High Risk of Secondary Infections Following Thrombotic Complications in Patients With COVID-19

Background. This study’s primary aim was to evaluate the impact of thrombotic complications on the development of secondary infections. The secondary aim was to compare the etiology of secondary infections in patients with and without thrombotic complications. Methods. This was a cohort study (NCT04318366) of coronavirus disease 2019 (COVID-19) patients hospitalized at IRCCS San Raffaele Hospital between February 25 and June 30, 2020. Incidence rates (IRs) were calculated by univariable Poisson regression as the number of cases per 1000 person-days of follow-up (PDFU) with 95% confidence intervals. The cumulative incidence functions of secondary infections according to thrombotic complications were compared with Gray’s method accounting for competing risk of death. A multivariable Fine-Gray model was applied to assess factors associated with risk of secondary infections. Results. Overall, 109/904 patients had 176 secondary infections (IR, 10.0; 95% CI, 8.8–11.5; per 1000-PDFU). The IRs of secondary infections among patients with or without thrombotic complications were 15.0 (95% CI, 10.7–21.0) and 9.3 (95% CI, 7.9–11.0) per 1000-PDFU, respectively (P = .017). At multivariable analysis, thrombotic complications were associated with the development of secondary infections (subdistribution hazard ratio, 1.788; 95% CI, 1.018–3.140; P = .043). The etiology of secondary infections was similar in patients with and without thrombotic complications. Conclusions. In patients with COVID-19, thrombotic complications were associated with a high risk of secondary infections

Description of the topographical changes associated to the different stages of the DsbA catalytic cycle

This paper provides a description of the surface topography of DsbA, the bacterial disulfide-bond forming enzyme, in the different phases of its catalytic cycle. Three representative states, that is, oxidized and reduced protein and a covalent complex mimicking the DsbA-substrate disulfide intermediate, have been investigated by a combination of limited proteolysis experiments and mass spectrometry methodologies. Protease-accessible sites are largely distributed in the oxidized form with a small predominance inside the thioredoxin domain. Proteolysis occurs even in secondary structure elements, revealing a significant mobility of the protein. Many cleavage sites disappear in the reduced form and most of the remaining ones appear with strongly reduced kinetics. The protein within the complex shows an intermediate behavior. This variation of flexibility in DsbA is probably the determining factor for the course of its catalytic cycle. In particular, the great mobility of the oxidized protein might facilitate the accommodation of its various substrates, whereas the increasing rigidity from the complexed to the reduced form could help the release of oxidized products. The formation of the complex between PID peptide and DsbA does not significantly protect the enzyme against proteolysis, reinforcing the results previously obtained by calorimetry concerning the weakness of their interaction. The few cleavage sites observed, however, are in favor of the presence of the peptide in the binding site postulated from crystallographic studies. As for the peptide itself, the proteolytic pattern and the protection effect exerted by DsbA could be explained by a preferential orientation within the binding site

Investigation of the DsbA Mechanism through the Synthesis and Analysis of an Irreversible Enzyme−Ligand Complex

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Early intermediates in the PDI-assisted folding of ribonuclease A

The oxidative refolding of ribonuclease A has been investigated in several experimental conditions using a variety of redox systems. All these studies agree that the formation of disulfide bonds during the process occurs through a nonrandom mechanism with a preferential coupling of certain cysteine residues. We have previously demonstrated that in the presence of glutathione the refolding process occurs through the reiteration of two sequential reactions: a mixed disulfide with glutathione is produced first which evolves to form an intramolecular S-S bond. In the same experimental conditions, protein disulfide isomerase (PDI) was shown to catalyze formation and reduction of mixed disulfides with glutathione as well as formation of intramolecular S-S bonds. This paper reports the structural characterization of the one-disulfide intermediate population during the oxidative refolding of Ribonuclease A under the presence of PDI and glutathione with the aim of defining the role of the enzyme at the early stages of the reaction. The one-disulfide intermediate population occurring at the early stages of both the uncatalyzed and the PDI-catalyzed refolding was purified and structurally characterized by proteolytic digestion followed by MALDI-MS and LC/ESIMS analyses. In the uncatalyzed refolding, a total of 12 disulfide bonds out of the 28 theoretical possible cysteine couplings was observed, confirming a nonrandom distribution of native and nonnative disulfide bonds. Under the presence of PDT, only two additional nonnative disulfides were detected. Semiquantitative LC/ESIMS analysis of the distribution of the S-S bridged peptides showed that the most abundant species were equally populated in both the uncatalyzed and the catalyzed process. This paper shows the first structural characterization of the one-disulfide intermediate population formed transiently during the refolding of ribonuclease A in quasi-physiological conditions that mimic those present in the ER lumen. At the early stages of the process, three of the foul native disulfides are detected, whereas the Cys26-Cys84 pairing is absent. Most of the nonnative disulfide bonds identified are formed by nearest-neighboring cysteines. The presence of PDI does not significantly alter the distribution of S-S bonds, suggesting that the ensemble of single-disulfide species is formed under thermodynamic control

Aspartate aminotransferase from the anctarctic bacterium Pseudolteromonas haloplanktis TAC 125. Clonong, expression, properties, and molecular modelling.

The gene encoding aspartate aminotransferase from the psychrophilic bacterium Pseudoalteromonas haloplanktis TAC 125 was cloned, sequenced and overexpressed in Escherichia coli. The recombinant protein (PhAspAT) was characterized both at the structural and functional level in comparison with the E. coli enzyme (EcAspAT), which is the most closely related (52% sequence identity) bacterial counterpart. PhAspAT is rapidly inactivated at 50 degrees C (half-life = 6.8 min), whereas at this temperature EcAspAT is stable for at least 3 h. The optimal temperature for PhAspAT activity is approximate to 64 degrees C, which is some 11 degrees C below that of EcAspAT. The protein thermal stability was investigated by following changes in both tryptophan fluorescence and amide ellipticity; this clearly suggested that a first structural transition occurs at approximate to 50 degrees C for PhAspAT. These results agree with the expected thermolability of a psychrophilic enzyme, although the observed stability is much higher than generally found for enzymes isolated from cold-loving organisms. Furthermore, in contrast with the higher efficiency exhibited by several extracellular psychrophilic enzymes, both k(cat) and k(cat)/K-m of PhAspAT are significantly lower than those of EcAspAT over the whole temperature range. This behaviour possibly suggests that the adaptation of this class of endocellular enzymes to a cold environment may have only made them less stable and not more efficient. The affinity of PhAspAT for both amino-acid and 2-oxo-acid substrates decreases with increasing temperature. However, binding of maleate and 2-methyl-L-aspartate, which both inhibit the initial steps of catalysis, does not change over the temperature range tested. Therefore, the observed temperature effect may occur at any of the steps of the catalytic mechanism after the formation of the external aldimine. A molecular model of PhAspAT was constructed on the basis of sequence homology with other AspATs. Interestingly, it shows no insertion or extension of loops, but some cavities and a decrease in side chain packing can be observed

Aspartate aminotransferase from the anctarctic bacterium Pseudolteromonas haloplanktis TAC 125. Clonong, expression, properties, and molecular modelling.

The gene encoding aspartate aminotransferase from the psychrophilic bacterium Pseudoalteromonas haloplanktis TAC 125 was cloned, sequenced and overexpressed in Escherichia coli. The recombinant protein (PhAspAT) was characterized both at the structural and functional level in comparison with the E. coli enzyme (EcAspAT), which is the most closely related (52% sequence identity) bacterial counterpart. PhAspAT is rapidly inactivated at 50 degrees C (half-life = 6.8 min), whereas at this temperature EcAspAT is stable for at least 3 h. The optimal temperature for PhAspAT activity is approximate to 64 degrees C, which is some 11 degrees C below that of EcAspAT. The protein thermal stability was investigated by following changes in both tryptophan fluorescence and amide ellipticity; this clearly suggested that a first structural transition occurs at approximate to 50 degrees C for PhAspAT. These results agree with the expected thermolability of a psychrophilic enzyme, although the observed stability is much higher than generally found for enzymes isolated from cold-loving organisms. Furthermore, in contrast with the higher efficiency exhibited by several extracellular psychrophilic enzymes, both k(cat) and k(cat)/K-m of PhAspAT are significantly lower than those of EcAspAT over the whole temperature range. This behaviour possibly suggests that the adaptation of this class of endocellular enzymes to a cold environment may have only made them less stable and not more efficient. The affinity of PhAspAT for both amino-acid and 2-oxo-acid substrates decreases with increasing temperature. However, binding of maleate and 2-methyl-L-aspartate, which both inhibit the initial steps of catalysis, does not change over the temperature range tested. Therefore, the observed temperature effect may occur at any of the steps of the catalytic mechanism after the formation of the external aldimine. A molecular model of PhAspAT was constructed on the basis of sequence homology with other AspATs. Interestingly, it shows no insertion or extension of loops, but some cavities and a decrease in side chain packing can be observed