Dna lobind 1.5 ml tubes

PMAxx^TM (Biotium) was dissolved in water to obtain 4 mM solution and stored protected from light at −20°C. Platinum (IV) chloride (PtCl₄) (Acros Organics, Morris Plains, NJ, United States), was dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich) at 50 mM concentration and stored at −20°C for later use.
Both PMAxx^TM and PtCl₄ were initially evaluated on HEV RNA purified using the NucleoSpin^® RNA virus kit. In particular, PMAxx (1.9 μl) at 50, 100 and 250 μM was initially incubated with HEV RNA (150 μl) in DNA LoBind 1.5 ml tubes (Eppendorf) at room temperature (RT) for 10 min in a shaker at 150 rpm. Then, samples were immediately exposed to 15 min photoactivation using a photo-activation system (Led-Active Blue, GenIUL). Similarly, purified HEV RNA (150 μl) was incubated with PtCl₄ (1.5 μl) at 50, 100, 500, and 1000 μM in DNA LoBind 1.5 ml tubes at 5°C for 30 min in a shaker at 150 rpm (Fraisse et al., 2018 (link)). Each experiment was performed in triplicate. HEV RNA (150 μL) without viability marker was used as a positive control. After viability pretreatments, RNA was purified again using the NucleoSpin^® RNA virus and quantified by RT-qPCR as reported above.

The determination of 260 nm absorbing material leakage from microbial cells induced by the peptides was based on a method described by Carson et al. [92 (link)] and Yasir et al. [93 (link)]. Microbial cell suspensions were prepared as described above, in Section 4.6.1. A total of 200 μL of these microbial suspensions were added to PCR grade DNA LoBind^® 1.5 mL tubes (Eppendorf, Hamburg, Germany), containing 200 μL of peptides, diluted in sterile saline solution. Final inocula were of 1 × 10⁸ CFU/mL and MICs and MBCs or MFCs (or 2x MICs when peptides had no bactericidal or fungicidal activity) were tested for peptide analogs 1, 5, 6, 7, and 8. 50 μL of these mixtures were withdrawn immediately after the addition of the cell suspensions to the peptide solutions (T0), diluted in saline solution (1:10), and filtered through 0.22 μM cellulose acetate filters. A total of 200 μL of these filtrates were then added in UV-STAR^® UV-transparent microplates (Greiner Bio-One, Frickenhausen, Germany). The same procedure was done after an incubation time of 120 min at room temperature. The 260 nm absorbance values were measured with a BioTek Synergy H1 spectrophotometer (Agilent, Santa Clara, CA, USA). Three independent repetitions, including three technical replicates, were conducted for each microorganism and peptide concentration.