Objective To investigate toil-like receptor 4 (TLR4) expression and activity in peripheral blood mononuclear cells(PBMCs) of obese patients.Methods PBMCs from 16 obese patients and 16 normal control subjects were collected.TLR4 and IκBα protein concentrations were measured in PBMCs by western blotting.TLR4 and interleukin-6 (IL-6) mRNA expression levels in PBMCs were measured by quantitative realtime PCR.The blood levels of glucose,insulin,free fatty acids (FFA),IL-6 and tmnor necrosis factor-α (TNF-α) were measured after an overnight fast.Results The levels of FFA,IL-6,TNF-α and homeostasis model assessment of insulin resistance index (HOMA-IR) in obese patients were higher than that of control group (FFA:(879 ± 64) μmol/L vs.(458 ± 48) μmol/L; IL-6:(2.20 ± 0.35) ng/L vs.(1.26 ± 0.25) ng/L; TNF-α:(1.96±0.32) ng/Lvs.(1.38 ±0.24) ng/L;HOMA-IR:(1.8±0.2) vs.(0.4±0.1) ;t=24.613,P=0.000;t =14.993,P =0.000;t =9.128,P =0.000;t =32.254,P =0.000).Increased TLR4 gene and protein expression were observed in PBMCs of obese patients compared with control group(TLR4 mRNA:(3.13±0.21) vs.(0.99 ± 0.03),t =54.758,P ＜ 0.05; TLR4 protein:(7.04 ± 0.42) vs.(2.53 ± 0.17),t =77.450,P ＜0.05).IκBα protein concentration in PBMCs of obese patients (2.52 ±0.16) was lower than in control group (4.00 ± 0.30,t =23.284,P ＜ 0.05),indicating elevated IKKβ/NF-κB signaling.The increase in TLR4 and NF-κB signaling was accompanied by elevated expression of the NFκB-regulated gene IL-6 ((2.55 ±0.15) vs.(1.03 ±0.11),t =53.981,P ＜0.05).Conclusion TLR4 expression and activity are increased in the PBMCs of patients with obesity and might involve in the development and progress of insulin resistance.

The effects of elevated levels of glucose and (or) free fatty acids on insulin secretion were studied in obese rats by intravenous glucose tolerance test and isolated pancreas perfusinn. The results showed that both glucose- and arginine-stimulated insulin secretions were severely impaired by glucolipotoxicity and the production of ketone was increased dramatically.

Objective To investigate thyroid hormone nuclear receptor expression in peripheral blood mononuclear cells (PBMCs) and its relation with injury severity in patients with severe multiple trauma.Methods Twenty-eight patients with severe muhiple trauma and another 26 healthy subjects as control were enrolled in the study.At 2 days after injury,the levels of free triiodothyronine (FT3),free thyroxine (FT4) and thyroid-stimulating hormone (TSH) in peripheral venous blood were determined by using immunoradiometry,and the expressions of mRNA of the thyroid nuclear receptors (TRα and TRβ) in PBMCs were measured by using real-time RT-PCR.Results FT3 concentration in peripheral venous blood was significantly decreased in patients with severe multiple trauma versus healthy subjects.Concurrently,the expressions of TR mRNA (TRα and TRβ) were significantly decreased in PBMCs of patients with severe multiple trauma compared with healthy subjects (TRα mRNA:3.86 ±0.54 vs.5.24 ± 1.17,P ＜0.05;TRβ mRNA:9.86±2.27 vs.13.57 ±2.45,P ＜0.05).Pearson correlation analysis showed that FT3 concentration and the expression of TRβ mRNA correlated negatively with injury severity score (ISS) in patients with severe multiple trauma (r=-0.445,-0.496,P=0.018,0.007).Conclusions These data provide the evidence of a lowered activity of the thyroid signaling pathway in PBMC and a significantly negative correlation between FT3 concentration and the expression of TRβ mRNA in respect of injury severity in patients with severe multiple trauma.Additional investigations are needed to further determine the roles of the thyroid signaling pathway in adverse outcomes in the wake of severe multiple trauma.

Objective To develop an HPLC-MS/ MS method for quantitative determination of PA-824 in rat plasma and to study the pharmacokinetics of PA-824 in rat after oral administration. Methods An HPLC-MS/ MS method was developed and validated for determination of PA-824 in rat plasma using metronidazole as internal standard.The proteins in plasma samples were precipitated with methanol,and PA-824 was enriched for analysis by HPLC-MS/ MS.An Inertsil? ODS3 C18 column (150 mm×4.6 mm,5 μm) was applied with mobile phase composed of methanol- 0.03% triethylamine (TEA) in water (90:10) ,at a flow rate of 0. 5 mL ? min-1 and column temperature of 30 ℃ . Quantitation was performed on a triple quadrupole mass spectrometer applying electrospray ionization technique and operating in multiple reaction monitoring (MRM) and positive ion mode with transitions at 360.1/ 175.0 for PA-824 and 172.0/ 128.0 for metronidazole.The concentration of PA-824 in plasma was tested after oral administration at various time points and the data were processed with software DAS.2.0. Results The standard calibration curve for spiked rat plasma containing PA-824 was linear over the range of 0. 1 - 10. 0 μg?mL-1 . The recoveries obtained for PA-824 were greater than 92.13%.Intra-day and inter-day coefficient of variation were less than 6.6%.After oral administration,the main pharmacokinetic parameters were AUC(0-t) : ( 3 297. 503 ± 320. 958) mg ? L-1 ? min-1 , AUC(0-∞ ) :(3 558.315±338.860)mg?L-1?min-1 ,tmax:(360.000±64.143)min,Cmax:(3.5±0.3)μg?mL-1 . Conclusion The method is rapid,accurate,simple,and successfully applied in a pharmacokinetic study of fixed dose oral administration of PA-824 in rats.

OBJECTIVETo study the effect of palmitate on toll-like receptor 4 (TLR4) expression and signaling in vascular endothelial cells.

METHODSPig iliac endothelial cells (PIECs) were incubated with palmitate. TLR4 gene expression levels were measured by quantitative real-time PCR, and TLR4 and IκBα protein expressions by Western blotting. The expression levels of TLR4 protein on the surface of PIECs were quantified using flow cytometry. ELISA was employed to detect tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) concentrations in the cell medium.

RESULTSPalmitate treatment significantly increased TLR4 mRNA and protein expression levels in PIECs compared with those in the control cells (4.73∓0.61 vs 1.25∓0.90, P<0.05; 5.79∓0.05 vs 4.07∓0.31, P<0.05). The expression levels of TLR4 on the cell surface significantly increased (38.070∓3.907 vs 29.390∓1.072, P<0.05), while IκBα protein level was significantly lowered in PIECs after palmitate treatment as compared with those in the control cells (2.04∓0.22 vs 3.98∓0.18, P<0.05). Palmitate treatment significantly elevated TNF-α (2.52∓0.30 vs 1.38∓0.26, P<0.05) and IL-6 (IL-6: 3.28∓0.32 vs 1.44∓0.28, P<0.05) concentrations in the cell culture medium.

CONCLUSIONPalmitate can enhance TLR4 expression and signaling in porcine vascular endothelial cells.

Animals ; Blotting, Western ; Endothelial Cells ; drug effects ; metabolism ; I-kappa B Proteins ; metabolism ; Interleukin-6 ; metabolism ; NF-KappaB Inhibitor alpha ; Palmitates ; pharmacology ; RNA, Messenger ; Real-Time Polymerase Chain Reaction ; Signal Transduction ; Swine ; Toll-Like Receptor 4 ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism

Objective:Objective To explore the clinical values of next-generation sequencing (NGS) in bacterial 16S rRNA region and fungal ITS region for diagnosing and treating urinary tract infection (UTI) in renal transplant recipients.Methods:A total of 90 mid-stream clean-catch urine samples were collected from renal transplant recipients who were diagnosed with UTI at Hospital from January 2017 to December 2019. Each sample was equally divided and tested via NGS method and traditional urine culture separately. The results of pathogen test and detection rate were analyzed and compared.Results:And 21/90 sample were considered to be contaminated due to the identification of three or more kinds of microorganisms by culture. And among the remaining 69 samples, 36 (52.17%) cases tested positive by 16S rRNA sequencing, 25 (36.23%) positive by urine bacterial culture; meanwhile, 34(49.28%) tested positive by ITS sequencing and 4(5.80%) positive by urine fungal culture.Conclusions:The detection rate of both bacteria and fungi in NGS microorganism testing is higher than that in traditional urine culture ( P< 0.05). For renal transplant recipients with UTI, NGS microorganism testing is an effective supplement for traditional urine culture. Improving the detection rate and accuracy of etiology may enable an optimization of individualized treatment.