D mannose

Roswell Park Memorial Institute 1640 medium (RPMI 1640) and inactivated fetal bovine serum (BSF) were obtained from Gibco^®, Life Technologies (Carlsbad, CA, USA). Prozima Prolav 750 was purchased from Prozyn Biosolutions (São Paulo, SP, Brazil). Ethylenediaminetetraacetic acid (EDTA), l-fucose, d-xylose, d-galactose, d-mannose, d-glucose, d-arabinose, d-rhamnose, d-glucuronic acid, N-d-acetylglucosamine, d-galactosamine, d-glucosamine, and silver nitrate were all obtained from Sigma Aldrich Co. (St. Louis, MO, USA). The other solvents and chemical reagents used in this study were of analytical grade.

The CdrA-mediated aggregation assay was performed as previously described (12 (link)). d-Mannose, l-rhamnose, alginic acid, and sodium alginate from Sigma-Aldrich and uronic acids from Carbosynth LLC were used at 5 mg/mL. Bacterial alginate (Dextra Laboratories Ltd.) was used at 0.5 mg/mL due to poor solubility. Aggregation was visually evaluated and then measured by optical density readings taken at 600 nm. Percent relative aggregation was calculated by taking the difference between the OD₆₀₀ of the P_BAD-cdrAB strain and that of its corresponding vector control strain, dividing by the OD₆₀₀ of the vector control strain, and multiplying by 100%.

To determine which cell surface molecules are responsible for virus’ attachment, competition experiments were designed, or SAs residues were enzymatically removed from the surface of cells. Cells were grown on coverslips for 48 h, washed twice with PBS and incubated for 30 min at 37 °C with type II neuraminidase (NA, 100–200 mU/mL) from Vibrio cholerae (N6514, Sigma-Aldrich, Poznan, Poland), N-acetylneuraminic acid (Neu5Ac 40–80 mM, A0812 Sigma-Aldrich, Poznan, Poland), heparan sulfate (HS, 10–100 µg/mL), d-(+)-galactose (Gal, 50 mM), d-(+)-glucose (Glu, 50 mM), d-(+)-mannose (Man, 50 mM), or N-acetyl-d-glucosamine (NAc, 50 mM) in PBS. Following incubation, cells were cooled, washed thrice with ice-cold PBS and overlaid with viral stocks. In case of Neu5Ac virus was additionally preincubated with the compound for 1 h at 4 °C before adsorption. Sugar moieties, Neu5Ac and HS but not NA were present during adsorption. Cells were incubated for 2 h at 4 °C, washed twice with ice-cold PBS and fixed with 4% formaldehyde. Analysis was carried out with flow cytometry or confocal microscopy.

D-mannose (purity ≥ 99%, Cat.#M2069) and ethanol (purity≥ 99.8%, 51976) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Rapamycin (purity = 99.30%, S1039), LY294002 (purity = 99.84%, S1105), 740 Y-P (purity = 98.38%, S7865) and MHY1485 (purity = 99.09%, S7811) were purchased from Selleck Chemicals (Houston, Texas, USA). Liquid Standard Diet (TP4020C), Lieber-DeCarli Control Liquid Diet (TP4030C), and Lieber-DeCarli ethanol Liquid Diet (TP4030D) were supplied by TROPHIC Animal Feed High-Tech Co. Ltd (Hai’an, Jiangsu, China). The antibodies against SREBP1c (AF-6283), PPARα (AF5301), ACC1 (AF6421), P110 of PI3K (AF-5112) were all from Affinity (Ancaster, ON, Canada). Anti-CPT1A (15184-1-AP), anti-ACOX1 (10957-1-AP), anti-FASN (10624-1-AP), anti-P85 (60225-1-Ig), anti-PPARγ (16643-1-AP), anti-PPARα (15540-1-AP) used in Figure 6D were obtained from Proteintech (Chicago, IL, USA). The antibodies against Akt (4691), phosphor-Akt (4060), mTOR (2983), phosphor-mTOR (5536) were from Cell Signaling Technology (Danvers, MA, USA).

G. sinensis and G. microphylla seeds were supplied by Shexian Forestry Bureau in Hebei, China. The samples were collected in December 2012. They were manually separated and kept in a cool and dry place for further use. Moisture content of the whole seed was dried at (105 ± 2 °C) for constant weight (10.0% (w/w)). The standard monosaccharides (l-rhamnose, l-arabinose, d-glucose, d-galactose, d-mannose, and d-xylose) were purchased from Sigma-Aldrich (Saint Quentin Fallavier, France). Dextran standards (DXT3k, DXT25k, DXT160k, DXT760k, and DXT1185k) with an average molecular weight range of 3.7 × 10³~1.2 × 10⁶ Da were purchased from TosoHaas (Tokyo, Japan). Absolute ethanol and isopropanol were purchased from Beijing Chemical Works (Beijing, China). Calcium carbonate and concentrated sulfuric acid were all purchased from China National Pharmaceutical Group Corporation (Shanghai, China). Syringe filters were supplied by Tianjin branch billion Lung Experiment Equipment Co. Ltd., (Tianjin, China).

Yeast agglutination assays were performed to investigate the expression of type 1 fimbriae by the K. pneumoniae strains. The assays were conducted on Kline concavity slides as described previously, with minor modifications (Gomes et al., 2021 (link)). Overnight cultures of the bacterial strains were inoculated (1:100) into fresh LB broth supplemented or not with C8-HSL and cultured until they reached O.D._600nm of 0.6. Bacteria were then mixed with 5% (w/v) suspension of Saccharomyces cerevisiae cells (Sigma-Aldrich) prepared in PBS. The intensity of the agglutination was documented using a digital camera. The agglutination of the yeast cells is specifically mediated by type 1 fimbriae since these fimbriae have great affinity for mannose, a highly abundant residue on yeast cell-surface. Therefore, the assays were also performed in the presence of 5% D-(+)-Mannose (Sigma-Aldrich) to confirm if the agglutination was indeed mediated by the type 1 fimbriae. Assays were carried out in triplicate for each K. pneumoniae strain.