Direct q uv5

Approximately 1 g of lichen thalli from each sampling site was washed using autoclaved Milli-Q ultrapure water from Direct-Q UV 5 (Merck Millipore, Burlington, MA, USA). After washing, the lichen thalli were cut into small pieces using flame-sterilized scissors and ground using an autoclaved mortar and pestle. Bulk DNA was extracted from the ground thalli by bead-beating using the ISOIL Large for Beads ver.2 (Nippon Gene, Tokyo, Japan) and precipitated in 70% ethanol with the precipitation-facilitator Ethachinmate (Nippon Gene, Tokyo, Japan) [29 (link)]. The DNA precipitate was resuspended in sterilized ultrapure water, assessed for purity and quantity using a NanoDrop 2000c (Thermo Fisher Scientific, Waltham, MA, USA) and stored at −20 °C until PCR amplification.

All
reagents and solvents were purchased
from Merck, Kanto Chemical, Tokyo Kasei, or FUJIFILM Wako Pure Chemical
Corporation. Anhydrous dimethylacetamide (DMAc) and methanol were
used as solvents for the synthesis of polymers and solvent casting,
respectively. Telechelic poly(ethylene glycol) (M_n = 2000 g/mol) and 2,2-bis(hydroxymethyl)propionic acid
were dried in vacuo at 100 °C for 1 h before use. Isophorone
diisocyanate was distilled under a vacuum and stored over molecular
sieves at 4 °C. Isophorone diamine was dried over molecular sieves
at r.t. for 1 day before use. Deionized water for hydrogel preparation
was obtained using a Merck Direct-Q UV5.

To prepare the 2% CyA ophthalmic solution, Sandimmun^® Oral Solution 10% (Novartis, Switzerland) has been used [7 (link), 8 (link)]. Therefore, we purchased Sandimmun^® Oral Solution 10% from Novartis Pharma K.K. (Japan). Olive oil was purchased from KENEI Pharmaceutical Co., Ltd. (Japan) because pure olive oil is often used for diluting Sandimmun^® Oral Solution from 10 to 2% [3 (link)]. The purchased olive oil was sterilized using dry-heat sterilization. For filtering the prepared 2% CyA ophthalmic solution, we used the 0.45-µm membrane filter (Millex^®-HV Syringe Filter Unit, 0.45 μm, PVDF, 33 mm, gamma sterilized; Merck Millipore Ltd., Ireland). To measure the concentration of CyA, Ecrusis Cyclosporine was purchased from Roche Diagnostics K.K. (Japan). In addition, the 2% CyA ophthalmic solution has to be diluted to measure the concentration of CyA; ethanol purchased from KENEI Pharmaceutical Co., Ltd. (Japan) and Milli-Q water (Direct-Q^® UV 5; Merck KGaA, Germany) were used as diluent solvents. To perform the sterility test for bacterial contamination of the 2% CyA ophthalmic solutions prepared without the 0.45-µm filter operation, we purchased BD Bactek™ 23 F aerobic resin bottle and BD Bactek™ 22 F anaerobic resin bottle (Nippon Becton Dickinson Company, Ltd., Japan), which can confirm the presence of aerobic and anaerobic bacteria.