Lps e coli serotype 055 b5

Forty-eight healthy female 8-wk-old C57BL/6J mice with a mean weight of 19.9±1.9 g were used in this study. All animal protocols were approved by the Ethics Committee for the Care and Use of Laboratory Animals at the Fourth Military Medical University. The animals were housed in plastic cages at a constant temperature (22±2°C) and humidity (55±10%) with 12 h–12 h light–dark conditions. The animals were allowed free access to food and water before the experiment. A peripheral injection of LPS was administered to evoke neuroinflammation in mice as previously described [15] (link). Briefly, experimental Groups Mice were randomly divided into four groups. In the sham control group (Normal) mice were injected intraperitoneally (i.p.) with saline (3 mg/kg) was allowed free access to food and water without any treatment. The control group (LPS) was intraperitoneally (i.p.) injected with a single dose of saline (3 mg/kg) and vehicle (normal saline) 1 h before the 1.0 mg/kg LPS (E.coli, serotype 055:B5, Sigma) injection. The PQQ treatment groups: LPS+PQQ3 and LPS+PQQ10 were administered two doses of PQQ (3 or 10 mg/kg) once 1 h prior to LPS injection respectively. Their body weights were monitored daily; in a subset of animals, core body temperature was also monitored (DC temperature controller, World Precision Instrument).

Microcalorimetric measurements of the binding of thanatin or divalent cations (Mg²⁺, Ca²⁺) to LPS were performed on a MicroCal Auto-ITC200 instrument (Malvern Instruments, Malvern, UK)^{35 (link)}. LPS (E. coli serotype 055:B5, Sigma, USA) was dissolved in 20 mM Tris-HCl (pH = 6.8) or 10 mM PBS (pH = 7.4), vortexed vigorously for 15 min, and sonicated for 15 min at 60 °C. The LPS solution was sonicated for 5 min prior to use. Thanatin was dissolved in Tris-HCl (pH = 6.8) and titrated into LPS in Tris-HCl (pH = 6.8). Divalent cations (Mg²⁺, Ca²⁺) were dissolved in PBS (pH = 7.4) and titrated into LPS in PBS (pH = 7.4). All samples were degassed for 10 min in a sonication bath before the experiments. These experiments were performed at 25 °C. The generated peaks were integrated using Origin 7.0 software. The errors for all the reported thermodynamic parameters were estimated through Monte Carlo simulation with the standard errors of three experiments.

A total of 250 healthy H. cumingii with an average weight of 15 g, ~1 year old, were purchased from an aquaculture farm in Wuhu City, Anhui Province, China. The freshwater mussels were maintained in aerated freshwater and allowed to acclimate at room temperature for 7 days before processing.
LPS (E. coli serotype 055:B5) and PGN (Staphylococcus staphylolyticus) were purchased from Sigma (St. Louis, MO, USA). Staphylococcus aureus, Micrococcus luteus, Bacillus subtilis, Vibrio anguillarum, E. coli (maintained and available in our laboratory), and Vibrio parahaemolyticus (ATCC 17802, Microbial Culture Collection Center, Beijing, China) bacteria were grown in Luria–Bertani (LB) at 37°C. Aeromonas hydrophila (ATCC 7966, Microbial Culture Collection Center, Beijing, China) was grown in LB broth and incubated at a temperature of 28°C.

C57BL/6J mice were purchased from Charles River Laboratories (Portage, MI, USA), and FAAH KO mice were kind gift from Dr. Cravatt’s laboratory. All experiments in this study were carried out in accordance with the guiding principles for the care and use of animals approved by the National Institute on Alcohol Abuse and Alcoholism (LMS-HK-13, LMS-HK-41). Mice 8–12 weeks of age received intraperitoneal (i.p.) injections of saline or 1.0 mg/kg LPS (E. coli, serotype 055:B5, Sigma) followed by synaptamide (2 or 5 mg/kg). At 2 h after final injection, mice were deeply anesthetized with isoflurane and perfused quickly with chilled phosphate-buffer saline (PBS) for RNA isolation or chilled PBS containing 4 % paraformaldehyde for immunostaining.

The animal use protocol for this study was approved by the Committee on Animal Care of the Institute of Subtropical Agriculture, Chinese Academy of Sciences. Thirty healthy pigs (Landrace, 21 ± 2 d, barrow, 5.86 ± 0.18 kg) were chosen and randomly assigned to three groups (n = 10): (1) nonchallenged control (CON); (2) LPS-challenged control (LPS, E. coli serotype 055:B5; Sigma Chemical, St. Louis, MO, USA); (3) LPS+0.60% HMB-Ca treatment (LPS+HMB; HMB-Ca, purity = 99.2%, Ca = 13.6%, Sipu Biochemical Co. LTD, Zhangjiagang, China). The HMB-Ca and LPS doses were used in accordance with our previous studies [12 (link)]. On days 1, 3, 5, 7, 9, 11, 13, and 15 of the trial, overnight fasted piglets of the LPS and LPS+HMB groups were intraperitoneally administered LPS, whereas piglets in the CON group were injected with the same volume of sterile saline as previously described [12 (link)]. Piglets were raised individually in cages (1.80 × 1.10 m pen) and had ad libitum access to diets and clean drinking water. Diets were formulated to meet the nutritional needs for piglets according to the National Research Council (NRC, Supplementary Table 1) [18 ]. The experiment lasted for 15 days.

LPS (E. coli, serotype 055:B5), anti-Flag antibody (F3165), anti-Myc antibody (M4439) and anti-β-actin antibody (A2228) were purchased from Sigma. Poly(I:C), R-848 and ODN 1585 were from Enzo Life Sciences. DAPI (D1306) were purchased from Invitrogen. Antibody to p-IKKβ (2694), IKKβ (2678), p-JNK (4668), JNK (9252), p-ERK (4376), ERK (4695), p-IκBα (9246), IκBα (4812), MyD88 (4283), HA (3724), TRAF6 (4743; immunofluorescence analysis), and Sufu (2522) were purchased from Cell Signaling. Anti-His (sc-8036), anti-TRAF6 (sc-8409) (immunoprecipitation and immunoblot analysis), anti-IRAK1 (sc-5288) and anti-IRAK4 (sc-374349) were purchased from Santa Cruz. Goat anti-mouse IgG (31430) and goat anti-rabbit IgG (31460) were purchased from Thermo Fisher Scientific. C25-140 (HY-120934) was purchased from MedChemExpress.