Sialic acid

Dynabeads Antibody Coupling Kit protocol was used to attach sialic acid (Sigma A0812) or ICAM-5 (recombinant human ICAM-5 Fc Chimera 1950-M5-050) to magnetic bead resin (Thermo Fisher cat no. 14311D). Antibody/bead complex was incubated with the 1.11 or 1.20 g/cm³ viral densities for 1 h at 33°C, then overnight at 4°C. A magnet was used to separate the bead complex from the supernatant (supernatant was discarded). The magnetic beads plus any bound virus (approximately 1 mg beads per sample) were rinsed 2× for 10 min in 1,000 μL sterile PBS, with agitation. The rinsed beads (plus any bound virus) were then mixed with 180 μL of PBS plus 0.1% NP-40 and agitated for 5 min before the beads and supernatant were added to TCID₅₀ plates for viral titer analysis.

The dried EBN was purchased from Kuantan, Malaysia, and provided by Xiamen Si Nong Co. Ltd. It was made into homogenate by the following method. Briefly, 9 g dried EBN was immersed in the distilled water for 5 h. Then the soaked EBN was stewed in the water for 20 min with the solid-liquid ratio at 1 : 20 (g/mL). After high-pressure sterilization, the EBN homogenate was prepared for the experiment.
Meanwhile, N-acetylneuraminic acid (purchased from Sigma-Aldrich Co. LLC), which constitutes the large majority compound in sialic acid, was dissolved in distilled water at the concentration of 0.5 mg/mL. Concentration of sialic acid was detected as described below. To verity the stability of EBN during storage, sialic acid was detected after 42 d storage. Additionally, BDNF antibody was purchased from Sigma Chemicals Co. Kits for measurement of SOD, MDA, AChE, and ChAT were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China).

Mtb H37Rv Pasteur strain was cultured on Middlebrook 7H11 agar plates (BD Diagnostics) at 37°C. After 3 weeks, mycobacterial cells were harvested and extracted with chloroform, methanol and mixtures of chloroform‐methanol. The extracts were pooled, dried and partitioned with chloroform/methanol/water (8:4:2 v/v). Aqueous phase and chloroform phase were separated and evaporated to dryness. Presence of free GM3, sialyllactose and sialic acid in these extracts was analyzed by thin layer chromatography developed with chloroform/methanol/water (with 0.2% of CaCl₂) (5:4:1 v/v). Thin layer chromatography plates were revealed with resorcinol, a reagent that specifically stains sialic acid or sialic acid‐derivatives with a distinctive brown‐violet or blue‐violet colour (Waters et al., 1992). Presence of total sialic acid in Mtb H37Rv chloroform and aqueous extracts was determined by acid hydrolysis with 1N HCl in methanol at 80°C for 2 h and thin layer chromatography analysis as explained above. Purified bovine milk ganglioside GM3, sialyllactose and sialic acid working standards (0.025 mg) were obtained from Sigma‐Aldrich.

dextran sulfate (DS) 5,000, DS 50,000, DS 500,000, dextran, heparin grade II, condroitin sulfate A, and C, heparan sulfate, keratan sulfate, glycophorin, asialoglycophorin, orosomucoid, fetuin, asialofetuin, laminin, heparin sulfate proteoglycan, maltose, galactose, trehalose, manose, glucose and N-acetyl-galactosamine, and sialic acid were from Sigma Chemical Co. (Saint Louis, MO). 3′-sialyl Lewis X, 3′-sialyl Lewis A, 3′-sulfated Lewis A, 3′-sialyl lactose, 6′-sialyl lactose, tetrasacharide -A, -B, and -C, di-sialyl lacto-tetraose, 3′-sialyl fucosil lactose, monosialoganglioside (GM3), disialoganglioside (GD1a), disialoganglioside (GD1b), trisialoganglioside (GT1B), tetrasialoganglioside (GM1), and tetrasialoganglioside (GQ1B) were from Oxford GlycoSytems (Oxford, UK) or from Carbosynth (Berkshire, UK). Other reagents were from Sigma Chemical Co (Saint Louis, MO).