Molecular Chaperones in the Pathogenesis of Amyotrophic Lateral Sclerosis: The Role of HSPB1

open15siGenetic discoveries in amyotrophic lateral sclerosis (ALS) have a significant impact on deciphering molecular mechanisms of motor neuron degeneration but, despite recent advances, the etiology of most sporadic cases remains elusive. Several cellular mechanisms contribute to the motor neuron degeneration in ALS, including RNA metabolism, cellular interactions between neurons and nonneuronal cells, and seeding of misfolded protein with prion-like propagation. In this scenario, the importance of protein turnover and degradation in motor neuron homeostasis gained increased recognition. In this study, we evaluated the role of the candidate gene HSPB1, a molecular chaperone involved in several proteome-maintenance functions. In a cohort of 247 unrelated Italian ALS patients, we identified two variants (c.570G>C, p.Gln190His and c.610dupG, p.Ala204Glyfs*6). Functional characterization of the p.Ala204Glyfs*6 demonstrated that the mutant protein alters HSPB1 dynamic equilibrium, sequestering the wild-type protein in a stable dimer and resulting in a loss of chaperone-like activity. Our results underline the relevance of identifying rare but pathogenic variations in sporadic neurodegenerative diseases, suggesting a possible correlation between specific pathomechanisms linked to HSPB1 mutations and the associated neurological phenotype. Our study provides additional lines of evidence to support the involvement of HSPB1 in the pathogenesis of sporadic ALS.openCapponi, Simona; Geuens, Thomas; Geroldi, Alessandro; Origone, Paola; Verdiani, Simonetta; Cichero, Elena; Adriaenssens, Elias; De Winter, Vicky; Bandettini di Poggio, Monica; Barberis, Marco; Chiò, Adriano; Fossa, Paola; Mandich, Paola; Bellone, Emilia; Timmerman, VincentCapponi, Simona; Geuens, Thomas; Geroldi, Alessandro; Origone, Paola; Verdiani, Simonetta; Cichero, Elena; Adriaenssens, Elias; De Winter, Vicky; BANDETTINI DI POGGIO, MONICA LAURA; Barberis, Marco; Chiò, Adriano; Fossa, Paola; Mandich, Paola; Bellone, Emilia; Timmerman, Vincen

Low grade endotoxemia and oxidative stress in offspring of patients with early myocardial infarction

Background and aims: Offspring of patients with early myocardial infarction are at higher cardiovascular risk, but the underlying physio-pathological mechanism is unclear. NADPH oxidase-type 2 (NOX-2) plays a pivotal role as mediator of oxidative stress and could be involved in activating platelets in these patients. Furthermore, altered intestinal permeability and serum lipopolysaccharide (LPS) could be a trigger to promote NOX-2 activation and platelet aggregation. This study aims to evaluate the behavior of low grade endotoxemia, oxidative stress and platelet activation in offspring of patients with early myocardial infarction. Methods: We enrolled, in a cross-sectional study, 46 offspring of patients with early myocardial infarction and 86 healthy subjects (HS). LPS levels and gut permeability (assessed by zonulin), oxidative stress (assessed by serum NOX-2-derived peptide (sNOX2-dp) release, hydrogen peroxide (H2O2) production and isoprostanes), serum nitric oxide (NO) bioavailability and platelet activation (by serum thromboxane B2 (TXB2) and soluble P-Selectin (sP-Selectin)) were analyzed. Results: Compared to HS, offspring of patients with early myocardial infarction had higher values of LPS, zonulin, serum isoprostanes, sNOX2-dp H2O2, TXB2, p-selectin and lower NO bioavailability. Logistic regression analysis showed that the variables associated with offspring of patients with early myocardial infarction were LPS, TXB2 and isoprostanes. The multiple linear regression analysis confirmed that serum NOX-2, isoprostanes, p-selectin and H2O2 levels were significantly associated to LPS. Furthermore, serum LPS, isoprostanes and TXB2 levels were significantly associated with sNOX-2-dp. Conclusions: Offspring of patients with early myocardial infarction have a low grade endotoxemia that could generate oxidative stress and platelet activation increasing their cardiovascular risk. Future studies are needed to understand the role of dysbiosis in this population

Case Report: Interindividual variability and possible role of heterozygous variants in a family with deficiency of adenosine deaminase 2: are all heterozygous born equals?

Deficiency of adenosine deaminase 2 (DADA2) is a rare systemic autoinflammatory disease, typically with autosomal recessive inheritance, usually caused by biallelic loss of function mutations in the ADA2 gene. The phenotypic spectrum is broad, generally including fever, early-onset vasculitis, stroke, and hematologic dysfunction. Heterozygous carriers may show related signs and symptoms, usually milder and at an older age. Here we describe the case of two relatives, the proband and his mother, bearing an ADA2 homozygous pathogenic variant, and a heterozygous son. The proband was a 17-year-old boy with intermittent fever, lymphadenopathies, and mild hypogammaglobulinemia. He also had sporadic episodes of aphthosis, livedo reticularis and abdominal pain. Hypogammaglobulinemia was documented when he was 10 years old, and symptoms appeared in his late adolescence. The mother demonstrated mild hypogammaglobulinemia, chronic pericarditis since she was 30 years old and two transient episodes of diplopia without lacunar lesions on MRI. ADA2 (NM_001282225.2) sequencing identified both mother and son as homozygous for the c.1358A>G, p.(Tyr453Cys) variant. ADA2 activity in the proband and the mother was 80-fold lower than in the controls. Clinical features in both patients improved on anti-tumor necrosis factor therapy. An older son was found to be heterozygous for the same mutation post-mortem. He died at the age of 12 years due to a clinical picture of fever, lymphadenitis, skin rash and hypogammaglobulinemia evolving toward fatal multiorgan failure. Biopsies of skin, lymph nodes, and bone marrow excluded lymphomas and vasculitis. Despite being suspected of symptomatic carrier, the contribution of an additional variant in compound heterozygosity, or further genetic could not be ruled out, due to poor quality of DNA samples available. In conclusion, this familiar case demonstrated the wide range of phenotypic variability in DADA2. The search for ADA2 mutations and the assessment of ADA2 activity should be considered also in patients with the association of hypogammaglobulinemia and inflammatory conditions, also with late presentation and in absence of vasculitis. Furthermore, the clinical picture of the deceased carrier suggests a possible contribution of heterozygous pathogenic variants to inflammation

Fatality rate and predictors of mortality in an Italian cohort of hospitalized COVID-19 patients

Clinical features and natural history of coronavirus disease 2019 (COVID-19) differ widely among different countries and during different phases of the pandemia. Here, we aimed to evaluate the case fatality rate (CFR) and to identify predictors of mortality in a cohort of COVID-19 patients admitted to three hospitals of Northern Italy between March 1 and April 28, 2020. All these patients had a confirmed diagnosis of SARS-CoV-2 infection by molecular methods. During the study period 504/1697 patients died; thus, overall CFR was 29.7%. We looked for predictors of mortality in a subgroup of 486 patients (239 males, 59%; median age 71 years) for whom sufficient clinical data were available at data cut-off. Among the demographic and clinical variables considered, age, a diagnosis of cancer, obesity and current smoking independently predicted mortality. When laboratory data were added to the model in a further subgroup of patients, age, the diagnosis of cancer, and the baseline PaO2/FiO2 ratio were identified as independent predictors of mortality. In conclusion, the CFR of hospitalized patients in Northern Italy during the ascending phase of the COVID-19 pandemic approached 30%. The identification of mortality predictors might contribute to better stratification of individual patient risk

Glass transition dynamics of ultra-thin H-bonding. Liquids studied by dielectric spectroscopy.

The work reported in this dissertation aimed to study the structural relaxation dynamics in ultrathin films of H-bonding liquids approaching the glass transition. In that respect, our work was focused on detecting deviations from bulk behavior in films of glycerol, xylitol and threitol, by two different experimental approaches. On the one hand, we searched for deviations from bulk behavior induced by one-dimensional confinement upon systematic reduction of the thickness of the films, trying to discern between pure finite-size effects and interface effects. The first ones are predicted to occur when the system is geometrically constrained at dimensions approaching the characteristic correlation length of the dynamics, while interface effects introduce additional perturbations due to intermolecular interactions between the supercooled liquid and the solid walls of the confining host. To tune the thickness of the films with a resolution of few molecular layers, we developed a novel experimental method, which exploits organic molecular beam deposition for the preparation of the films, and employs broadband dielectric spectroscopy to probe the dynamics.On the other hand, we also investigated deviations in the structural dynamics of ultrathin films grown in the glassy state by physical vapour deposition, monitoring them after annealing above the glass transition temperature, Tg. In this case deviations from bulk behavior were induced by the specific preparation method, and they can be explained assuming the existence of hidden liquid phases, originated by the strong intermolecular interactions among the polyols molecules, favored by the preparation technique. The goal of this part of our work was to provide further experimental evidence of the presence of crystal-like regions with medium range orientational order (MRCOs) in a supercooled liquid, as predicted by the two-order parameter model [Nature Physics, 2, 2006, 200-205]. To this end, we aimed to produce liquid films enriched in MRCOs with respect to the ordinary liquid. We thus searched evidence of the consequent increase in bond orientational order by monitoring deviations in the dielectric response in these systems and comparing them with the response of films deposited above Tg.Concerning the first part of our work we stress that, despite succeeding in investigating films as thin as 3 molecular layers, no deviations from bulk behavior ascribable to pure finite-size effects have been found [J. Chem. Phys. C, 114, 2010, 16696-16699]. Conversely, experiments revealed a slight slowing down of the dynamics in 1.6 nm thick layers of supercooled glycerol, in contrast with the enhancement of the molecular mobility expected under confinement. The slower dynamics observed for the thinnest films has been attributed to the presence of a reduced mobility layer in the neighborhood of the lower interface, due to the attractive interactions between the glycerol molecules and the quartz substrate of the interdigitated sensor used to perform dielectric measurements. The analysis of the relaxation time and the shape of the dielectric loss have allowed us to separate the size effects from the interface contributions, also relying on models previously proposed by our group [Macromolecules, 41, 2008, 1061-1063]. The absence of pure finite-size confinement effects in our experiments, in contrast with other experiments on liquids confined in nanopores, may be due to the following reasons:* Ultrathin films are confined only along one dimension, while systems hosted in nanopores are forced in a 2 or 3 dimensional spatial constraint.* The deviations observed in nanopores could be actually induced by variations in the density and in the internal pressure of the system, as a result of the curvature of the walls of the host.As regards the investigation of ultrathin films deposited below Tg, the most remarkable experimental finding is a strong enhancement in the dielectric strength with respect to the values observed for films grown in the liquid state, far above Tg. The extent of such increase is higher for films below 20 nm (up to a factor of 4.5 for the thinnest film), and it reaches a plateau of a factor of 3.7 for the thickest films. Additionally, an increase in the structural relaxation time by a factor of 5 was observed. We argument that these findings are indeed ascribable to an enhancement in the overall bond orientational order inside the film, and thus they are consistent with the scenario of a liquid phase enriched in MRCOs. The stability of this phase was investigated against annealing by acquiring isothermal dielectric spectra at increasing temperatures, and monitoring the evolution of the dielectric strength Δε. We observed that Δε progressively decreased upon heating, indicating the metastable character of the ordered liquid phase, namely, the MRCO phase. We measured the rate tc of the conversion to the ordinary phase from the time evolution of Δε during isothermal measurements assuming a growth front-like transformation process. We found a surprising stability of the ordered liquid phase, the value of tc exceeding by a few orders of magnitude the structural relaxation time at each annealing temperature. The origin of this surprising stability needs further investigations and additional studies in order to gain insights into the actual spatial molecular arrangement inside the ordered regions.status: publishe

1D confinement stabilizes non-equilibrium liquid phase with enhanced orientational order

Recent models of the glass transition rationalize the relevant features of vitrification by introducing the concept of medium range order in supercooled liquids. In particular, a crucial role is assigned to the orientational order induced by intermolecular bonding (bond orientational order, BOO). Although this idea is very appealing, severe difficulties limit the experimental determination of the BOO in molecular liquids: because of the small difference in entropy and in form factor between the isotropic liquid and the phase rich in orientational order, calorimetry and scattering techniques cannot be used to assess the BOO. In this chapter we describe an innovative model to investigate BOO via broadband dielectric spectroscopy. We verified that ultrathin films of polyols deposited by physical vapor deposition below their glass transition temperature show an extraordinary enhancement in bond orientational, indicated by a huge enhancement of the dielectric strength with respect to the bulk values. Hint of an underlying phase transition was found from a liquid phase enriched in bond orientational order, with a metastable character, towards an ordinary liquid phase. The kinetic stability of the metastable phase could be tuned by varying the deposition conditions and the molecular size. The impact of film thickness and of the presence of a solid substrate on the persistence of enhanced BOO were also investigated. We demonstrate that confinement stabilizes the non-equilibrium character of our supercooled liquids with enhanced orientational order.SCOPUS: ch.binfo:eu-repo/semantics/publishe

[INVITED Colloquium] Glassy dynamics of soft matter under 1D confinement: How irreversible adsorption affects molecular packing, mobility gradients and orientational polarization in thin films

The structural dynamics of polymers and simple liquids confined at the nanometer scale has been intensively investigated in the last two decades in order to test the validity of theories on the glass transition predicting a characteristic length scale of a few nanometers. Although this goal has not yet been reached, the anomalous behavior displayed by some systems - e.g. thin films of polystyrene exhibit reductions of Tg exceeding 70K and a tremendous increase in the elastic modulus - has attracted a broad community of researchers, and provided astonishing advancement of both theoretical and experimental soft matter physics. 1D confinement is achieved in thin films, which are commonly treated as systems at thermodynamic equilibrium where free surfaces and solid interfaces introduce monotonous mobility gradients, extending for several molecular sizes. Limiting the discussion to finite-size and interfacial effects implies that film thickness and surface interactions should be sufficient to univocally determine the deviation from bulk behavior. On the contrary, such an oversimplified picture, although intuitive, cannot explain phenomena like the enhancement of segmental mobility in proximity of an adsorbing interface, or the presence of long-lasting metastable states in the liquid state. Based on our recent work, we propose a new picture on the dynamics of soft matter confined in ultrathin films, focusing on non-equilibrium and on the impact of irreversibly chain adsorption on the structural relaxation. We describe the enhancement of dynamics in terms of the excess in interfacial free volume, originating from packing frustration in the adsorbed layer (Guiselin brush) at t < 1, where t* is the ratio between the annealing time and the time scale of adsorption. Prolonged annealing at times exceeding the reptation time (usually t* ≫ 1 induces densification, and thus reduces the deviation from bulk behavior. In this Colloquium, after reviewing the experimental approaches permitting to investigate the structural relaxation of films with one, two or no free surfaces by means of dielectric spectroscopy, we propose several methods to determine gradients of mobility in thin films, and then discuss on the unexploited potential of analyses based on the time, temperature and thickness dependence of the orientational polarization via the dielectric strength. © 2013 EDP Sciences, SIF, Springer-Verlag Berlin Heidelberg.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Scavi archeologici, ricerca e valorizzazione di Hadrianopolis, Antigonea e dei siti della valle del Drino

Ricerche storico-archeologiche e valorizzazione dei siti della valle del Drin

Scavi archeologici, ricerca e valorizzazione di Paleokastër (Regione di Gjirokaster)

Ricerche storico archeologiche finalizzate allo studio e valorizzazione della fortificazione di Paleokaste