Objective To establish a specific,sensitive,simple human respiratory adenovirus detection method to put a good experimental foundation for developing universal and specific diagnostic reagents of human respiratory adenovirus.Methods The nucleotide sequences of 10 serotype of human respiratory adenovirus were obtained from GenBank.Highly consewed five pairs of universal and specific adenovirus primers were designed on the evaluation of multiple sequence alignment of the 10 full genomic sequences with the software DNAMAN 5.2.2,Gene Runner 3.05,BLAST,and to ensure that the polymerase chain reaction(PCR)products were type-specific.NP-40 sample lytic method was employed to prepare the template.The effectiveness,specificity and sensitivity of primers were evaluated.And PCR was carried out to test 64 samples of throat swabs of acute respiratory infection children.The positive PCR products were sequenced directly to identify the adenovirus serotypes.The positive specimens was inoculated on HeLa cells to observe cytopathie effect(CPE)under light microscope,and virus morphology were observed under electron microscope.Results BLAST results indicated that the five pairs of primers were specific adenovirus primers with low homology to the others.The primers were identified as PCR positive fragments obtained by using five pairs of primers to amplify the human adenovirus type 3 DNA,which did not react to the respiratory syncytial virus and Coxsackie virus.the effectiveness and specificity of the primer were thus indicated.PCR sensitive results showed 10-5 dilutin of adenovirus culture and DNA sample could be deteeted which meant the method was sensitive and stable.Two PCR positive specimens were detected in 64 clinical samples.the positive rate was 3.13%(2/64).Using the two PCR positive specimens to inoculate the HeLa cells,the typical adenovirus CPEs of rounded and aggregated,detatched cells were observed under light microscope.And a large number of adenovirus typical particles with characteristic lattice arrangement were observed in the infected cells under electron microscope.PCR product sequencing results showed that these two isolated adenoviruses were typed in human adenovims group B.Conclusions Universal and specific adenovirus PCR primers with the serotype specificity of the PCR products were designed successfully.The PCR primers was sensitive and specific and could be routinely applied in clinical adenovirus diagnosis for respiratory specimens.

OBJECTIVETo assess the role of methylated mismatch repair (MMR) genes (hMLH1, hMSH2 and hMSH3) in the carcinogenesis and progression of hepatocellular carcinoma (HCC).

METHODSSamples of 38 cases of HCC along with their corresponding noncancerous tissues, 2 samples of donated normal tissue and 6 cell lines were collected and subject to the methylation-specific PCR (MSP) to examine promoter methylation status of MLH1, MSH2 and MSH3. Six tumor cell lines were analyzed before and after 5-aza-2'-deoxycytidine treatment. In addition, alterations of mRNA expression of MMRs were investigated by quantitative reverse transcription-PCR.

RESULTSCpG island methylation of hMLH1 and hMSH2 was observed in 13.2% (5 of 38 samples) and 68.4% (26 of 38 samples) respectively in HCC, 2.6% (1 of 38 samples) and 55.3% (21 of 38) respectively in corresponding noncancerous tissues, but not in normal control tissues. Promoter methylation of the hMSH2 gene was present in 83.3% of cell lines tested (5/6), but none were observed for the hMLH1 gene. Promoter methylation of the hMSH3 gene was not identified in any tissue samples or cell lines. After 5-aza-2'-deoxycytidine treatment, hMSH2 methylation was induced or completely reversed, and its mRNA expression was increased in most cell lines.

CONCLUSIONSOur results suggest that promoter hypermethylation of hMLH1 and hMSH2 genes is common in HCC. Particularly, there is a high frequency of methylation of hMSH2 in both cancer and noncancerous tissues, but not in normal control tissue. Therefore, hypermethylation of MMR genes, especially hMSH2, may be involved in the carcinogenesis of HCC and may serve as an early diagnostic marker for HCC. The close correlation between hMSH2 methylation and low expression of its mRNA suggests that hMSH2 methylation is an important pathway in the regulation of gene expression.

Adaptor Proteins, Signal Transducing ; Azacitidine ; analogs & derivatives ; pharmacology ; Base Pair Mismatch ; genetics ; Carcinoma, Hepatocellular ; genetics ; Carrier Proteins ; biosynthesis ; genetics ; Cell Line, Tumor ; DNA Methylation ; DNA Modification Methylases ; antagonists & inhibitors ; DNA Repair ; genetics ; Gene Expression Regulation, Neoplastic ; Humans ; Liver Neoplasms ; genetics ; MutL Protein Homolog 1 ; MutL Proteins ; Neoplasm Proteins ; biosynthesis ; genetics ; Nuclear Proteins ; biosynthesis ; genetics ; RNA, Messenger ; biosynthesis ; genetics

Tumor microenvironment contributes to poor prognosis of pancreatic adenocarcinoma (PAAD) patients. Proper regulation could improve survival. Melatonin is an endogenous hormone that delivers multiple bioactivities. Here we showed that pancreatic melatonin level is associated with patients' survival. In PAAD mice models, melatonin supplementation suppressed tumor growth, while blockade of melatonin pathway exacerbated tumor progression. This anti-tumor effect was independent of cytotoxicity but associated with tumor-associated neutrophils (TANs), and TANs depletion reversed effects of melatonin. Melatonin induced TANs infiltration and activation, therefore induced cell apoptosis of PAAD cells. Cytokine arrays revealed that melatonin had minimal impact on neutrophils but induced secretion of Cxcl2 from tumor cells. Knockdown of Cxcl2 in tumor cells abolished neutrophil migration and activation. Melatonin-induced neutrophils presented an N1-like anti-tumor phenotype, with increased neutrophil extracellular traps (NETs) causing tumor cell apoptosis through cell-to-cell contact. Proteomics analysis revealed that this reactive oxygen species (ROS)-mediated inhibition was fueled by fatty acid oxidation (FAO) in neutrophils, while FAO inhibitor abolished the anti-tumor effect. Analysis of PAAD patient specimens revealed that CXCL2 expression was associated with neutrophil infiltration. CXCL2, or TANs, combined with NET marker, can better predict patients' prognosis. Collectively, we discovered an anti-tumor mechanism of melatonin through recruiting N1-neutrophils and beneficial NET formation.