For pharmacokinetic experiments, an N-terminal selenomethionine probe was attached to Ω76. The peptide’s MBC against A. baumannii (table S5) remained unaffected by this alteration.

Six- to 8-week-old BALB/c mice (20 g weight) were used for these experiments. Mice were anesthetized with 2 mg of ketamine/0.16 mg of xylazine suspended in 200 μl of saline and injected intraperitoneally. Three types of samples were collected: (i) Cardiac punctures on anesthetized but untreated mice were first performed to assay baseline serum selenium content. (ii) Calibration controls were created by mixing known quantities of Nselmet-Ω76 into untreated mouse blood extracted via cardiac puncture. (iii) Later, mice were injected with Nselmet-Ω76 (70 mg/kg; corresponding to an Ω76 dose of 64 mg/kg), and cardiac punctures were performed at chosen time points in a 0- to 10-min range. It should be noted that, because of the difficulty involved in locating the mouse heart, time points could not be evenly sampled. Once blood was drawn from the heart, the time point for that sample was taken to be the mean time between the start and end of blood collection.

All blood samples were collected in clotting Vacutainer tubes (yellow top). After allowing the blood to clot for 1 hour, the serum from all blood samples was extracted by centrifuging at 6000 rpm/20 min and weighed out. Typically, 200 to 800 μl of blood were obtained from each mouse, which corresponded to 100 to 400 μl of serum. The mass for each sample was made up to 500 mg by diluting in high-performance liquid chromatography (HPLC)–grade DW. All samples were sent to Ramaiah Advanced Testing Lab (Bangalore) for quantification of selenium via ICP-MS. The serum concentration of Nselmet-Ω76 was then back-calculated from elemental selenium concentrations.

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