Ampicillin

MES (M3671), BSA (9048-46-8), BES (10191-18-1), BCA (B9643), CuSO4 (C2284), ThS (T1892), MTT (M2128) and Rose Bengal (330000) were purchased from Sigma. IPTG (420322) and DTT (3870) were purchased from Calbiochem. Other chemicals such as Ampicillin (2007081), NaCl (194848), KCl (194844), Na₂HPO₄ (191437), KH₂PO₄ (19142), EGTA (194823), MgCl₂ (191421), PMSF (195381), Ammonium acetate (191404), Heparin (904108) and DMSO were from purchased from MP biomedicals and protease inhibitor cocktail was from Roche (11697498001). Copper coated carbon grids were purchased from Ted Pella (01814.F, carbon type-B, 400 mesh Cu), DMEM advanced F12 media (12634010), Fetal Bovine Serum (16000044), Pensterp cocktail (04693159001) and Anti-anti (15240062) were purchased from Gibco. Tubulin (Thermo PA1-41331), actin (Thermo MA5-15739) were procured from thermo, total Tau antibody K9JA was purchased from Dako (K9JA, Dako A0024). The secondary antibodies Alexa Fluor 488 (A11034) and Alexa Fluor 555 (A32727) were purchased from thermo.

To further investigate the effect of dietary SM on atherosclerosis in the absence of the potentially confounding effects of a high fat diet, female apoE^-/- mice were randomly allocated into four groups and maintained for 19 weeks on a: (i) normal chow diet (n = 10), (ii) normal chow diet supplemented with 1.2% (wt/wt) egg SM (n = 9), (iii) normal chow diet plus antibiotics (n = 10), or (iv) normal chow diet supplemented with 1.2% (wt/wt) egg SM and antibiotics (n = 12) (Table 1).The SM-supplemented diet was prepared by adding 1.2% (wt/wt) egg SM (BOC Sciences, USA) to normal chow.
The chow diet was composed of wheat, lupins, barley, soya meal, mixed vegetable oils, canola oil, sodium chloride, calcium carbonate, di-calcium phosphate, magnesium oxide and a vitamin and trace mineral premix. It contained 4.8% total fat, 4.8% crude fibre, 20% protein, 7.6% acid detergent fibre, 16.4% neutral detergent fibre and 59.4% digestible energy.
The antibiotic solution was prepared by dissolving 1 g/L neomycin sulfate (Biomatik Corporation, Canada), 1 g/L ampicillin (MP Biomedicals, Australia), 1 g/L metronidazole (MP Biomedicals, Australia), and 0.5 g/L vancomycin HCl (Biomatik Corporation, Canada) in autoclaved water. The solution was filtered (0.22 μm) and stored for a maximum of 2 weeks at 4°C before use.

Fraction 430F-F5 and isolated compounds of S. terebinthifolia1-3, were evaluated for minimum inhibitory concentrations (MICs) against the four agr groups, the biofilm test strain (UAMS-1) and NRS249 (for δ-toxin quantification experiments) following the Clinical Laboratory Standards Institute (CLSI) M100-S23 guidelines for microtiter broth dilution testing^{45 (link)}. Fraction, compounds 1-3 and vehicle were tested at a concentration range of 4 to 563 μM, using 2-fold serial dilution. The optical density (OD_600nm) was measured with a BioTek Cytation 3 multimode plate reader, to calculate percent inhibition of growth. The IC₅₀ for growth was defined as the lowest concentration at which an extract displayed ≥ 50% inhibition and MIC (IC₉₀) at ≥ 90% inhibition. Control included the vehicle (DMSO) and antibiotic (Ampicillin, MP Biomedicals Inc). All tests were performed in triplicate and repeated using a new stock of bacteria on a separate day.

Equal parts (45.0 g; together 315 g) of seven whole sea cucumbers E. fraudatrix including the body wall were used to obtain the extracts. All pieces of cucumbers before lysis were treated carefully three times with a buffer containing mixture of antibiotics (10,000 U Pen/mL penicillin, 10,000 µg Strep/mL ampicillin, 25 µg Amphotericin B/mL Streptomycin; SKU:091674049) from MP bio (Irvine, CA, USA), to inactivate and remove possible bacteria on their surface. Then pieces of whole sea cucumbers were homogenized using a buffer containing 10 mM Tris-HCl, pH 8.0; 1.0 M NaCl, 1.0 mM DTT, 1.0 mM EDTA, and the above antibiotics, in a volume ratio of 1:10. The same method was used to prepare extracts of combined samples of isolated gonads, lungs, and intestines (10 g) from 18 to 20 sea cucumbers.

For all experiments, zebrafish embryos were initially derived germ-free using previously described gnotobiotic procedures with slight modification [75 (link)]. Briefly, fertilized eggs from adult mating pairs were harvested and incubated in sterile embryo media (EM) containing ampicillin (100 μg/mL), gentamicin (10 μg/mL), amphotericin B (250 ng/mL; MP Biomedicals, Santa Ana, CA), tetracycline (1 μg/mL; Sigma-Aldrich, St. Louis, MO), and chloramphenicol (1 μg/mL; Amresco, Solon, OH) for approximately 6 hours. Embryos were then washed in EM containing 0.1% polyvinylpyrrolidone-iodine (Syndel, Ferndale, WA) followed by EM containing 0.003% sodium hypochlorite (Fisher Scientific, Hampton, NH). Surface sterilized embryos were distributed into T25 tissue culture flasks (TPP, Trasadingen, Switzerland) containing 15 mL sterile EM at a density of 1 embryo/mL and kept in a temperature-controlled room at 28°C to 30°C with a 14 hours/ 10 hours light/dark cycle. The germ-free status of larval zebrafish was assessed before every experiment by visually inspecting flask water for microbial contaminants using an inverted microscope. Culture-based assessment of germ-free status was done as needed by plating 100 μL flask water on rich media (e.g., tryptic soy agar). Embryos were sustained on yolk-derived nutrients and not fed prior to or during any experiments.