Study on droplet microfluidic chips for drug screening against Candida albicans / 药学学报

A droplet microfluidic chip system was developed for drug screening against Candida albicans. The microfluidic chip was designed and prepared for the formation of droplets. Alamar blue was selected as an indicator for its characteristic of fluorescence mission in live cells. Four antifungal drugs (amphotericin B, caspofungin, 5-fluorocytosine, terbinafine) and a new drug (iodiconazole) were selected as model drugs to test the microfluidic chip approach. At the same time, 96-well microplate method was performed to verify the applicability of the chip method. The results showed that the developed droplet microfluidic chip platform was able to complete the antifungal susceptibility test within 2 h. In comparison with the 96-well microplate method, the microfluidic chip method showed a consistence of 100% with regard to the minimum inhibition concentrations and less reagent consumption. The new droplet microfluidic chip method is simple, rapid and suitable for rapid screening of antifungal drugs.

Activation of transient receptor potential vanilloid 1 inhibits RhoA/Rho kinase and improves vasorelaxation dysfunction mediated by high-fat diet in mice / 中国医学科学院学报

<p><b>OBJECTIVE</b>To investigate the role of dietary capsaicin in activating transient receptor potential vanilloid 1 (TRPV1) and thus influencing the vascular dysfunction mediated by high-fat diet and the potential mechanisms.</p><p><b>METHODS</b>A total of 80 male C57BL/6J mice aged 10 weeks were equally divided into four groups, in which the mice were fed with normal diet (ND), normal diet plus capsaicin (NC), high-fat diet (HD), or high-fat diet plus capsaicin (HC) for 20 weeks. Tail-cuff blood pressure (BP), vascular function of mice aortic rings, expressions of voltage-gated potassium-channel Kv1.4, RhoA and Rho kinase in aorta were examined.</p><p><b>RESULTS</b>Compared with ND group, both nitroglycerin [(18.9 +/- 13)% vs. 100%, P < 0.01] and acetylcholine [(26 +/- 12)% vs. 100%, P < 0.01] induced vasorelaxation of aortic rings were significantly reduced in HD group. Both endothelium dependent and independent aortic rings vasorelaxation in HC group were significantly improved compared with that in HD group [acetylcholine: (69 +/- 15)%; nitroglycerin: (46.5 +/- 6)%, P < 0.05], but still reduced compared with that in ND group (P < 0.05, P < 0.01). High fat diet induced the expression of RhoA and Rho kinase. Dietary capsaicin down-regulated the expression of RhoA and Rho kinase but up-regulated the expression of Kv1.4 in aorta in mice fed with normal or high fat diet (all P < 0.05).</p><p><b>CONCLUSION</b>Dietary capsaicin can ameliorate vasorelaxation dysfunction mediated by high-fat diet. The potential mechanisms may be related with TRPV1 activation, which in turn stimulates potassium channel and inhibits RhoA and Rho kinase in the vasculature.</p>

A modified template matching algorithm for chromatographic fingerprint of S. miltiorrhiza Bge, a traditional Chinese herb / 第二军医大学学报

Objective: To introduce a new template peak matching algorithm for calculating the similarity of chromatographic fingerprint of traditional Chinese herbs. Methods: Tanshinone II A in S. miltiorrhiza Bge of different batches were determined by HPLC and the chromatograms of them were obtained. We designated a new peak matching algorithm based on the previous algorithms, which employed a certain real chromatographic fingerprint as their template. In the new algorithm, we arranged the retention times of chromatographic peaks of all chromatograms into an ascending order, forming a template. The sorting procedure complied with the following 2 rules. First, the same peak matches the nearest corresponding peak. Second, one peak does not appear twice in the same chromatogram. Results: The new algorithm avoided the shortcomings of previous algorithm, such as mismatching and missed matching, and obtained a satisfactory outcome. Conclusion: Our new algorithm provides a basis for improving the reliability of retention time-based peak matching algorithm.

Preparation of artificial antigen and egg yolk-derived immunoglobulin (IgY) of citrinin for enzyme-linked immunosorbent assay / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To prepare artificial antigens and anti-citrinin egg yolk-derived immunoglobulin (IgY) to build an enzyme-linked immunosorbent assay (ELISA) for citrinin (CTN).</p><p><b>METHODS</b>CTN was conjugated with bovine serum albumin (BSA), ovalbumin (OVA) with formaldehyde condensation method to prepare artificial antigens and identified by ultraviolet (UV) spectrometry and Infrared (IR) spectrometry. Artificial antigens for CTN and anti-CTN IgY were purified with polyethylene glycol two-step precipitation method and identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). ELISA with IgY was established. Cross-reactivity of IgY with various structural similarities to CTN and possible co-occurrence with CTN in agricultural commodities were studied.</p><p><b>RESULTS</b>UV and IR absorption spectra suggested that CTN was correlated with the carrier protein of BSA or OVA. SDS-PAGE patterns showed that the anti-CTN IgY was almost pure with a molecular weight of approximate 100 KD. The indirect competitive ELISA showed that the detection limit of CTN was 10 ng x mL(-1), with a good linearity ranging 20-640 ng x mL(-1).</p><p><b>CONCLUSION</b>Artificial antigens of CTN can be successfully synthesized. The established ELISA can be used to determine CTN- contaminated samples.</p>

Effect of chronic enhanced external counterpulastion on gene expression profiles of arterial endothelial cells of pigs fed with high-cholesterol diet / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the effect of chronic enhanced external counterpulastion (EECP) on gene expression profiles of arterial endothelial cells (ECs) of pigs fed with high-cholesterol diet.</p><p><b>METHODS</b>Eight male pigs were fed with high-cholesterol diet for 12 weeks to induce arteriosclerosis and subjected to EECP for accumulative 36 h (2 h every other day for 18 sessions). Another 8 pigs on cholesterol-enriched diet and 6 normally fed pigs served as the arteriosclerosis model group and normal control group, respectively, and the high-cholesterol diet was maintained until the end of EECP treatment. The coronary artery was then isolated for transmission electro microscopy, and the abdominal aorta was observed using Sudan III staining. The gene expression profiles in ECs from the thoracic aorta using cDNA microarrays.</p><p><b>RESULTS</b>Macrophages and foam cells were detected beneath the ECs in the coronary artery of pigs in the model group, but not in the other two groups. The ratios of Sudan III-positive area in the celiac aorta were significantly lower in normal control and EECP groups than in the model control group (P<0.05). Compared with the normal control group, the gene expressions of integrins-beta1 and CTGF were up-regulated in the model group. Compared with the model group, the expressions of integrins-beta1, CTGF and VCAM-1 were down-regulated and eNOS up-regulated in EECP group.</p><p><b>CONCLUSION</b>Chronic EECP may reduce endothelial injury, down-regulate the gene expression level of integrin-beta1, CTGF and VCAM-1, lower cholesterol uptake and attenuate arterial endothelial inflammation to protect the pigs fed with high-cholesterol diet from arteriosclerosis.</p>

MLST typing of Streptococcus suis isolated from clinical patients in Guangdong Province in 2005 / 南方医科大学学报

Intensive surveillance of human S.suis infection was carried out in July and August of 2005 in Guangdong Province, which coincided with the Sichuan outbreak. Five isolated cases of human infections were identified during this period, from which 5 S. suis serotype 2 isolates were recovered. MLST analysis showed that these 5 isolates shared identical sequences of 6 MLST housekeeping genes except for one point mutation found within the thrA gene fragment, a neutral mutation (TTA to TTG) in the third nucleotide (360 nt) of the codon for leucine. MLST analysis identified 2 sequence types in the Guangdong sporadic infection. Three Guangdong isolates L-SS002, L-SS003 and L-SS005 belonged to ST7, while the other two isolates L-SS004 and L-SS006 belonged to ST1, but they all belonged to ST1 clonal complex. This finding represents a striking feature that differs from the Sichuan outbreak caused by a single ST7 SS2 clone. The 3 isolates of ST7 were probably imported from Sichuan Province, while the origin of the other 2 isolates of ST1 still remain to be clarified.

Preparation and Analytical Method in the Study of Micrbial Metabolomics / 微生物学通报

Microbial metabolomics is a subject that chiefly studying all the low molecular weight metabolites in an organism or cells during their growing process. The progress of analytical technology promotes microbial metabolomics to make advancement. In this paper, the commonly used analytical technology, sample preparation and its application were discussed and the prospects of the analytical methods were also discussed.

Comparison of two kinds of operations for high anal fistula / 中华胃肠外科杂志

<p><b>OBJECTIVE</b>To investigate the more reliable and effective operations for high anal fistula.</p><p><b>METHODS</b>From Jan. 2002 to Oct. 2004, 117 cases suffering from high anal fistula were divided into two groups, and received tying therapy on main fistula with external anal fistulae excision (62 cases) or tying therapy on main fistula with external anal fistulae laid aside (55 cases). The curing time and recurrence were compared between the two groups.</p><p><b>RESULTS</b>There were no significant differences in basic clinical data between the two groups. There were 37 cases of high simple fistula, and 25 cases of complicated fistulae in resection group, while 39 cases of simple fistula and 16 cases of complicated fistulae in laying aside group. The curing time was 15-20 (17+/-2) days and no recurrence occurred after follow-up for half a year in resection group. The curing time was 25-55 (35+/-8) days and recurrence occurred in 6 cases (10.9%) in laying aside group including one case of high simple anal fistula and five cases of high complicated anal fistulae. There was statistical significance in treatment efficacy for high complicated anal fistulae (chi2=6.23, P=0.013), and the overall efficacy (chi2=5.06, P=0.024) between the two groups.</p><p><b>CONCLUSION</b>Tying therapy on main fistula with external anal fistulae excision is a more effective treatment for high complicated anal fistulae.</p>

Determination of three constituents in Radix Astragali by HPLC-MS / 药学学报

<p><b>AIM</b>To determine calycosin-7-O-beta-D-glucoside, astragaloside IV and formononetin in Radix Astragali and other relative samples by HPLC-MS.</p><p><b>METHODS</b>HPLC was carried out with Agilent 1100LC/MSD, equipped with Agilent Zorbax SB C18 column (250 mm x 4.6 mm ID, 5 microm) and mass spectrum detector. The mobile phase (CH3CN-H2O) was eluted in gradient mode.</p><p><b>RESULTS</b>The calibration curves of calycosin-7-O-beta-D-glucoside, astragaloside IV and formononetin were linear in the range of 0.03 - 1.21 microg x mL(-1), 0.35 - 13.86 microg x mL(-1) and 0.38 - 15.22 microg x mL(-1), respectively. These recoveries of samples were from 95% to 105% with RSD less than 1.5%.</p><p><b>CONCLUSION</b>The method was employed to analyse 25 samples of Radix Astragali and other relative samples, including Radix Astragali slice, Radix Astragali Preparata, Hedysarum polybotrys Hand. -Mazz, Astragalus ernestii Comb. The contents of three constituents vary greatly because of the species, place of collection and season of harvesting. This method could apply to evaluate the quality of Radix Astragali and it is simple, sensitive and reliable.</p>

Apoptosis induced by DNA primase inhibitor 3,3'-diethyl-9-methylthia-carbocyanine iodide in human leukemia HL-60 cells / 药学学报

<p><b>AIM</b>To investigate apoptosis induced by 3,3'-diethyl-9-methylthia-carbocyanine iodide (DMTCCI), an inhibitor of DNA primase found in our previous study, and the mechanism of DMTCCI in human myelogenous leukemia HL-60 cells.</p><p><b>METHODS</b>HL-60 cells were cultured in RPMI-1640 medium and treated with different concentrations of DMTCCI. MTT assay was used to detect growth inhibition. Flow cytometry and DNA ladders were used to detect apoptosis. Western blotting was used to observe the expression of survivin, Bcl-xL, Bad, Bax, Bcl-2, caspase-9, caspase-3, caspase-6, PARP, DFF45 and lamin B protein. Caspase-3 activity was measured by ApoAlert Caspase-3 Assay Kit.</p><p><b>RESULTS</b>DMTCCI inhibited proliferation of human leukemia HL-60 cells with IC50 value of 0.24 micromol x L(-1). The results of flow cytometry and DNA ladders showed that DMTCCI could induce apoptosis of HL-60 cells. The expression levels of protein survivin and Bcl-xL were down-regulated, Bad and Bax were up-regulated, while Bcl-2 protein had no change in response to DMTCCI treatment in HL-60 cells. Treatment of HL-60 cells with DMTCCI induced the proteolytic cleavage of caspase-9, caspase-3, caspase-6, PARP, DFF45 and lamin B protein. Caspase-3 activity apparently increased at 3 h and reached a peak at 12 h after exposure to 1 micromol x L(-1) of DMTCCI in HL-60 cells.</p><p><b>CONCLUSION</b>DMTCCI inhibited proliferation and induced apoptosis of human leukemia HL-60 cells. Bcl-2 family proteins, survivin and caspases family proteins might play a role in the apoptosis process induced by DMTCCI.</p>

Comparative proteomic analysis of B. henselae Houston and B. henselae Marseille by two-dimensional gel electrophoresis / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To compare the protein difference between B. henselae Houston and B. henselae Marseille by two-dimensional gel electrophoresis.</p><p><b>METHOD</b>Protein samples were prepared by vorterx, ultrasonic treatment, and centrifugation. Protein concentrations were determined by Bradford method. Protein difference was compared by the first IEF and the second SDS-polyacrylamide gel electrophoresis.</p><p><b>RESULTS</b>Protein concentrations in samples of Bartonella henselae Houston and Bartonella henselae Marseille were 2.117 microg/microL and 2.200 microg/microL respectively. Sample protein of 40 microg for IPG strips loading was perfect. The results of 2-DE in pH 4 to 7 IPG strips showed that the total protein spots of Bartonella henselae Houston and Bartonella henselae Marseille were 375 and 379 respectively, 95% of the spots were the same between the two strains of Bartonella henselae.</p><p><b>CONCLUSION</b>The procedure of 2-DE may prove successful for the proteomic analysis of Bartonella henselae. Bartonella henselae Houston and Bartonella henselae Marseille are different genotypes.</p>

The preparation of endostatin protein and the measurement of its biologic activity / 国际眼科杂志(Guoji Yanke Zazhi)

·AIM: To investigate the preparation of endostatin protein and its biologic activity on vascular endothelial cell.· METHODS: pBlast-hEndostatin and pBlast-Mcs were identified by digesting with Nhe Ⅰ and Sal Ⅰ, by PCR reaction, by sequencing, and by Alignments of PCR products with gene bank using NCBIBLAST software. The identified pBlast-hEndostatin as well as pBlast-Mcs were then purified with QIAGEN Endofree plasmid maxi kit.The purified plasmids transfected human fibroblasts. The expression of endostatin was detected by RT-PCR, Westem-Blot and immunohistochemistry. The endostatin prorein produced by transfected fibroblasts was purified by ultrafiltration and affinity chromatography. The inhibitory action of endostatin on human umbilical vein endothelium was measured by MTT assay.· RESULTS: pBlast-hEndostatin was found to contain human endostatin gene. Endostatin protein was produced by transfected fibroblasts. The inhibitory ratio of 2.5,5,10,20,40,80mg/L endostatin on human umbilical vein endothelium for 48h were 8.5%,13.1%,27.7%,38.1%,56.7%,63.8% respectively. IC50 value was 34.5mg/L.No inhibition action was found on fibroblasts.·CONCLUSIONS: Endostatin protein can be produced by the transfected fibroblasts. The produced endostatin has inhibitory action on human umbilical vein endothelium and has no inhibition action on fibroblasts.

Loss of p53 gene and amplification of HER-2 oncogene in primary hepatocellular carcinoma and their clinical significance / 中华病理学杂志

<p><b>OBJECTIVE</b>To investigate the deletion of p53 gene and amplification of HER-2 oncogene at chromosome 17 in primary hepatocellular carcinoma (HCC) and the clinical significance.</p><p><b>METHODS</b>Interphase dual fluorescence in situ hybridization (FISH) was applied to detect the ratio of the number of p53 gene copy or HER-2 oncogene copy to that of chromosome 17 copy, to determine the p53 gene deletion and HER-2 oncogene amplification in nuclei prepared from 42 surgical specimens of HCC. Statistical analysis for their clinical significance was performed.</p><p><b>RESULTS</b>Loss of p53 gene and amplification of HER-2 oncogene were detected in 27 (64.3%) and 9 (21.4%) of the 42 HCC respectively including 4 cases with low and 5 with high copy amplification. Six (14.3%) of 42 HCC showed simultaneously p53 gene deletion and HER-2 oncogene amplification. 61.9% (26/42) of HCC were polysomy 17, which correlated positively with p53 gene deletion (chi(2) = 12.286, P < 0.001). No close correlation between p53 gene loss and HER-2 oncogene amplification was found (chi(2) = 0.00, P = 1.00). Loss of p53 gene was related to the serum alpha-fetoprotein (AFP) level and the tumor size (P < 0.05). The postoperative 2-year survival rate (18.5%) of HCC patients with p53 gene deletion was significantly lower than postoperative 2-year survival rate (60.0%) of those without p53 gene loss (chi(2) = 7.467, P = 0.006). Meanwhile, HER-2 oncogene amplification showed a tendency of correlation with the tumor size (chi(2) = 2.973, P = 0.085), and the postoperative 2-year survival rate (0/9) of HCC patients with HER-2 oncogene amplification was significantly lower than those (42.4%) without HER-2 oncogene amplification (chi(2) = 3.977, P = 0.046).</p><p><b>CONCLUSION</b>There were a high frequency of p53 gene deletion and a low frequency of HER-2 oncogene amplification in primary HCC, which might be involved in initiation and development of a subset of primary HCC.</p>

Studies on purification of methamidophos monoclonal antibodies and comparative immunoactivity of purified antibodies / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To purify Methamidophos (Met) monoclonal antibodies with two methods and compare immune activity of purified antibodies.</p><p><b>METHOD</b>Caprylic acid ammonium sulphate precipition (CAASP) method and Sepharose protein-A (SPA) affinity chromatography method were used to purify Met monoclonal antibodies, UV spectrum scanning was used to determine protein content and recovery of purified antibodies, sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) was used to analyze the purity of purified antibodies, and enzyme-linked immunosorbent assay (ELISA) was used to determine immune activity of purified antibodies.</p><p><b>RESULTS</b>Antibody protein content and recovery rate with CAASP method were 7.62 mg/mL and 8.05% respectively, antibody protein content and recovery rate with SPA method were 6.45 mg/mL and 5.52% respectively. Purity of antibodies purified by SPA method was higher than that by CAASP method. The half-maximal inhibition concentration (IC50) of antibodies purified by SPA to Met was 181.26 microg/mL, and the linear working range and the limit of quantification (LOD) were 2.43-3896.01 microg/mL and 1.03 microg/mL, respectively. The IC50 of antibodies purified by CAASP to Met was 352.82 microg/mL, and the linear working range and LOD were 10.91-11412.29 microg/mL and 3.42 microg/mL, respectively.</p><p><b>CONCLUSION</b>Antibodies purified by SPA method are better than those by CAASP method, and Met monoclonal antibodies purified by SPA method can be used to prepare gold-labelled testing paper for analyzing Met residue in vegetable and drink water.</p>