Avicel PH-101

Eight high purity (>95% cellulose in all cases except for Solka-Floc, which was >93%) celluloses were used in this study. Bacterial microcrystalline cellulose (BMCC) was prepared from Gluconacetobacter hansenii (American Type Culture Collection (ATCC) 10821) in our laboratory [16 (link)]. The seven other celluloses were commercially available: Sigmacell 50 (S5504), Sigmacell 20 (S3504), Avicel PH-101 (11365), Fluka cellulose (22183), α-cellulose (C8002) (all purchased from Sigma-Aldrich, St. Louis, MO, USA), Solka-Floc (International Fiber Corporation (North Tonawanda, NY, USA) and JT Baker cellulose (1529) (Mallinckrodt Baker, Phillipsburg, NJ, USA). Ball milled cellulose was prepared by milling Avicel PH-101 (1.5 g) for 20 minutes in a cryogenic impact mill (6770 Freezer Mill; Spex, Metuchen, NJ, USA) cooled by liquid nitrogen.

The P. oxalicum strains used in this study were maintained on potato–dextrose agar (PDA) plates at 4 °C. The fungal spores used for reproduction were harvested after 6 days on PDA plates at 28 °C, resuspended in buffer containing 0.1% Tween 80, and adjusted to a concentration of 1 × 10⁸ per milliliter.
For the transcriptomic (RNA-Seq) analysis of gene expression in P. oxalicum strain HP7-1 (China General Microbiological Culture Collection [CGMCC] no. 10781), 1 mL of spore suspension (1.0 × 10⁸/mL) was used to inoculate 100 mL of modified minimal medium (MMM [g/L]: (NH₄)₂SO₄ 4.0, KH₂PO₄ 4.0, CaCl₂ 0.6, MgSO₄·7H₂O 0.60, FeSO₄·7H₂O 0.005, MnSO₄ 0.0016, ZnCl₂ 0.0017, CoCl₂ 0.002, and 1 mL of Tween 80) containing glucose (1.0 g), wheat bran (4.0 g) (purchased from a local farmer’s market in Nanning, China), or wheat bran (4.0 g) plus Avicel^® PH-101 (2.0 g) (Cat No. 11365-1KG; Sigma-Aldrich, Darmstadt, Germany) as the sole carbon source, respectively. The inoculated cultures were placed in a shaker at 28 °C, and shaken at 180 rpm for 72 h. Mycelia were collected from the cultures with four-layer filter fabrics, and washed with diethyl-pyrocarbonate-treated water before RNA extraction.
The constructed P. oxalicum mutants of the candidate TF genes were inoculated into 100 mL of MMM containing a final concentration of 2% (w/v) Avicel. The inoculated medium was incubated at 28 °C, with shaking at 180 rpm for 6 days. The supernatant was collected by centrifugation at 11,300×g for 10 min to assay the FPase activity.
To measure the growth profiles, ΔPoxCxrA (ΔPOX01167, CGMCC no. 12965), ΔPoxCxrB (ΔPOX04420, CGMCC no. 12966), ΔPoxNsdD (ΔPOX08415, CGMCC no. 12967), and the parental strain ΔPoxKu70 (CGMCC no. 3.15650) [5 (link)] were inoculated into MMM containing 1% (w/v) glucose or 2% (w/v) Avicel and incubated at 28 °C with shaking at 180 rpm. The mycelia were collected every 12 h until 72 h.
A shift experiment was performed followed by an RT-qPCR analysis of gene expression and an enzymatic activity assay of ΔPoxCxrA, ΔPoxCxrB, ΔPoxNsdD, and the parental strain ΔPoxKu70 [5 (link)]. Approximately 1.0 × 10⁸/mL spores from the P. oxalicum mutant strains were grown in 100 mL of MMM containing 1% (w/v) glucose at 28 °C with shaking at 180 rpm for 24 h. Equal portions of the harvested and washed mycelial samples were then aseptically inoculated into fresh MMM containing 2% (w/v) Avicel as the sole carbon source. After culture for 4, 12, 24, or 48 h, the mycelia were harvested, and the total RNA was extracted for RT-qPCR.
For the enzymatic activity assay of the three mutants ΔPoxCxrA, ΔPoxCxrB, and ΔPoxNsdD under induction conditions, the pregrown mycelia of these strains were cultured for 2–4 days in MMM containing Avicel as the sole carbon source, as described above.

OSX, BiWX, Avicel PH-101, xylo-oligosaccharides (DP1-5) and β-(1 → 4)-linked gluco-oligosaccharides (DP1-5) were obtained from Sigma-Aldrich (Steinheim, Germany). WAX (medium viscosity), β-(1 → 3, 1 → 4)-linked glucan from barley (medium viscosity) and oat spelt (medium viscosity) were purchased from Megazyme (Bray, Ireland). Xyloglucan (XG; from tamarind seed) was obtained from Dainippon Sumitomo Pharma (Osaka, Japan). Regenerated amorphous cellulose (RAC) was prepared from Avicel PH-101 by adopting a method described elsewhere [42 (link)]. Briefly, Avicel PH-101 (100 mg) was moistened with 0.6 mL water. Next, 10 mL 86.2% (w/v) ortho-phosphoric acid was slowly added followed by rigorous stirring for 30 min until the cellulose was completely dissolved. The dissolved cellulose precipitated during stepwise addition of 40 mL of water. After centrifugation (4,000g, 12 min, 4°C), the pellet obtained was washed twice with water and neutralized (pH 7.0) with 2 M sodium carbonate. The pellet was washed again with water (three times) and the final pellet was suspended in water to a dry matter content of 1.4 ± 0.1% (w/w) RAC suspension.

Cellulose contents of developed cotton fibers were measured by the modified Updegraff method [28 (link)]. Five cotton bolls were randomly selected from each biological replicate samples of the cultivated G. arboreum (A₂-100 and SXY1) and G. hirsutum (TM-1, SG-747, and DP-5690) producing long fibers. Two to six cotton bolls were also randomly selected from each biological replicate samples of the G. raimondii (D₅-6 and D₅-31) and G. hirsutum mutants (Li₁ and Li₂) generating short fibers. Dried fiber samples of the selected bolls were manually harvested, and cut into small pieces. Ten milligrams of the blended fibers were placed in 5 mL Reacti-Vials^TM (Thermol Fisher Scientific, Waltham, MA), and hydrolyzed with acetic-nitric reagent (a mixture of 73% acetic acid, 9% nitric acid and 18% water). The remaining cellulose was hydrolyzed with 67% sulfuric acid (v/v) and measured by a colorimetric assay with anthrone with Avicel PH-101 (FMC, Rockland, ME, USA) as a cellulose standard. Mean cellulose content of each cotton variety was obtained by measuring the randomly selected cotton bolls from two biological replicates.

Release studies were
performed with a tablet formulation of the ASD to circumvent wetting
issues with the ASD powder. The tablet (75 mg) contained ASD powder
(equivalent to 5 mg DLM) mixed with excipients (sodium starch glycolate,
4 mg; croscarmellose sodium, 4 mg; silica colloidal hydrate, 0.6 mg;
magnesium stearate, 0.6 mg; Avicel PH 101, q.s. 75 mg).^{15 (link)} Release testing was conducted at 150 rpm, 37
°C, using a USP apparatus II (Hanson, Billerica, MA). Release
studies were performed in a single-stage medium (phosphate buffer,
pH 6.5) or using pH shift experiments, with the first dissolution
stage at an acidic pH (0.02 M HCl solution, pH 1.6, or phosphate buffer
at pH 3.0 or pH 5.0) for 60 min, followed by dissolution in phosphate
buffer pH 6.5 for an additional 30 min. The low pH media were converted
to pH 6.5 by adding concentrated phosphate buffer pH 7.3, the composition
of which is presented in Table 2. The dose concentration in the dissolution medium was 100
μg/mL.
Drug release as a function of time was determined
using an in situ
dissolution monitoring system (Pion Rainbow Instrument, Billerica,
MA) based on fiber optic UV spectroscopy. Drug concentration was calculated
from analysis of second derivative spectra by determining the area
under the curve over the wavelength range of 330–350 nm. Calibration
curves of DLM in different buffers were built over the concentration
range of 1–100 μg/mL.
Polymer release was evaluated
at different time points by withdrawing
1.5 mL of dissolution medium, which was replaced with fresh media,
followed by centrifugation at 14,800 rpm, 37 °C for 3 min to
remove undissolved ASDs. Polymer concentration was determined by the
HPLC-ELSD method as described above.