Bingjie Zou

A Pharmacometabonomic Approach To Predicting Metabolic Phenotypes and Pharmacokinetic Parameters of Atorvastatin in Healthy Volunteers.

Genetic polymorphism and environment each influence individual variability in drug metabolism and disposition. It is preferable to predict such variability, which may affect drug efficacy and toxicity, before drug administration. We examined individual differences in the pharmacokinetics of atorvastatin by applying gas chromatography-mass spectrometry-based metabolic profiling to predose plasma samples from 48 healthy volunteers. We determined the level of atorvastatin in plasma using liquid chromatography-tandem mass spectrometry. With the endogenous molecules, which showed a good correlation with pharmacokinetic parameters, a refined partial least-squares model was calculated based on predose data from a training set of 36 individuals and exhibited good predictive capability for the other 12 individuals in the prediction set. In addition, the model was successfully used to predictively classify individual pharmacokinetic responses into subgroups. Metabolites such as tryptophan, alanine, arachidonic acid, 2-hydroxybutyric acid, cholesterol, and isoleucine were indicated as candidate markers for predicting by showing better predictive capability for explaining individual differences than a conventional physiological index. These results suggest that a pharmacometabonomic approach offers the potential to predict individual differences in pharmacokinetics and therefore to facilitate individualized drug therapy.

Sensitive and specific colorimetric DNA detection by invasive reaction coupled with nicking endonuclease-assisted nanoparticles amplification

Colorimetric DNA detection is preferable to methods in clinical molecular diagnostics, because no expensive equipment is required. Although many gold nanoparticle-based colorimetric DNA detection strategies have been developed to analyze DNA sequences of interest, few of them can detect somatic mutations due to their insufficient specificity. In this study, we proposed a colorimetric DNA detection method by coupling invasive reaction with nicking endonuclease-assisted nanoparticles amplification (IR-NEANA). A target DNA firstly produces many flaps by invasive reaction. Then the flaps are converted to targets of nicking reaction-assisted nanoparticles amplification by ligation reaction to produce the color change of AuNPs, which can be observed by naked eyes. The detection limit of IR-NEANA was determined as 1pM. Most importantly, the specificity of the method is high enough to pick up as low as 1% mutant from a large amount of wild-type DNA backgrounds. The EGFR gene mutated at c.2573 T>G in 9 tissue samples from non-small cell lung cancer patients were successfully detected by using IR-NEANA, suggesting that our proposed method can be used to detect somatic mutations in biological samples.

Dye‐Free MicroRNA Quantification by Using Pyrosequencing with a Sequence‐Tagged Stem–loop RT Primer

MicroRNAs (miRNAs) are a class of endogenous, ~22-nucleotide (nt) noncoding RNAs that play an important role in the control of the developmental processes of cells by negative regulation of protein-coding gene expression. To date, there are 17 341 mature miRNAs, including 1048 human miRNAs, in the University of Manchester miRNA database (http://www. mirbase.org/). Although miRNAs represent a relatively abundant class of transcripts, their expression levels vary greatly in different tissue types and species. Analyzing miRNA expression levels in tissues or cells can supply valuable information for investigating the biological functions of miRNAs; however conventional techniques to amplify miRNAs for detection and quantification present a significant challenge because of the short length of these molecules; thus, a number of straightforward methods without the use of amplification have been developed for miRNA detection. Northern blotting 5] is the widely used standard method for analyzing miRNAs; however, relatively large amounts of starting material (RNA) are required for an assay. To improve the sensitivity of miRNA quantification, a method based on splinted ligation was developed. This exhibits approximately 50 times greater sensitivity than Northern blotting, but radioactive P labels are needed. A single-molecule method, based on the hybridization of two spectrally distinguishable LNA–DNA oligonucleotide probes (for the miRNA of interest), offers a direct miRNA assay as sensitive as 500 fm, but an expensive single-molecule detection instrument is required. For sensitive miRNA detection, amplification techniques are thus necessary. By skillfully designing detection probes, a modified “Invader” assay was developed for the quantification of miRNAs. Although 20 000 miRNAs were detected, accurate quantification of miRNAs among samples is difficult because the initial target concentration is proportional to the steady-state reaction rate of “invasive” amplification. In contrast, an miRNA assay based on real-time quantitative PCR with a stem–loop reverse transcription (RT) primer was much more quantitative, as the Ct (cycle threshold) value is inversely proportional to the amount of initial target. However, PCRs of the sample and reference targets are performed separately, and a small difference in amplification efficiency between the sample and the reference yields a large difference in the amount of final product ; this results in large inter-PCR variations. Recently, a simple and sensitive miRNA quantification method that used branched rolling-circle amplification (BRCA) was reported, but quantification based on endpoint readout seems challengeable because of the time-dependent amplification efficiency of BRCA. To achieve accurate quantification of a target miRNA in a sample, real-time monitoring of signal intensities from both a sample and a reference (quantification standard) is necessary, because the reaction rate slows down as the reaction proceeds. As the real-time detection requires a sophisticated instrument, quantification using endpoint data is preferable. In the present study, we have developed a pyrosequencing-based method for absolute quantification, and for comparing the relative miRNA expression levels in biological samples. Pyrosequencing is a well-developed technology for DNA sequencing. It uses cascade enzymatic reactions to monitor the release of inorganic pyrophosphate that results from dNTP incorporation. Because of its highly quantitative performance, pyrosequencing has been widely used for genotyping, and the analysis of DNA methylation and gene expression. Here we employed pyrosequencing technology to quantify microRNAs by quantitatively detecting sequence labels that were artificially tagged into the RT products of miRNA. Unlike mRNA, miRNA is very short and can be easily synthesized; synthesized molecules with known concentration could thus be used as a reference for quantifying miRNA in a sample. As shown in Figure 1, sequence labels for discriminating the sources of miRNA (sample or reference) are designed into the loop near to the 3’-end of the miRNA-specific RT primer, so that the 5’ end of the primer can offer a universal priming site for the following PCR. The structure of the miRNA-specific stem–loop RT primer is the same as that used by Chen’s group. After reverse transcription with the sequence-tagged RT primers, cDNA from the different sources (sample and reference) were similarly labeled with different sequences (thus, different colors in a fluorescence-based assay). To avoid PCR-bias resulting from Tm differences, the labels were designed from the same base species but with different base order. We labeled the sample-miRNA and the reference-miRNA with the sequences “catg” and “gatc” respectively ; hence, in a pyrogram (Figure 1), [a] H. Jing, Prof. Q. Song, Z. Chen, B. Zou, Prof. G. Zhou Huadong Research Institute for Medicine and Biotechnics Nanjing 210002 (China) Fax: (+ 86) 25-8451-4223 E-mail : ghzhou@nju.edu.cn [b] Prof. Q. Song, B. Zou School of Life Science and Technology, China Pharmaceutical University Nanjing 210009 (China9 [c] C. Chen, Prof. M. Zhu Model Animal Research Centre, Nanjing University Nanjing 210093 (China) [d] Z. Chen, Prof. G. Zhou Medical School, Nanjing University Nanjing 210093 (China) [e] T. Kajiyama, Prof. H. Kambara Central Research Laboratory, Hitachi, Ltd. Tokyo 185-8601 (Japan) Supporting information for this article is available on the WWW under http ://dx.doi.org/10.1002/cbic.201100023.

An internal amplification control for quantitative nucleic acid analysis using nanoparticle-based dipstickbiosensors

Quantitative analysis of virus nucleic acids is essential for monitoring the efficacy of medical treatment based on the copy numbers of viruss RNA or DNA in blood. To quantitatively detect virus nucleic acids in blood, here an internal amplification control (IAC) coupled with a nanoparticle-based DNA biosensor was proposed. The IACs with a specific sequence were designed and spiked into serum before nucleic acids extraction. Sequences of the IACs and the targets only differ in the base order of one PCR priming site; thus, the IACs and the targets are identical in Tm, giving the same amplification efficiency during PCR. To visually detect amplicons, a dipstick biosensor based on streptavidin-functionalized nanoparticles is employed. By comparing color densities of a test zone with an IAC zone on the biosensor, the content of the target in serum can be semi-quantitatively analyzed. This approach has achieved the detection of HBV DNA at approximately 100 copies of the pathogen load. The feasibility of this method is demonstrated by successful semi-quantification of pathogen load in 30 clinical samples from HBV-infected patients. These data indicate that the introduction of an IAC and nanoparticle-based dipstick-type biosensor could be a powerful tool in point of care testing (POCT).

Pyrosequencing-based barcodes for a dye-free multiplex bioassay

A novel dye-free labeling method for a multiplex bioassay was proposed by using short sequence-based barcodes consisting of a reporter base and repeats of two stuffer bases; then, the barcodes were quantitatively decoded by a single pyrosequencing assay without any pre-separation.

Assessing Fungal Population in Soil Planted with Cry1Ac and CPTI Transgenic Cotton and Its Conventional Parental Line Using 18S and ITS rDNA Sequences over Four Seasons.

Long-term growth of genetically modified plants (GMPs) has raised concerns regarding their ecological effects. Here, FLX-pyrosequencing of region I (18S) and region II (ITS1, 5.8S, and ITS2) rDNA was used to characterize fungal communities in soil samples after 10-year monoculture of one representative transgenic cotton line (TC-10) and 15-year plantation of various transgenic cotton cultivars (TC-15mix) over four seasons. Soil fungal communities in the rhizosphere of non-transgenic control (CC) were also compared. No notable differences were observed in soil fertility variables among CC, TC-10, and TC-15mix. Within seasons, the different estimations were statistically indistinguishable. There were 411 and 2 067 fungal operational taxonomic units in the two regions, respectively. More than 75% of fungal taxa were stable in both CC and TC except for individual taxa with significantly different abundance between TC and CC. Statistical analysis revealed no significant differences between CC and TC-10, while discrimination of separating TC-15mix from CC and TC-10 with 37.86% explained variance in PCoA and a significant difference of Shannon indexes between TC-10 and TC-15mix were observed in region II. As TC-15mix planted with a mixture of transgenic cottons (Zhongmian-29, 30, and 33B) for over 5 years, different genetic modifications may introduce variations in fungal diversity. Further clarification is necessary by detecting the fungal dynamic changes in sites planted in monoculture of various transgenic cottons. Overall, we conclude that monoculture of one representative transgenic cotton cultivar may have no effect on fungal diversity compared with conventional cotton. Furthermore, the choice of amplified region and methodology has potential to affect the outcome of the comparison between GM-crop and its parental line.

Expression and purification of ATP sulfurylase from Saccharomyces cerevisias in Escherichia coli and its application in pyrosequencing

ATP sulfurylase (ATPS,EC 2.7.7.4) reversibly catalyzes the reaction between ATP and sulfate to produce APS and pyrophosphate (PPi), and has been used in pyrosequencing. The gene coding ATP sulfurylase was amplified from the genomic DNA of Saccharomyces cerevisias (CICC 1202), and cloned into prokaryotic expression plasmid pET28a( + ) to provide a recombinant expression plasmid pET28a( + )-ATPS. Upon IPTG induction, ATP sulfurylase was produced by E. coli BL21 (DE3) harboring the recombinant expression plasmid pET28a( + )-ATPS. The relative molecular weight of recombinant ATP sulfurylase with His tag was about 60 kD. The recombinant ATP sulfurylase with electrophoretic pure grade was obtained only by two purification steps: His * Bind Resin affinity chromatography and ultrafiltration. The specific activity of the purified recombinant ATP sulfurylase was as high as 5.1 x 10(4) u/mg. The successful application of the enzyme in pyrosequencing was also demostrated.

Prenatal diagnosis of trisomy 21, 18 and 13 by quantitative pyrosequencing of segmental duplications

Chromosomal aberration mostly occurs in chromosomes 21, 18 and 13, with an incidence approximately 1 out of 160 live births in humans, therefore making prenatal diagnosis necessary in clinics. Current methods have drawbacks such as time consuming, high cost, complicated operations and low sensitivity. In this paper, a novel method for rapid and accurate prenatal diagnosis of aneuploidy is proposed based on pyrosequencing, which quantitatively detects the peak height ratio (PHR) of different bases of segmental duplication. A direct polymerase chain reaction (PCR) approach was undertaken, where a small volume of amniotic fluid was used as the starting material without DNA extraction. Single‐stranded DNA was prepared from PCR products and subsequently analyzed using pyrosequencing. The PHR between target and reference chromosome of 2.2 for euploid and 3:2 for a trisomy fetus were used as reference. The reference intervals and z scores were calculated for discrimination of aneuploidy. A total of 132 samples were collected, within trisomy 21 (n = 11), trisomy 18 (n = 3), trisomy 13 (n = 2), and unaffected controls (n = 116). A set of six segmental duplications were chosen for analysis. This method had consistent results with karyotyping analysis, a correct diagnosis with 100% sensitivity and 99.9% specificity.

Signal amplification of microRNAs with modified strand displacement-based cycling probe technology

Micro ribose nucleic acids (miRNAs) play an important role in biological processes such as cell differentiation, proliferation and apoptosis. Therefore, miRNAs are potentially a powerful marker for monitoring cancer and diagnosis. Here, we present sensitive signal amplification for miRNAs based on modified cycling probe technology with strand displacement amplification. miRNA was captured by the template coupled with beads, and then the first cycle based on SDA was repeatedly extended to the nicking end, which was produced by the extension reaction of miRNA. The products generated by SDA are captured by a molecular beacon (MB), which is designed to initiate the second amplification cycle, with a similar principle to the cycling probe technology (CPT), which is based on repeated digestion of the DNA-RNA hybrid by the RNase H. After one sample enrichment and two steps of signal amplification, 0.1 pM of let-7a can be detected. The miRNA assay exhibits a great dynamic range of over 100 orders of magnitude and high specificity to clearly discriminate a single base difference in miRNA sequences. This isothermal amplification does not require any special temperature control instrument. The assay is also about signal amplification rather than template amplification, therefore minimising contamination issues. In addition, there is no need for the reverse transcription (RT) process. Thus the amplification is suitable for miRNA detection.

Multiplex PCR based on a universal biotinylated primer to generate templates for pyrosequencing.

Pyrosequencing is a powerful tool widely used in genetic analysis, however template preparation prior to pyrosequencing is still costly and time-consuming. To achieve an inexpensive and labor-saving template preparation for pyrosequencing, we have successfully developed a single-tube multiplex PCR including a pre-amplification and a universal amplification. In the process of pre-amplification, a low concentration of target-specific primers tagged with universal ends introduced universal priming regions into amplicons. In the process of universal amplification, a high concentration of universal primers was used for yielding amplicons with various SNPs of interest. As only a universal biotinylated primer and one step of single-stranded DNA preparation were required for typing multiple SNPs located on different sequences, pyrosequencing-based genotyping became time-saving, labor-saving, sample-saving, and cost-saving. By a simple optimization of multiplex PCR condition, only a 4-plex and a 3-plex PCR were required for typing 7 SNPs related to tamoxifen metabolism. Further study showed that pyrosequencing coupled with an improved multiplex PCR protocol allowed around 30% decrease of either typing cost or typing labor. Considering the biotinylated primer and the optimized condition of the multiplex PCR are independent of SNP locus, it is easy to use the same condition and the identical biotinylated primer for typing other SNPs. The preliminary typing results of the 7 SNPs in 11 samples demonstrated that multiplex PCR-based pyrosequencing could be promising in personalized medicine at a low cost.