Polymyxin b sulphate

Wt, Map3k8^−/−, and Map3k8^KD C57BL/6 J mice were maintained in an animal house in accordance with the Institutional Guidelines for the Care and Use of Laboratory Animals in Research, the relevant European Council Directive (2010/63/EU) and Spanish law (R.D. 1201/2005) with approval of the Ethics Committee of the Consejo Superior de Investigaciones Científicas. All the mice used were males that were five weeks old at the time of BM transplantation, and the analyses were conducted ten weeks later. Two weeks before irradiation, recipient Wt and Map3k8^−/− mice were given sterilized acidic water supplemented with 10 μg/ml polymyxin B sulphate (Sigma-Aldrich, P4932) and 0.1 mg/ml neomycin (Sigma-Aldrich, N112). This water supply was maintained for five weeks, and the animals were then switched to acidic water without antibiotics for the remainder of the study. BM aplasia was induced by irradiating the mice with one dose of 10 Gy. On the same day, 2 × 10⁶ BM cells (Map3k8^−/− and DsRed⁺Wt cells at a 1:1 ratio) were injected into the mice. BM DsRed⁺Wt cells were isolated from C57BL/6 mice transgenic for DsRed expressed under control of the β-actin promoter^{52 (link)}. Ten weeks after BM transplantation, blood was collected from the facial vein, and the percentages of circulating DsRed⁺Wt and Map3k8^−/− myeloid cells were determined.

E. coli DH5α was used for cloning (Clontech) while E. coli OverExpress C43(DE3) (Lucigen) was applied for heterologous expression. Catalase from bovine, testosterone 1, (2hydroxypropyl)-β-cyclodextrin and polymyxin B sulphate were obtained from Sigma Aldrich. Other chemicals were of analytical grade and purchased from commercial sources.

The roots of infected samples at each time point were vigorously grinded in a mortar for 1 g of roots per 1 ml distilled water, and the serial dilutions of extracted suspensions were made with dilution multiple from 10³ to 10⁸_, and 0.1 ml aliquots were spread on the surface of a CPG medium then cultured in a CPG medium. The medium is made of 10 g bacto peptone (Difco), 5 g Glucose, 1 g casamino acid (Difco), 15 g bacto agar (Difco), and 1 L distilled water. This medium was supplemented with 1% polymyxin B sulphate (Sigma), 1% crystal violet, 1% bacitracin (Sigma), and 1% cycloheximide (Sigma). After incubating plates at 28°C for 48 hours, colonies of R. solanacearum were counted and cfu were calculated per gram of roots. Three replicates were prepared for each sample.

Tetracycline hydrochloride (Sigma), gentamicin sulphate (Sigma), Muller-Hinton broth (Oxoid), Muller Hinton agar (Oxoid), Resazurin (Sigma), rhamnolipids JBR325 comprising approx. 3:1 dirhamnolipids to monorhamnolipids respectively (Jeneil Biotech), Rhamnolipids (produced in-house from Burkholderia thailandensis E264 predominantly comprising C₁₄–C₁₄ dirhamnolipid with a minor component of monorhamnolipid), lactonic sophorolipids (LSL) mainly comprising the diacetylated congener of molecular weight 688, acidic sophorolipids (ASL) comprising different chain length acetylated congeners, Polymyxin B sulphate comprising a mixture of poymyxin B1 and B2 (Sigma).

Bacterial lipopolysaccharides (LPS) from Escherichia coli (TLR4 agonist) (Sigma, O111:B4) was dissolved in 1X phosphate- buffered saline (PBS). Polymyxin B sulphate (TLR4 antagonist) (Sigma-81334) was dissolved in water. The LPS-polymyxin B combination was pre-incubated at 37°C for 2 hours before treating cells. Methyl pyruvate (Sigma-371173) was dissolved in 1XPBS and used at 8.8 mM final concentration. Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) (Sigma-C2920) was dissolved in 95% ethanol at 10 μM and used as a positive control for the mitochondrial membrane potential assay.

Blood was collected from the brachial vein in the wing from nearly 750 wild-caught adult birds using sterile syringes, into EDTA-microfil tubes. Plasma was separated from the cellular part by centrifugation of the blood samples. Resulting plasma was stored at − 70°C. Additionally, faecal samples were collected for screening of an active virus excretion. Flaviviruses have been detected in birds from cloacae swab samples in previous studies [25 (link), 26 (link)]. Faecal samples were taken from 300 birds, and placed in virus transport medium (Hanks balanced salt solution containing 0.5% lactalbumin, 10% glycerol, 200 U/ml penicillin, 200 μg/ml streptomycin, 100 U/ml polymyxin B sulphate, and 250 μg/ml gentamycin, and 50 U/ml nystatin; Sigma) in 2011. Faecal samples were further collected from 202 birds in 2013 and placed into empty tubes and frozen immediately to − 70°C.