The branched M33 antimicrobial peptide was previously shown to be very active against Gram-negative bacterial pathogens, including multidrug-resistant strains. In an attempt to produce back-up ...molecules, we synthesized an M33 peptide isomer consisting of D-aminoacids (M33-D). This isomeric version showed 4 to 16-fold higher activity against Gram-positive pathogens, including Staphylococcus aureus and Staphylococcus epidermidis, than the original peptide, while retaining strong activity against Gram-negative bacteria. The antimicrobial activity of both peptides was influenced by their differential sensitivity to bacterial proteases. The better activity shown by M33-D against S. aureus compared to M33-L was confirmed in biofilm eradication experiments where M33-L showed 12% activity with respect to M33-D, and in vivo models where Balb-c mice infected with S. aureus showed 100% and 0% survival when treated with M33-D and M33-L, respectively. M33-D appears to be an interesting candidate for the development of novel broad-spectrum antimicrobials active against bacterial pathogens of clinical importance.
Receptors for endogenous regulatory peptides, like the neuropeptide neurotensin, are overexpressed in several human cancers and can be targets for peptide-mediated tumor-selective therapy. Peptides, ...however, have the main drawback of an extremely short half-life in vivo. We showed that neurotensin and other endogenous peptides, when synthesized as dendrimers, retain biological activity and become resistant to proteolysis. Here, we synthesized the neurotensin functional fragment NT(8-13) in a tetrabranched form linked to different units for tumor therapy or diagnosis. Fluorescent molecules were used to monitor receptor binding and internalization in HT29 human adenocarcinoma cells and receptor binding in HT29 tumor xenografts in nude mice. Linking of chemotherapic molecules like chlorin e6 and methotrexate to dendrimers resulted in a dramatic increase in drug selectivity, uptake of which by target cells became dependent on peptide receptor binding. When nude mice carrying human tumor xenografts were treated with branched NT(8-13)-methotrexate, a 60% reduction in tumor growth was observed with respect to mice treated with the free drug.
A synthetic antimicrobial peptide was identified as a possible candidate for the development of a new antibacterial drug. The peptide, SET-M33L, showed a MIC90 below 1.5 μM and 3 μM for Pseudomonas ...aeruginosa and Klebsiella pneumoniae, respectively. In in vivo models of P. aeruginosa infections, the peptide and its pegylated form (SET-M33L-PEG) enabled a survival percentage of 60-80% in sepsis and lung infections when injected twice i.v. at 5 mg/Kg, and completely healed skin infections when administered topically. Plasma clearance showed different kinetics for SET-M33L and SET-M33L-PEG, the latter having greater persistence two hours after injection. Bio-distribution in organs did not show significant differences in uptake of the two peptides. Unlike colistin, SET-M33L did not select resistant mutants in bacterial cultures and also proved non genotoxic and to have much lower in vivo toxicity than antimicrobial peptides already used in clinical practice. The characterizations reported here are part of a preclinical development plan that should bring the molecule to clinical trial in the next few years.
The peptide sequence KKIRVRLSA was synthesized in a dimeric structure (SET‐M33DIM) and evaluated as a candidate drug for infections due to multidrug‐resistant (MDR) Gram‐negative pathogens. ...SET‐M33DIM showed significant antibacterial activity against MDR strains of Klebsiella pneumoniae, Acinetobacter baumannii, and Escherichia coli (Minimal Inhibitory Concentration MICs, 1.5‐11 µM), and less activity against Pseudomonas aeruginosa (MICs, 11‐22 µM). It showed very low toxicity in vitro, ex vivo, and in vivo; in cytotoxicity tests, its EC50 was as much as 22 times better than that of SET‐M33, a peptide with the same amino‐acid sequence, but synthesized in tetra‐branched form (638 vs 28 µM). In in vivo and ex vivo experiments, SET‐M33DIM cleared P. aeruginosa infection, significantly reducing signs of sepsis in animals, and restoring cell viability in lung tissue after bacterial challenge. It also quelled inflammation triggered by LPS and live bacterial cells, inhibiting expression of inflammatory mediators in lung tissue, cultured macrophages, and bronchial cells from a cystic fibrosis patient.
We describe the nonnatural antimicrobial peptide KKIRVRLSA (M33) and its capacity to neutralize LPS-induced cytokine release, preventing septic shock in animals infected with bacterial species of ...clinical interest. M33 showed strong resistance to proteolytic degradation when synthesized in tetrabranched form with 4 peptides linked by a lysine core, making it suitable for use in vivo. HPLC and mass spectrometry demonstrated its stability in serum beyond 24 h. M33 was found to be very selective for gram-negative bacteria. Minimal inhibitory concentration (MIC) ranged from 0.3 to 3 μM for multidrug resistant clinical isolates of several pathogenic species, including Pseudomonas aeruginosa, Klebsiella pneumoniae, and Acinetobacter baumannii. M33 neutralized LPS derived from P. aeruginosa and K. pneumoniae, and prevented TNF-α release from LPS-activated macrophages, with an EC₅₀ of 3.8e-8 M and 2.8e-7 M, respectively, as detected by sandwich ELISA. M33 activity was also tested in sepsis animal models. It averted septic shock symptoms due to Escherichia coli and P. aeruginosa in doses compatible with clinical use (5-25 mg/kg). These properties make tetrabranched M33 peptide a good candidate for the development of a new antibacterial drug.--Pini, A., Falciani, C., Mantengoli, E., Bindi, S., Brunetti, J., Iozzi, S., Rossolini, G. M., Bracci, L. A novel tetrabranched antimicrobial peptide that neutralizes bacterial lipopolysaccharide and prevents septic shock in vivo.
The tetra-branched peptide M33 (Pini et al. in FASEB J 24:1015–1022,
2010
) is under evaluation in animal models for its activity as antimicrobial agent in lung infections and sepsis. The preclinical ...development of a new drug requires medium-scale manufacture for tests of efficacy, biodistribution, pharmacokinetics and toxicity. In order to produce the most suitable peptide form for these purposes, we evaluated the behaviour of the peptide M33 obtained with different counter-ions. We compared activity and toxicity in vitro and in vivo of the peptide M33 produced as trifluoroacetate salt (TFacetate) and as acetate salt. The two forms did not differ substantially in terms of efficacy in vitro or in vivo but showed different toxicities for human cells and in animals. M33-TFacetate proved to be 5–30% more toxic than M33-acetate for cells derived from normal bronchi and cells carrying ΔF508 mutation in the CFTR gene, the most frequent variant in cystic fibrosis. M33-TFacetate produced manifest signs of in vivo toxicity immediately after administration, whereas M33-acetate only generated mild signs, which disappeared within a few hours. The peptide M33-acetate proved more suitable for the development of a new drug, and was therefore chosen for further characterization.
We propose a P wave based procedure for the rapid estimation of the radiated seismic energy, and a novel relationship for obtaining an energy‐based local magnitude (MLe) measure of the earthquake ...size. We apply the new procedure to the seismic sequence that struck Central Italy in 2016. Scaling relationships involving seismic moment and radiated energy are discussed for the Mw 6.0 Amatrice, Mw 5.9 Ussita, and Mw 6.5 Norcia earthquakes, including 35 ML > 4 aftershocks. The Mw 6.0 Amatrice earthquake shows the highest apparent stress, and the observed differences among the three main events highlight the dynamic heterogeneity with which large earthquakes can occur in Central Italy. Differences between estimates of MLe and Mw allows identification of events which are characterized by a higher proportion of energy being transferred to seismic waves, providing important real‐time indications of earthquakes shaking potential.
Key Points
An energy‐based local magnitude scale (MLe) for measuring the earthquake size during real‐time operations is proposed
In the analysis of the 2016 Central Italy sequence, large values for the apparent stress are observed, extending from about 0.5 to 25 MPa
Mw and MLe can be used together for better capturing the shaking potential of earthquakes in real‐time applications
In this article, we propose a monitoring procedure based on a multilayer neural network with multivalued neurons (MLMVN) capable of preventing catastrophic failures of dc-dc converters. The neural ...classifier allows both the detection of any malfunction and its localization. Thanks to the low computational complexity, the proposed method operates online, estimating the deviations of the passive components from their nominal values: this allows control strategies to be promptly adopted and operation of the dc-dc converter to be kept in high efficiency and reliability conditions. Since measuring the voltage and current on each component increases the complexity of the system, a testability analysis is proposed with the aim of identifying the minimum number of measurements needed to distinguish the classes of failure. To make the testability phase easier and more intuitive, a graphical representation is proposed. As a case study, prognostic analysis has been applied to prevent catastrophic failures in a synchronous Zeta converter. Several fault conditions have been analyzed through simulations and experimental tests. The obtained results confirm the ability of the proposed method to prevent failures and, also, show that the application of MLMVN results in a better performance than classic solutions available in the literature, such as support vector machine.
SUMMARY
3‐D shear wave velocity images are of particular interest for engineering seismology. To obtain information about the local subsoil structure, we present a one‐step inversion procedure based ...on the computation of high‐frequency correlation functions between stations of a small‐scale array deployed for recording ambient seismic noise. The calculation of Rayleigh wave phase velocities is based on the frequency‐domain SPatial AutoCorrelation technique. Constitutively, a tomographic inversion of the traveltimes estimated for each frequency is performed, allowing the laterally varying 3‐D surface wave velocity structure below the array to be retrieved. We test our technique by using simulations of seismic noise for a simple realistic site and by using real‐world recordings from a small‐scale array performed at the Nauen test site (Germany). The results imply that the cross‐sections from passive seismic interferometry provide a clear image of the local structural heterogeneities and the shear wave velocities are satisfactorily reproduced. The velocity structure is also found to be in good agreement with the results of geoelectrical measurements, indicating the potential of the method to be easily applied for deriving the shallow 3‐D velocity structure in urban areas and for monitoring purposes.
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