ABSTRACT
CT protocols that diagnose COVID-19 vary in regard to the associated radiation exposure and the desired image quality (IQ). This study aims to evaluate CT protocols of hospitals participating in the RACOON (Radiological Cooperative Network) project, consolidating CT protocols to provide recommendations and strategies for future pandemics. In this retrospective study, CT acquisitions of COVID-19 patients scanned between March 2020 and October 2020 (RACOON phase 1) were included, and all non-contrast protocols were evaluated. For this purpose, CT protocol parameters, IQ ratings, radiation exposure (CTDIvol), and central patient diameters were sampled. Eventually, the data from 14 sites and 534 CT acquisitions were analyzed. IQ was rated good for 81% of the evaluated examinations. Motion, beam-hardening artefacts, or image noise were reasons for a suboptimal IQ. The tube potential ranged between 80 and 140 kVp, with the majority between 100 and 120 kVp. CTDIvol was 3.7 ± 3.4 mGy. Most healthcare facilities included did not have a specific non-contrast CT protocol. Furthermore, CT protocols for chest imaging varied in their settings and radiation exposure. In future, it will be necessary to make recommendations regarding the required IQ and protocol parameters for the majority of CT scanners to enable comparable IQ as well as radiation exposure for different sites but identical diagnostic questions.
ABSTRACT
Structurally different and functionally diverse natural compounds - antitumour agents pyrrolo[1,4]benzodiazepines, bacterial hormone hormaomycin, and lincosamide antibiotic lincomycin - share a common building unit, 4-alkyl-L-proline derivative (APD). APDs arise from L-tyrosine through a special biosynthetic pathway. Its generally accepted scheme, however, did not comply with current state of knowledge. Based on gene inactivation experiments and in vitro functional tests with recombinant enzymes, we designed a new APD biosynthetic scheme for the model of lincomycin biosynthesis. In the new scheme at least one characteristic in each of five final biosynthetic steps has been changed: the order of reactions, assignment of enzymes and/or reaction mechanisms. First, we demonstrate that LmbW methylates a different substrate than previously assumed. Second, we propose a unique reaction mechanism for the next step, in which a putative γ-glutamyltransferase LmbA indirectly cleaves off the oxalyl residue by transient attachment of glutamate to LmbW product. This unprecedented mechanism would represent the first example of the C-C bond cleavage catalyzed by a γ-glutamyltransferase, i.e., an enzyme that appears unsuitable for such activity. Finally, the inactivation experiments show that LmbX is an isomerase indicating that it transforms its substrate into a compound suitable for reduction by LmbY, thereby facilitating its subsequent complete conversion to APD 4-propyl-L-proline. Elucidation of the APD biosynthesis has long time resisted mainly due to the apparent absence of relevant C-C bond cleaving enzymatic activity. Our proposal aims to unblock this situation not only for lincomycin biosynthesis, but generally for all above mentioned groups of bioactive natural products with biotechnological potential.
ABSTRACT
The immediate post-condensation steps in lincomycin biosynthesis are reminiscent of the mycothiol-dependent detoxification system of actinomycetes. This machinery provides the last proven lincomycin intermediate, a mercapturic acid derivative, which formally represents the 'waste product' of the detoxification process. We identified and purified new lincomycin intermediates from the culture broth of deletion mutant strains of Streptomyces lincolnensis and tested these compounds as substrates for proteins putatively involved in lincomycin biosynthesis. The results, based on LC-MS, in-source collision-induced dissociation mass spectrometry and NMR analysis, revealed the final steps of lincomycin biosynthesis, i.e. conversion of the mercapturic acid derivative to lincomycin. Most importantly, we show that deacetylation of the N'-acetyl-S-cysteine residue of the mercapturic acid derivative is required to 'escape' the detoxification-like system and proceed towards completion of the biosynthetic pathway. Additionally, our results, supported by l-cysteine-13C3, 15N incorporation experiments, give evidence that a different type of reaction catalysed by the homologous pair of pyridoxal-5'-phosphate-dependent enzymes, LmbF and CcbF, forms the branch point in the biosynthesis of lincomycin and celesticetin, two related lincosamides.
