Factors associated with prolonged duration of viral clearance in non-severe SARS-CoV-2 patients in Osaka, Japan.

BACKGROUND: We investigated factors associated with prolonged viral clearance of SARS-CoV-2 among non-severe adult patients in Osaka, Japan. A total of 706 laboratory-confirmed COVID-19 patients were enrolled in this longitudinal observational study between 29 January 2020 and 31 May 2020, across 62 hospitals and three non-hospital recuperation facilities. METHODS: Logistic regression analysis was performed to investigate the factors associated with prolonged (29 days: upper 25% in duration) viral clearance of SARS-CoV-2. Linear regression analysis was conducted to assess these factors 14 days after symptom onset. RESULTS: The median duration of viral clearance was 22 days from symptom onset. After adjustment for sex, age, symptoms, comorbidity, and location of recuperation, comorbidities were associated with prolonged duration: (OR, 1.77 [95% CI, 1.11-2.82]) for one, (OR, 2.47 [95% CI, 1.32-4.61]) for two or more comorbidities. Viral clearance 14 days after symptom onset was 3 days longer for one comorbidity and 4 days longer for two or more comorbidities compared to clearance when there was no comorbidity. CONCLUSION: The presence of comorbidity was a robust factor associated with a longer duration of viral clearance, extending by 3 to 4 days compared to patients with no comorbidity.

Crystal structure of LysK, an enzyme catalyzing the last step of lysine biosynthesis in Thermus thermophilus, in complex with lysine: Insight into the mechanism for recognition of the amino-group carrier protein, LysW.

LysK is an M20 peptidase family enzyme that hydrolyzes the isopeptide bond between the carrier protein LysW and lysine in order to release lysine, which is the last step of lysine biosynthesis in Thermus thermophilus. In the present study, we determined the crystal structure of LysK in complex with lysine at a resolution of 2.4 Å. The α-amino group of the bound lysine was oriented toward the catalytic center, which was composed of the residues coordinating divalent metal ions for the hydrolysis of the isopeptide bond. An 11 Å-long path was observed from the active site binding lysine to the protein surface, which may be responsible for recognizing the C-terminal extension domain of LysW with the conserved EDWGE sequence. A positively-charged surface region was detected around the exit of the path, similar to other lysine biosynthetic enzymes using LysW as the carrier protein. Mutational studies of the surface residues provided a plausible model for the electrostatic interaction with LysW.

The characteristics and clinical course of patients with COVID-19 who received invasive mechanical ventilation in Osaka, Japan.

OBJECTIVE: To describe the detailed clinical course of patients with coronavirus disease 2019 (COVID-19) who received invasive mechanical ventilation. METHODS: We conducted a case series of patients with COVID-19 who received invasive mechanical ventilation in Osaka, Japan, between January 29 and May 28, 2020. We describe the patient characteristics and clinical course from onset. Additionally, we fitted logistic regression models to investigate the associations between patient characteristics and the 30-day mortality rate. RESULTS: A total of 125 patients who received invasive mechanical ventilation (median age [interquartile range], 68 [57-73] years; male, 77.6%) were enrolled. Overall, the 30-day mortality was 24.0%, and the median (interquartile range) length of ICU stay and length of invasive mechanical ventilation use were 16 (12-29) days and 13 (9-26) days, respectively. From clinical onset, 121 patients (96.8%) were intubated within 14 days. In multivariable logistic regression analysis, age of 65 years or older (odds ratio, 3.56; 95% confidence interval, 1.21-10.49; P = 0.02) and male sex (odds ratio, 3.75; 95% confidence interval, 1.00-11.24, P = 0.04) were significantly associated with a higher 30-day mortality rate. CONCLUSIONS: In this case series of patients with COVID-19 who received invasive mechanical ventilation in Japan, the 30-day mortality rate was 24.0%, and age 65 years or older and male sex were associated with higher 30-day mortality rate.

Arabidopsis CYP90B1 catalyses the early C-22 hydroxylation of C27, C28 and C29 sterols.

Arabidopsis dwf4 is a brassinosteroid (BR)-deficient mutant, and the DWF4 gene encodes a cytochrome P450, CYP90B1. We report the catalytic activity and substrate specificity of CYP90B1. Recombinant CYP90B1 was produced in Escherichia coli, and CYP90B1 activity was measured in an in vitro assay reconstituted with NADPH-cytochrome P450 reductase. CYP90B1 converted campestanol (CN) to 6-deoxocathasterone, confirming that CYP90B1 is a steroid C-22 hydroxylase. The substrate specificity of CYP90B1 indicated that sterols with a double bond at positions C-5 and C-6 are preferred substrates compared with stanols, which have no double bond at the position. In particular, the catalytic efficiency (k(cat)/K(m)) of CYP90B1 for campesterol (CR) was 325 times greater than that for CN. As CR is more abundant than CN in planta, the results suggest that C-22 hydroxylation of CR before C-5alpha reduction is the main route of BR biosynthetic pathway, which contrasts with the generally accepted route via CN. In addition, CYP90B1 showed C-22 hydroxylation activity toward various C(27-29) sterols. Cholesterol (C27 sterol) is the best substrate, followed by CR (C28 sterol), whereas sitosterol (C29 sterol) is a poor substrate, suggesting that the substrate preference of CYP90B1 may explain the discrepancy between the in planta abundance of C27/C28/C29 sterols and C27/C28/C29 BRs.

C-23 hydroxylation by Arabidopsis CYP90C1 and CYP90D1 reveals a novel shortcut in brassinosteroid biosynthesis.

Brassinosteroids (BRs) are biosynthesized from campesterol via several cytochrome P450 (P450)-catalyzed oxidative reactions. We report the functional characterization of two BR-biosynthetic P450s from Arabidopsis thaliana: CYP90C1/ROTUNDIFOLIA3 and CYP90D1. The cyp90c1 cyp90d1 double mutant exhibits the characteristic BR-deficient dwarf phenotype, although the individual mutants do not display this phenotype. These data suggest redundant roles for these P450s. In vitro biochemical assays using insect cell-expressed proteins revealed that both CYP90C1 and CYP90D1 catalyze C-23 hydroxylation of various 22-hydroxylated BRs with markedly different catalytic efficiencies. Both enzymes preferentially convert 3-epi-6-deoxocathasterone, (22S,24R)-22-hydroxy-5alpha-ergostan-3-one, and (22S,24R)-22-hydroxyergost-4-en-3-one to 23-hydroxylated products, whereas they are less active on 6-deoxocathasterone. Likewise, cyp90c1 cyp90d1 plants were deficient in 23-hydroxylated BRs, and in feeding experiments using exogenously supplied intermediates, only 23-hydroxylated BRs rescued the growth deficiency of the cyp90c1 cyp90d1 mutant. Thus, CYP90C1 and CYP90D1 are redundant BR C-23 hydroxylases. Moreover, their preferential substrates are present in the endogenous Arabidopsis BR pool. Based on these results, we propose C-23 hydroxylation shortcuts that bypass campestanol, 6-deoxocathasterone, and 6-deoxoteasterone and lead directly from (22S,24R)-22-hydroxy-5alpha-ergostan-3-one and 3-epi-6-deoxocathasterone to 3-dehydro-6-deoxoteasterone and 6-deoxotyphasterol.