This version of the article has been accepted for publication, after peer review (when applicable) and is subject to Springer Nature's AM terms of use, but is not the Version of Record and does not reflect post-acceptance improvements, or any corrections.Nonribosomal depsipeptides are natural products composed of amino and hydroxy acid residues. The hydroxy acid residues often derive from α-keto acids, reduced by ketoreductase domains in the depsipeptide synthetases. Biochemistry and structures reveal the mechanism of discrimination for α-keto acids and a remarkable architecture: flanking intact adenylation and ketoreductase domains are sequences separated by >1100 residues that form a split "pseudoAsub" domain, structurally important for the depsipeptide module's synthetic cycle.Nonribosomal peptide synthetases (NRPSs) are multi-domain enzymes that produce a vast array of biologically-active compounds, including clinically-used therapeutics such as the antibiotic daptomycin, the immunosuppressant cyclosporin and the antifungal caspofungin 1 . NRPSs are composed of a series of modules, sets of domains that work together to add a building block substrate to the growing peptide chain, in a manner analogous to assembly lines.Depsipeptides contain both amino acid residues and hydroxy acid residues, linked by amide and ester bonds, respectively. Important depsipeptides include the piscicide antimycin, the K+ ionophores cereulide 2,3 and valinomycin 4,5 (Supplementary Fig. 1), the anticancer agent cryptophycin 6 , the antimicrobial kutzneride 7 , the antifungal hectochlorin 8 and the insecticide mycotoxins bassianolide and beauvericin 9 . The hydroxy acid residues in fungal compounds bassianolide and beauvericin originate from direct selection and incorporation of α-hydroxy acids, but the hydroxy acid residues in the bacterial compounds antimycin, hectochlorin, kutzneride, cryptophycin, cereulide and valinomycin are derived from α-keto acid substrates 10 .In bacterial depsipeptide synthetases, modules responsible for adding hydroxy acids include condensation (C) (if an elongation module), adenylation (A), ketoreductase (KR), and peptidyl carrier protein (PCP) domains (Fig. 1a and Supplementary Fig. 1) [11][12][13][14] . These A domains select α-keto acids using a hitherto unknown mechanism to differentiate them from α-amino and α-hydroxy acids.Intriguingly, the aspartate which contacts the α-amino group in amino acid-selecting A domains is altered to a hydrophobic residue in α-keto acid-selecting A domains, not to a positive or polar residue 7,10,11 . Depsipeptide synthetase A domains adenylate the α-keto acid, then transfer it to the PCP domain. The PCP domain transports the α-keto acyl moiety to the KR domain for stereoselective reduction. After reduction, the α-hydroxyl acyl-PCP moves to that module's C domain for condensation, making an ester bond (or goes directly to the downstream C domain in the case of A-KR-PCP initiation modules), and synthesis continues as in the canonical case.Ketoreducing depsipeptide modul...

New lineages of SARS-CoV-2 are of potential concern due to higher transmissibility, risk of severe outcomes, and/or escape from neutralizing antibodies. Lineage B.1.1.7 (the Alpha variant) became dominant in early 2021, but the association between transmissibility and risk factors, such as age of primary case and viral load remains poorly understood. Here, we used comprehensive administrative data from Denmark, comprising the full population (January 11 to February 7, 2021), to estimate household transmissibility. This study included 5,241 households with primary cases; 808 were infected with lineage B.1.1.7 and 4,433 with other lineages. Here, we report an attack rate of 38% in households with a primary case infected with B.1.1.7 and 27% in households with other lineages. Primary cases infected with B.1.1.7 had an increased transmissibility of 1.5–1.7 times that of primary cases infected with other lineages. The increased transmissibility of B.1.1.7 was multiplicative across age and viral load.

Key Points• Anoxia combined with glucose supplementation maintains viability of neutrophils for 20 hours without affecting their functions.• Such conditioned neutrophils are suitable for efficient DNA transfection and transfusion.Functional studies of human neutrophils and their transfusion for clinical purposes have been hampered by their short life span after isolation. Here, we demonstrate that neutrophil viability is maintained for 20 hours in culture media at 37°C under anoxic conditions with 3 mM glucose and 32 mg/mL dimethyloxalylglycine supplementation, as evidenced by stabilization of Mcl-1, proliferating cell nuclear antigen (PCNA), and pro-caspase-3. Notably, neutrophil morphology (nucleus shape and cell-surface markers) and functions (phagocytosis, degranulation, calcium release, chemotaxis, and reactive oxygen species production) were comparable to blood circulating neutrophils. The observed extension in neutrophil viability was reversed upon exposure to oxygen. Extending neutrophil life span allowed efficient transfection of plasmids (40% transfection efficiency) and short interfering RNA (interleukin-8, PCNA, and Bax), as a validation of effective and functional genetic manipulation of neutrophils both in vitro and in vivo. In vivo, transfusion of conditioned neutrophils in a neutropenic guinea pig model increased bacterial clearance of Shigella flexneri upon colonic infection, strongly suggesting that these conditioned neutrophils might be suitable for transfusion purposes. In summary, such conditioning of neutrophils in vitro should facilitate their study and offer new opportunities for genetic manipulation and therapeutic use. (Blood. 2016;128(7):993-1002)

In early 2021, the SARS-CoV-2 lineage B.1.1.7 became dominant across large parts of the world. In Denmark, comprehensive and real-time test, contact-tracing, and sequencing efforts were applied to sustain epidemic control. Here, we use these data to investigate the transmissibility, introduction, and onward transmission of B.1.1.7 in Denmark. In a period with stable restrictions, we estimated an increased B.1.1.7 transmissibility of 58% (95% CI: [56%,60%]) relative to other lineages. Epidemiological and phylogenetic analyses revealed that 37% of B.1.1.7 cases were related to the initial introduction in November 2020. Continuous introductions contributed substantially to case numbers, highlighting the benefit of balanced travel restrictions and self-isolation procedures coupled with comprehensive surveillance efforts, to sustain epidemic control in the face of emerging variants.