Peg 6000

A 16–20 h culture of C. difficile LIBA-5763 in BHI broth (Oxoid) was induced with mitomycin C (3 μg/mL, Roche) and the supernatant was filtered through a 0.22 μm membrane filter. This filtrate was tested for plaque production on six C. difficile isolates from human and animal sources (Supplementary Table 2). Log (3–4 h) and stationary phase (18–20 h) cultures were used as previously described (Goh et al., 2005 (link)). Single plaques were propagated on susceptible host isolates to obtain crude phage suspensions of ~1 × 10⁹ PFU/ml, which were semi-purified for electron microscopy and DNA extraction (Goh et al., 2005 (link)). Semi purification of phage involved removal of contaminating host nucleic acid by digestion with DNase I (100 μg/mL, Thermo Scientific) and RNase A (100 μg/mL, Roche) at 37°C for 30 min, overnight concentration of phage particles with PEG 6000 (10% w/v, Sigma), and 1 M NaCl (Sigma) at 4°C, and release of phage from PEG 6000 with 1 M KCl (Sigma). Phage particles were resuspended in phage buffer [0.15 M NaCl, 10 mM Tris (pH 6.5), 10 mM MgSO₄, 1 mM CaCl₂]_.

For the PEGylation, PEG 3000 (for molecular biology; Sigma Aldrich, Darmstadt, Germany) was available in monodisperse solution, while PEG 6000 (for synthesis) and PEG 8000 (for synthesisUSP) (Sigma Aldrich, Budapest, Hungary) were commercially sold in solid form. At first, these power-based agents were dissolved in 50% ethanol–water mixture the final PEG content was set to 10 w/w%.
PBNP-MB solutions (2 mg/mL) were prepared by adding distilled water (Milli-Q) to the stock MB-labelled PBNP solution. After 15 min incubation time at room temperature, different PEG solutions (PEG 3000, PEG 6000, and PEG 8000) were added to the PBNP-MB solutions and dialyzed for 24 h (14 kDa filter) (Sigma-Aldrich, Budapest, Hungary) in phosphate buffer saline solution (pH = 6.8; Ph.Eur. 8.). Different v/v% concentration compositions (PEG 3000: 1.47-1.96-2.44-3.85-9.09, PEG 6000: 1.47-1.96-2.44-3.85, and PEG 8000: 1.47-1.96-2.44) were prepared and characterized in the following with DLS and Zetasizer instruments. Other details are available in the Supplemental Information.

In vitro plants of Beauregard (CIP440132) and Tanzania (CIP440166) were obtained from the International Potato Center (CIP) Genebank and nodes were cultured to form plantlets on MPB medium (Murashige and Skoog salts, 3% sucrose, 2 mg/L calcium pantothenate, 100 mg/L l‐arginine, 200 mg/L ascorbic acid, 20 mg/L putrescine‐HCl, 10 mg/L GA₃, 0.3% Phytagel, pH 5.7, autoclaved at 121°C for 15 min as recommended by the supplier) in culture tubes (three per tube). Shoot cultures were kept in a growth room at 27 ± 2°C, 3,000 lx, and 12 hr light/12 hr dark.
Polyethylene glycol (PEG 6000, Sigma) concentrations were chosen to create osmotic pressure levels between field capacity (−0.033 mpa) and permanent wilting (−1.5 mpa), as estimated based on osmotic pressure values reported for different PEG 6000 concentrations (http://www.plantstress.com/methods/peg.htm; Michel & Kaufmann, 1973). After 21 days of growth in solid MPB media, the plants were transferred to 35 ml of 15%, 20%, and 25% PEG6000 liquid MPB media, representing osmotic pressure values of −0.295, −0.491, and −0.735 mpa, respectively. Three replicates were used. Cultures were maintained in a growth chamber under the conditions described above and stress symptoms were evaluated at 4, 24, 48 hr, and 21 days after stress application.

For PEG precipitation human sera were mixed with PEG 6000 (Sigma‐Aldrich) in PBS at a final concentration of 10% PEG 6000. After overnight incubation at 4°C, ICs were precipitated by centrifugation at 2,000 g for 30 min at 4°C, pellets were washed once with PEG 6000 and then centrifuged at 2,000 g for 20 min at 4°C. Supernatants were harvested and precipitates re‐suspended in pre‐warmed PBS for 1 h at 37°C. IgG concentrations of serum, precipitates, and supernatants obtained after precipitation were quantified by Nanodrop (Thermo Scientific™) measurement.

PEG 6000, (Sigma-Aldrich) coated SPIONs samples were prepared using ex situ coating technicality. Here, PEG 6000 (Sigma-Aldrich) was used as a dispersing agent to prevent the aggregation of nanoparticles [25 (link)]. Briefly, different loading concentrations (25, 50, 100, 500, 1000, 1500, 2000, and 2500 mg/L) of PEG 6000 dissolved in 10 mL water were sonicated with 0.2 g of the suspended SPIONs for 20 min. The mixed solution was shacked for 24 h at room temperature. After shaking and agitation, the suspended yield was separated using an external magnet and then washed with water and ethanol. The synthesized PEG functionalized Fe₃O₄ (PEG-coated SPIONs) was dried at 80°C for 6 h.