neurons. Moreover, MC1568 treatment upregulated the expression of the neurotrophic factor, BMP2, and its downstream transcription factor, SMAD1. In addition, MC1568 or siRNAs targeting HDAC5 or HDAC9 led to an increase in Smad-dependent GFP expression in a reporter assay. Furthermore, MC1568 treatment of cultured rat dopaminergic neurons increased cellular levels of phosphorylated Smad1, which was prevented by the BMP receptor inhibitor, dorsomorphin. Dorsomorphin treatment prevented the neurite growth-promoting effects of siRNAs targeting HDAC5, as did overexpression of dominant-negative Smad4 or of the inhibitory Smad7, demonstrating a functional link to BMP signaling. Supplementation with BMP2 prevented the neurite growth-inhibitory effects of nuclear-restricted HDAC5. Finally, we report that siRNAs targeting HDAC5 or HDAC9 promoted neurite growth in cells overexpressing wild-type or A53T-α-synuclein and that MC1568 protected cultured rat dopaminergic neurons against the neurotoxin, MPP+. These findings establish HDAC5 and HDAC9 as novel regulators of BMP-Smad signaling, that additionally may be therapeutic targets worthy of further exploration in iPSC-derived human DA neurons and in vivo models of Parkinson’s disease