Objective To establish a sensitive,specific,simple and high-throughput method for detection of the epidermal growth factor receptor (EGFR) mutation in the plasma samples of patients with non-small cell lung cancer (NSCLS) by the use of mutant-enriched liquid chip (MEL) assay.Methods The specific probes for the EGFR exon19 E746-750 deletion,exon 21 L858R mutation and wild-type sequence were designed and coupled to the microspheres coding with different fluorescent dye.The probe coupling efficiency was verified by crossing hybridization test with biotin-labeled reverse sequence.A blood-based MEL approach which integrates a sensitive mutant-enriched PCR and quantitative high throughput liquid chip assay for assessment of EGFR mutations was developed.The sensitivity and specificity of MEL was further evaluated using the mixture with different copy numbers of mutant and wild-type plasmids as template.The mutations of exon 19 and 21 of EGFR gene in plasma samples from 201 patients with stage ⅢB or Ⅳ NSCLC who enrolled in the First Affiliated Hospital of Guangzhou Medical College from September 2008 to April 2010 were analyzed by both the MEL and the mutant-enriched PCR assay.The result comparison was made between direct sequencing and MEL in 50 cases whose EGFR gene type had been tested by MEL and mutantenriched PCR.The correlation of EGFR gene mutation and the response to the Geftinib treatment was analyzed in 16 patients with lung adenocarcinoma as well.Results The probes were successfully coupled to the microspheres encoding with different fluorescent dye,and could be specifically recognized by the corresponding target sequence.The MEL was capable of detecting as few as 10 copies of EGFR mutants (sensitivity was 0.1％).Among the enrolled 201 cases of advanced NSCLC,the detection rate of the EGFR exon19 E746-750 del and exon 21 L858R was 55.7％ (112/201) by MEL assay.Conpared with mutantenriched PCR[58.2％ (117/201)],the coincidence rate was 97.5％ (196/201).There was no statistically significant difference between the results of mutant-enriched PCR and MEL (x2 =3.20,P ＞ 0.05).The mutations detection rate was 22.0％ (11/50) by directing sequencing,which was significantly lower than by MEL[50.0％ (25/50),x2 =12.07,P ＜ 0.05].Among the 16 patients treated with Gefitinib,9 cases who had EGFR mutation showed a higher response rate(P =0.041)and prolonged progression-free survival (x2 =6.76,P =0.009) after the treatment compared to those 7 who without EGFR mutation.Conclusions A new method of MEL with accuracy,specificity,fast and high-throughput is established for the detection of EGFR 19 E746-750 deletion and exon 21 L858R mutations in plasma from advanced NSCLC patients.It has the ability to provide the most direct and valuable guidance for clinicians to make decision on EGFR tyrosine kinase inhibitors therapy in the advanced NSCLC paticnts.

Isatis indigotica Fort. (Ban-Lan-Gen) is an herbal medicine prescribed for influenza treatment. However, its active components and mode of action remain mostly unknown. In the present study, erucic acid was isolated from Isatis indigotica Fort., and subsequently its underlying mechanism against influenza A virus (IAV) infection was investigated in vitro and in vivo. Our results demonstrated that erucic acid exhibited broad-spectrum antiviral activity against IAV resulting from reduction of viral polymerase transcription activity. Erucic acid was found to exert inhibitory effects on IAV or viral (v) RNA-induced pro-inflam-matory mediators as well as interferons (IFNs). The molecular mechanism by which erucic acid with antiviral and anti-inflammatory properties was attributed to inactivation of NF-kB and p38 MAPK signaling. Furthermore, the NF-kB and p38 MAPK inhibitory effect of erucic acid led to diminishing the transcriptional activity of interferon-stimulated gene factor 3 (ISGF-3), and thereby reducing IAV-triggered pro-inflammatory response amplification in IFN-β-sensitized cells. Additionally, IAV- or vRNA-triggered apoptosis of alveolar epithelial A549 cells was prevented by erucic acid. In vivo, erucic acid administration consistently displayed decreased lung viral load and viral antigens expression. Meanwhile, erucic acid markedly reduced CD8+cytotoxic T lymphocyte (CTL) recruitment, pro-apoptotic signaling, hyperactivity of multiple signaling pathways, and exacerbated immune inflammation in the lung, which resulted in decreased lung injury and mortality in mice with a mouse-adapted A/FM/1/47-MA(H1N1) strain infection. Our findings provided a mechanistic basis for the action of erucic acid against IAV-mediated inflammation and injury, suggesting that erucic acid may have a therapeutic potential in the treatment of influenza.