In order to develop a rapid detection kit for novel avian influenza virus (AIV) subtype H7N9, two sets of specific primers and probes were designed based on the nucleotide sequences of hemagglutinin antigen (HA) and neuraminidase antigen (NA) of novel H7N9 virus (2013) available in GenBank to establish the method of TaqMan probe-based multiplex real-time RT-PCR for rapid detection of AIV subtype H7N9. The primer and probe of HA were for all H7 subtype AIVs, while the primer and probe of NA were only for novel N9 subtype AIVs. The results showed that this method had high sensitivity and specificity. This method was applicable to the testing of positive standard sample with a minimum concentration of 10 copies/microL; it not only distinguished H7 subtype from H1, H3, H5, H6, and H9 subtypes, but also distinguished novel N9 subtype from traditional N9 subtype. A total of 2700 samples from Zhuhai, China were tested by this method, and the results were as expected. For the advantages of sensitivity and specificity, the method holds promise for wide application.
Animals ; Birds ; virology ; Influenza A Virus, H7N9 Subtype ; genetics ; isolation & purification ; physiology ; Influenza in Birds ; prevention & control ; virology ; Real-Time Polymerase Chain Reaction ; methods ; Species Specificity ; Taq Polymerase ; metabolism ; Time Factors

Objective　To explore the risk factors and indications for perioperative tracheostomy in patients with myasthenia gravis. Methods　A total of 174 patients with myasthenia gravis undergoing thymectomy were reviewed retrospectively between April 1980 and August 1999. Perioperative tracheostomy was performed on 44(25.3%) cases. Myasthenic or cholinergic crisis happened in 38 cases (21.8%). The relationship of the crisis incidence and Osserman classification, state of illness, present of thymoma, preoperative pulmoary function and the dose of anticholinergic agents given preoperatively were analyzed. At the same time the indications of perioperative tracheostomy were discussed. Results　The highest incidence of myasthenic or cholinergic crisis and perioperative tracheostomy were found in cases with long history of the disease, high dose of anticholinergic agents administration, Osserman classification over stageⅡb, with infiltrated thymoma, and preoperative pulmoary function impairment. Conclusion Perioperative tracheostomy and artificial ventilation are most important approaches when myasthenic or cholinergic crisis after thymectomy appeared. But the indications for postoperative prophylactic tracheostomy and artificial ventilation must be strictly selected.

Objective　To explore the effect of video-assi sted thoracic surgery (VATS) on common diseases of chest. Methods　Video-assisted thoracoscopic surgery was performed on 131 patients with ches t diseases from April 1994 to December 2000 in which 109 cases were spontane ous pneumothorax and hemothorax, 10 pulmonary tuberculoma, 5 pulmonary carcinoma , 3 esophageal carcinoma, 2 localized benign mesothelioma, 1 pulmonary hamartoma and 1 myasthenia gravis. Results　There was no operative death in all cases. Four patients with spontaneous pneumothorax complicated persist ent air leak(more than 7 d) and 1 patient with hemopneumothorax formed hemoth orax after the operation, which was stopped by the second VATS. The others recov ered well without any postoperative complications. Conclusion　 VATS is characterized by safety and mild tissue injury in the operation, and les s pain, fewer complications, rapid recovery and short duration of hospitalizatio n after the operation.

In order to study the proliferation inhibition effect of H5N1 subtype avian influenza virus (AIV) with small interfere RNA (siRNA), a total of 4 siRNAs were designed in accordance with the NP and PA genes of H5N1 subtype AIV, the siRNAs were then transfected to chicken embryo fibroblast(CEF), CEF was infected with H5N1 subtype AIV after 6 hrs. Virus titer of cell supernatant was tested at 16-56hrs post infection, and pathological changes of the cells was observed; mRNA levels of NP, PA, HA and p13-actin gene were tested at 36hrs post infection. The results showed that these 4 siRNAs could inhibit the prolif-eration of H5N1 subtype AIV in CEF in varying degrees, and one siRNA targeting PA was best per-formed. The experimental results also showed that the inhibition effect was decreased with the time prolonged. This research provides a basis for further studying RNAi on AIV prevention and control.
Actins ; genetics ; Animals ; Chick Embryo ; DNA Primers ; genetics ; Fibroblasts ; virology ; Hemagglutination ; Hemagglutinin Glycoproteins, Influenza Virus ; genetics ; Hemagglutinins ; genetics ; Humans ; Influenza A Virus, H5N1 Subtype ; genetics ; growth & development ; physiology ; RNA Interference ; RNA Replicase ; genetics ; RNA, Small Interfering ; chemical synthesis ; genetics ; RNA-Binding Proteins ; genetics ; Real-Time Polymerase Chain Reaction ; Specific Pathogen-Free Organisms ; Transfection ; Viral Core Proteins ; genetics ; Viral Proteins ; genetics ; Virus Replication

Objective: “Same treatment for different diseases” is a unique treatment strategy under the guidance of traditional Chinese medicine (TCM) theory. Codonopsis Radix (Codonopsis pilosula, Dangshen in Chinese) with spleen-fortifying effect was employed to understand the strategy of “Same treatment for different diseases”, based on its common mechanism in the treatment of gastric diseases including gastric ulcer, gastritis and gastric cancer via network pharmacology research. Methods: Network pharmacology research methods were used to analyze the interaction network and potential mechanisms of Dangshen in treating gastric ulcer, gastritis and gastric cancer. The active components and their target proteins of Dangshen were integrated from TCMSP, BATMAN-TCM databases. The targets of gastric ulcer, gastritis and gastric cancer were collected through GeneCards, PubMed, TDD and DisGeNET Database. Through screening, the key components and the key targets of Dangshen in treating gastric ulcer, gastritis and gastric cancer were obtained. After KEGG pathway analysis and GO analysis, the important pathways and biological processes were analyzed. Results: Through data and literature mining, the common and specific pharmaceutical effects and mechanism of Dangshen were summarized in these three gastric lesions. It was shown that Dangshen mainly acted on gastric ulcer, gastritis and gastric cancer through the overall regulation of the PI3K-AKT signaling pathway. With the development of the disease, it will gradually increase the control of inflammation through TNF, NF-κB and other inflammation-related signaling pathways to reduce inflammatory damage. For tumorigenesis, it pays more attention to inhibiting the ErbB signaling pathways to reduce the proliferation and migration of tumor cells. In addition, Dangshen's regulation of HIF-1 signaling pathway may also be beneficial for the treatment of gastric ulcer, gastritis and gastric cancer. Conclusion: Dangshen achieves spleen-fortifying effect on gastric diseases including gastric ulcer, gastritis and gastric cancer through multiple targets in multiple pathways, especially PI3K-AKT pathway and HIF-1 pathway. It could provide a scientific basis for understanding the strategy of “Same treatment for different diseases” in traditional Chinese medicine.

OBJECTIVETo screen the antisense oligodeoxynucleotides (asONs) which could hybridize with KDR (kinase insert domain-containing receptor) mRNA in an effective and specific way and to explore their anti-tumor effects on breast cancer MCF-7 cell line in vitro.

METHODSThe asONs were firstly selected using oligodeoxynucleotides library hybridization or computer prediction, then their hybridization ability with KDR mRNA was further tested with oligonucleotide microarray. The asONs with strong hybridization intensity were selected. Their inhibitory effects on MCF-7 cells proliferation and KDR expression were assayed by MTT, RT-PCR and Western blotting assay, respectively.

RESULTSIn 13 asONs selected with oligodeoxynucleotides library hybridization, 8 (8/13, 61.5%) showed strong hybridization signals, while such was only 1 in 17 asONs designed by computer prediction. 9 asONs with strong hybridization intensity were selected and synthesized with phosphorothioated modification. All these asONs inhibited the MCF-7 cells proliferation in a dose-dependent manner, in which asON4 and asON7 screened by oligodeoxynucleotides library in combination with oligonucleotide microarray were the most effective, with inhibitory rates of 51.6% and 62.2% at 0.8 micromol/L, respectively. The KDR expression at mRNA and protein levels was reduced by both the two asONs, in a dose-dependent manner.

CONCLUSIONasONs screened by oligodeoxynucleotides library hybridization are well consistent with that chosen with oligonucleotide microarray. The combination of oligodeoxynucleotides library with oligonucleotide microarray is an effective approach of asONs screening. The asONs targeting KDR mRNA showed prominent anti-tumor activity on breast cancer MCF-7 cells.

Breast Neoplasms ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Dose-Response Relationship, Drug ; Female ; Gene Library ; Humans ; In Situ Hybridization ; Oligodeoxyribonucleotides, Antisense ; genetics ; pharmacology ; RNA, Messenger ; biosynthesis ; genetics ; Transfection ; Vascular Endothelial Growth Factor Receptor-2 ; biosynthesis ; genetics

OBJECTIVETo investigate the diagnostic accuracy and clinical value of electron-beam CT (EBCT) single flow mode study (EBCTSF) in combination with EBCT coronary angiography (EBCTCA) and three dimensional reconstruction using medial axis reformation (MAR) for diagnosis of coronary in-stent stenosis.

METHODSElectrocardiogram-gated EBCT single coronary scanning (without and with contrast medium) was performed in 25 consecutive coronary heart disease (CHD) patients during a short breathhold. EBCTSF was then performed at the level nearly distal to stent. Three-dimensional coronary images were reformed using MAR. EBCT findings were compared with that of conventional coronary angiography (CAG).

RESULTSThirty-five intracoronary stents were implanted in thirty-one diseased vessel segments. EBCTSF procedure was unsuccessful in 2 patients (successful rate was 92.0%, 23/25). There was a significant decrease in flow peak value (Dp), increased value (Deltad) and area under curve (A), and a significant increase in prolonged peak time (Td) in stenosed stents compared to normal stents (P < 0.05). EBCTCA was successful for all patients. Seven stenosed stents (5 in left anterior descending branch and 2 in right coronary) were correctly evaluated with EBCT. Compared with CAG, EBCTSF in combination with EBCTCA images and MAR reconstruction images had a diagnostic sensitivity of 85.0% (6/7) and a specificity of 92.9% (26/28) for detecting significant in-stent stenosis (> 50% lumen diameter). Positive and negative predictive value were 75.0% (6/8) and 96.5% (26/27) respectively. Compared with EBCT cross-section images alone, or cross-section images and three-dimensional images, the diagnostic accuracy increased from 80.0% and 88.6% to 91.4% (32/35).

CONCLUSIONSNoninvasive EBCTSF can be used to quantitatively analyze coronary flow characteristics. This technique, used in combination with EBCTCA and three dimensional reconstruction using MAR, seems to be an effective imaging modality in identifying coronary in-stent stenosis. For stent-implanted patients with atypical and nonischemic chest pain after coronary intervention, the above-mentioned technique is of important value for evaluating therapeutic effect and follow-up results.

Coronary Angiography ; methods ; Coronary Artery Disease ; diagnostic imaging ; Coronary Restenosis ; diagnostic imaging ; Female ; Follow-Up Studies ; Humans ; Male ; Middle Aged ; Stents ; Tomography, X-Ray Computed ; methods

Adult ; Aged ; Female ; Humans ; Intervertebral Disc Displacement ; therapy ; Lumbar Vertebrae ; Male ; Manipulation, Spinal ; methods ; Middle Aged

The alpha-amylase (EC 3.2.1.1) from the Gram-positive Alicyclobacillus acidocaldarius was one kind of thermoacidophilic enzyme, with optimal temperature and pH of 75 degrees C and 3, respectively. The nucleotide sequence of the gene amy was cloned by PCR. The gene amy was 3901bp long, comprising one open reading frame encoding a polypeptide of 1301 amino acids. The calculated molecular weight of the alpha-amylase AMY was about 140kD. The gene amy was expressed in E. coli BL21 (DE3) and Pichia pastoris respectively, and both of the cloned proteins had bioactivity. The activity of amylase expressed in P. pastoris was further testified by amylase activity staining. The alpha-amylase expressed in P. pastoris had been purified and characterized. The apparent molecular weight of that was about 160kD according to SDS-PAGE. The optimum of pH for the enzyme was pH 3.2 as the native enzyme was; but the optimum of temperature was 65 degrees C and a little lower than that of the native enzyme. Above 50% of relative activity remained after incubation for 30 minutes in 70 degrees C. So the enzyme expressed by P. pastoris was also thermoacidophilic. Moreover some sequence was cloned by PCR, which ranged from + 1174 bp to + 3288 bp in the gene amy, encoding 705 amino acids with the calculated molecular weight of 79kD. The truncated gene amy' was expressed in E. coli BL21 (DE3) induced by 1 mmol/L IPTG, and the expressed enzyme also retained alpha-amylase activity.
Bacillus ; enzymology ; genetics ; Bacterial Proteins ; genetics ; isolation & purification ; metabolism ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; genetics ; Pichia ; genetics ; metabolism ; alpha-Amylases ; genetics ; isolation & purification ; metabolism

Hypothalamic-pituitary-adrenal(HPA)axis-related factors in patients with type 2 diabetes were studied according to different levels of HbA1C.It showed that HPA axis was normal in HbA1C≤ 7% group[ACTH (18.03±8.39)ng/L,blood cortisol(49.22±8.68)μg/L],hyperactive in 7%11% group with weak feedback regulation[ACTH(26.08±15.41)ng/L,blood cortisol(55.64±24.27)μg/L].These results suggest that HPA axis-related factors in type 2 diabetic patients are different with different grades of glucose metabolic turbulence.