Mncl2 4h2o

To detect the assimilation of selected neurotransmitters, we used a modified M9 minimal medium (pH 7.4) [43 ], consisting of 0.6% Na₂HPO₄ (Sigma Aldrich, USA), 0.3% KH₂PO₄ (Sigma-Aldrich, USA), 0.05% NaCl (Fisher Scientific, USA), 0.1% NH₄Cl (Merck, Germany), and 0.15% agar in Milli-Q water. To the autoclaved and cooled medium were added 5 mL of 40% (w/v) glucose, and 1 mL of microelements [3 mM (NH₄)₆Mo₇O₂₄ × 4H₂O (Sigma Aldrich, USA), 400 mM H₃BO₃ (Merck, Germany), 30 mM CoCl₂ × 6H₂O (Merck, Germany), 10 mM CuSO₄ × 5H₂O (Merck, Germany), 80 mM MnCl₂ × 4H₂O (Sigma Aldrich, USA), 10 mM ZnSO₄ × 7H₂O (Sigma Aldrich, USA), 1 mL of 1 M MgSO₄ (Carlo Erba, Milano, Italy), 100 µL of 1 M CaCl₂ (Gram-mol, Zagreb, Croatia), and 200 µL of 5 mM FeSO₄ (Sigma Aldrich, USA)]. After autoclaving, we added filter-sterilized 0.1 M of one of the selected neurotransmitters: acetylcholine (ACh), γ-aminobutyric acid (GABA), glycine (Gly), glutamate (Glu), and dopamine (DA) (all Sigma-Aldrich, USA). M9 minimal medium (pH 7.4) without added neurotransmitters was used as a control. Plates were inoculated with 10 µL of cell suspension or plugs of mycelium in three replicates and incubated for one month at 24 °C and 37 °C. Assimilation was assessed by comparing growth on plates with and without added neurotransmitters.

An isotonic solution of MnCl₂·4H₂O (203734; Sigma-Aldrich) was prepared at a concentration of 100 mM in 100 mM bicine solution, as previously described (24 (link)). Then, the MnCl₂ solution was diluted to 50 mM with saline, and the pH was adjusted to 7.4.
We intraperitoneally administered a 50 mM solution of MnCl₂ immediately after the initial MRI acquisition, twice at 1-h intervals, to get a total dose of 66 mg/kg. MEMRI acquisition was performed 24 h after the first administration of MnCl₂ based on the kinetics of intracerebral Mn²⁺ concentration (11 (link)). The imaging area was adjusted to the same position as in the initial MRI to facilitate comparison of T1 values at the same ROI.

The reactants MnCl ₂·4H ₂O, Ca(NO ₃) ₂, La(NO ₃) ₃, KMnO ₄ and KOH were purchased from Sigma Aldrich Co., Madrid, Spain.

The fluorescence emitted was recorded by employing GloMax Discover (Promega, USA) using the excitation and emission filters at 485 and 520 nm. This assay records the synthesis of dsRNA in a reaction using ATP (Sigma Aldrich) as a nucleotide substrate and a poly-U (Sigma Aldrich) molecule as a template using fluorescent dye SYTO 9 (Invitrogen), which binds only to dsRNA [24 (link)]. The standard reaction contained 50 mM Tris-HCl (pH 8.0), 5 mM MnCl₂·4H₂O (Sigma Aldrich), 50 mM NaCl (Samchun), 4 mM DTT (TaKaRa), 3 mM ATP, 13 μg/ml poly-U, and 0.25 μM SYTO 9. The assay was initiated by adding 1 μg/μl RdRp/NSP7/NSP8 (SARS-CoV-2) Complex (BPS Bioscience, USA), and the fluorescence was recorded over 20 min at 30°C. The reaction was conducted in black, 96-well, flat-bottomed plates. For the compound used in this assay, NPP B1 with a purity of over 80% on HPLC was used, and ampicillin, kanamycin, and amphotericin B were purchased from Sigma Aldrich.

Tetramethylammoniumhydroxide (TMAH), HCl suprapure, and ammonium citrate were purchased from Merck (Darmstadt, Germany). Argon_liqud (Ar) was purchased from Air-Liquide (Düsseldorf, Germany) and gaseous Ar was gained at the vaporizer at the tank. FeCl₃ · 6H₂O standard, 2,6-pyridine dicarboxylic acid (dipicolinic acid, PDCA) and Manganese-acetate [Mn (II)] was from Sigma Aldrich Chemie (Steinheim, Germany). FeCl₂ · 4H₂O was purchased from AppliChem GmbH (Darmstadt, Germany). Sodium deoxycholate, NP-40, MnCl₂ · 4H₂O, phenylmethane sulfonyl fluoride or phenylmethylsulfonyl fluoride (PMSF), fetal calf serum, glutamine, phosphate buffered saline (PBS) and orthovanadate were purchased from Sigma-Aldrich (Taufkirchen, Germany). Dulbecco's modified Eagle's medium (DMEM) was delivered from Thermo Fisher Scientific (München, Germany). The cOmplete™ Protease Inhibitor Cocktail was from Roche, Mannheim, Germany. Chemicals were of highest available purity, i.e., all chemicals in the speciation laboratory were bought in ultrapure quality. All solutions were prepared by using MilliQ® water (18.2 mΩcm, Merck-Millipore, Darmstadt, Germany).