Fastprep 24 system

Five-week-old female BALB/c mice were anesthetized by intraperitoneal injection of a mixture of Ketamine and Xylazine (100 μg and 5 μg per gram of body weight), prior to intranasal administration of either PBS or 100 micrograms of peptide-conjugated phosphorodiamidate morpholino oligomers (PPMO) mix (50 micrograms of PPMO1 and 2 each) in 40 μl of PBS, on Day -2 and Day -1. On Day 0, Mice were challenged intranasally with 40 PFU of PR8 IAV (LD50 = 50 PFU) in 40μl PBS. Mice were monitored daily for weight loss and clinical signs. Mice lungs were harvested on Day 3 and Day 6 post infection for measuring viral titers (5 mice per condition). Lung homogenates were prepared using a FastPrep24 system (MP Biomedicals). After addition of 800 μl of PBS containing 0.3% BSA, lungs were subjected to two rounds of mechanical treatment for 10 s each at 6.5 m/s. Tissue debris was removed by low-speed centrifugation, and virus titers in supernatants were determined by plaque assay. A group of mice (5 per condition) were monitored until day 14 post infection for survival.

Fungal genomic DNA was extracted as previously described (Abdolrasouli et al., 2015 (link)). Briefly, gDNA was extracted with an optimized MasterPure yeast DNA purification kit (Epicentre Biotechnologies, Cambridge, United Kingdom) with an additional bead-beating step included. Harvested conidia were homogenized using 1.0-mm-diameter zirconia/silica beads (BioSpec Products, Bartlesville, OK, United States) in a FastPrep-24 system (MP Biomedicals, Solon, OH, United States) at 4.5 m/s for 45 s. DNA samples were stored at −80°C for molecular testing.

KDPG aldolase activity was quantified by a lactate dehydrogenase (LDH) coupled assay where the production of pyruvate is related to the NADH consumption, as described in [29 (link)]. 2 ml of R. jostii RHA1 RM or MMGls cultures were harvested and resuspended in 1 ml of buffer TrisHCl 100 mM pH 7.5, NaCl 300 mM, EDTA 1 mM, DTT 1 mM and PMSF 1 mM. The cells were lysed using 0.2 mm silica beads and a Fast Prep-24 system (MP Biomedicals) for 3 cycles of 60 s and centrifuged at 100,000g for 25 min at 4 °C. 150 μl aliquots of the resulting RM or MMGls total extracts were then treated with 1 μl of LDH (5 U/μL), 0.70 μl of NADH (50 mM) and 1 μl of KDPG (50 mM). Decrease in NADH absorbance at 340 nm was measured in quartz microcuvettes (150 μl) in a UV-1603 spectrophotometer (Shimadzu) for 5 min. Total protein concentration was determined by Bradford assays using BSA as standard. KDGP activity was calculated as moles of NADH consumed per mg of total protein per second (mol/s/mg).

DNA was extracted using the FastDNA SPIN kit for soil (MP Biomedicals, Illkirch, France). The extraction included two bead beating steps of 45 s at 6.5 m s⁻¹ on a FastPrep-24 system (MP Biomedicals) and one additional washing step of the binding matrix with 1 ml 5.5 M guanidine thiocyanate (Carl Roth, Karlsruhe, Germany). DNA extraction failed from a phase 2 rhizosphere sample from the control treatment with straw amendment; therefore, there were only three replicates from this treatment combination.

Co-immunoprecipitation was performed as described in [22 (link)]. Δswi6 cells (YTP1847) were co-transformed with pRep42-Lsw1-3Flag and either pGP4110 or pGP4110-Lsw1. The pGP4110 and pRep42 plasmid markers are LEU2 or ura4⁺, respectively. Plasmid-containing cells were cultured in EMM2 without thiamine for 23 h at 30 °C. The cell pellets were suspended in lysis buffer (50 mM HEPES pH7.5, 1 mM EDTA, 200 mM NaCl, 1 mM β-mercaptoethanol, 10% glycerol, 0.5% Triton X-100), and cell lysates were prepared using a FastPrep-24 system (MP-Biomedicals, Irvine, CA, USA) with glass beads. The cell lysates were then incubated with anti-GFP antibody (Roche, Mannheim, Germany, 1814460) for 2 h at 4 °C followed by addition of Dynabeads α-Mouse IgG (VERITAS) for 3 h at 4 °C. The beads were washed with lysis buffer and analyzed by Western blotting using anti-GFP (Roche, 1814460) and anti-M2 (SIGMA, St. Louis, MO, USA, F3165-0.2MG).