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Biomedical subjects

Rong Shi

Publications and source records attributed to Rong Shi.

At least 19 recordsLinked to original sources

MAPU: Max-Planck Unified database of organellar, cellular, tissue and body fluid proteomes.

Mass spectrometry (MS)-based proteomics has become a powerful technology to map the protein composition of organelles, cell types and tissues. In our department, a large-scale effort to map these proteomes is complemented by the Max-Planck Unified (MAPU) proteome database. MAPU contains several body fluid proteomes; including plasma, urine, and cerebrospinal fluid. Cell lines have been mapped to a depth of several thousand proteins and the red blood cell proteome has also been analyzed in depth. The liver proteome is represented with 3200 proteins. By employing high resolution MS and stringent validation criteria, false positive identification rates in MAPU are lower than 1:1000. Thus MAPU datasets can serve as reference proteomes in biomarker discovery. MAPU contains the peptides identifying each protein, measured masses, scores and intensities and is freely available at http://www.mapuproteome.com using a clickable interface of cell or body parts. Proteome data can be queried across proteomes by protein name, accession number, sequence similarity, peptide sequence and annotation information. More than 4500 mouse and 2500 human proteins have already been identified in at least one proteome. Basic annotation information and links to other public databases are provided in MAPU and we plan to add further analysis tools.

Animals↗

Three-dimensional modeling of cytomegalovirus DNA polymerase and preliminary analysis of drug resistance.

Cytomegalovirus (CMV) is the leading cause of congenital infection and a frequent opportunistic agent in immunocompromised hosts such as transplant recipients and AIDS patients. CMV DNA polymerase, a member of the polymerase B family, is the primary target of all available antivirals (ganciclovir, cidofovir, and foscarnet) and certain variations of this enzyme could lead to drug resistance. However, understanding the drug resistance mechanisms at the atomic level is hampered by the lack of its three-dimensional (3D) structure. In the present work, 3D models of two different conformations (closed and open) for CMV DNA polymerase have been built based on the crystal structures of bacteriophage RB69 DNA polymerase (a member of the polymerase B family) by using the 3D-Jury Meta server and the program MODELLER. Most of the variations on CMV DNA polymerase pertinent to ganciclovir/cidofovir and foscarnet resistance can be explained well based on the open and closed conformation models, respectively. These results constitute a first step towards facilitating our understanding of drug resistance mechanisms for CMV and the interpretation of novel viral mutations.

Antiviral Agents↗

A dataset of human fetal liver proteome identified by subcellular fractionation and multiple protein separation and identification technology.

A high throughput process including subcellular fractionation and multiple protein separation and identification technology allowed us to establish the protein expression profile of human fetal liver, which was composed of at least 2,495 distinct proteins and 568 non-isoform groups identified from 64,960 peptides and 24,454 distinct peptides. In addition to the basic protein identification mentioned above, the MS data were used for complementary identification and novel protein mining. By doing the analysis with integrated protein, expressed sequence tag, and genome datasets, 223 proteins and 15 peptides were complementarily identified with high quality MS/MS data.

Cell Membrane↗

[Cross-sectional investigation of prevalence of type 2 diabetes in Shanghai].

OBJECTIVE: To investigate the prevalence of type 2 diabetes and impaired glucose regulation among the permanent urban and rural inhabitants in Shanghai. METHODS: Questionnaire survey, physical examination, and laboratory testing were conducted among 14401 urban and rural inhabitants aged 15 - 74 in Shanghai selected by multistage cluster random sampling. The data about the prevalence of type 2 diabetes and impaired glucose regulation were collected and analyzed. RESULTS: The respondent rate of this investigation was 80.5% (11,589/14,401). The prevalence rates of type 2 diabetes, impaired glucose tolerance (IGT) and impaired fasting glucose (IFG) were 8.6% (1000/11,589), 6.9% (802/11,589), and 1.0% (120/11,589) respectively (the standardized rates were 6.2%, 5.1% and 0.8%). The prevalence rates of type 2 diabetes, IGT, and IFG were significantly higher in those with overweight, obesity, central obesity, and hypertension. The prevalence rates of type 2 diabetes, IGT, and IFG in men were 8.9% (412/4621), 6.4% (296/4621), and 1.0% (47/4621) respectively, all not significantly different from those of the women, i.e. e., 8.4% (588/6968), 7.3% (506/6968), and 1.0% (73/6968) respectively (all P > 0.05). The prevalence rates of type 2 diabetes and IGT in the urban area were 11.2% (730/6500) and 6.4% (419/6500) respectively, both significantly higher than those in the rural area, i.e., 5.3% (270/5089) and 7.5% (383/5089) respectively (both P < 0.05). The prevalence rate of IFG in the urban area was 1.2% (77/6500), not significantly different from that in the rural area (0.8%, 43/5039, P > 0.05). The prevalence of type 2 diabetes, IGT, and IFG increased apparently with age, body mass index, waist hip ratio, and waist circumference. The ratios of undiagnosed type 2 diabetes in the urban area and rural area were 39.6% (289/730) and 69.3% (187/270) respectively (chi(2) = 74.07, P < 0.01). CONCLUSION: The prevalence rate of type 2 diabetes in Shanghai is higher than the mean national level and shows an increasing tendency. Screening of type 2 diabetes in rich rural areas should be emphasized.

Adolescent↗

CT colonography: influence of 3D viewing and polyp candidate features on interpretation with computer-aided detection.

PURPOSE: To retrospectively determine if three-dimensional (3D) viewing improves radiologists' accuracy in classifying true-positive (TP) and false-positive (FP) polyp candidates identified with computer-aided detection (CAD) and to determine candidate polyp features that are associated with classification accuracy, with known polyps serving as the reference standard. MATERIALS AND METHODS: Institutional review board approval and informed consent were obtained; this study was HIPAA compliant. Forty-seven computed tomographic (CT) colonography data sets were obtained in 26 men and 10 women (age range, 42-76 years). Four radiologists classified 705 polyp candidates (53 TP candidates, 652 FP candidates) identified with CAD; initially, only two-dimensional images were used, but these were later supplemented with 3D rendering. Another radiologist unblinded to colonoscopy findings characterized the features of each candidate, assessed colon distention and preparation, and defined the true nature of FP candidates. Receiver operating characteristic curves were used to compare readers' performance, and repeated-measures analysis of variance was used to test features that affect interpretation. RESULTS: Use of 3D viewing improved classification accuracy for three readers and increased the area under the receiver operating characteristic curve to 0.96-0.97 (P<.001). For TP candidates, maximum polyp width (P=.038), polyp height (P=.019), and preparation (P=.004) significantly affected accuracy. For FP candidates, colonic segment (P=.007), attenuation (P<.001), surface smoothness (P<.001), distention (P=.034), preparation (P<.001), and true nature of candidate lesions (P<.001) significantly affected accuracy. CONCLUSION: Use of 3D viewing increases reader accuracy in the classification of polyp candidates identified with CAD. Polyp size and examination quality are significantly associated with accuracy.

Adult↗

[Research of the spermatozoal gene expression with gene microarrays].

OBJECTIVE: To perform the detection of spermatozoal gene expression in order to accelerate the study of spermatozoal molecular biology. METHODS: To collect the healthy adults sperm and lymphocytes respectively, and then to extract the total RNAs from them by RNeasy mini kit (QIAGEN) or Trizol reagent. Corresponding cDNAs were produced, digested, ligated, finally labeled with Cy3 (sperm) and CyS (lymphocyte) in the course of RD amplifying reactions. Hybridization with self-made microarrays contained 560 probes was carried out after the labeled cDNAs pured by PCR Product Purification Kit. RESULTS: Among the 560 probes, 72 genes were up-regulated, 321 genes were down-regulated, the others had no different expression. Furthermore, genes associated with replication, transcription, translation and regulative functions were non-different expression or down-regulated, and those belonged to the spermatogenesis associated, sperm associated antigen were up-regulated, but those involved in the glycolysis were up-regulated, in the oxidative phosphorylation were down-regulated. CONCLUSION: It had successfully confirmed that there were a plenty of genes expressed in sperm, furthermore the genes expressed were accorded to spermatozoal functions and characteristics.

Adult↗

[Effects of microRNA miR-181a on gene expression profiles of K562 cells].

OBJECTIVE: To investigate the effect of microRNA on the gene expression profile of human leukemia K562 cells using microarray technique. METHODS: miR-181a RNA duplexes were designed and synthesized according to the mature sequence of miR-181a. Forty-eight hours after transfection of in vitro cultured K562 cells using Oligofectamine, gene expression profiles of the cells were studied and analyzed using Agilent Human 1A Oligo microarray. RESULTS: Totalling 228 differentially expressed genes were identified from the 20,173 screened genes, including 59 up-regulated ones (consisting of metabolism-associated genes, tumor suppressor genes, signal transduction-associated genes, immunity and defense-associated genes etc), and 169 down-regulated ones (consisting of oncogenes, DNA-binding and transcription genes, metabolism-associated genes, signal transduction-associated genes, cell cycle and development-associated genes etc.) in the transfected K562 cells as compared with the control K562 cells. Changes in expressions of CTCF, ZAP70, SEMA4C and RALA were confirmed by semi-quantitative reverse transcription-polymerase chain reaction. CONCLUSIONS: miR-181a transfection for 48 h induces gene expression profile changes in K562 cells, indicating the functionality of the miR-181a. These differentially expressed genes are related to the functions of the microRNA, and may also be the basis of the regulation model of posttranscriptional gene silencing. These findings provide an evidence for further study of the machineries and functions of the microRNA in mammalian cells.

Gene Expression Profiling↗

[Study on retention time shift correction of fingerprint chromatograms of soybean isoflavones].

A method has been developed to improve the reproducibility of retention time on the fingerprint chromatograms of soybean isoflavones by high performance liquid chromatography (HPLC). Alltech C18 column and Diamonsil C18 column were used on LC-10AT system and Aglient 1100 system respectively, the mobile phase being acetic acid solution (pH 3.2)-methanol, flow rate being 0.6 mL/min, detection wavelength at 261 nm. All experiments were performed at room temperature. Under the chromatographic conditions of soybean isoflavone fingerprints, five substances as standards were used to establish the calibration curves. The shift of retention time of the fingerprint chromatogram in which peak area percentage is more than 1.5% was corrected by linear equation. The results show that the absolute error of retention times is diminished from 5.868 min to 0.854 min after calibration. This method is a good way to correct the shift of retention time for chromatographic fingerprints obtained from different C18 columns or different HPLC systems. In addition, the calibration results improve the reproducibility greatly and can be applied in different laboratories conveniently.

Chromatography, High Pressure Liquid↗

[Phenotypic analysis of luxS gene deletion mutants and its application in virulence regulation research in group B Streptococcus].

OBJECTIVE: To explore the mechanism of virulence regulation in group B streptococcus (BGS) by studying LuxS-related AI-2 quorum-sensing pathway in GBS. METHODS: luxS gene deletion mutants of GBS (Delta lusX) were characterized by reverse transcription (RT)-PCR, colony immunoblot analysis, growth curve measurement, and cAMP determination. Functional analysis of luxS in the deletion mutants was conducted by bioluminescence assay. RESULTS: Genetic analysis results showed that the luxS deletion in the mutant 515-Delta lusX caused upregulation of scpB gene expression. Phenotypic analysis revealed that, in comparison with the wild strain, 515-Delta lusX mutant grew slowly in DCM media but quickly in THY media. An approximately two-fold decrement in bioluminescence was detected in the luxS deletion mutants as compared with the wild strain. CONCLUSION: This study confirms the importance of LuxS molecule in the AI-2 quorum-sensing pathway in GBS and provides new insights into the virulence regulation mechanism of GBS.

Bacterial Proteins↗

Coronary artery calcium outperforms carotid artery intima-media thickness as a noninvasive index of prevalent coronary artery stenosis.

OBJECTIVE: Increased carotid artery intima-media thickness (IMT) and increased coronary artery calcium (CAC) are noninvasive surrogate indices of prevalent coronary artery disease (CAD). We compared CAC to IMT for noninvasive detection of prevalent CAD in participants whose coronary status was identified by coronary angiography. METHODS AND RESULTS: Male and female CAD patients (> or =50% stenosis in one or more coronary artery, n=79) and controls (no lumen irregularities, n=93) were identified using coronary angiography. Mean maximum carotid IMT was quantified using B-mode ultrasound and total CAC was measured using ECG-gated helical computed tomography (HCT). Carotid IMT was approximately 20% higher in CAD cases compared with controls (P<0.001), whereas mean CAC was 1000% higher in CAD cases than controls (P<0.0001). In multivariable models adjusted for age and sex, IMT greater than the median (1.13 mm) was associated with 2-fold increase in likelihood of prevalent CAD compared with scores below that cut point (P=0.015). CAC scores that exceeded the median score of 92 were associated with 28-fold increase in likelihood of prevalent CAD (P<0.0001). Although associations of increased IMT with prevalent CAD were similar in males and females, CAC scores above the median in females were associated with 39-fold increase in odds of prevalent CAD, whereas males with elevated CAC had 19-fold risk of CAD. CONCLUSIONS: HCT-measured CAC compares favorably with carotid IMT measured by B-mode ultrasound as a noninvasive index of prevalent CAD.

Age Distribution↗

Pericardial and visceral adipose tissues measured volumetrically with computed tomography are highly associated in type 2 diabetic families.

RATIONALE AND OBJECTIVES: Pericardial and visceral adipose tissue volumes can provide new insight into the complex relation between obesity, adult-onset diabetes, and cardiovascular disease. We describe a new method for quantifying pericardial adipose tissue volumes with computed tomography (CT), and present its precision and relation to established measures of adiposity. METHODS: Eighty subjects randomly selected from a family study of sibling pairs concordant for type 2 diabetes and unaffected siblings, 69 with diabetes, had 2 cardiac CT scans with electrocardiographic gating and 1 abdominal scan as part of an examination designed to measure calcified atherosclerotic plaque. Pericardial adipose tissue and visceral adipose tissue were measured using a 3-dimensional analysis technique. Body mass index, waist circumference, waist-to-hip ratio, and percent fat by dual x-ray absorptiometry were measured during the same visit. RESULTS: Pericardial adipose tissue volumes measured independently and in a random order from the 2 sequential cardiac CT scans obtained during the same examination were highly correlated (Spearman R = 0.99; P < 0.0001). The mean +/- standard deviation (median) pericardial adipose tissue volume was 320.5 +/- 147.3 (281.7) mL. Pericardial adipose tissue was highly correlated with total abdominal visceral adipose tissue (R = 0.81; P < 0.0001). CONCLUSION: The significant association between pericardial and visceral adipose tissue volumes in this preliminary study suggests that pericardial, like visceral adipose tissue, may be an important predictor or risk factor for cardiovascular disease and other related illnesses and warrants further evaluation.

Absorptiometry, Photon↗

[A new fluorescent labeling technique in microarray studies: universal primer U2 labeling].

OBJECTIVE: To develop a new method for fluorescent labeling technique, universal primer U(2) labeling (UPL), for microarray studies. METHOD: Influenza virus RNA was labeled with four labeling methods, namely UPL, random primer, restriction display incorporation labeling method and reverse transcription coupled random primer spiking labeling method (RT-PSL), respectively, and hybridized to influenza virus oligonucleotide microarray. The signals extracted from the microarrays were analyzed with SPSS 10.0 software to compare the efficiency and reproducibility of the labeling methods. RESULTS: The fluorescence intensity, signal-to-noise ratio (SNR), true positive ratio (TPR) of the probes and reproducibility of labeling with UPL were comparable with those RD-labeling method, and higher than those of RT-PSL method. UPL reduced the complexity of the procedures in comparison with the other labeling methods. CONCLUSION: UPL labeling method can be used in research and development of the microarray technique.

Fluorescent Dyes↗

[Construction and preliminary identification of subtracted cDNA library of leukemia cell line K562].

OBJECTIVE: Subtractive hybridization technology is a common method to screen and clone differentially expressed genes. This study was to construct subtracted cDNA library of leukemia cell line K562, and screen for differentially expressed genes. METHODS: cDNA fragments of K562 cells (tester), prepared by restriction display (RD), were subtracted with the Sau3A I-digested cDNA fragments of normal lymphocytes (driver). The subtracted cDNA fragments were re-amplified, and cloned into pMD18-T vectors. Positive clones were selected by blue-white screening. The inserts in plasmid were amplified by polymerase chain reaction (PCR), and some of which were sequenced. RESULTS: The subtracted library contained 360 positive clones with cDNA fragments distributed mainly from 200 to 800 bp. The 50 randomly sequenced clones were derived from 42 known genes. CONCLUSION: Specific subtracted cDNA library of K562 cells was successfully constructed with reliable quality, and may be used to further screen and clone differentially expressed genes of K562 cells.

Cloning, Molecular↗

[c-myc gene silencing in K562 cells with RNA interference].

OBJECTIVE: To study the inhibitory effect of small interfering RNA (siRNA) targeting c-myc gene in K562 cells. METHODS: siRNAs targeting the site 1357 of c-myc mRNA was designed and synthesized. In vitro cultured K562 cells were transfected with lipofectamine 2000 and the inhibitory effect was detected by reverse transcriptase (RT)-PCR, cell count, MTT assay and fluorescence-activated cell sorting. RESULTS: Compared with the negative and blank control group, the transfection group showed marked decrease in the c-myc expression and the K562 cells exhibited increased apoptosis rate. CONCLUSION: RNA interference can effectively inhibit c-myc expression and induce apoptosis in K562 cells.

Apoptosis↗

A novel sample labeling method with restriction display PCR for 60-mer oligonucleotide microarray.

OBJECTIVE: To investigate the value of restriction display PCR (RD-PCR) as a novel and expedient sample labeling method for high-density 60-mer oligonucleotide microarray. METHODS: Peripheral blood samples from three volunteers were collected and the total RNA was extracted from the peripheral blood mononuclear cells and labeled with RD-PCR protocol, followed by hybridization with Agilent Human 1B oligonucleotide microarrays in a two-color comparison format. The RNA from the same subject was divided into two aliquot and labeled with Cy3 and Cy5 respectively. The spots with significant difference between the foreground and local background intensities and those without significant difference between Cy5 and Cy3 signal intensities were selected for analysis. SPSS software was used to perform the statistical tests and plot generation. VSN packages were used under R language to remove the systematic array and dye biases. RESULTS: Totally 8744 common spots of the 3 microarrays were evaluated. The results demonstrated that RD-PCR could be a promising novel method for efficient labeling of microarray samples. Further analysis indicated the presence of adjustable biases derived from the array and incorporated dye in the labeling processes. The RD-PCR labeling showed better performance than the conventional approaches in regards to reproducibility of the quantitative signals for gene intensity and capability to label RNAs of lowly expressed genes. CONCLUSION: Given the evidence of the feasibility of using RD-PCR labeling in the field of high-density long oligonucleotide microarray, further optimization of the protocol may unleash the full potential of this novel labeling method.

Humans↗

[Detection of quorum-sensing pathway and construction of LuxS gene deletion mutants of group B Streptococcus].

OBJECTIVE: To detect the possible presence of AI-2 quorum-sensing pathway and construct group B Streptococcus (GBS) mutants with deletion of LuxS gene related to quorum-sensing pathway. METHOD: V. harveyi BB170 was employed as the reporter strain to detect AI-2 pathway in GBS, and identification of LuxS homologous gene in GBS type V strain 2603 was performed by software-based analysis. LuxS gene deletion mutant Delta LusX was then constructed in GBS by means of allelic exchange and verified by Southern hybridization analysis. RESULTS: A component in the secretions of GBS could induce bioluminescence activity in the reporter strain, suggesting the presence of AI-2 quorum-sensing pathway in GBS. luxS homologous gene was detected in GBS and LuxS gene deletion mutants was successfully constructed in GBS Ia 515 and V 2603 strains. CONCLUSION: This study establishes a bacterial model for studying the role of LuxS molecule in AI-2 quorum-sensing pathway in GBS and provides new insights into virulence regulation mechanism of GBS.

Bacterial Adhesion↗

Cofactor hydrogen bonding onto the protein main chain is conserved in the short chain dehydrogenase/reductase family and contributes to nicotinamide orientation.

Human estrogenic 17beta-hydroxysteroid dehydrogenase (17beta-HSD1), a member of the short chain dehydrogenase/reductase (SDR) family, is responsible for the biosynthesis of all active estrogens. The crystal structures of two C19-steroid ternary complexes (17beta-HSD1-androstanedione-NADP and 17beta-HSD1-androstenedione-NADP) reveal the critical role of Leu149 in regulating the substrate specificity and provide novel insight into the different fates of a conserved glutamate residue in the estrogen-specific proteins upon the binding of the keto and hydroxyl groups of steroids. The whole NADP molecule can be unambiguously defined in the NADP binary complex, whereas both ternary complexes show that the nicotinamide moiety of NADP cannot be located in the density maps. In both ternary complexes, the expected position of carboxamide oxygen of NADP is occupied by a water molecule, which makes a bifurcated hydrogen bond with the O3 of C19-steroid and the main chain nitrogen of Val188. These results demonstrate that the hydrogen bonding interaction between the main chain amide group and the carboxamide group of NAD(P)(H) plays an important role in anchoring the nicotinamide ring to the enzyme. This finding is substantiated by structural analyses of all 33 NAD(P)(H) complexes of different SDR proteins, because 29 structures of 33 show this interaction. This common feature reveals a general mechanism among the SDR family, providing a rational basis for inhibitor design against biologically relevant SDR targets.

17-Hydroxysteroid Dehydrogenases↗