Establishment of a forward primers-superposed amplification analysis for accurate aspirin pharmacogenomic measurement.

Genotyping of gDNA rs12041331 (PEAR1), rs6065 (GP1BA), and rs730012 (LTC4S) can provide systematic guidance on the use of aspirin. However, an accurate, reliable and economical approach to simultaneous detection of the above single nucleotide polymorphisms (SNPs) is not reported. Herein, we designed and substantiated an allele-specific (AS) forward primer-superposed amplification analysis for measurement of the SNPs in PEAR1, GP1BA and LTC4S genes, in which the values of ∆Cq (differences in threshold cycles between the wild-type forward primer-based assay and the mutated-type forward primer-based assay) were employed to decide genotype. Mismatch AS forward primers were screened with the singleplex amplification analysis. Moreover, Cq extension optimized by AS forward primer superposition was observed in the selected forward primer-based triplex analysis. Further, robustness assessment of the triplex analysis showed the amplification efficiency ranging from 0.9 to 1.1. Precision test demonstrated the coefficient of variation of less than 2%. And the detective results of 189 DNA samples was completely concordant with that of commercial Sanger sequencing. In summary, we developed a simple, accurate and economical approach to genotyping of rs12041331 (PEAR1), rs6065 (GP1BA) and rs730012 (LTC4S) to provide a valuable pharmacogenomics tool for guidance of aspirin delivery.

New insight into the classification and evolution of glucose transporters in the Metazoa.

Because glucose is an essential energy source for living organisms, glucose transporters (GLUTs) are present in all species worldwide. Encoded by the solute carrier family 2 gene family, the GLUT proteins generally have 12 transmembrane helices (TMHs). In total, 14 GLUT proteins have been identified in humans (hGLUTs), and they are divided into 3 classes on the basis of their transport characteristics and sequence similarities. Herein, we report the use of protein sequence similarity networks (SSNs) to visualize the sequence trends of 4101 GLUT proteins across the Metazoa. The SSNs separated the metazoan proteins into 3 new classes that were different from the traditional classification system. In the new system, 9 of the 14 hGLUTs (hGLUT1-5, 7, 9, 11, and 14) were grouped into class I, 3 (hGLUT10, 12, and 13) were grouped into class II, and 2 (hGLUT6 and 8) were grouped into class III, as also supported by the phylogenetic tree. Multiple sequence alignments further showed that the conserved residues in each class were different. Furthermore, the hGLUTs in each class showed unique evolutionary characteristics, with similar nonsynonymous-to-synonymous divergence ratios and similar regions under conservative selection pressure. Of note, GLUTs with 3, 6, 18, 24, and 36 TMHs were identified among the metazoan genomes, and 1 Chinese hamster protein with 6 TMHs showed GLUT activity. In summary, this large-scale sequence analysis provided new insights into the classification and evolution of GLUTs and further showed that gene duplication and fusion could have been important drivers during the evolution of these transporter molecules.-Jia, B., Yuan, D. P., Lan, W. J., Xuan, Y. H., Jeon, C. O. New insight into the classification and evolution of glucose transporters in the Metazoa.

Surface-decorated S. cerevisiae for flow cytometric array immunoassay.

Our laboratory had developed a cell-based bio-bead for protein quantification. However, the selection of antibody in the above immunoassay is limited. This study describes a surface-decorated Saccharomyces cerevisiae for flow cytometric array immunoassay. S. cerevisiae was labeled with fluorescein isothiocyanate (FITC) and oxidized by sodium periodate, in which the saccharide group on the cytoderm outer layer was converted to an aldehyde group. In succession, adipic dihydrazide was bio-conjugated to the aldehyde group and glutaraldehyde bound to the hydrazide group. Phycoerythrin (PE)-labeled goat anti-mouse polyclonal antibody was used to assess the conjugation of mouse anti-human monoclonal antibody to surface-decorated S. cerevisiae. Cytokeratin 19 fragment (Cyfra21-1) and neuron-specific enolase (NSE) antigens were also employed to evaluate the flow cytometric array immunoassay based on surface-decorated S. cerevisiae. Flow cytometry demonstrated that FITC-barcoded S. cerevisiae as two legible populations. PE-labeled polyclonal antibody validated the coating of surface-decorated S. cerevisiae with the monoclonal antibody. The flow cytometric array immunoassays for Cyfra21-1 and NSE documented that the limit of detection (LOD) was at least 0.4 ng/mL. Precision and accuracy assessments appeared that the relative standard deviation (R.S.D.) was <15%, and the relative error (R.E.) ranged from 0.9 to 1.1. The correlation coefficient between this immunoassay and electrochemiluminescence immunoassay was 0.9622 for serum Cyfra21-1 and 0.9918 for serum NSE. In conclusion, the surface-decorated S. cerevisiae may be of use in flow cytometric array immunoassay.

Detection of exogenous gene doping of IGF-I by a real-time quantitative PCR assay.

Gene doping can be easily concealed since its product is similar to endogenous protein, making its effective detection very challenging. In this study, we selected insulin-like growth factor I (IGF-I) exogenous gene for gene doping detection. First, the synthetic IGF-I gene was subcloned to recombinant adeno-associated virus (rAAV) plasmid to produce recombinant rAAV2/IGF-I-GFP vectors. Second, in an animal model, rAAV2/IGF-I-GFP vectors were injected into the thigh muscle tissue of mice, and then muscle and blood specimens were sampled at different time points for total DNA isolation. Finally, real-time quantitative PCR was employed to detect the exogenous gene doping of IGF-I. In view of the characteristics of endogenous IGF-I gene sequences, a TaqMan probe was designed at the junction of exons 2 and 3 of IGF-I gene to distinguish it from the exogenous IGF-I gene. In addition, an internal reference control plasmid and its probe were used in PCR to rule out false-positive results through comparison of their threshold cycle (Ct) values. Thus, an accurate exogenous IGF-I gene detection approach was developed in this study.

[Addition effects of co-expansion of two bamboos on plant diversity in broad-leaved forests]. / ç«¹å­æ‰©å¼ å¯¹é˜”å¶æž—ç‰©ç§å¤šæ ·æ€§çš„å½±å“ï¼šä¸¤ç«¹ç§çš„å åŠ æ•ˆåº”.

The effects of Phyllostachys edulis and Oligostachyum oedogonatum expansion on species diversity of broad-leaved forests were investigated in Wuyishan National Nature Reserve, Jiangxi Province, China. Ph. edulis and/or O. oedogonatum expansion changed community structure and species composition. The co expansion of the two bamboos in high intensity (Ph. edulis was 30-50 ind·100 m-2, O. oedogonatum was 300-500 ind·100 m-2) changed the density ratio of arbor and shrub from 2:8 to 1:9, and density ratio of bamboo and tree from 0:10 to 9:1. The main effects of Ph. edulis and O. oedogonatum on the species diversity were significant. Ph. edulis mainly influe-nced arbor layer, while O. oedogonatum influenced shrub layer more, and the interaction effect of two bamboos was not remarkable. There existed addition effects between Ph. edulis and O. oedogonatum on species diversity. The co-occurrence of two bamboos in high intensity decreased the Shannon index of community by 91.3%. Ph. edulis and O. oedogonatum did not compete obviously unless in high density. It was addition effect rather than interaction effect that changed the community structure and reduced species diversity when broad leaved forest suffered invasion by Ph. edulis and O. oedogonatum.

[Establishment and identification of the near-infrared fluorescence labeled exosomes in breast cancer cell lines].

Exosomes, a population of extracellular membrane vesicles of 30-100 nm in diameter, play important roles in cell biological functions, intercellular signal transduction and especially in cancer diagnosis and therapy. To better apply exosomes in mechanistic study of breast cancer signal transduction, we constructed recombinant eukaryotic expression vector expressing the near-infrared fluorescence protein and CD63 fusion protein through cloning iRFP682 gene and exosomal marker protein CD63 gene into plasmid containing the ITR of AAV. The constructed plasmids were co-transfected with helper plasmid in AAV-293 cell lines and were packaged into rAAV. After titer measurement, the recombinant plasmids were transfected into breast cancer cell lines. The cell lines that stably expressing near-infrared fluorescence protein were selected by fluorescence. Through isolation, purification and identification, we finally obtained a new biomarker: iRFP682 labeled exosomes secreted by breast cancer cell lines, which could be used in further studies of the distribution and signal transduction of exosomes in breast cancer microenvironment.

An on-bacterium flow cytometric immunoassay for protein quantification.

The polystyrene bead-based flow cytometric immunoassay has been widely reported. However, the preparation of functional polystyrene bead is still inconvenient. This study describes a simple and easy on-bacterium flow cytometric immunoassay for protein quantification, in which Staphylococcus aureus (SAC) is used as an antibody-antigen carrier to replace the polystyrene bead. The SAC beads were prepared by carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling, paraformaldehyde fixation and antibody binding. Carcinoembryonic antigen (CEA) and cytokeratin-19 fragment (CYFRA 21-1) proteins were used as models in the test system. Using prepared SAC beads, biotinylated proteins, and streptavidin-phycoerythrin (SA-PE), the on-bacterium flow cytometric immunoassay was validated by quantifying CEA and CYFRA 21-1 in sample. Obtained data demonstrated a concordant result between the logarithm of the protein concentration and the logarithm of the PE mean fluorescence intensity (MFI). The limit of detection (LOD) in this immunoassay was at least 0.25 ng/ml. Precision and accuracy assessments appeared that either the relative standard deviation (R.S.D.) or the relative error (R.E.) was <10%. The comparison between this immunoassay and a polystyrene bead-based flow cytometric immunoassay showed a correlation coefficient of 0.998 for serum CEA or 0.996 for serum CYFRA 21-1. In conclusion, the on-bacterium flow cytometric immunoassay may be of use in the quantification of serum protein.

Duplexed on-microbead binding assay for competitive inhibitor of epidermal growth factor receptor by quantitative flow cytometry.

Upon binding agonist, the epidermal growth factor receptor (EGFR) is dimerized and auto-phosphorylated to activate downstream pathway that induces diverse physiology and pathology processes. Conventional methods for evaluation of EGFR inhibitors are limited. This study describes a duplexed on-microbead binding assay allowing competitive EGFR inhibitors to be quantificationally evaluated in vitro. Polystyrene microbeads barcoded by fluoresceine isothiocyanate fluorescence as high brightness and low brightness microspheres were coated with receptor tyrosine kinase (RTK) ligand-epidermal growth factor (EGF)/stem cell factor (SCF) and ATP/GTP, respectively. High and low brightness microbeads were mixed and incubated with EGFR and its competitive inhibitor in binding assay buffer. Phycoerythrin (PE) fluorescence-labelled antibody was employed to report the level of EGFR binding to EGF/SCF and ATP/GTP. Values were numbered via PE molecules assessed by quantitative flow cytometry. Results from this study demonstrated that incubation with EGFR identified by PE-labelled antibody can make EGF- and ATP-coated microbeads luminous. And EGF or ATP-competitive EGFR inhibitors, respectively, alleviated this in a concentration-dependent manner. Coating microbeads with SCF or GTP as a negative control cannot capture EGFR. The duplexed on-microbead binding assay in this study might be useful for discovering ligand- and ATP-competitive EGFR inhibitors in a rapid and quantificational approach.

Geniposide enhances melanogenesis by stem cell factor/c-Kit signalling in norepinephrine-exposed normal human epidermal melanocyte.

Geniposide is an iridoid glycoside isolated from the fruit of Gardenia jasminoides Ellis used as a Chinese traditional medicine for treatment of generalized vitiligo. Stem cell factor from keratinocyte recognizes and activates its receptor c-kit carried by melanocyte to potent enhance melanocytic melanogenesis that can be suppressed by norepinephrine. This study addresses the action and mechanism of geniposide enhancing melanogenesis in norepinephrine-exposed normal human epidermal melanocyte. Flow cytometry results from this study exhibited the augmentation effect of geniposide on production of c-kit receptor by norepinephrine-exposed normal human epidermal melanocyte. However, geniposide did not affect the production of stem cell factor by norepinephrine-exposed normal human epidermal keratinocyte assessed by cellular enzyme-linked immunosorbent assay (ELISA). ELISA indicated that at the presence of stem cell factor, geniposide was capable of elevating the level of extracellular signal-regulated kinase 1/2 phosphorylation within norepinephrine-exposed normal human epidermal melanocyte, which is known to be involved in stem cell factor/c-kit downstream signalling. And inhibition of c-kit with inhibitory antibody K44.2 completely blocked the increase in geniposide-stimulated extracellular signal-regulated kinase 1/2 phosphorylation. In addition, spectrophotometry demonstrated the enhancement effect of geniposide on melanogenesis (tyrosinase activity and melanin production) in norepinephrine-exposed normal human epidermal melanocyte at the presence of stem cell factor, which was blocked by c-kit inhibitory antibody K44.2. Data from this study suggest that geniposide can enhance melanogenesis by stem cell factor/c-kit signalling in which the expression of c-kit receptor is augmented in norepinephrine-exposed normal human epidermal melanocyte.

Activity of Ginkgo biloba extract and quercetin on thrombomodulin expression and tissue-type plasminogen activator secretion by human umbilical vein endothelial cells.

OBJECTIVE: In order to investigate the pharmacological properties of Ginkgo biloba extract (GBE) on improving blood circulation, the regulating action of GBE and quercetin (a main flavonoid ingredient in GBE) on thrombomodulin (TM) expression and tissue-type plasminogen activator (t-PA) secretion was studied. METHODS: Using flow cytometer and gel image system respectively, we evaluated the TM expression and the t-PA secretion by human umbilical vein endothelial cells (HUVECs) in vitro. RESULTS: The increase of TM expression on HUVECs surface was induced by GBE rather than quercetin in a dose- and time-dependent manner. Both GBE and quercetin increased the t-PA release significantly. CONCLUSION: The effect of GBE on improving blood circulation may be partly attributed to its promoting TM expression and t-PA secretion by endothelial cells, and quercetin participated in the effect of GBE on t-PA secretion. However, the action of GBE on increasing TM expression needs further study.

Regulative effects of hawthorn leave flavonoids on cytotoxicity, NO and Ca2+ in hypoxia-treated human umbilical vein endothelial cells.

OBJECTIVE: To evaluate the potential effect of HLF (Hawthorn leave flavonoids, w/w, 80% flavonoids) against thrombus formation, effect of HLF on hypoxia-treated human umbilical vein endothelial cell (HUVECs) was studied. METHOD: The levels of cytotoxicity and NO upon HUVECs were studied by flow cytometry. Moreover, the level of calcium ion in HUVECs was examined through laser scanning confocal microscopy. RESULT: Data from this study showed that HLF at concentrations of 5 micrograms/ml and 10 micrograms/ml decreased the cytotoxicity of hypoxia to HUVECs (P<0.05, P<0.01). The intracellular levels of NO and calcium ion were downregulated by HLF at concentrations of 5 micrograms/ml (P<0.01; P<0.01) and 10 micrograms/ml (vs control, P<0.01; P<0.01) too. CONCLUSION: Results observed suggest that HLF protect HUVECs from hypoxia partly through its regulative effect on NO and calcium ion levels.