Glass bead beater

Some mice were euthanized on days 6, 8 and 10 after intranasal inoculation with influenza A as we have previously reported ^{33 (link)}; lungs were collected and rinsed in sterile water to lyse excess red blood cells. Lungs were placed in Dulbecco's modified Eagle's medium (DMEM) and homogenized using a glass bead beater (Biospec Products, Bartlesville, OK). Samples were diluted in DMEM containing 0.05 % trypsin (Worthington Biochemical, Lakewood, NJ), were centrifuged for 10 min at 9,000 rpm, and supernatants were serially diluted in 96-well round-bottom plates (Fisher Scientific, Atlanta, GA). Samples were then transferred to 96-well round-bottom plates containing Madin Darby canine kidney (MDCK) cell monolayers. Lung dilutions and MDCK cells were incubate for 4 days, and then visualized for characteristic adherence of turkey red blood cells (Fitzgerald Industries, Concord, MA).

For AST assay, purified rPdxR was washed and reconstituted in 50 mM sodium phosphate buffer, pH 7.4, using Protein Desalting Spin Columns (#89849, Pierce Biotechnology, Rockford, IL). The S. mutans whole cell lysates were prepared from mid-exponential phase cultures grown in BHI and homogenized using a glass bead beater (Biospec, Bartlesville, OK) as described previously (Burne et al., 1999 (link); Wen & Burne, 2002a (link)). AST activity was measured in a coupled assay with malate dehydrogenase and aspartate plus 2-oxoglutarate as substrates (Karmen et al., 1955 (link); Ziak et al., 1990 ). Malate dehydrogenase catalyzed oxidation of NADH, which is proportional to AST product, oxaloacetate, was monitored by the change of absorbance at 340 nm using a Bio-Tek microplate reader (Synergy II™; Bio-Tek, Winooski, VT). Porcine AST (Sigma, St Louis, MO) was used as a positive control, and maltose-binding protein domain (MalE) (New England Biolabs) expressed and purified using similar strategies, was used as a negative control. Protein concentration was assayed using a BCA™ protein assay kit (Pierce) as recommended by the manufacturer. AST activity was further normalized by protein concentration and is expressed as units per milligram of protein.