The novel Mechanical Ventilator Milano for the COVID-19 pandemic.

This paper presents the Mechanical Ventilator Milano (MVM), a novel intensive therapy mechanical ventilator designed for rapid, large-scale, low-cost production for the COVID-19 pandemic. Free of moving mechanical parts and requiring only a source of compressed oxygen and medical air to operate, the MVM is designed to support the long-term invasive ventilation often required for COVID-19 patients and operates in pressure-regulated ventilation modes, which minimize the risk of furthering lung trauma. The MVM was extensively tested against ISO standards in the laboratory using a breathing simulator, with good agreement between input and measured breathing parameters and performing correctly in response to fault conditions and stability tests. The MVM has obtained Emergency Use Authorization by U.S. Food and Drug Administration (FDA) for use in healthcare settings during the COVID-19 pandemic and Health Canada Medical Device Authorization for Importation or Sale, under Interim Order for Use in Relation to COVID-19. Following these certifications, mass production is ongoing and distribution is under way in several countries. The MVM was designed, tested, prepared for certification, and mass produced in the space of a few months by a unique collaboration of respiratory healthcare professionals and experimental physicists, working with industrial partners, and is an excellent ventilator candidate for this pandemic anywhere in the world.

Interaction of Escherichia coli translation-initiation factor IF-1 with ribosomes.

The interaction between Escherichia coli translation-initiation factor IF-1 and ribosomes was studied in binding experiments by Airfuge centrifugation. IF-1 binds to the 30S, but not to the 50S, ribosomal subunit and its binding is strongly stimulated by IF-3 and IF-2, either alone or in combination. From the dependence of the Kd of the 30S-subunit--IF-1 complex on ionic strength, it can be concluded that IF-1 binds primarily via an ionic interaction, most likely with the 16S rRNA, with the minimum number of ion pairs involved being 2.7-3.6. The 30S-subunit--IF-1 interaction is unaffected by temperature changes between 11 degrees C and 44 degrees C and is thus accompanied by a negligible enthalpy change. It is concluded that the interaction is an entropy-driven process triggered mainly by the release of counter ions from the RNA phosphates. Titration of 30S-subunit--IF-1 complexes with 50S subunits causes the ejection of the factor indicating that IF-1 is released from the ribosomes during the subunit association step which marks the transition from a 30S-initiation-complex to a 70S initiation complex.

Site-directed mutagenesis of Escherichia coli translation initiation factor IF1. Identification of the amino acid involved in its ribosomal binding and recycling.

Starting from a synthetic modular gene (infA) encoding Escherichia coli translation initiation factor IF1, we have constructed mutants in which amino acids are deleted from the carboxyl terminus or in which His29 or His34 are replaced by Tyr or Asp residues. The mutant proteins were overproduced, purified and tested in vitro for their properties in several partial reactions of the translation initiation pathway and for their capacity to stimulate MS2 RNA-dependent protein synthesis. The results allow for the conclusion that: (i) Arg69 is part of the 30S ribosomal subunit binding site of IF1 and its deletion results in the substantial loss of all IF1 function; (ii) neither one of its two histidines is essential for the binding of IF1 to the 30S ribosomal subunit, for the stimulation of fMet-tRNA binding to 30S or 70S ribosomal particles or for MS2 RNA-dependent protein synthesis; but (iii) His29 is involved in the 50S subunit-induced ejection of IF1 from the 30S ribosomal subunit.

Clavin, a type-1 ribosome-inactivating protein from Aspergillus clavatus IFO 8605. cDNA isolation, heterologous expression, biochemical and biological characterization of the recombinant protein.

We describe the cloning and expression of a new cDNA from the filamentous fungus Aspergillus clavatus IFO 8605. This cDNA contains an open reading frame (ORF) that predicts a putative ribonuclease precursor with high similarity to the alpha-sarcin family of ribosome-inactivating proteins (RIPs). The cDNA encoding the mature protein was expressed in Escherichia coli, and the recombinant protein, a 17-kDa polypeptide designated clavin was purified and characterized. Clavin shows typical type-1 RIP properties: specific cleavage of ribosomal and synthetic RNA and inhibition of protein synthesis in cell-free and cellular systems. When selectively targeted to a tumour cell antigen by coupling to a monoclonal antibody (mAb) clavin was able to inhibit protein synthesis at nanomolar concentration. Pharmacokinetics analysis in mice indicated an elimination half-life (t1/2 beta) of 7.4 h with no particular accumulation in major organs. Liver toxicity was very limited and transient while no alteration of kidney function was observed. Clavin induced a late and very low antibody response in mice. The in vitro and in vivo biological characteristics of clavin, together with its availability in large amounts, suggest the usefulness of this toxin in the production of toxic chemical conjugates.