Lung ultrasound to evaluate aeration changes in response to recruitment maneuver and prone positioning in intubated patients with COVID-19 pneumonia: preliminary study.

BACKGROUND: This single-center preliminary prospective observational study used bedside ultrasound to assess the lung aeration modifications induced by recruitment maneuver and pronation in intubated patients with acute respiratory disease syndrome (ARDS) related to coronavirus 2019 disease (COVID-19). All adult intubated COVID-19 patients suitable for pronation were screened. After enrollment, patients underwent 1 h in a volume-controlled mode in supine position (baseline) followed by a 35-cmH2O-recruitment maneuver of 2 min (recruitment). Final step involved volume-controlled mode in prone position set as at baseline (pronation). At the end of the first two steps and 1 h after pronation, a lung ultrasound was performed, and global and regional lung ultrasound score (LUS) were analyzed. Data sets are presented as a median and 25th-75th percentile. RESULTS: From January to May 2022, 20 patients were included and analyzed. Global LUS reduced from 26.5 (23.5-30.0) at baseline to 21.5 (18.0-23.3) and 23.0 (21.0-26.3) at recruitment (p < 0.001) and pronation (p = 0.004). In the anterior lung regions, the regional LUS were 1.8 (1.1-2.0) following recruitment and 2.0 (1.6-2.2) in the supine (p = 0.008) and 2.0 (1.8-2.3) in prone position (p = 0.023). Regional LUS diminished from 2.3 (2.0-2.5) in supine to 2.0 (1.8-2.0) with recruitment in the lateral lung zones (p = 0.036). Finally, in the posterior lung units, regional LUS improved from 2.5 (2.3-2.8) in supine to 2.3 (1.8-2.5) through recruitment (p = 0.003) and 1.8 (1.3-2.2) with pronation (p < 0.0001). CONCLUSIONS: In our investigation, recruitment maneuver and prone positioning demonstrated an enhancement in lung aeration when compared to supine position, as assessed by bedside lung ultrasound. TRIAL REGISTRATION: www. CLINICALTRIALS: gov , Number NCT05209477, prospectively registered and released on 01/26/2022.

Recruitment of distinct UDP-glycosyltransferase families demonstrates dynamic evolution of chemical defense within Eucalyptus L'Hér.

The economic and ecologically important genus Eucalyptus is rich in structurally diverse specialized metabolites. While some specialized metabolite classes are highly prevalent across the genus, the cyanogenic glucoside prunasin is only produced by c. 3% of species. To investigate the evolutionary mechanisms behind prunasin biosynthesis in Eucalyptus, we compared de novo assembled transcriptomes, together with online resources between cyanogenic and acyanogenic species. Identified genes were characterized in vivo and in vitro. Pathway characterization of cyanogenic Eucalyptus camphora and Eucalyptus yarraensis showed for the first time that the final glucosylation step from mandelonitrile to prunasin is catalyzed by a novel UDP-glucosyltransferase UGT87. This step is typically catalyzed by a member of the UGT85 family, including in Eucalyptus cladocalyx. The upstream conversion of phenylalanine to mandelonitrile is catalyzed by three cytochrome P450 (CYP) enzymes from the CYP79, CYP706, and CYP71 families, as previously shown. Analysis of acyanogenic Eucalyptus species revealed the loss of different ortholog prunasin biosynthetic genes. The recruitment of UGTs from different families for prunasin biosynthesis in Eucalyptus demonstrates important pathway heterogeneities and unprecedented dynamic pathway evolution of chemical defense within a single genus. Overall, this study provides relevant insights into the tremendous adaptability of these long-lived trees.

A flavin-dependent monooxygenase catalyzes the initial step in cyanogenic glycoside synthesis in ferns.

Cyanogenic glycosides form part of a binary plant defense system that, upon catabolism, detonates a toxic hydrogen cyanide bomb. In seed plants, the initial step of cyanogenic glycoside biosynthesis-the conversion of an amino acid to the corresponding aldoxime-is catalyzed by a cytochrome P450 from the CYP79 family. An evolutionary conundrum arises, as no CYP79s have been identified in ferns, despite cyanogenic glycoside occurrence in several fern species. Here, we report that a flavin-dependent monooxygenase (fern oxime synthase; FOS1), catalyzes the first step of cyanogenic glycoside biosynthesis in two fern species (Phlebodium aureum and Pteridium aquilinum), demonstrating convergent evolution of biosynthesis across the plant kingdom. The FOS1 sequence from the two species is near identical (98%), despite diversifying 140 MYA. Recombinant FOS1 was isolated as a catalytic active dimer, and in planta, catalyzes formation of an N-hydroxylated primary amino acid; a class of metabolite not previously observed in plants.

Crystallographic and X-ray absorption spectroscopic characterization of Helicobacter pylori UreE bound to Ni²âº and Zn²âº reveals a role for the disordered C-terminal arm in metal trafficking.

The survival and growth of the pathogen Helicobacter pylori in the gastric acidic environment is ensured by the activity of urease, an enzyme containing two essential Ni²âº ions in the active site. The metallo-chaperone UreE facilitates in vivo Ni²âº insertion into the apoenzyme. Crystals of apo-HpUreE (H. pylori UreE) and its Ni⁺- and Zn⁺-bound forms were obtained from protein solutions in the absence and presence of the metal ions. The crystal structures of the homodimeric protein, determined at 2.00 Å (apo), 1.59 Å (Ni²âº) and 2.52 Å (Zn²âº) resolution, show the conserved proximal and solvent-exposed His¹°² residues from two adjacent monomers invariably involved in metal binding. The C-terminal regions of the apoprotein are disordered in the crystal, but acquire significant ordering in the presence of the metal ions due to the binding of His¹5². The analysis of X-ray absorption spectral data obtained using solutions of Ni²âº- and Zn²âº-bound HpUreE provided accurate information of the metal-ion environment in the absence of solid-state effects. These results reveal the role of the histidine residues at the protein C-terminus in metal-ion binding, and the mutual influence of protein framework and metal-ion stereo-electronic properties in establishing co-ordination number and geometry leading to metal selectivity.

Unraveling the Helicobacter pylori UreG zinc binding site using X-ray absorption spectroscopy (XAS) and structural modeling.

The pathogenicity of Helicobacter pylori depends on the activity of urease for pH modification. Urease activity requires assembly of a dinickel active site that is facilitated in part by GTP hydrolysis by UreG. The proper functioning of Helicobacter pylori UreG (HpUreG) is dependent on Zn(II) binding and dimerization. X-ray absorption spectroscopy and structural modeling were used to elucidate the structure of the Zn(II) site in HpUreG. These studies independently indicated a site at the dimer interface that has trigonal bipyramidal geometry and is composed of two axial cysteines at 2.29(2) Å, two equatorial histidines at 1.99(1) Å, and a solvent-accessible coordination site. The final model for the Zn(II) site structure was determined by refining multiple-scattering extended X-ray absorption fine structure fits using the geometry predicted by homology modeling and ab initio calculations.

Model structures of Helicobacter pylori UreD(H) domains: a putative molecular recognition platform.

The analysis of the sequence of Helicobacter pylori UreD(H), an accessory protein involved in the activation of urease through the assembly of the Ni(2+)-containing active site, revealed the presence of two domains. The structure of these domains was calculated using threading and modeling algorithms. A search for putative binding sites on the protein surface was carried out using dedicated algorithms sensitive to either sequence conservation or structural similarity based on geometry and physicochemical properties. The results suggest that UreD(H) acts as a multifunctional molecular recognition platform facilitating the interaction between apo-urease and the ancillary proteins UreG, UreF, and UreE, responsible for nickel trafficking and delivering.

Helicobacter pylori UreE, a urease accessory protein: specific Ni(2+)- and Zn(2+)-binding properties and interaction with its cognate UreG.

The persistence of Helicobacter pylori in the hostile environment of the human stomach is ensured by the activity of urease. The essentiality of Ni(2+) for this enzyme demands proper intracellular trafficking of this metal ion. The metallo-chaperone UreE promotes Ni(2+) insertion into the apo-enzyme in the last step of urease maturation while facilitating concomitant GTP hydrolysis. The present study focuses on the metal-binding properties of HpUreE (Helicobacter pylori UreE) and its interaction with the related accessory protein HpUreG, a GTPase involved in the assembly of the urease active site. ITC (isothermal titration calorimetry) showed that HpUreE binds one equivalent of Ni(2+) (Kd=0.15 microM) or Zn(2+) (Kd=0.49 microM) per dimer, without modification of the protein oligomeric state, as indicated by light scattering. Different ligand environments for Zn(2+) and Ni(2+), which involve crucial histidine residues, were revealed by site-directed mutagenesis, suggesting a mechanism for discriminating metal-ion-specific binding. The formation of a HpUreE-HpUreG protein complex was revealed by NMR spectroscopy, and the thermodynamics of this interaction were established using ITC. A role for Zn(2+), and not for Ni(2+), in the stabilization of this complex was demonstrated using size-exclusion chromatography, light scattering, and ITC experiments. A calculated viable structure for the complex suggested the presence of a novel binding site for Zn(2+), actually detected using ITC and site-directed mutagenesis. The results are discussed in relation to available evidence of a UreE-UreG functional interaction in vivo. A possible role for Zn(2+) in the Ni(2+)-dependent urease system is envisaged.

The Ni2+ binding properties of Helicobacter pylori NikR.

The binding constants between Ni2+ and Helicobacterpylori NikR have been determined using isothermal titration microcalorimetry in order to rationalize the role of this protein as a nickel-dependent biological sensor.