Loss of Nuclear Envelope Integrity and Increased Oxidant Production Cause DNA Damage in Adult Hearts Deficient in PKP2: A Molecular Substrate of ARVC.

BACKGROUND: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is characterized by high propensity to life-threatening arrhythmias and progressive loss of heart muscle. More than 40% of reported genetic variants linked to ARVC reside in the PKP2 gene, which encodes the PKP2 protein (plakophilin-2). METHODS: We describe a comprehensive characterization of the ARVC molecular landscape as determined by high-resolution mass spectrometry, RNA sequencing, and transmission electron microscopy of right ventricular biopsy samples obtained from patients with ARVC with PKP2 mutations and left ventricular ejection fraction >45%. Samples from healthy relatives served as controls. The observations led to experimental work using multiple imaging and biochemical techniques in mice with a cardiac-specific deletion of Pkp2 studied at a time of preserved left ventricular ejection fraction and in human induced pluripotent stem cell-derived PKP2-deficient myocytes. RESULTS: Samples from patients with ARVC present a loss of nuclear envelope integrity, molecular signatures indicative of increased DNA damage, and a deficit in transcripts coding for proteins in the electron transport chain. Mice with a cardiac-specific deletion of Pkp2 also present a loss of nuclear envelope integrity, which leads to DNA damage and subsequent excess oxidant production (O2.- and H2O2), the latter increased further under mechanical stress (isoproterenol or exercise). Increased oxidant production and DNA damage is recapitulated in human induced pluripotent stem cell-derived PKP2-deficient myocytes. Furthermore, PKP2-deficient cells release H2O2 into the extracellular environment, causing DNA damage and increased oxidant production in neighboring myocytes in a paracrine manner. Treatment with honokiol increases SIRT3 (mitochondrial nicotinamide adenine dinucleotide-dependent protein deacetylase sirtuin-3) activity, reduces oxidant levels and DNA damage in vitro and in vivo, reduces collagen abundance in the right ventricular free wall, and has a protective effect on right ventricular function. CONCLUSIONS: Loss of nuclear envelope integrity and subsequent DNA damage is a key substrate in the molecular pathology of ARVC. We show transcriptional downregulation of proteins of the electron transcript chain as an early event in the molecular pathophysiology of the disease (before loss of left ventricular ejection fraction <45%), which associates with increased oxidant production (O2.- and H2O2). We propose therapies that limit oxidant formation as a possible intervention to restrict DNA damage in ARVC.

Cellular pathways of calcium transport and concentration toward mineral formation in sea urchin larvae.

Sea urchin larvae have an endoskeleton consisting of two calcitic spicules. The primary mesenchyme cells (PMCs) are the cells that are responsible for spicule formation. PMCs endocytose sea water from the larval internal body cavity into a network of vacuoles and vesicles, where calcium ions are concentrated until they precipitate in the form of amorphous calcium carbonate (ACC). The mineral is subsequently transferred to the syncytium, where the spicule forms. Using cryo-soft X-ray microscopy we imaged intracellular calcium-containing particles in the PMCs and acquired Ca-L2,3 X-ray absorption near-edge spectra of these Ca-rich particles. Using the prepeak/main peak (L2'/ L2) intensity ratio, which reflects the atomic order in the first Ca coordination shell, we determined the state of the calcium ions in each particle. The concentration of Ca in each of the particles was also determined by the integrated area in the main Ca absorption peak. We observed about 700 Ca-rich particles with order parameters, L2'/ L2, ranging from solution to hydrated and anhydrous ACC, and with concentrations ranging between 1 and 15 M. We conclude that in each cell the calcium ions exist in a continuum of states. This implies that most, but not all, water is expelled from the particles. This cellular process of calcium concentration may represent a widespread pathway in mineralizing organisms.

Mixed-Sandwich Titanium(III) Qubits on Au(111): Electron Delocalization Ruled by Molecular Packing.

Organometallic sandwich complexes are versatile molecular systems that have been recently employed for single-molecule manipulation and spin sensing experiments. Among related organometallic compounds, the mixed-sandwich S = 1/2 complex (η8-cyclooctatetraene)(η5-cyclopentadienyl)titanium, here [CpTi(cot)], has attracted interest as a spin qubit because of the long coherence time. Here the structural and chemical properties of [CpTi(cot)] on Au(111) are investigated at the monolayer level by experimental and computational methods. Scanning tunneling microscopy suggests that adsorption occurs in two molecular orientations, lying and standing, with a 3:1 ratio. XPS data evidence that a fraction of the molecules undergo partial electron transfer to gold, while our computational analysis suggests that only the standing molecules experience charge delocalization toward the surface. Such a phenomenon depends on intermolecular interactions that stabilize the molecular packing in the monolayer. This orientation-dependent molecule-surface hybridization opens exciting perspectives for selective control of the molecule-substrate spin delocalization in hybrid interfaces.

Pathophysiological and Molecular Basis of the Side Effects of Ticagrelor: Lessons from a Case Report.

Ticagrelor is currently considered a first-line choice in dual antiplatelet therapy (DAPT) following revascularization of acute coronary syndrome (ACS). However, its use is correlated with an increased incidence of two side effects, dyspnea and bradyarrhythmias, whose molecular mechanisms have not yet been defined with certainty and, consequently, neither of the therapeutic decisions they imply. We report the case of a patient with acute myocardial infarction treated with ticagrelor and aspirin as oral antithrombotic therapy after primary percutaneous coronary intervention (PCI), manifesting in a significant bradyarrhythmic episode that required a switch of antiplatelet therapy. Starting from this case report, this article aims to gather the currently available evidence regarding the molecular mechanisms underlying these side effects and propose possible decision-making algorithms regarding their management in clinical practice.

Shedding Light on Osteosarcoma Cell Differentiation: Impact on Biomineralization and Mitochondria Morphology.

Osteosarcoma (OS) is the most common primary malignant bone tumor and its etiology has recently been associated with osteogenic differentiation dysfunctions. OS cells keep a capacity for uncontrolled proliferation showing a phenotype similar to undifferentiated osteoprogenitors with abnormal biomineralization. Within this context, both conventional and X-ray synchrotron-based techniques have been exploited to deeply characterize the genesis and evolution of mineral depositions in a human OS cell line (SaOS-2) exposed to an osteogenic cocktail for 4 and 10 days. A partial restoration of the physiological biomineralization, culminating with the formation of hydroxyapatite, was observed at 10 days after treatment together with a mitochondria-driven mechanism for calcium transportation within the cell. Interestingly, during differentiation, mitochondria showed a change in morphology from elongated to rounded, indicating a metabolic reprogramming of OS cells possibly linked to an increase in glycolysis contribution to energy metabolism. These findings add a dowel to the genesis of OS giving new insights on the development of therapeutic strategies able to restore the physiological mineralization in OS cells.

Metabolomic and transcriptomic signatures of chemogenetic heart failure.

The failing heart is characterized by elevated levels of reactive oxygen species. We have developed an animal model of heart failure induced by chemogenetic production of oxidative stress in the heart using a recombinant adeno-associated virus (AAV9) expressing yeast d-amino acid oxidase (DAAO) targeted to cardiac myocytes. When DAAO-infected animals are fed the DAAO substrate d-alanine, the enzyme generates hydrogen peroxide (H2O2) in the cardiac myocytes, leading to dilated cardiomyopathy. However, the underlying mechanisms of oxidative stress-induced heart failure remain incompletely understood. Therefore, we investigated the effects of chronic oxidative stress on the cardiac transcriptome and metabolome. Rats infected with recombinant cardiotropic AAV9 expressing DAAO or control AAV9 were treated for 7 wk with d-alanine to stimulate chemogenetic H2O2 production by DAAO and generate dilated cardiomyopathy. After hemodynamic assessment, left and right ventricular tissues were processed for RNA sequencing and metabolomic profiling. DAAO-induced dilated cardiomyopathy was characterized by marked changes in the cardiac transcriptome and metabolome both in the left and right ventricle. Downregulated transcripts are related to energy metabolism and mitochondrial function, accompanied by striking alterations in metabolites involved in cardiac energetics, redox homeostasis, and amino acid metabolism. Upregulated transcripts are involved in cytoskeletal organization and extracellular matrix. Finally, we noted increased metabolite levels of antioxidants glutathione and ascorbate. These findings provide evidence that chemogenetic generation of oxidative stress leads to a robust heart failure model with distinct transcriptomic and metabolomic signatures and set the basis for understanding the underlying pathophysiology of chronic oxidative stress in the heart.NEW & NOTEWORTHY We have developed a "chemogenetic" heart failure animal model that recapitulates a central feature of human heart failure: increased cardiac redox stress. We used a recombinant DAAO enzyme to generate H2O2 in cardiomyocytes, leading to cardiomyopathy. Here we report striking changes in the cardiac metabolome and transcriptome following chemogenetic heart failure, similar to changes observed in human heart failure. Our findings help validate chemogenetic approaches for the discovery of novel therapeutic targets in heart failure.

Discordance between eNOS phosphorylation and activation revealed by multispectral imaging and chemogenetic methods.

Nitric oxide (NO) synthesized by the endothelial isoform of nitric oxide synthase (eNOS) is a critical determinant of vascular homeostasis. However, the real-time detection of intracellular NO-a free radical gas-has been difficult, and surrogate markers for eNOS activation are widely utilized. eNOS phosphorylation can be easily measured in cells by probing immunoblots with phosphospecific antibodies. Here, we pursued multispectral imaging approaches using biosensors to visualize intracellular NO and Ca2+ and exploited chemogenetic approaches to define the relationships between NO synthesis and eNOS phosphorylation in cultured endothelial cells. We found that the G protein-coupled receptor agonists adenosine triphosphate (ATP) and histamine promoted rapid increases in eNOS phosphorylation, as did the receptor tyrosine kinase agonists insulin and Vascular Endothelial Growth Factor (VEGF). Histamine and ATP also promoted robust NO formation and increased intracellular Ca2+ By contrast, neither insulin nor VEGF caused any increase whatsoever in intracellular NO or Ca2+-despite eliciting strong eNOS phosphorylation responses. Our findings demonstrate an unexpected and striking discordance between receptor-modulated eNOS phosphorylation and NO formation in endothelial cells. Previous reports in which phosphorylation of eNOS has been studied as a surrogate for enzyme activation may need to be reassessed.

The role of (bio)degradability on the management of petrochemical and bio-based plastic waste.

In order to mitigate the social and ecological impacts of post-consumer plastic made of conventional petrochemical polymers, the market of (bio)degradable plastics have recently become more widespread. Although (bio)degradable plastics could be an environmentally friendly substitute of petrochemical ones, the consequences of their presence in the waste management system and in the environment (if not correctly disposed) are not always positive and plastic pollution is not automatically solved. Consequently, this work aims to review how plastic (bio)degradability affects the municipal solid waste management cycle. To this end, the state-of-the-art of the intrinsic (bio)degradability of conventional and unconventional petrochemical and bio-based polymers has been discussed, focusing on the environment related to the waste management system. Then, the focus was on strategies to improve polymer (bio)degradability: different types of eco-design and pre-treatment approach for plastics has been investigated, differently from other works that focused only on specific topics. The information gathered was used to discuss three typical disposal/treatment routes for plastic waste. Despite many of the proposed materials in eco-design have increased the plastics (bio)degradability and pre-treatments have showed interesting results, these achievements are not always positive in the current MSW management system. The effect on mechanical recycling is negative in several cases but the enhanced (bio)degradability can help the treatment with organic waste. On the other hand, the current waste treatment facility is not capable to manage this waste, leading to the incineration the most promising options. In this way, the consumption of raw materials will persist even by using (bio)degradable plastics, which strength the doubt if the solution of plastic pollution leads really on these materials. The review also highlighted the need for further research on this topic that is currently limited by the still scarce amount of (bio)degradable plastics in input to full-scale waste treatment plants.

Effects of cellulose-based bio-plastics on the aerobic biological stabilization treatment of mixed municipal solid waste: A lab-scale assessment.

The aim of this work is to assess how the presence of cellulose-based bio-plastics influence the biological stabilization of mixed Municipal Solid Waste (MSW). For the scope, two cellulose acetate bio-plastics have been mixed with a synthetic mixed waste to create samples with and without bio-plastics. A self-induced biostabilization has been carried out for 7 and 14 days where temperature and off-gas have been monitored continuously. Results about temperature evolution, O2 consumption, CO2 production and respiratory quotient did not show a substantial difference regarding both the duration of the process and the presence of cellulose-based bio-plastics on the mixture. On the average, the temperature peak and the maximum daily O2 consumption and CO2 production were 52.2 °C, 35.81 g O2/kg DM *d and 48.95 g CO2/kg DM *d respectively. Disintegration of bio-plastics samples after 7 and 14 days were comparable (on the average 23.13%). The self-induced biostabilization gave its main contribution after 4 days and resulted almost finished at the end of the day 7 of the process. Results showed that cellulose-based bio-plastics did not give a negative effect on mixed MSW biological stabilization and suggest a possible management, aiming at energy recovery of the outputs.

Quantum dynamics of a single molecule magnet on superconducting Pb(111).

Magnetic materials interfaced with superconductors may reveal new physical phenomena with potential for quantum technologies. The use of molecules as magnetic components has already shown great promise, but the diversity of properties offered by the molecular realm remains largely unexplored. Here we investigate a submonolayer of tetrairon(III) propeller-shaped single molecule magnets deposited on a superconducting lead surface. This material combination reveals a strong influence of the superconductor on the spin dynamics of the single molecule magnet. It is shown that the superconducting transition to the condensate state switches the single molecule magnet from a blocked magnetization state to a resonant quantum tunnelling regime. Our results open perspectives to control single molecule magnetism via superconductors and to use single molecule magnets as local probes of the superconducting state.

Substrate mediated interaction of terbium(III) double-deckers with the TiO2(110) surface.

A terbium(iii)-bis(phthalocyaninato) neutral complex was deposited on the rutile TiO2(110) surface, and their interaction was studied by Scanning Tunneling Microscopy (STM) and X-ray Photoelectron Spectroscopy (XPS). It was found that the TiO2 rutile surface favours the adsorption of isolated molecules adopting a lying down configuration with the phthalocyanine planes tilted by about 30° when they lie in the first layer. The electronic and chemical properties of the molecules on the surface were studied by XPS as a function of the TiO2(110) substrate preparation. This study evidences that strong molecule-substrate interactions are present and a charge transfer process occurs from the molecule to the surface.

Native-state imaging of calcifying and noncalcifying microalgae reveals similarities in their calcium storage organelles.

Calcium storage organelles are common to all eukaryotic organisms and play a pivotal role in calcium signaling and cellular calcium homeostasis. In most organelles, the intraorganellar calcium concentrations rarely exceed micromolar levels. Acidic organelles called acidocalcisomes, which concentrate calcium into dense phases together with polyphosphates, are an exception. These organelles have been identified in diverse organisms, but, to date, only in cells that do not form calcium biominerals. Recently, a compartment storing molar levels of calcium together with phosphorous was discovered in an intracellularly calcifying alga, the coccolithophore Emiliania huxleyi, raising a possible connection between calcium storage organelles and calcite biomineralization. Here we used cryoimaging and cryospectroscopy techniques to investigate the anatomy and chemical composition of calcium storage organelles in their native state and at nanometer-scale resolution. We show that the dense calcium phase inside the calcium storage compartment of the calcifying coccolithophore Pleurochrysis carterae and the calcium phase stored in acidocalcisomes of the noncalcifying alga Chlamydomonas reinhardtii have common features. Our observations suggest that this strategy for concentrating calcium is a widespread trait and has been adapted for coccolith formation. The link we describe between acidocalcisomal calcium storage and calcium storage in coccolithophores implies that our physiological and molecular genetic understanding of acidocalcisomes could have relevance to the calcium pathway underlying coccolithophore calcification, offering a fresh entry point for mechanistic investigations on the adaptability of this process to changing oceanic conditions.

Calcite as a Precursor of Hydroxyapatite in the Early Biomineralization of Differentiating Human Bone-Marrow Mesenchymal Stem Cells.

Biomineralization is the process by which living organisms generate organized mineral crystals. In human cells, this phenomenon culminates with the formation of hydroxyapatite, which is a naturally occurring mineral form of calcium apatite. The mechanism that explains the genesis within the cell and the propagation of the mineral in the extracellular matrix still remains largely unexplained, and its characterization is highly controversial, especially in humans. In fact, up to now, biomineralization core knowledge has been provided by investigations on the advanced phases of this process. In this study, we characterize the contents of calcium depositions in human bone mesenchymal stem cells exposed to an osteogenic cocktail for 4 and 10 days using synchrotron-based cryo-soft-X-ray tomography and cryo-XANES microscopy. The reported results suggest crystalline calcite as a precursor of hydroxyapatite depositions within the cells in the biomineralization process. In particular, both calcite and hydroxyapatite were detected within the cell during the early phase of osteogenic differentiation. This striking finding may redefine most of the biomineralization models published so far, taking into account that they have been formulated using murine samples while studies in human cell lines are still scarce.

Stabilization of an Enantiopure Sub-monolayer of Helicene Radical Cations on a Au(111) Surface through Noncovalent Interactions.

In the past few years, the chirality and magnetism of molecules have received notable interest for the development of novel molecular devices. Chiral helicenes combine both these properties, and thus their nanostructuration is the first step toward developing new multifunctional devices. Here, we present a novel strategy to deposit a sub-monolayer of enantiopure thia[4]helicene radical cations on a pre-functionalized Au(111) substrate. This approach results in both the paramagnetic character and the chemical structure of these molecules being maintained at the nanoscale, as demonstrated by in-house characterizations. Furthermore, synchrotron-based X-ray natural circular dichroism confirmed that the handedness of the thia[4]helicene is preserved on the surface.

Multifunctionality of structural nanohybrids: the crucial role of carbon nanotube covalent and non-covalent functionalization in enabling high thermal, mechanical and self-healing performance.

This study proposes new kinds of functionalization procedures able to preserve specific properties of carbon nanotubes (CNTs) and to improve compatibility with the epoxy matrix. Through a covalent approach, for the first time, CNTs are functionalized with the same hardener agent, 4,4'-diaminodiphenyl sulfone, employed to solidify the epoxy matrix and capable to fulfill mechanical requirements of industrial structural resins. The same CNTs are non-covalently modified through the polymer wrapping mechanism with benzoxazine (Bz) terminated polydimethylsiloxane (PDMS). The comparison between electrical and mechanical properties of the nanocomposites highlights the success of the non-covalent functionalization in determining an increase in the glass transition temperature (Tg) and in better preserving the unfunctionalized CNT electrical conductivity. Besides, tunneling atomic force microscopy (TUNA), powerful to catch ultra-low currents, has been used for revealing the morphology on nanoscale domains and detecting the conductivity on the same location of CNT/epoxy resins. No electrical contacts to the grounds have been used for the TUNA analysis; a procedure that does not alter the results on the interface domains which experience contact areas with strong differences in their properties. The effectiveness of performed CNT functionalizations as a route to impart self-healing efficiency to the resin formulations has also been proved.

Reversal of heart failure in a chemogenetic model of persistent cardiac redox stress.

We previously described a novel "chemogenetic" animal model of heart failure that recapitulates a characteristic feature commonly found in human heart failure: chronic oxidative stress. This heart failure model uses a chemogenetic approach to activate a recombinant yeast d-amino acid oxidase in rat hearts in vivo to generate oxidative stress, which then rapidly leads to the development of a dilated cardiomyopathy. Here we apply this new model to drug testing by studying its response to treatment with the angiotensin II (ANG II) receptor blocker valsartan, administered either alone or with the neprilysin inhibitor sacubitril. Echocardiographic and [18F]fluorodeoxyglucose positron emission tomographic imaging revealed that valsartan in the presence or absence of sacubitril reverses the anatomical and metabolic remodeling induced by chronic oxidative stress. Markers of oxidative stress, mitochondrial function, and apoptosis, as well as classical heart failure biomarkers, also normalized following drug treatments despite the persistence of cardiac fibrosis. These findings provide evidence that chemogenetic heart failure is rapidly reversible by drug treatment, setting the stage for the study of novel heart failure therapeutics in this model. The ability of ANG II blockade and neprilysin inhibition to reverse heart failure induced by chronic oxidative stress identifies a central role for cardiac myocyte angiotensin receptors in the pathobiology of cardiac dysfunction caused by oxidative stress.NEW & NOTEWORTHY The chemogenetic approach allows us to distinguish cardiac myocyte-specific pathology from the pleiotropic changes that are characteristic of other "interventional" animal models of heart failure. These features of the chemogenetic heart failure model facilitate the analysis of drug effects on the progression and regression of ventricular remodeling, fibrosis, and dysfunctional signal transduction. Chemogenetic approaches will be highly informative in the study of the roles of redox stress in heart failure providing an opportunity for the identification of novel therapeutic targets.

Notch signaling modulates the electrical behavior of cardiomyocytes.

Notch receptor signaling is active during cardiac development and silenced in myocytes after birth. Conversely, outward K+ Kv currents progressively appear in postnatal myocytes leading to shortening of the action potential (AP) and acquisition of the mature electrical phenotype. In the present study, we tested the possibility that Notch signaling modulates the electrical behavior of cardiomyocytes by interfering with Kv currents. For this purpose, the effects of Notch receptor activity on electrophysiological properties of myocytes were evaluated using transgenic mice with inducible expression of the Notch1 intracellular domain (NICD), the functional fragment of the activated Notch receptor, and in neonatal myocytes after inhibition of the Notch transduction pathway. By patch clamp, NICD-overexpressing cells presented prolonged AP duration and reduced upstroke amplitude, properties that were coupled with reduced rapidly activating Kv and fast Na+ currents, compared with cells obtained from wild-type mice. In cultured neonatal myocytes, inhibition of the proteolitic release of NICD with a γ-secretase antagonist increased transcript levels of the Kv channel-interacting proteins 2 (KChIP2) and enhanced the density of Kv currents. Collectively, these results indicate that Notch signaling represents an important regulator of the electrophysiological behavior of developing and adult myocytes by repressing, at least in part, repolarizing Kv currents. NEW & NOTEWORTHY We investigated the effects of Notch receptor signaling on the electrical properties of cardiomyocytes. Our results indicate that the Notch transduction pathway interferes with outward K+ Kv currents, critical determinants of the electrical repolarization of myocytes.

3D reconstruction of magnetization from dichroic soft X-ray transmission tomography.

The development of magnetic nanostructures for applications in spintronics requires methods capable of visualizing their magnetization. Soft X-ray magnetic imaging combined with circular magnetic dichroism allows nanostructures up to 100-300 nm in thickness to be probed with resolutions of 20-40 nm. Here a new iterative tomographic reconstruction method to extract the three-dimensional magnetization configuration from tomographic projections is presented. The vector field is reconstructed by using a modified algebraic reconstruction approach based on solving a set of linear equations in an iterative manner. The application of this method is illustrated with two examples (magnetic nano-disc and micro-square heterostructure) along with comparison of error in reconstructions, and convergence of the algorithm.

Clathrin regulates lymphocyte migration by driving actin accumulation at the cellular leading edge.

Lymphocyte migration, which is essential for effective immune responses, belongs to the so-called amoeboid migration. The lymphocyte migration is up to 100 times faster than between mesenchymal and epithelial cell types. Migrating lymphocytes are highly polarized in three well-defined structural and functional zones: uropod, medial zone, and leading edge (LE). The actiomyosin-dependent driving force moves forward the uropod, whereas massive actin rearrangements protruding the cell membrane are observed at the LE. These actin rearrangements resemble those observed at the immunological synapse driven by clathrin, a protein normally involved in endocytic processes. Here, we used cell lines as well as primary lymphocytes to demonstrate that clathrin and clathrin adaptors colocalize with actin at the LE of migrating lymphocytes, but not in other cellular zones that accumulate both clathrin and actin. Moreover, clathrin and clathrin adaptors, including Hrs, the clathrin adaptor for multivesicular bodies, drive local actin accumulation at the LE. Clathrin recruitment at the LE resulted necessary for a complete cell polarization and further lymphocyte migration in both 2D and 3D migration models. Therefore, clathrin, including the clathrin population associated to internal vesicles, controls lymphocyte migration by regulating actin rearrangements occurring at the LE.