Detection and subtyping of influenza A virus based on a short oligonucleotide microarray.

We report the design and characterization of a microarray with 46 short virus-specific oligonucleotides for detecting influenza A virus of 5 subtypes: H1N1, H1N2, H3N2, H5N1, and H9N2. A unique combination of 3 specific modifications was introduced into the microarray assay: (1) short probes of 19 to 27 nucleotides, (2) simple amplification of full-length hemagglutinin and neuraminidase cDNAs with universal primers, and (3) Klenow-mediated labeling and further amplification of the samples before hybridization. The assay correctly and specifically detected and subtyped 11 different influenza A isolates from human, avian, and swine species representing the 5 subtypes. When tested with 225 clinical samples, 20 were detected to be positive using our microarray-based assay, whereas only 10 were positive by the conventional culture method. The entire analysis was completed within 7 h. Thus, these modifications result in a specific, sensitive, and rapid microarray assay and may be used for constructing microarrays for the detection of all influenza subtypes and strains.

Effects of exogenous IGF- Ⅰ on intestinal mucosal epithelial cell apoptosis in rats with sever acute pancreatitis / 中华急诊医学杂志

Objective To investigate the effects of exogenous IGF- Ⅰon apoptosis, bax,bcl-2 and caspase-3 gene mRNA transcription in intestinal mucosal epithelial cell of SAP rats. Method Seventy-two male Wistar rats were randomly divided into sham operation group (SO),SAP group(SAP) and IGF-Ⅰ treatment (SAP + IGF-Ⅰ) group.Every group was randomly divided into 3 time units (6,12,24 h),8 rats as each time unit. SAP was induced in the rats by injecting adversely 5.0 % sodium taurocholate into biliary-pancreafic duct. The SO rats were infused with NS by the same way. The rats in IGF-Ⅰgroup were injected with IGF-Ⅰ by subcutano at half an hour before operation and three hours after operation,respectively. Animals in each group were killed separately at 6,12 and 24 hours after operation.The apoptosis in mucesal cells of small intestine was detected by TUNEL, and histo pathological changes of the small intestine was observed. The expressions of bax and bcl-2 and caspase-3 mR-NA gene in small intestine were measured by reverse transcription polymerase chain reaction(RT-PCR). Results Compared with the SAP group,the serum amylase were lower in IGF-Ⅰ group,and there existed significant at 12 h and24 h (P < 0.05).The pathological score of small intestinal was significantly reduced in IGF-Ⅰ group com-pared with SAP group,and there were statistical differences at 12 h and 24 h.ln IGFo-Ⅰ group,the apoptosis index of intestinal epithelial decreased significantly compared with SAP group[6 h: (13.88±1.73) vs. (19.00±2.78) ;12h:(10.13±1.55) vs. (17.63±.60);24 h:(9.50±1.07) vs. (17.25±2.76)] (P <0.05); the histopathdogical changes were more improved compared wit SAP group under the electronic microscope; the expres-sion of bax mRNA [6 h:(1.35±0.18) vs.(0.85±0.12);12 h:(1.21±0.21) vs. (0.86±0.24);24 h:(1.14±0.24) vs. (0.95±0.22)] and caspese-3 mRNA[6 h:(0.78±0.01) vs. (0.55±0.04);12 h:(0.79±0.04) vs. (0.57±0.05) ;24 h: (0.81±0.06) vs. (0.55±0.01) (P < 0.01)] were higher in three time units in SAP group than those in SO group (P < 0.01) ,and in IGF-1 group it was weakened significantly compared with the SAP group at each time point (P <0.05). bcl-2 mRNA expression was weak and have no difference between the SO group and SAP group (P > 0.05), but increased signifycantly in the IGF-± group at each time point [6 h:(0.65±0.07) vs. (0.54±0.04) vs. (0.57±0.06);12 h:(0.69±0.04) vs. (0.56±0.05) vs. (0.53±0.05);24h:(0.72±0.05) vs. (0.54±0.07) vs. (0.58±0.08)] (P <0.05). Conclusions Exogenous IGF-Ⅰ could rivalry SAP induced apoptosis to mucosal cells of small intestine , then could alleviate SAP induced injury to intestinal mucosal, It may be associated with the mechanisms that IGF-Ⅰ could improve the expression of bcl-2 mRNA and inhibit the expression of bax,caspase-3 mRNA.

Optimal purification method for Herpes-based viral vectors that confers minimal cytotoxicity for systemic route of vector administration.

Herpes simplex virus (HSV)-1 amplicon vectors could be packaged in the presence of replication-competent helper virus or in a helper virus-free system. In the latter system, cytotoxicity due to the expression of de novo viral gene expression is greatly reduced due to the absence of helper virus. However, the titers produced are relatively low in the range of 10(7) and 10(8)TU/ml after sucrose gradient concentration. This may become a limitation to certain gene transfer applications, such as brain disorder studies since the volume of vectors that could be administered is restricted. In contrast, amplicon viral vectors of high titers can be easily generated in the presence of helper viruses. Despite the potential cytotoxicity caused by the presence of helper virus in the latter method of viral packaging, studies involving vector targeting would still require the complementing function of helper virus for the generation of recombinant HSV-1 amplicon vectors with modified viral envelopes. In view of this, the optimal method of purifying Herpes-based viral vectors that confers minimal cytotoxicity for systemic route of viral vector administration is examined. Parameters such as the ratio of amplicon versus helper viruses, the percentage of viral lost, and the extent of liver cytotoxicity induced by these viral vectors purified using different methods were investigated. In addition, the maximum recombinant HSV-1 viral dosage was also determined in vivo. Taken together, these findings may be of importance to the efficient production of contaminant-free HSV-1 amplicon viral vectors required for animal and human studies.

Comparative proteome analysis of differentially expressed proteins induced by Al toxicity in soybean.

Phytotoxic aluminum (Al) is a limiting factor for crop production on acid soils. The molecular mechanism, however, underlying Al toxicity and responses in plants is still not well understood. We report here the characterization of comparative proteome of aluminum-stress-responsive proteins in a known Al-resistant soybean cultivar, Baxi 10 (BX10). To investigate time-dependent responses, 1-week-old soybean seedlings were exposed to 50 microM AlCl3 for 24, 48 and 72 h, and total proteins extracted from roots were separated by two-dimensional electrophoresis. More than 1200 root proteins of the soybean BX10 seedling were reproducibly resolved on the gels. A total of 39 differentially expressed spots in abundance were identified by mass spectrometry, with 21 upregulated, 13 newly induced and 5 downregulated. The heat shock protein, glutathione S-transferase, chalcone-related synthetase, GTP-binding protein and ABC transporter ATP-binding protein were previously detected at the transcriptional or translational level in other plants. Other proteins, identified in this study, are new Al-induced proteins. Soybean BX10 roots under aluminum stress could be characterized by the cellular activities involved in stress/defense, signal transduction, transport, protein folding, gene regulation, and primary metabolisms, which are critical for plant survival under Al toxicity. This present study expands our understanding of differentially expressed proteins associated with aluminum stress on soybean BX10.

An Efficient and Safe Herpes Simplex Virus Type 1 Amplicon Vector for Transcriptionally Targeted Therapy of Human Hepatocellular Carcinomas.

Our previous studies have shown that transgene expression could be targeted to proliferating cells when cell cycle transcriptional regulatory elements were incorporated into herpes simplex virus type 1 (HSV-1) amplicon backbone vectors. In the study reported here, we further demonstrated the transcriptional activation of transgene expression in association with the onset of cellular proliferation using the mouse partial hepatectomy model. Moreover, transcriptional regulation could be rendered specific to human hepatocellular carcinoma (HCC) cells by inserting the chimeric gene Gal4/NF-YA under the regulation of the HCC-specific hybrid promoter. The hybrid promoter, which consists of four copies of the apolipoprotein E (ApoE) enhancer element inserted upstream of the human α1-antitrypsin(hAAT) promoter, induced an higher level of transcription than other liver-specific promoters such as alpha-fetoprotein (AFP) and albumin (Alb) promoter. As a consequence, the enhancement of tissue-specific expression in the context of Gal4/NF-YA fusion proteins enabled the monitoring of transgene expression using a bioluminescence imaging system. Furthermore, these vectors have been shown to be non-toxic and exhibited potent infectivity for proliferating primary HCC cells and HCC cell lines. Together, these results demonstrated that the new hybrid vectors could provide options for the design of safe and efficient systemic gene therapeutic strategies for human HCC.