Regulation of Carbapenemase Gene Conjugation in Escherichia coli Clinical Isolates.

Background: The purpose of this study is to raise awareness of the hazards of carbapenemase epidemics and provide theoretical support for preventing the spread of carbapenemase-producing organisms. Methods: A total of 893 non-duplicate E. coil strains were recruited from three major local hospitals. The carbapenemase genotype of each imipenem-resistant strain was analyzed. Molecular typing and homology analysis of the main carbapenemase-producing strains reveal the transmission mode of resistance genes. Through the conjugation experiment, the potential spreading risk of carbapenemase genes was analyzed. Extended-spectrum beta-lactamase genes and replicon detection of the conjugant carrying plasmid were performed. The unannotated Escherichia coli bacterial small non-coding RNAs (sRNAs) interacting with sdiA were predicted through a bioinformatics tool. The sRNAs overexpression and knockout strains were constructed, and the effect of sRNA on conjugation was analyzed. Results: A total of 8 carbapenemase-producing strains were detected (0.90%, 8/893). The main carbapenemase genotype was blaKPC -2 (7 strains). Multilocus sequence typing indicated that 7 E. coli isolates belonged to ST-10, ST-101, ST-131, ST-405, ST-410, and ST-1193, ST-2562, respectively. Homologous cluster analysis revealed that the sequence types among the 7 E. coli were high diversity. The blaKPC -2 genes were successfully transferred from these isolates to EC600 by conjugation. All transconjugant cells exhibited significantly reduced susceptibility to the imipenem. IncFII was the most common conjugative plasmid type (85.7%, 6/7). Bioinformatics predicted the interaction between RydB and sdiA. Further experiments found that the interaction between RydB and sdiA improved the bacterial conjugation rate between MG1655 and EC600. The regulation effect of RydB on E. coli conjugation was not affected by the replicon type and/or harboring resistance coding genotype in conjugative plasmids. Conclusion: Our findings emphasized the epidemiological characteristics of carbapenemase-resistant E. coli. A functional phenotype of the new sRNA RydB was identified, and the regulation effect of RydB on E. coli conjugation was improved.

Ibuprofen-mediated potential inhibition of biofilm development and quorum sensing in Pseudomonas aeruginosa.

AIMS: Pseudomonas aeruginosa is one of the leading causes of opportunistic and hospital-acquired infections worldwide, which is frequently linked with clinical treatment difficulties. Ibuprofen, a widely used non-steroidal anti-inflammatory drug, has been previously reported to exert antimicrobial activity with the specific mechanism. We hypothesized that inhibition of P. aeruginosa with ibuprofen is involved in the quorum sensing (QS) systems. MAIN METHODS: CFU was utilized to assessed the growth condition of P. aeruginosa. Crystal violent staining and acridine orange staining was used to evaluate the biofilm formation and adherence activity. The detection of QS virulence factors such as pyocyanin, elastase, protease, and rhamnolipids were applied to investigation the anti-QS activity of ibuprofen against P. aeruginosa. The production of 3-oxo-C12-HSL and C4-HSL was confirmed by liquid chromatography/mass spectrometry analysis. qRT-PCR was used to identify the QS-related gene expression. Furthermore, we explored the binding effects between ibuprofen and QS-associated proteins with molecular docking. KEY FINDINGS: Ibuprofen inhibits P. aeruginosa biofilm formation and adherence activity. And the inhibitory effects of ibuprofen on C4-HSL levels were concentration-dependent (p < 0.05), while it has no effect on 3-oxo-C12-HSL. Moreover, ibuprofen attenuates the production of virulence factors in P. aeruginosa (p < 0.05). In addition, the genes of QS system were decreased after the ibuprofen treatment (p < 0.05). Of note, ibuprofen was binding with LuxR, LasR, LasI, and RhlR at high binding scores. SIGNIFICANCE: The antibiofilm and anti-QS activity of ibuprofen suggest that it can be a candidate drug for the treatment of clinical infections with P. aeruginosa.

APOC3 promotes TNF-α-induced expression of JAM-1 in endothelial cell via PI3K-IKK2-p65 pathway.

Atherosclerosis is a chronic inflammatory disease with lipid accumulation. Apolipoprotein C3 (APOC3), which is an important regulator of human lipid metabolism, is associated with multiple vascular mechanisms in atherosclerosis and proinflammatory responses. We have previously reported that the expression of inflammatory cytokine TNF-α is elevated in human endothelial cells (HUVECs) after APOC3 treatment. This study investigates the APOC3 signaling pathway involved in TNF-α-mediated expression of JAM-1 in HUVECs. Cultured HUVECs were exposed to APOC3 (50 µg/ml) for 16 h. Mechanistic studies were carried out by silencing TNF-α gene with lentiviral TNF-α-shRNA. Our study was based on the eight signaling pathway inhibitors to block the effect of APOC3 in HUVECs. The expression of JAM-1 was determined by qRT-PCR, Western blotting, and flow cytometry. IKK2 degradation and NF-κB p65 phosphorylation were determined by Western blotting. Our results showed that APOC3 significantly promoted the TNF-α-induced expression of JAM-1 in HUVECs. Inhibiting APOC3 reversed the TNF-α-induced overexpression of JAM-1. Moreover, APOC3 induced the expression of NF-κB p65 and degraded IκB. In conclusion, APOC3 promoted the expression of JAM-1 via the NF-κB, IKK2, and PI3K signaling pathway.

APOC3 induces endothelial dysfunction through TNF-α and JAM-1.

BACKGROUND: The fatality rate for cardiovascular disease (CVD) has increased in recent years and higher levels of triglyceride have been shown to be an independent risk factor for atherosclerotic CVD. Dysfunction of endothelial cells (ECs) is also a key factor of CVD. APOC3 is an important molecule in lipid metabolism that is closely associated with hyperlipidemia and an increased risk of developing CVD. But the direct effects of APOC3 on ECs were still unknown. This study was aimed at determining the effects of APOC3 on inflammation, chemotaxis and exudation in ECs. METHODS: ELISA, qRT-PCR, immunofluorescence, flow cytometry and transwell assays were used to investigate the effects of APOC3 on human umbilical vein endothelial cells (HUVECs). SiRNA-induced TNF-α and JAM-1 silencing were used to observe how APOC3 influenced the inflammatory process in the ECs. RESULTS: Our results showed that APOC3 was closely associated with the inflammatory process in ECs, and that this process was characterized by the increased expression of TNF-α. Inflammatory processes further disrupted the tight junctions (TJs) between HUVECs by causing increased expression of JAM-1. JAM-1 was involved in maintaining the integrity of TJs, and it promoted the assembly of platelets and the exudation of leukocytes. Changes in its expression promoted chemotaxis and the exudation of ECs, which contributed to atherosclerosis. While the integrity of the TJs was disrupted, the adhesion of THP-1 cells to HUVECs was also increased by APOC3. CONCLUSIONS: In this study, we describe the mechanism by which APOC3 causes inflammation, chemotaxis and the exudation of ECs, and we suggest that controlling the inflammatory reactions that are caused by APOC3 may be a new method to treat CVD.

Plasma miR-185 is decreased in patients with esophageal squamous cell carcinoma and might suppress tumor migration and invasion by targeting RAGE.

The receptor for advanced-glycation end products (RAGE) is upregulated in various cancers and has been associated with tumor progression, but little is known about its expression and regulation by microRNAs (miRNAs) in esophageal squamous cell carcinoma (ESCC). Here, we describe miR-185, which represses RAGE expression, and investigate the biological role of miR-185 in ESCC. In this study, we found that the high level of RAGE expression in 29 pairs of paraffin-embedded ESCC tissues was correlated positively with the depth of invasion by immunohistochemistry, suggesting that RAGE was involved in ESCC. We used bioinformatics searches and luciferase reporter assays to investigate the prediction that RAGE was regulated directly by miR-185. Besides, overexpression of miR-185 in ESCC cells was accompanied by 27% (TE-11) and 49% (Eca-109) reduced RAGE expression. The effect was further confirmed in RAGE protein by immunofluorescence in both cell lines. The effects were reversed following cotransfection with miR-185 and high-level expression of the RAGE vector. Furthermore, the biological role of miR-185 in ESCC cell lines was investigated using assays of cell viability, Ki-67 staining, and cell migration and invasion, as well as in a xenograft model. We found that overexpression of miR-185 inhibited migration and invasion by ESCC cells in vitro and reduced their capacity to develop distal pulmonary metastases in vivo partly through the RAGE/heat shock protein 27 pathway. Interestingly, in clinical specimens, the level of plasma miR-185 expression was decreased significantly (P = 0.002) in patients with ESCC [0.500; 95% confidence interval (CI) 0.248-1.676] compared with healthy controls (2.410; 95% CI 0.612-5.671). The value of the area under the receiver-operating characteristic curve was 0.73 (95% CI 0.604-0.855). In conclusion, our findings shed novel light on the role of miR-185/RAGE in ESCC metastasis, and plasma miR-185 has potential as a novel diagnostic biomarker in ESCC.

[The diagnostic value of quantitative measurement of a proliferation-inducing ligand mRNA in sputum samples from lung cancer patients].

OBJECTIVE: To establish a real-time fluorescence quantitative polymerase chain reaction (RFQ-PCR) method for quantifying a proliferation-inducing ligand (APRIL) mRNA in sputum samples from patients with non-small cell lung cancer (NSCLC), and to evaluate its role in the diagnosis of NSCLC. METHODS: Seventy-one cases of NSCLC and 62 cases of benign pulmonary disease were enrolled in this study from August 2007 to May 2008 in Affiliated Hospital of Nantong University, Jiangsu. Sixty-five healthy volunteers served as the control. The fluorescence of the PCR products was detected continuously during the amplification by RFQ-PCR. According to the standard curves created by plasmid DNA, the expression level of target genes in clinical samples was determined using software. The results were presented as the ratios of target genes to beta(2)-microglobulin (beta(2)-M) mRNA, and compared with those obtained by conventional cytological method. RESULTS: The detection range of the assay was from 38 copies/microl to 3.8 x 10(6) copies/microl. The coefficients of variation values of both intra-experimental and inter-experimental reproducibility were 8.5% and 13.6%, respectively. The expression of APRIL mRNA in tumor sputum was higher than that in benign pulmonary disease and healthy volunteers (t = 10.50, 11.32, P < 0.01). The positive rate for APRIL mRNA expression was 81.7% (58 of 71) in sputum samples of NSCLC, 3.2% (2/62) in benign pulmonary disease and 1.5% (1/65) in healthy volunteers when cut-off values for positivity were set at the x(-) +/- 2 s of mRNA expression in health volunteers. The level of APRIL mRNA of NSCLC was not related to sex, age, smoking status, TNM stage and lymph node metastasis (P > 0.05, respectively), but was related to pathology subtype and the location of tumors (P < 0.05, respectively). The APRIL mRNA assay (82%) produced a higher detection rate than conventional cytological method (14%) (chi(2) = 67.68, P < 0.01). CONCLUSION: Measurement of the expression of APRIL mRNA in sputum by RFQ-PCR showed high sensitivity and specificity, which maybe useful in diagnosing NSCLC.