Beadbug

Cecal tissues (25 mg) were removed from RNAlater and transferred to pre-filled 2-ml tube containing Triple-Pure™ 1.5-mm zirconium beads. RLT lysis buffer (600 μl) from the RNeasy mini kit (Qiagen) was added, and the tissue was homogenized for 1–2 min at 4,000 rpm in a Bead Bug microtube homogenizer (Benchmark Scientific, Inc., Edison, NJ, USA). Total RNA was extracted from the homogenized lysates according to the manufacturer’s instructions, eluted with 50 μl RNase-free water, and stored at −80°C until qRT-PCR analyses were performed. RNA was quantified and the quality evaluated using a spectrophotometer (NanoDrop Products, Wilmington, DE, USA).

Between December 2014–June 2019, a total of 17 samples from 12 clinical cases were diagnosed as WCS by Poultry Health Services (PHS, Airdrie, AB, Canada). Clinical samples, such as liver and intestines, were obtained from affected dead-in-shell embryos and young birds and tested by the Animal Health Laboratory (University of Guelph, Guelph, ON, Canada) for CAstV using quantitative polymerase chain reaction (qPCR) [23 (link),26 (link)]. Affected tissues from some cases were also submitted to the same laboratory for histopathology examination for confirmation of diagnosis, and 17 samples from these clinical cases were held at −80 °C for further processing.
The liver and intestine samples were placed into sterile tubes prefilled with 1.0 mm zirconium beads (Benchmark Scientific Sayreville, NJ, USA), and 0.5 mL 1× phosphate buffered saline (PBS) (Gibco, Waltham, MA, USA) on ice, and homogenized (BeadBug, Benchmark Scientific, Sayreville, NJ, USA) during three series of 30 seconds each at 300 rounds per minute (RPM). Samples were incubated on ice for 3 minutes (min) in between series. Following disruption, the samples were centrifugated at 7500× g for 30 min at 4 °C and the supernatant filtered using a 0.2 µM syringe filter (Millipore Sigma, Burlington, MA, USA) and kept on ice for further processing.

Total RNA was extracted from the right hemi-striatum using the RNeasy Lipid Tissue Mini Kit (Qiagen). Tissue was homogenized in 500 μL of Qiazol Lysis Reagent for one minute at 4,000 rpm using a homogenizer (BeadBug; Benchmark Scientific). Aqueous and organic phases were separated by the addition of 100 μL of chloroform. All subsequent steps in were performed according to the manufacturer’s protocol. Reverse transcription was performed using the Superscript III First Strand Synthesis System (Life Technologies) following the manufacturer’s protocol. Quantitative real-time polymerase chain reaction (QRT-PCR) was conducted with the resulting cDNA using the Real-Time StepOne System (Applied Biosystems). Transcripts were analyzed by the relative quantitation standard curve method and normalized to a transcript unregulated by mutant Htt expression (Actb). All taqman probes were purchased from Life Technologies and were as follows: Drd1a: Mm02620146\_s1, Drd2: Mm00438545\_m1, Cnr1: Mm01212171\_s1, Darpp32: Mm00454892\_m1, Penk: Mm01212875\_m1, N4bp2: Mm01208882\_m1, Islr2: Mm00623260\_s1, Pde10a: Mm00449329\_m1, H60b: Mm04243254\_m1, and Scn4b: Mm01175562\_m1.

This study was approved and performed in accordance with the recommendations and guidelines of the Western Sydney Local Health District Animal Ethics Committee, Department of Animal Care, Westmead Hospital (protocol number 4254). Female C57BL/6 mice (20 to 22 g) were obtained from the Animal Resource Centre, Floreat Park, Western Australia, Australia. C. neoformans strains were grown overnight in YPD broth, washed twice with phosphate-buffered saline (PBS), and resuspended in PBS. Prior to infection, mice were transiently anesthetized by inhalation of 3% isoflurane, then 5x10⁵ cells in 20 μl of PBS were delivered into the nares using a pipette. For the survival analysis, 10 mice in each group were monitored daily and euthanized by CO₂ asphyxiation when they had lost in excess of 20% of their pre-infection weight or if showing debilitating symptoms of infection. Differences in survival were determined using a log rank test. For organ burden analysis at the time of death, lungs and brain were then removed, weighed, and mechanically disrupted in 2 ml sterile PBS using a BeadBug (Benchmark Scientific). Serial dilutions of blood and the organ samples were plated onto SDA agar plates and incubated at 30°C for 2 days. Colony counts were performed and adjusted to reflect the total number of CFU per gram of tissue or ml of blood.

The total antioxidant capacity and reduced glutathione (GSH) activity of Renuspore^® and L. rhamnosus GG were quantified by colorimetric analysis at 570 nm and 420 nm, respectively, using the total antioxidant capacity (TAC) (MAK187, Sigma-Aldrich, Ireland) (Ibrahim et al., 2021 (link)) and Reduced Glutathione Assay Kits (E-BC-K030-S, Elabscience , China). For these studies, both strains were grown in the TSB medium at 37°C until a final concentration of 1x10⁸ CFU/mL was reached. The culture was then centrifuged at 6,000 RPM for 15 min, and the supernatant was discarded. The cell pellet was washed and resuspended in 1X PBS three times. The washed cell pellet was resuspended in 1.5 mL 1x PBS and homogenized three times at 3,500 RPM for 30 s using a microtube homogenizer (Beadbug, Benchmark, Ireland). The homogenized sample was centrifuged at 6,000 RPM for 15 min at 4°C. The cell debris was discarded, and the cell lysate was recovered for testing. 1X PBS was used as a negative control. The TAC and reduced glutathione assay were performed following the manufacturer's instructions.

To prepare hypothalamus samples for analysis, 10 µL of 1 µg/mL D6-morphine in-ternal standard solution was added to hypothalamus samples and matrix-matched standards. Here, matrix-matched standards were hypothalamus samples collected from untreated mice spiked with a morphine standard. Hypothalamus samples/standards were then homogenized for 10 s using a BeadBug™ (Benchmark Scientific, Inc. Sayreville, NJ, USA) 3 Position Bead Microtube homogenizer in 400 µL ice-cold acetonitrile. Samples were then centrifuged 14,000 rpm for 5 min and supernatant was added to Phenomenex Strata-X-Drug B solid phase columns. Columns were washed with 2 mL 0.1% formic acid in water and 2 mL methanol. Columns were then dried for 10 min prior to elution. Two successive aliquots of a solution containing 50% acetonitrile, 42% methanol, and 8% 7N ammonium in methanol were used to elute samples from the columns. Eluents were collected into a clean glass test tube and dried under nitrogen at 40 °C. Dried eluents were reconstituted with 200 µL 95:5% water:acetonitrile and transferred to autosampler vials fitted with 400 μL glass inserts. The limit of detection was 0.2 ng/mL morphine.