D mannitol

Hydroxypropyl cellulose LFP (HPC-LFP) was obtained from Nisso HPC (Nisso Chemical Europe GmbH, Düsseldorf, Germany). Bovine intestinal alkaline phosphatase (BIAP), bovine serum albumin (BSA), ammediol (2-Amino-2-methyl-1,3-propanediol), HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), and triethyl citrate were obtained from Sigma-Aldrich (St. Louis, MO, USA). D-mannitol was purchased from VWR Chemicals, BDH, USA. Eudragit S100 and Eudragit L100-55 were gifts from Evonik Operation GmbH (Essen, Germany). Macrogolum 6000 (PEG6000) and talc were obtained from BUFA (IJsselstein, The Netherlands). Croscarmellose sodium (AcDiSol) was obtained from FMC BioPolymer (Philadelphia, PA, USA). Inulin (4 kDa) was a generous gift from Sensus (Roosendaal, The Netherlands). Methylene blue was obtained from Interpharm B.V. (Rotterdam, The Netherlands; now discontinued).

Mesoporous magnesium carbonate (MMC) was kindly provided by Disruptive Materials AB (Uppsala, Sweden). Celecoxib was purchased from 3Way Pharm Inc. (Shanghai, China). D-mannitol, ethanol absolute >99.8%, ammonia 28%, and tetraethyl orthosilicate 98% (TEOS) were purchased from VWR International (Stockholm, Sweden). Monobasic sodium phosphate, dibasic sodium phosphate, hexadecyltrimethylammonium bromide (CTAB), and polyvinyl alcohol 4-88 (PVA) were all purchased from Sigma-Aldrich (Stockholm, Sweden).

Growth curves were performed using a minimal medium based on a previously described complete defined minimal media (CDMM), but lacking D-glucose as used by Cartman et al. and adjusted to pH 7.4 [33 (link), 72 (link)]. The base medium was supplemented with 30 mM D-glucose (Sigma-Aldrich), 30 mM D-fructose (Fisher), 30 mM D-mannose (BD Difco), 30 mM D-mannitol (Amresco), 30 mM N-acetylglucosamine (Chem-Impex), or 30 mM ethanolamine-HCl (Sigma-Aldrich), as noted. Growth curves in minimal medium (MM) were carried out as follows: log-phase cultures were grown to an OD₆₀₀ of 0.5 in BHIS medium, then diluted 5-fold into MM. Diluted cultures were then used to inoculate minimal medium broth for growth assays at a starting OD₆₀₀ of ~0.01 (2 ml into 23 ml of MM). Growth curve data were analyzed by two-way ANOVA with Dunnett’s test for multiple comparisons, comparing each strain to 630Δerm at each time point. Doubling times were analyzed by one-way ANOVA with Dunnett’s test for multiple comparisons or by Student’s t test, as indicated.

Seeds were surface sterilized in 4 % (v/v) sodium hypochlorite, and rinsed five times with sterile water. The seeds were sown on MS basal medium as mentioned above, with addition of NaCl or D-mannitol (Amresco, Solon, OH, USA) at indicated concentrations for assaying salt and osmotic stress responses. The seeds were stratified at 4 °C for 3 day, and transferred to 20 °C under long-day cycle (16 h/8 h light/dark) for phenotypic analysis of germination and post-germination growth.

The phenotypic analysis was performed essentially as described previously (Shang et al., 2010 (link)). For the seedling growth assay, two approaches were used to test the effects of ABA, NaCl, or mannitol on early seedling growth. The first approach is that seeds were sown on basal MS medium or MS medium supplemented with different concentrations of (±)-ABA (Sigma, A1049, Saint Louis, MO, USA), NaCl (Amresco, 0241, OH, USA), or d-mannitol (Amresco, 0122, OH, USA) medium. After 3 d of stratification at 4 °C, they were placed in a light incubator for 10 d, and the root length was recorded. The second approach is that seeds were planted in ABA-free MS medium and, after 3 d of stratification at 4 °C, they were placed in a light incubator for 60 h, and then young seedlings were transferred to (±)-ABA-containing MS medium and continued to grow for 10 d before the root length was recorded. For the cotyledon greening assay, seeds were sown on either (±)-ABA-free MS medium or MS medium supplemented with different concentrations of (±)-ABA for 3 d of stratification at 4 °C, and then the germinating seeds/young seedlings were placed in a light incubator for 10 d before the cotyledon greening rates were recorded. Cotyledon greening was defined as obvious cotyledon expansion and turning green.

Pancreatic Min6 beta cells were purchased from the Cell Bank of the Chinese Academy of Sciences. Min6 cells were routinely cultured in RPMI-1640 (SH30809.01; GE Healthcare, Salt Lake City, UH, USA) supplemented with 20% fetal bovine serum (FBS), 1% glutamine, 1% β-mercaptoethanol, and 10 mM HEPES. Cells were cultured in a 37 °C with 5% CO₂ incubator. The medium was refreshed every 24 h. Palmitic acid (P0500, PA; Sigma-Aldrich, Carlsbad, CA, USA) was conjugated with fatty-acid-free bovine serum albumin (BSA) (A8020; Solarbio, Beijing, China) before addition to cell culture. PA was dissolved in 99% ethanol and then mixed with 10% BSA in serum-free DMEM (SH30022.01; GE Healthcare, Salt Lake City, UT, USA) to make a 50 mM PA stock solution. Different concentrations of glucose and Palmitic acid were prepared in RPMI-1640 medium. The osmotic pressure was adjusted with D-mannitol (0122; Amresco, Seattle, Washington, USA). Min6 cells were treated with glucose (5.6,16.7 , and 33.3 mmol/l) and Palmitic acid (0.2, 0.5, and 1.0 mmol/l) for 24 h, 36 h, and 48 h.