Microarray technology are being performed more widely than ever before on many areas in lifescience,although the technology is still evolving,the challenge of performing a microarray experiment is no longer in the data generation,but in extracting useful information and utilizing it to get the results with biological meanings.Some methods and tools used for expressional microarray data mining based on previous work were summarized.These methods include gene clustering,GO analysis,regulating pathway analysis,and related algorithm.We hope this can be helpful for those researchers who are implementing expressional microarray for biological analysis.

Objective: To establish a multiplex PCR-microarray method for detecting important enteropahogens.Methods: Uniplex and multiplex PCR were performed to obtain the best primer sets for identifying the target bacteria at species and multi-species level.Fluorescent dyes were mixed into PCR reaction to determine whether it can affect the efficiency of amplification.To improve the efficiency of microarray,a 35 pairs primer-labeling system was optimized based on the hybridization results to find the best combination to avoid false negative results.Results: Specific PCR products were all obtained using species-specific primer sets.More preferential amplification may happen when more primer pairs were added to the reaction.The hybridization results showed a positive association between the efficiency of multiplex-PCR and signal intensity.Conventional PCR yielded more products than fluorescent dyes labeled PCR.Thirty-five primers were divided into three different combinations to label target respectively,hybridization results showed a high specificity.Conclusion: Mixing fluorescent dyes into PCR may reduce the efficiency of amplification and hybridization,but may have no effect on the analysis of hybridization results.The hybridization efficiency of microarray depends on the amplification efficiency of multiplex PCR.For microarray target labeling,three primer sets could be used to avoid negative hybridization led by preferential amplification of multiplex-PCR.It indicates that the multiplex PCR-microarray method is an attractive diagnosis tool for the high-throughput identification of enteropathogenic organisms especially for multiple causative agents and epidemiological investigations.

Objective To compare the differences of clinical characteristics between genotype B and C chronic hepatitis B(CHB)patients and to summarize clinical factors related to genotype C hepa- titis B virus(HBV)infection.Methods Seventy eight CHB patients who were diagnosed with genotype B or C infection by liver puncture biopsy and genotyping were enrolled.Their serum HBV DNA levels were detected.Severe hepatitis,liver cirrhosis,hepatocellular carcinoma and HBeAg positive rate were analyzed to determine the pathologic inflammation and fibrosis degree of liver tissue.Chi square test and Logistic multiple regression analysis were employed for the statistical analysis.Results The serum albumin and pre-protein were lower in genotype C CHB patients than that in genotype B.The alanine aminotrans- ferase,total bilirubin and prothrombin time were higher in genotype C CHB patients than that in genotype B.The rates of genotype C patients increased significantly with the grade of liver necroin- flammation progressing from GO to G4(1.8%,11.1%,20.4%,33.3%,33.3%) and the stage of liver fibrosis progressing from SO to S4(5.6%,5.6%,14.8%,33.3%,40.7%),but the rates of genotype B patients did not change significantly with the grade of liver necroinflammation(16.7%, 25.0%,25.0%,20.8%,12.5%)and stage of liver fibrosis progressing(16.7%,29.2%%,20.8%, 16.7%,16.7%).There was statistical significance in grades of liver necroinflammation(X~2= 11.49,P=0.022)and stages of liver fibrosis(X~2=13.56,P=0.006)between genotype B and gen- otype C patients.The rates of genotype C CHB patients were higher than,similar with and lower than the rates of genotype B patients of HBV DNA level above 1.0?10~6 copy/mL,between 5.0?10~2-1.0?10~6 copy/mL and under 5.0?10~2 copy/mL,respectively(51.8% vs 12.5%,35.2% vs 45.8% and 13.0% vs 41.7%).There was statistical significance of HBV loads between genotype B and genotype C patients(X~2=13.25,P=0.001).HBeAg positive rate in genotype C patients was significantly higher than that in genotype B patients(61.1% vs 25.0%,X~2=8.67,P=0.003).The rates of decompensated cirrhosis,compensated cirrhosis and no-cirrhosis in genotype C patients were higher than,similiar with and lower than the rates in genotype B patients,respectively(40.7% vs 4.2%,22.2% vs 20.8% and 37.0% vs 75.0%).There was statistical significance of the rate of cirrhosis between genotype B and genotype C patients (X~2=12.47,P=0.002).Conclusions The degree of liver necroinflammation and fibrosis,the HBeAg positive rate and the incidence of cirrhosis are all related with genotype C HBV infection.

To investigate the role and mechanism of ceramide (Cer) regulation in alcohol-induced neuronal proliferation and the newborn neurons formation, we used sphingomyelin synthase 2 (predominant enzyme of Cer metabolism) knockout (SMS2(-/-)) and wild type (WT) female mice to establish the model of prenatal alcohol exposure. In 24 h after being given birth (postnatal day 0, P0), the offspring of model mice received blood sphingomyelin (SM) measurement with enzymatic method. On P0, P7, P14 and P30, the proliferation of granule cells in the dentate gyrus and newborn neurons were investigated with immunofluorescent labeling. The expression of protein kinase Cα (PKCα) in the hippocampus was tested with Western blot analysis. The results showed that the SM level of blood in SMS2(-/-) pups was significantly lower than that in WT pups. No matter in SMS2(-/-) or WT mice, the prenatal alcohol exposure down-regulated the SM levels in pups with dose-dependency. In both SMS2(-/-) and WT pups, the number of proliferative neurons and newborn neurons in the dentate gyrus gradually decreased with the growing age. Compared with the WT pups, SMS2(-/-) pups showed significantly more proliferative neurons and newborn neurons in the dentate gyrus. Notably, prenatal alcohol exposure dose-dependently increased proliferative neurons and newborn neurons in the dentate gyrus in both WT and SMS2(-/-) pups. The hippocampal expression of PKCα protein in SMS2(-/-) mice was lower than that in WT mice, and prenatal alcohol exposure could up-regulate the PKCα protein expression in both WT and SMS2(-/-) mice with dose dependency. These results suggest that alcohol exposure during pregnancy can induce the compensatory neural cell proliferation and the production of newborn neurons in offspring, and the Cer-ceramide-1-phosphate (C1P) pathway is involved in alcohol-induced neural cell proliferation. The activation of PKCα may be a key step to start the Cer-C1P pathway and up-regulate the alcohol-induced neural cell proliferation and the newborn neurons formation.
Animals ; Animals, Newborn ; Cell Proliferation ; drug effects ; Cells, Cultured ; Ceramides ; metabolism ; Dentate Gyrus ; cytology ; Ethanol ; toxicity ; Female ; Mice ; Mice, Knockout ; Neurons ; cytology ; Pregnancy ; Prenatal Exposure Delayed Effects ; physiopathology ; Protein Kinase C-alpha ; metabolism ; Signal Transduction ; Transferases (Other Substituted Phosphate Groups) ; genetics

OBJECTIVEThe quality of microarray data influences the accuracy of comparative genomic analyses to a large extent. To ensure that the results obtained by using an in situ synthesized microarray are accurate, data quality is to be assessed by evaluating the melting temperature (Tm) of probes, probability of false synthesis rates, and fragmentation of labeled targets.

METHODSDNA from the Yersinia pestis vaccine strain EV76 was used for microarray analyses. Microarray results were confirmed by PCR. Statistical and bioinformatics methods were employed to perform microarray data analyses and evaluation.

RESULTSCorrelation coefficients of the three datasets were above 0.95 after two-time stripping and hybridization with a labeled DNA with the size of fragmentation being 200 bp - 2 kb, which showed that the hybridization results were highly reproducible. Correlation coefficients were lower with the values ranging from 0.87 to 0.92 between the datasets generated from hybridization with different sizes of the labeled DNA fragment. For the relationship between Tm and signal intensity, there was a different distribution of Tm in the lowest 300 or 3,000 probes with a range of 70 °C-72 °C and the highest 300 or 3,000 probes with a range of 72 °C-74 °C.

CONCLUSIONThe results of this study suggest that the initial microarray design may affect the accuracy of final analyses and that the probe Tm and the size of the labeled fragment may be the two factors of the greatest importance.

Cluster Analysis ; Comparative Genomic Hybridization ; methods ; standards ; DNA Fragmentation ; DNA, Bacterial ; genetics ; Genome, Bacterial ; Oligonucleotide Array Sequence Analysis ; methods ; standards ; Polymerase Chain Reaction ; Reproducibility of Results ; Yersinia pestis ; genetics

OBJECTIVETo assess the value of compound anesthesia of transcutaneous electrical point stimulation and Remifentanil and the efficacy of this method on postoperative acute pain.

METHODSSixty cases with vertebral lamina internal fixation decompression operation were selected and randomly divided into 2 groups, an observation group and a control group, 30 cases in each group. The patients in the observation group received compound anesthesia of transcutaneous electrical point stimulation at Hegu (LI 4), Laogong (PC 8), Neiguan (PC 6) and Waiguan (TE 5) 30 min before anesthesia induction with HANS stimulator and then Remifentanil anesthesia. During the operation, the stimulation was lasted for 30 min and ceased for 30 min until the end of operation. The patients in the control group received simple Remifentanil anesthesia. The dosage of the narcotic, changes of both blood pressure and heart rate during operation, before and after extubation and the pain degree, etc. were investigated in the two groups.

RESULTS(1) The dosage of Isoflurane, (0.52 +/- 0.33)vol%, in the observation group was significantly lower than (1.12 +/- 0.18) vol% in the control group (P < 0.01). (2) Both blood pressure and heart rate during operation, before and after extubation in the observation group were lower than those before operation (P < 0.01), and both the blood pressure and heart rate during operation in the control group were lower than those before operation (P < 0.01). The blood pressure after extubation in the observation group was significantly lower than that of the control group (P < 0.01), and the heart rate before and after extubation in the observation group was significantly lower than that of the control group (P < 0.01). (3) The time of extubation and palinesthesia in the observation group were significantly shorter than those in the control group (P < 0.01). (4) In the observation group, the VAS scores after palinesthesia in 26 cases were < 4, and in 4 cases were > or = 5, while in the control group, the scores in 4 cases were < 4 and in 20 cases > or = 5, with a significant difference between the two groups (P < 0.01).

CONCLUSIONCompound anesthesia of transcutaneous electrical point stimulation and Remifentanil can reduce the dosage of narcotics, shorten the time of palinesthesia and effectively prevent and treat acute pain after Remifentanil anesthesia.

Acupuncture Analgesia ; Adult ; Anesthesia Recovery Period ; Anesthetics, Intravenous ; administration & dosage ; Electroacupuncture ; Female ; Humans ; Male ; Middle Aged ; Pain, Postoperative ; prevention & control ; therapy ; Piperidines ; administration & dosage ; Spine ; surgery

OBJECTIVETo develop a technique for simultaneous detection of various target genes in Roundup Ready soybean by combining multiplex PCR and low-density DNA microarray.

METHODSTwo sets of the multiplex PCR system were used to amplify the target genes in genetically modified (GM) soybean. Seventeen capture probes (PCR products) and 17 pairs of corresponding primers were designed according to the genetic characteristics of Rroundup Ready soybean (GTS40-3-2), maize (Mon810, Nk603, GA21), canola (T45, MS1/RF1), and rice (SCK) in many identified GM crops. All of the probes were categorized and identified as species-specific probes. One negative probe and one positive control probe were used to assess the efficiency of all reactions, and therefore eliminate any false positive and negative results. After multiplex PCR reaction, amplicons were adulterated with Cy5-dUTP and hybridized with DNA microarray. The array was then scanned to display the specific hybridization signals of target genes. The assay was applied to the analysis of sample of certified transgenic soybean (Roundup Ready GTS40-3-2) and canola (MS1/RF1).

RESULTSA combination technique of multiplex PCR and DNA microarray was successfully developed to identify multi-target genes in Roundup Ready soybean and MS1/RF1 canola with a great specificity and reliability. Reliable identification of genetic characteristics of Roundup Ready of GM soybean from genetically modified crops was achieved at 0.5% transgenic events, indicating a high sensitivity.

CONCLUSIONA combination technique of multiplex PCR and low-density DNA microarray can reliably detect and identify the genetically modified crops.

Base Sequence ; Cloning, Molecular ; Crops, Agricultural ; DNA Primers ; DNA Probes ; Oligonucleotide Array Sequence Analysis ; Plants, Genetically Modified ; Polymerase Chain Reaction ; methods

To decrease the hemolysis side effect of puerarin, the basic formula and preparation of puerarin submicron emulsion were optimized and the physicochemical properties were evaluated. Puerarin submicron emulsions were prepared by phase inversion-ultrasound combining with phospholipids complexes technology. The effects of preparative parameters, such as emulsification time, stirring velocity and ultrasound time, on mean diameter, span of dispersity, entrapment efficiency and overall desirability were investigated. The three dimensional response surface graphs were produced by second-order polynomial and liner equation, which predict the optimal experiment conditions. All response variables were found to be greatly dependent on three independent variables. Second-order polynomial equations were fitter than liner equations for this study. The optimal emulsification time, stirring velocity and ultrasound time was 15 min, 2 000 r x min(-1), 30 min, respectively. The mean diameter, span of dispersity, entrapment efficiency, drug content and zeta potential of emulsions prepared by the method were 228.23 nm, 0.628 4, 84. 32%, 9.98 mg x mL(-1), - 29.03 mV, respectively. Puerarin submicron emulsion was prepared by the optimized preparation method. The narrow particle diameter distribution, high envelopment efficacy and good stability were obtained. The physicochemical properties were suitable for the requirement of the intravenous emulsion.
Emulsions ; Isoflavones ; administration & dosage ; chemistry ; Particle Size

OBJECTIVETo investigate the correlation between sensitivity to tumor necrosis factor-related apoptosis inducing ligand (TRAIL) and expression level of death receptor 5 (DR5) on tumor cell surface.

METHODSAnti-DR5 mAb was used to detect expression level of DR5 on surface of tumor cells by flow cytometry. Sensitivity to apoptosis induced by TRAIL was determined by TRAIL apoptosis kit. The correlation between expression level of DR5 and sensitivity to TRAIL was analyzed.

RESULTSThe expression of DR5 on surface of tumor cells was approximately 97.9% in U937 cells, 95.1% in Jurkat cells, 93.8% in SW480 cells, 86.2% in HCT116 cells, 64.2% in HL-60 cells, 46.6% in HeLa cells and 13.1% in K562 cells, respectively. The apoptosis rate induced by TRAIL was 72.6% in U937 cells, 85.2% in Jurkat cells, 78.6% in SW480 cells, 70.2% in HCT116 cells, 60.1% in HL-60 cells, 45.4% in HeLa cells and 12.3% in K562 cells, respectively. There was a significant positive correlation between the expression level of DR5 with TRAIL-inducing apoptosis (r = 0.997, P < 0.001).

CONCLUSIONTRAIL-inducing apoptosis is related to the expression level of DR5 on surface of tumor cells. The results confirm the importance of DR5 expression for induction of apoptosis by TRAIL.

Antibodies, Monoclonal ; immunology ; Apoptosis ; drug effects ; Apoptosis Regulatory Proteins ; metabolism ; pharmacology ; Cell Line, Tumor ; Humans ; Jurkat Cells ; Membrane Glycoproteins ; metabolism ; pharmacology ; Receptors, TNF-Related Apoptosis-Inducing Ligand ; Receptors, Tumor Necrosis Factor ; immunology ; metabolism ; TNF-Related Apoptosis-Inducing Ligand ; Tumor Necrosis Factor-alpha ; metabolism ; pharmacology ; U937 Cells

Objective To determine the prevalence and distribution of type 2 diabetes mellitus (T2DM) and the relationship between maximum body mass index (MAXBMI) and T2DM. Methods From June to August, 2005, a stratified cluster sampling of 1071 permanent residents in communities, over 20 years old, from 4 districts and 1 county of Mudanjiang was chosen. The prevalence of T2DM, and the association between T2DM and different levels of the MAXBMI, current BMI were studied. Results The prevalence in the communities was 7.09% and in those with past maximum BMI≥28 kg/m~2, it was 12.10%. With the increase of past MAXBMI levels, the risk of T2DM patients also increased significantly(trend X~2=17.387 23, P<0.0001). Data from multifactor analysis showed that MAXBMI in the past was positively related to T2DM (OR=3.06, P=0.0013). In T2DM patients, the group with MAXBMI≥27.4 kg/m~2 had higher 2-hour postprandial blood glucose than those with lower MAXBMI (P=0.0408). When compared with low maximum BMI group in normal blood glucose population, the group with higher MAXBMI (≥ 25.4 kg/m~2) had higher blood glucose and greater change of BMI. Conclusion In both groups that patients with T2DM and with normal glucose, in order to control blood glucose better, researchers should not only concern about the influence of the MAXBMI in the past, but also pay attention to constantly keep BMI at the normal range.