Muographic monitoring of the volcano-tectonic evolution of Mount Etna.

At Mount Etna volcano, the focus point of persistent tectonic extension is represented by the Summit Craters. A muographic telescope has been installed at the base of the North-East Crater from August 2017 to October 2019, with the specific aim to find time related variations in the density of volcanic edifice. The results are significant, since the elaborated images show the opening and evolution of different tectonic elements; in 2017, a cavity was detected months before the collapse of the crater floor and in 2018 a set of underground fractures was identified, at the tip of which, in June 2019, a new eruptive vent started its explosive activity, still going on (February, 2020). Although this is the pilot experiment of the project, the results confirm that muography could be a turning point in the comprehension of the plumbing system of the volcano and a fundamental step forward to do mid-term (weeks/months) predictions of eruptions. We are confident that an increment in the number of telescopes could lead to the realization of a monitoring system, which would keep under control the evolution of the internal dynamic of the uppermost section of the feeding system of an active volcano such as Mount Etna.

Synthesis and chemical characterization of several perfluorinated sialic acid glycals and evaluation of their in vitro antiviral activity against Newcastle disease virus.

Newcastle Disease Virus (NDV), belonging to the Paramyxoviridae family, causes a serious infectious disease in birds, resulting in severe losses in the poultry industry every year. Haemagglutinin neuraminidase glycoprotein (HN) has been recognized as a key protein in the viral infection mechanism, and its inhibition represents an attractive target for the development of new drugs based on sialic acid glycals, with the 2-deoxy-2,3-didehydro-d-N-acetylneuraminic acid (Neu5Ac2en) as their backbone. Herein we report the synthesis of several Neu5Ac2en glycals and of their perfluorinated C-5 modified derivatives, including their respective stereoisomers at C-4, together with evaluation of their in vitro antiviral activity. While all synthesized compounds were found to be active HN inhibitors in the micromolar range, we found that their potency was influenced by the chain-length of the C-5 perfluorinated acetamido functionality. Thus, the binding modes of the inhibitors were also investigated by performing a docking study. Moreover, the perfluorinated glycals were found to be more active than the corresponding normal C-5 acylic derivatives. Finally, cell-cell fusion assays on NDV infected cells revealed that the addition of a newly synthesized C-4α heptafluorobutyryl derivative almost completely inhibited NDV-induced syncytium formation.

Structure and dynamics of the pore-lining helix of the nicotinic receptor: MD simulations in water, lipid bilayers, and transbilayer bundles.

Multiple nanosecond duration molecular dynamics simulations on the pore-lining M2 helix of the nicotinic acetylcholine receptor reveal how its structure and dynamics change as a function of environment. In water, the M2 helix partially unfolds to form a molecular hinge in the vicinity of a central Leu residue that has been implicated in the mechanism of ion channel gating. In a phospholipid bilayer, either as a single transmembrane helix, or as part of a pentameric helix bundle, the M2 helix shows less flexibility, but still exhibits a kink in the vicinity of the central Leu. The single M2 helix tilts relative to the bilayer normal by 12 degrees, in agreement with recent solid state NMR data (Opella et al., Nat Struct Biol 6:374-379, 1999). The pentameric helix bundle, a model for the pore domain of the nicotinic receptor and for channels formed by M2 peptides in a bilayer, is remarkably stable over a 2-ns MD simulation in a bilayer, provided one adjusts the pK(A)s of ionizable residues to their calculated values (when taking their environment into account) before starting the simulation. The resultant transbilayer pore shows fluctuations at either mouth which transiently close the channel. Proteins 2000;39:47-55.

Simulation studies of the interaction of antimicrobial peptides and lipid bilayers.

Experimental studies of a number of antimicrobial peptides are sufficiently detailed to allow computer simulations to make a significant contribution to understanding their mechanisms of action at an atomic level. In this review we focus on simulation studies of alamethicin, melittin, dermaseptin and related antimicrobial, membrane-active peptides. All of these peptides form amphipathic alpha-helices. Simulations allow us to explore the interactions of such peptides with lipid bilayers, and to understand the effects of such interactions on the conformational dynamics of the peptides. Mean field methods employ an empirical energy function, such as a simple hydrophobicity potential, to provide an approximation to the membrane. Mean field approaches allow us to predict the optimal orientation of a peptide helix relative to a bilayer. Molecular dynamics simulations that include an atomistic model of the bilayer and surrounding solvent provide a more detailed insight into peptide-bilayer interactions. In the case of alamethicin, all-atom simulations have allowed us to explore several steps along the route from binding to the membrane surface to formation of transbilayer ion channels. For those antimicrobial peptides such as dermaseptin which prefer to remain at the surface of a bilayer, molecular dynamics simulations allow us to explore the favourable interactions between the peptide helix sidechains and the phospholipid headgroups.

Peptide-bilayer interactions: simulations of dermaseptin B, an antimicrobial peptide.

Dermaseptins, a family of antimicrobial peptides, are believed to act by forming amphipathic alpha-helices which associate with the cell membrane, leading to its permeabilisation and disruption. A simple mean field method is described for simulation of the interactions of peptides with lipid bilayers which includes an approximate representation of the electrostatic effects of the head-group region of the bilayer. Starting from an atomistic model of a PC phospholipid bilayer we calculate an average electrostatic potential along the bilayer normal. By combining the interaction of the peptide with this electrostatic potential and with the hydrophobic core of the membrane we arrive at a more complete description of peptide-bilayer energetics than would be obtained using sidechain hydrophobicities alone. Using this interaction potential in MD simulations of the frog skin peptide dermaseptin B reveals that the lipid bilayer stabilises the alpha-helical conformation of the peptide. This is in agreement with FTIR data. A surface associated orientation thus appears to be the most stable arrangement of the peptide, at least at zero ionic strength and without taking account of possible peptide-peptide interactions.

The structure and organization within the membrane of the helices composing the pore-forming domain of Bacillus thuringiensis delta-endotoxin are consistent with an "umbrella-like" structure of the pore.

The aim of this study was to elucidate the mechanism of membrane insertion and the structural organization of pores formed by Bacillus thuringiensis delta-endotoxin. We determined the relative affinities for membranes of peptides corresponding to the seven helices that compose the toxin pore-forming domain, their modes of membrane interaction, their structures within membranes, and their orientations relative to the membrane normal. In addition, we used resonance energy transfer measurements of all possible combinatorial pairs of membrane-bound helices to map the network of interactions between helices in their membrane-bound state. The interaction of the helices with the bilayer membrane was also probed by a Monte Carlo simulation protocol to determine lowest-energy orientations. Our results are consistent with a situation in which helices alpha4 and alpha5 insert into the membrane as a helical hairpin in an antiparallel manner, while the other helices lie on the membrane surface like the ribs of an umbrella (the "umbrella model"). Our results also support the suggestion that alpha7 may serve as a binding sensor to initiate the structural rearrangement of the pore-forming domain.

Secondary structure of an isolated P-region from the voltage-gated sodium channel: a molecular modelling/dynamics study.

Conformational studies of synthetic peptides corresponding to the pore-forming regions of voltage-gated sodium channels show a high tendency for beta-sheet conformation when interacting with lipid vesicles, as revealed by circular dichroism and infrared spectroscopy. These observations have guided our choice of possible molecular models for the P-region peptide of domain II of voltage-gated sodium channels: three alternative beta-hairpins, with differing turn assignments, or an alpha-helical hairpin. After generation of models by distance geometry-based methods, molecular dynamics (MD) simulations were run. in the absence of explicit solvent molecules but employing three different dielectric constants, to explore possible conformational preferences. The simulations in the different dielectric environments suggest that a 4-residue turn with the sequence LCGE yields more stable beta-hairpins. The MD results suggest that the SS1 part of the peptide may be more stable as an alpha-helix, whereas the SS2 part tends to adopt a beta-conformation.

Pharmacologic, metabolic, and toxicologic profile of spirapril (SCH 33844), a new angiotensin converting inhibitor.

Spirapril (SCH 33844; 7-N-[1(S)-ethoxycarbonyl-3-phenylpropyl]-(S)-alanyl-1,4-dithia- 7-azaspiro[4,4]-nonane-8(S)-carboxylic acid) is a new angiotensin-converting enzyme (ACE) inhibitor. SCH 33844 diacid inhibited hydrolysis of hip-his-leu by rabbit lung ACE in a potent (Ki = 0.74 nM), selective, and noncompetitive fashion. SCH 33844 (0.03-1 mg/kg p.o.) produced dose-related inhibition of angiotensin I (AI) pressor responses in conscious rats with a duration of 24 h at the higher dose. SCH 33844 (0.3-30 mg/kg p.o.) reduced blood pressure in a dose-related manner in conscious SHR with a 24-h duration. Antihypertensive activity was enhanced in the presence of hydrochlorothiazide. The drug (1-10 mg/kg p.o.) also lowered blood pressure in conscious hydrochlorothiazide-treated normotensive dogs. In anesthetized dogs, SCH 33844 (1 mg/kg i.v.) reduced blood pressure and total peripheral vascular resistance and slightly increased cardiac output and stroke volume. These results suggest that peripheral vasodilation is the primary mechanism of the antihypertensive action. The metabolic profile of SCH 33844 was evaluated in dogs and rats. The compound was absorbed in a dose-proportional manner and excreted primarily as the diacid form. In contrast to captopril and enalapril, most of the drug (67%) was excreted into the feces following i.v. dosing. Chronic toxicological evaluation in dogs and rats demonstrated that the drug was relatively devoid of toxicity at oral doses as high as 400 and 450 mg/kg/day, respectively. Slight decreases in heart weight (rats) and increases in granularity of the juxtaglomerular apparatus were observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute and subchronic toxicity of a nonsulfhydryl angiotensin-converting enzyme inhibitor.

Acute and 1-month toxicity studies with SCH 31846, a nonsulfhydryl anti-hypertensive agent which acts by inhibiting angiotensin-converting enzyme, were initiated to evaluate its toxicity. The oral LD50s in mice and rats were approximately 1.8 and 2.5 g/kg, respectively, while the iv LD50 was approximately 450 mg/kg in mice and 150 mg/kg in rats. Signs of acute toxicity in rats and mice included salivation, hypoactivity, ataxia, prostration, and convulsions. In a 1-month dog study at oral doses of 25, 75, or 150 mg/kg, there was a dose-related increase in emesis between 1 and 2 hr after dosing. Absorption studies showed peak blood concentrations occurring in dogs between 0.3 and 1 hr after dosing. No other noteworthy antemortem changes were observed. In a 1-month rat study at oral doses of 30, 180, or 600 mg/kg, the hematocrit and hemoglobin values of the 600 mg/kg-dosed female rats were slightly but significantly (p less than 0.05) decreased and the blood urea nitrogen was slightly but significantly (p less than 0.05) increased in all SCH 31846-dosed male rats and the 600 mg/kg-dosed female rats. Absorption studies in male rats at doses of 30, 180, and 600 mg/kg indicate that SCH 31846 is well absorbed in rats. The 150 mg/kg-dosed dogs and the 180- and 600 mg/kg-dosed rats had a slight increase in the number of renin-containing granules in the renal juxtaglomerular cells. No other compound-related microscopic changes were observed. These data are similar to data reported for Captopril and suggest that in the dog and rat the toxicity of ACE inhibitors is not dependent upon the presence or absence of a sulfhydryl group.

Skin and mucous membrane ulceration in beagle dogs following oral dosing with an experimental aminoglycoside antibiotic.

G-418 is a unique aminoglycoside antibiotic that is structurally related to gentamicin; however, unlike gentamicin, G-418 inhibits growth of both procaryotic and eucaryotic cells. In a preliminary acute oral safety study, adult male and female beagles were given a single oral dose of either 2000, 1000, 500, 200, or 50 mg/kg of G-418. Ulceration of the lip, tongue, and/or gingiva occurred in all G-418-dosed dogs 1 to 9 days after dosing. Ulceration of the glans penis, penis sheath, and scrotum occurred 7 to 14 days after a single oral dose with 1000 and 500 mg/kg G-418, and ulceration of the vaginal mucosa of the 2000-, 1000-, 500-, and 50-mg/kg-dosed female dogs occurred 2 to 8 days after dosing. Ulcers of the lip and vaginal area began at the mucocutaneous border and were more severe at these borders. In some dogs a yellow membrane formed over these lesions. Ulceration of the oral and vaginal mucosa disappeared 10 days after the first occurrence and reoccurred 3-7 days later. All ulcers healed within 30 days after the single oral dose; however, at necropsy hemorhagic areas of the urinary bladder were observed in at least one of two dogs at each dose level. Similar lesions have not been reported in animals treated with any other aminoglycoside antibiotics. The etiology of these lesions is unknown.

Anchorage-independent growth of normal human mesothelial cells: a sensitive bioassay for EGF which discloses the absence of this factor in fetal calf serum.

This laboratory recently reported that normal human mesothelial cells require epidermal growth factor (EGF) and hydrocortisone (HC), in addition to fetal calf serum and a complex defined medium component, in order to grow optimally in surface culture. We report here that this normal cell type also forms large colonies at high efficiency in semi-solid medium, but exhibits more stringent serum and EGF requirements for anchorage-independent than for surface growth. Mesothelial cells are unable to divide at all in semi-solid medium without added EGF or with less than 2% serum, whereas they grow slowly but progressively in surface culture under such conditions. In semi-solid medium containing 20% serum and HC, mesothelial cells are stimulated to divide by the addition of as little as 30 pg/ml purified EGF. Human urine or male mouse plasma could substitute for purified EGF, yielding growth commensurate with the levels of EGF in these biological fluids previously measured by others using radioreceptor and radioimmune assays. Thus growth of mesothelial cells in semi-solid medium can serve as a highly sensitive assay of EGF biological activity which is unaffected by the presence of serum proteins. In addition, our results demonstrate that fetal calf serum does not provide mitogenic levels of EGF to cultured cells, raising the question of the identity of plasma and serum mitogens.

Kinetics of chick embryo cell types in culture.

The growth kinetics and population doubling limits of chick embryonic fibroblasts, chondroblasts, and retinal pigment cells were compared. Chondroblasts were found to have a cumulative population doubling level (37 +/- 3 PDL) similar (p = 0.05) to that of control fibroblasts (42 +/- 2 PDL), in individual and pooled clones. While both cell types have similar doubling potential, the proportion of tritium-labeled nuclei decreases, and differs significantly as doubling level increases. This age-associated decline is due to an extension in the population doubling time. Direct cell-cycle analysis shows this increase to occur in the G1 phase. Furthermore, cartilage colonies maintain their phenotypic expression (metachromasia) throughout their lifespan under conditions of subcloning at sparse density. When fibroblasts derived from 15 day chick embryos are compared with fibroblasts from 10 day embryos (41 +/- 2 PDL) there is no significant difference (p = 0.05) in cumulative PDL or percent labeled nuclei, indicating that fibroblasts of different embryonic age have similar potential. The addition of hydrocortisone and insulin to the medium significantly shortens (25 +/- 2 PDL) the lifespan of 10 day chick fibroblasts. Kinetics of retinal pigment cells show a population doubling potential (29 +/- 1 PDL) different from fibroblasts and chondroblasts, suggesting that different cell types may not have similar limits on doubling potential when first determined in embryogenesis. Hydrocortisone and insulin have no effect on the growth kinetics or lifespan of retinal pigment cells in culture.