Application of Matrix-assisted Laser Desorption Ionization Mass Spectrometry in Analysis of Traditional Chinese Medicine：A Comprehensive Review / 中国实验方剂学杂志

Traditional Chinese medicine （TCM）， which owns abundant chemical components and complex action pathways， has been widely recognized in the prevention and treatment of diseases. Some analysis methods have been emerged in order to ensure the quality of TCM and to develop new TCM drugs. Matrix-assisted laser desorption ionization mass spectrometry （MALDI-MS） is a soft ionization mass spectrometric technique with the advantages of high throughput， high sensitivity， low cost and so on. It provides technical support for the molecular level study on TCM. At present， this technique has been used in the field of composition analysis and metabonomics research of TCM， and plays an important role in the identification of Chinese herbal medicines， real-time molecular screening and the construction of metabolic network pathway of active ingredients. Among them， the selection of appropriate matrix and sample preparation technology is the key to ensure the detection effect of MALDI-MS. With the development and optimization of new matrix， the continuous improvement of sample preparation technology and the combination of MALDI-MS with various analytical methods will greatly improve the detection effect. Based on this， this paper discusses the application of MALDI-MS in TCM， including high-throughput detection of active ingredients in TCM， monitoring of the original medicines and their metabolites in vivo， and in situ visualization and characterization of tissue distribution information of active ingredients in TCM. It also discusses the application prospect and existing problems of MALDI-MS in TCM， so as to provide technical support for the identification of active ingredients in TCM， drug utilization and metabolism.

Study on Tissue Distribution of A Variety of Endogenous Metabolites by Air Flow Assisted Ionization-Ultra High Resolution Mass Spectrometry Imaging / 分析化学

As a promising new molecular imaging technique,mass spectrometry imaging(MSI) has attracted more and more attention in the field of biomedicine. A method of air flow assisted ionization-ultra high resolution mass spectrometry-based mass spectrometric imaging (AFAI-MSI) was developed to profile endogenous metabolites in rat kidney tissue in this study. Rat kidneys were collected and cut into frozen tissue sections,and then were analyzed on an AFAI-MSI system in positive ion mode using acetonitrile-isopmpyl alcohol-water (4:4:2,V/V,5 μL/min) as spray solvent,N2as spray gas(0.6 MPa) and air as assisting gas (45 L/min). The mass range and resolution were set to be 70-1000 Da and 70000, respectively. As a result,a total of 38 metabolites, including organic amines, sugars, vitamins, peptides, neurotransmitters, organic acids,phospholipids,sphingolipids,glyceride,and cholesterol esters, were identified and imaged to characterize their tissue-specific distribution in kidney tissues, and some metabolites, such as choline, acetylcoline,betaine,phoshocholine,and glycerophosphocholine were found to have distinct distribution along the cortex-medulla axis,which may be involved in the formation of osmotic pressure gradient in the kidney. The proposed ultra high resolution mass spectrometry based AFAI-MSI method could work without sample pretreatment, showed high sensitivity and wide metabolite coverage, and was expected to provide a new analytical approach for the research of in situ characterization and metabolic regulation mechanism of endogenous metabolites in kidney.

Construction and immunogenicity analysis of recombinant replication-defective human adenovirus type 5 bearing the porcine circovirus type 2 Cap protein gene / 病毒学报

To construct a recombinant replication-defective human adenovirus type 5 expressing Cap protein of PCV2 and test the immunological efficacy in mice. In this study, the recombinant replication-defective human adenovirus type 5, named as rAd5-Cap (wt-rAd5), was constructed through homologous recombination internally in the HEK293AD cells after co-transfection of the Pac I-linearized backbone plasmid and the shuttle plasmid pacAd5CMV-Cap containing the open reading frame (ORF2) of the porcine circovirus type 2 (PCV2) cap protein or pacAd5CMV without inserted fragment. Furthermore, the rAd5-Cap could induce the expression of PCV2 cap protein in the HEK293AD cells with high efficacy evaluated by the RT-PCR and indirect immunofluorescence assay (IFA). The virus titer of rAd5-Cap could reach up to 10(8.5) TCID50/mL similarly to that of wt-rAd5, indicating that there was little affect on the virus proliferation after the insertion of PCV2 cap protein gene. The humeral immune responses could be activated and detected 14 days after the inoculation of the mice with 10(7) TCID50 rAd5-Cap intramuscularly, and constantly in crease in another 14 days. These molecular biological and animal experiments results demonstrated that the PCV2 cap protein could be efficiently expressed by the recombinant adenovirus rAd5-Cap in eukaryotic cells and induce robust immune responses in mice, which laid a good foundation for the development of new type vaccine against porcine circovirus.

Immune efficacy of rabies virus glycoprotein expressed by baculovirus vector / 病毒学报

To construct a recombinant baculovirus expressing glycoprotein (GP) of RV SRV9 strain and test the immunological efficacy in mice, open reading frame of rabies virus GP gene of SRV9 strain was cloned into the shuttle vector Bacmid to construct the recombinant shuttle plasmid Bacmid-G and transfection was performed into S f9 cells with the recombinant shuttle plasmid. CPE appeared in cell cultures was identified by electronmicroscopy. Western-blot, IFA and immunity tests in mice were performed to identify the immunoreactivity and immunogenicity of the expression products. Our results showed a recombinant baculovirus expressing GP protein of rabies virus SRV9 was obtained. The expression products possessed a favorable immunogenicity and fall immunized mice could develop 100% protective level of anti-rabies neutralizing antibody. In conclusion, The SRV9 glycoprotein expressed by the recombinant baculovirus in this study had good immunogenicity and could induce anti-rabies neutralizing antibody, which laid the foundation of further development of rabies subunit vaccine.

Expression variation and significance of Skp2 and p27(kip1) during the proliferation of Jurkat cells / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the expression variation and significance of Skp2 and p27(kip1) during the proliferation of lymphoma cell line Jurkat cells.</p><p><b>METHODS</b>The binding of p27(kip1) and Skp2 in Jurkat cells were detected by immunoprecipitation. Jurkat cells were treated with serum starvation and release synchronization. The expression variation and subcellular localization of p27(kip1) and Skp2 were detected by subcellular fractionation, Western blot and double immunofluorescence labelling.</p><p><b>RESULTS</b>The results of immunoprecipitation suggested that p27(kip1) and Skp2 could bind each other in Jurkat cells. During the proliferation of Jurkat cells, the protein expression of p27(kip1) decreased and intranuclear p27(kip1) decreased significantly, while the Skp2 protein increased and cytoplasmic Skp2 increased significantly.</p><p><b>CONCLUSION</b>During the proliferation of Jurkat cells, the increased cytoplasmic synthesis of Skp2 may speed up p27(kip1) degradation via the ubiquitin-proteasome pathway, then intranuclear p27(kip1) decreases significantly, leading to an increased cell cycling activity.</p>

Gene mutation in the areas of PreC/C and BCP of HBV DNA / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To investigate the gene mutation in the areas of pre core/core (Pre C/C) and basic core promotor (BCP) of HBV DNA and its clinical significance.</p><p><b>METHODS</b>The nt 1 735-1 965 segment of HBV DNA was amplified with PCR in 54 cases with chronic hepatitis B and 10 cases with post-hepatitis cirrhosis. Then the PCR product was sequenced.</p><p><b>RESULTS</b>There were 168 site mutations in 48.5% (33/68) cases with hepatitis B. The first ten mutation sites were nt 1 764 (58.8%), 1 762 (48.5%), 1 799 (21.0%), 1 766 (14.7%), 1 896 (13.2%), 1 754 (8.8%), 1 899 (8.8%), 1 768 (7.4%), 1 814 (7.4%) and 1 913 (7.4%). Three rare mutations of nt 1907, 1 922 and 1 923 were also detected. The mutations of nt 1 896, 1 764 and 1 762 were found in 16.7%, 35.2% and 35.2% of chronic hepatitis, and in 30.0%, 60.0% and 60.0% respectively of post-hepatitis cirrhosis cases. There was statistical significance between the two groups (P < 0.01).</p><p><b>CONCLUSION</b>The mutations in the areas of Pre C/C and BCP of HBV DNA might possibly be associated with liver fibrosis. There are many mutation sites in HBV DNA and mutation occurs frequently, therefore gene sequencing is helpful to the design of gene chip and to clinical application.</p>