Q700 sonicator

Aqueous dispersions of Kraft birchwood cellulosic fibers (kindly provided by Sandra Tapin, FCBA, Grenoble, France) were adjusted to pH 5.2 with acetate buffer (50 mM) in a final reaction volume of 5 ml. Each GcLPMO9 enzyme was added to the fibers at a final concentration of 20 mg g⁻¹ in the presence of 1 mM of ascorbic acid. Enzymatic incubation was performed at 40 °C under mild agitation for 48 h. Samples were then dispersed using a Polytron PT 2100 homogenizer (Kinematica AG, Luzern, Switzerland) for 3 min, and ultrasonicated by means of a QSonica Q700 sonicator (20 kHz, QSonica LLC., Newtown, USA) at 350-W ultrasound power for 3 min as previously described [20 (link)]. The reference sample was submitted to the same treatment but did not contain the enzyme. Wood cellulose fibers (reference and GcLPMO9-treated) were deposited onto a glass slide and observed under a BX51 polarizing microscope (Olympus France S.A.S.) with a 4 × objective. Images were captured on a U-CMAD3 camera (Olympus, Tokyo, Japan).

TAC was suspended in a mix of water and propylene glycol (90:10), 3 to 5 days before the first administration and kept at 4 °C. The stability of the solution was validated for 15 days. TAC was first dissolved in propylene glycol, then the water was added. The suspension was homogenized using an ultrasonic homogenizer 3 times for 15 s (QSonica Q700 sonicator, QSonica LLC., USA). TAC concentration was always checked before the first and after the last gavage to control for the dose. TAC or the vehicle was administered once a day (always at the same period) by oral gavage. A dose of 3 mg of TAC/kg of body weight was administered for 5 days, except in the dose determination study where different escalating doses (from 0.1 to 10 mg/kg of body weight) were tested (4 days of treatment). Mice were fasted for minimum 4 h before the last gavage because preliminary experiments demonstrated a reduced variability in TAC PK in fasted state (data not shown).

Insoluble protein aggregates were isolated as described (Rand and Grant, 2006), with minor adjustments. The protocol was scaled up to suit the harvesting of 35 OD600 units of cells. Pefabloc SC (Roche) was used instead of phenylmethylsulfonyl fluoride in the lysis buffer. After freezing and thawing the cells they were immediately disrupted by a minibead beater (Fastprep) at 5.5 m/s for 4x30 sec at 4°C. Cells were kept on ice for 5 min between each beating. Removal of intact cells was accomplished by centrifugation at 1180 x g for 15 min. Protein concentration was determined using Pierce 660 nm Assay Reagent (Thermo Scientific) and adjusted before isolation of protein aggregates as well as verified by running reduced samples on Criterion XT Precast Gel 4–12% Bis-Tris (BioRad) and staining with Coomassie Brilliant Blue R-250 staining solution (BioRad). The subsequent centrifugations were all done at 21130 x g for 20 min. Insoluble fractions were resuspended in detergent washes by 5 x 5 sec sonication at amp 40 with 15 sec rest between intervals using Q700 Sonicator (QSonica, LLC. Newtown, USA). The insoluble fractions were added to reduced protein loading buffer, loaded on Criterion XT Precast Gel 4–12% Bis-Tris (BioRad) and visualized by silver staining with Pierce Silver Stain Kit (Thermo Scientific).

Upon thawing, purified α-syn was centrifuged at 100,000×g for 30 min at 4 °C to remove any aggregates that may have formed during the freeze-thaw process. Monomeric α-syn was then diluted to 50 μM (0.723 mg/ml) in 10 mM Tris pH 7.5, 150 mM NaCl in 0.2 mL PCR tubes (GeneMate) with about 10 zirconium oxide beads (1 mm diameter; Next Advance). PCR tubes were then placed in a Q700 sonicator (Qsonica) connected to a circulating water bath. Unless specified, the PMCA was carried out with repeated cycles of 10 s of sonication and 29 min 50 s of incubation at 37 °C. At various time points, 2 μL of sample was removed and was incubated with 198 μL thioflavin T solution (20 μM ThT, 50 mM glycine, pH 8.5) in a 96-well plate (Greiner Bio-One) for 5 min at room temperature. ThT readings were performed on a Tecan M200 plate reader with an optimized gain of 100 (excitation at 440 nm and emission at 480 nm). For seeded reactions, α-syn fibrils formed by PMCA were centrifuged and the pellet was resuspended in 10 mM Tris buffer, pH 7.5 to 10× of the desired final concentration. Seed was added to the PMCA reactions at a 1:10 ratio.

The extracted brain tissues of Ewsr1 KO and WT mice were transferred to tubes with an approximately 5× greater volume of lysis buffer [4% sodium dodecyl sulfate (SDS), 2 mM Tris(2-carboxyethyl) phosphine, and 0.1 M Tris–HCl (pH 7.5) in HPLC-grade water]. The lysed brain tissue was immediately homogenized using Q700 sonicator (QSONICA, CT, USA). After homogenization, all samples were heated in a heating block at 95 °C for 1 h for protein extraction and delipidation. The extracted proteins were additionally filtered with Costar Spin-X 0.22 µm centrifuge tube filter (Corning Inc., NY, USA) to remove the remaining lipids. The protein concentration was determined by tryptophan fluorescence (WF) assay^{18 (link)}. To remove contaminants and lipid contents from brain tissue lysate, 200 μg of protein was precipitated with acetone^{19 (link)}. Briefly, approximately five sample volumes of cooled (− 20 °C) acetone were added, the samples mixed by gentle tapping, and stored in LoBind tubes (Eppendorf, Hamburg, Germany) at − 20 °C overnight. The samples were then centrifuged for 10 min at 15,000 rpm at 4 °C. The supernatant was discarded, and the above steps repeated. After drying at 25 °C for 15–30 min, the supernatant was completely evaporated, and the precipitated samples stored at − 80 °C until FASP digestion.

The method for purification of MSP2N2 was adapted from a published protocol³⁴ . Cell suspensions were thawed and lysed by sonication on ice using a Q700 sonicator (Qsonica) (50% output amplitude, 30 cycles of 10 s on, 20 s off). The cell lysate was clarified by centrifugation using a SS34 rotor at 30,000 × g for 1 h at 4 °C. The supernatant was collected, syringe filtered through a 0.22 µm membrane (Merck Millipore Ltd.) and applied to a 1 mL Ni-NTA column (His-Trap^TM HP, Cytiva) equilibrated with 50 mM Tris-HCl (pH 8), 500 mM NaCl (=buffer A) + 1% v/v Triton X-100. The column was washed with 10 column volumes of buffer A + 1% v/v Triton X-100 followed by 10 column volumes of buffer A + 50 mM sodium cholate. Non-specifically bound proteins were washed with 10 column volumes of buffer A + 80 mM imidazole and finally MSP2N2 was eluted with buffer A + 400 mM imidazole. To obtain highly pure MSP2N2, the eluate from 1 mL Ni-NTA column was reapplied to a 5 mL Ni-NTA column (His-Trap^TM HP, Cytiva) and the same procedure was repeated. Pure MSP2N2 fractions were pooled and dialysed against 2 L of 10 mM MOPS (pH 7.5 at 4 °C), 50 mM KCl at 4 °C. Sample homogeneity was confirmed by SDS-PAGE, and the protein flash frozen and stored at −80 °C.