Publications (17)60.52 Total impact
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Article: Use of hGluc/tdTomato pair for sensitive BRET sensing of protease with high solution media tolerance.
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ABSTRACT: Due to the complicated media, monitoring proteases in real physiological environments is still a big challenge. Bioluminescence resonance energy transfer (BRET) is one of the promising techniques but its application is limited by the susceptibility to buffer composition, which might cause serious errors for the assay. Herein we report a novel combination of BRET pair with humanized Gaussia luciferase (hGluc) and highly bright red fluorescence protein tdTomato for sensitive and robust protease activity determination. As a result, the hGluc/tdTomato BRET pair showed much better tolerance to buffer composition, pH and sample matrices, and wide spectral separation (Δλ:∼110nm). With the protease sensor built with this pair, the detection limit for enterokinase reached 2.1pM in pure buffer and 3.3pM in 3% serum. The proposed pair would find broad use in both in vitro and in vivo assays, especially for samples with complicated matrix.Talanta 05/2013; 109:141-6. · 3.79 Impact Factor -
Article: Reversibly acetylated lysine residues play important roles on the enzymatic activity of Escherichia coli N-hydroxyarylamine O-acetyltransferase.
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ABSTRACT: CobB is a bacterial NAD+ - dependent protein deacetylase. Although progress has been made in functional studies of this protein in recent years, its substrates and biological functions are still largely unclear. Using proteome microarray technology, potential substrates of Escherichia coli CobB were screened and nine proteins were identified, including N-hydroxyarylamine O-acetyltransferase (NhoA). In vitro acetylation/deacetylation of NhoA was verified by Western blotting and mass spectrometry, and two acetylated lysine residues were identified. Site-specific mutagenesis experiments showed that mutation of each acetylated lysine decreased the acetylation level of NhoA in vitro. Further analysis showed that variant NhoA proteins carrying substitutions at the two acetylated lysine residues are involved in both the O-acetyltransferase (OAT) activity and the N-acetyltransferase (NAT) activity of NhoA. Structural analyses were also performed to explore the effects of the acetylated lysine residues on the activity of NhoA. These results suggest that reversible acetylation may play a role on the activity of Escherichia coli NhoA. © 2013 The Authors Journal compilation © 2013 FEBS.FEBS Journal 03/2013; · 3.79 Impact Factor -
Article: Existence of Separate Domains in Lysin PlyG for Recognizing Bacillus anthracis Spores and Vegetative Cells.
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ABSTRACT: As a potential antimicrobial, the bacteriophage lysin PlyG has been reported to specifically recognize Bacillus anthracis vegetative cells only and to kill B. anthracis vegetative cells and its germinating spores. However, how PlyG interacts with B. anthracis spores remains unclear. Herein, a 60-amino-acid domain in PlyG (residues 106 to 165), located mainly in the previously identified catalytic domain, was found able to specifically recognize B. anthracis spores but not vegetative cells. The exosporium of the spores was found to be the most probable binding target of this domain. This is the first time that a lysin for spore-forming bacteria has been found to have separate domains to recognize spores and vegetative cells, which might help in understanding the coevolution of phages with spore-forming bacteria. Besides providing new biomarkers for developing better assays for identifying B. anthracis spores, the newly found domain may be helpful in developing PlyG as a preventive antibiotic to reduce the threat of anthrax in suspected exposures to B. anthracis spores.Antimicrobial Agents and Chemotherapy 07/2012; 56(10):5031-9. · 4.84 Impact Factor -
Article: Buffer enhanced bioluminescence resonance energy transfer sensor based on Gaussia luciferase for in vitro detection of protease.
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ABSTRACT: Bioluminescence resonance energy transfer (BRET) has gained favors in recent years as a detection technology for protease activity due to its extreme reliability, high sensitivity and low intrinsic backgrounds. Because of the sensitivity of the donors, substrates and the acceptors, it is expected that BRET systems are sensitive to buffer environments. However, no systematic study has been reported on how buffer components would affect the BRET ratio, and thus affect the determination of protease activity based on BRET. We present here that several environmental factors, including buffer agents, pH and divalent metal ions, influenced BRET ratio significantly, when humanized Gaussia luciferase (hGluc) was utilized as the donor and enhanced yellow fluorescence protein (EYFP) as the acceptor. Based on these findings, an enhancing solution was optimized to improve the performance of the BRET sensor for analysis of enterokinase activity in vitro, resulting in 10-fold and 7-fold improvement of the sensitivity and the detection limit, respectively. We anticipate the system will be applicable for improving performance of other in vitro BRET protease sensors, especially when the optimal conditions for protease activity would severely affect the BRET signal.Analytica chimica acta 04/2012; 724:104-10. · 4.31 Impact Factor -
Article: Human cytomegalovirus UL94 is a nucleocytoplasmic shuttling protein containing two NLSs and one NES.
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ABSTRACT: The tegument protein UL94 is a human cytomegalovirus (HCMV) late protein and its function has yet to be determined. Using live cell fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) imaging, we found that UL94 is able to shuttle between the nucleus and cytoplasm. Analysis of UL94 mutants fused to EGFP showed that two newly characterized nuclear localization sequences (NLSs) and amino acid 343 play key roles in UL94 nuclear localization. Mutation of these sequences can alter the intracellular distribution of UL94 and disrupt its nucleocytoplasmic shuttling. Amino acid 343 of UL94 was also found to be crucial for its interaction with another HCMV tegument protein pp28. Furthermore, one nuclear export sequence (NES) was identified within UL94. Mutation of the key amino acids in the NES can also alter the intracellular distribution of UL94 and disrupt its shuttling function. Like other proteins containing a leucine-rich export signal, nuclear export of the UL94 was affected by leptomycin B, indicating that it is exported via the Crm1-dependent pathway. Our data provide a basis for further understanding the character and function of HCMV UL94.Virus Research 03/2012; 166(1-2):31-42. · 2.94 Impact Factor -
Article: Functionally orthologous viral and cellular microRNAs studied by a novel dual-fluorescent reporter system.
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ABSTRACT: Recent research raised the possibility that some viral microRNAs (miRNAs) may function as orthologs of cellular miRNAs. In the present work, to study the functional orthologous relationships of viral and cellular miRNAs, we first constructed a dual-fluorescent protein reporter vector system for the easy determination of miRNA function. By expressing the miRNAs and the indicator and internal control fluorescent proteins individually from a single vector, this simple reporter system can be used for miRNA functional assays that include visualizing miRNA activity in live cells. Sequence alignments indicated that the simian virus 40 (SV40) encoded miRNA sv40-mir-S1-5p contains a seed region identical to that of the human miRNA hsa-miR423-5p. Using the new reporter system, it was found that sv40-mir-S1-5p and hsa-miR423-5p downregulate the expression of common artificial target mRNAs and some predicted biological targets of hsa-miR423-5p, demonstrating that they are functional orthologs. The human immunodeficiency virus 1 (HIV-1) encoded hiv1-miR-N367 also contains a seed sequence identical to that of the human miRNA hsa-miR192. Functional assays showed that hiv1-miR-N367 and hsa-miR192 could downregulate common artificial and predicted biological targets, suggesting that these miRNAs may also act as functional orthologs. Thus, this study presents a simple and universal system for testing miRNA function and identifies two new pairs of functional orthologs, sv40-mir-S1-5p and hsa-miR423-5p as well as hiv-1-miR-N367 and hsa-miR192. These findings also expand upon our current knowledge of functional homology and imply that a more general phenomenon of orthologous relationships exists between viral and cellular miRNAs.PLoS ONE 01/2012; 7(4):e36157. · 4.09 Impact Factor -
Article: Encapsulation of gold nanoparticles by simian virus 40 capsids.
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ABSTRACT: Viral capsid-nanoparticle hybrid structures constitute a new type of nanoarchitecture that can be used for various applications. We previously constructed a hybrid structure comprising quantum dots encapsulated by simian virus 40 (SV40) capsids for imaging viral infection pathways. Here, gold nanoparticles (AuNPs) are encapsulated into SV40 capsids and the effect of particle size and surface ligands (i.e. mPEG and DNA) on AuNP encapsulation is studied. Particle size and surface decoration play complex roles in AuNP encapsulation by SV40 capsids. AuNPs ≥15 nm (when coated with mPEG750 rather than mPEG2000), or ≥10 nm (when coated with 10T or 50T DNA) can be encapsulated. Encapsulation efficiency increased as the size of the AuNPs increased from 10 to 30 nm. In addition, the electrostatic interactions derived from negatively charged DNA ligands on the AuNP surfaces promote encapsulation when the AuNPs have a small diameter (i.e. 10 nm and 15 nm). Moreover, the SV40 capsid is able to carry mPEG750-modified 15-nm AuNPs into living Vero cells, whereas the mPEG750-modified 15-nm AuNPs alone cannot enter cells. These results will improve our understanding of the mechanisms underlying nanoparticle encapsulation in SV40 capsids and enable the construction of new functional hybrid nanostructures for cargo delivery.Nanoscale 08/2011; 3(10):4275-82. · 5.91 Impact Factor -
Article: Aptamer beacons for visualization of endogenous protein HIV-1 reverse transcriptase in living cells.
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ABSTRACT: Direct visualization of endogenous proteins in living cells remains a challenge. Aptamer beacon is a promising technique to resolve this problem by combining the excellent protein binding specificity of the aptamer with the sensitive signal transduction mechanism of the molecular beacon. In this study, aptamer 93 del against HIV-1 reverse transcriptase (RT) was engineered into aptamer beacons to recognize and image HIV-1 RT. The constructed aptamer beacons could specifically bind to HIV-1 RT and the beacon-RT binding showed effective fluorescence signal transduction in homogeneous solution. In solutions with 1 μM of the aptamer beacon, the effective fluorescence signal increased with increasing concentration of HIV-1 RT from 0.5 μM to 5 μM. When the aptamer beacons were delivered into the living cells that transiently expressed HIV-1 RT, HIV-1 RT could be specifically labeled and imaged. The designed aptamer beacons were further successfully applied for RT imaging in HIV-1 integrated U1 cells. The method developed here may be extended to visualize many other endogenous proteins in living cells using appropriate aptamer beacons.Biosensors & bioelectronics 07/2011; 28(1):270-6. · 5.43 Impact Factor -
Article: Effects of microtubule modulators on HIV-1 infection of transformed and resting CD4 T cells.
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ABSTRACT: Previous studies have observed fluorescently labeled HIV particles tracking along microtubule networks for nuclear localization. To provide direct evidence for the involvement of microtubules in early steps of HIV infection of human CD4 T cells, we used multiple microtubule modulators such as paclitaxel (originally called taxol; 1 μM), vinblastine (1 and 10 μM), colchicine (10 and 100 μM), and nocodazole (10 and 100 μM) to disturb microtubule networks in transformed and resting CD4 T cells. Although these drugs disrupted microtubule integrity, almost no inhibition of HIV-1 infection was observed. Our results do not appear to support an essential role for microtubules in the initiation of HIV infection of CD4 T cells.Journal of Virology 03/2011; 85(6):3020-4. · 5.40 Impact Factor -
Article: DNA probe functionalized QCM biosensor based on gold nanoparticle amplification for Bacillus anthracis detection.
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ABSTRACT: The rapid detection of Bacillus anthracis, the causative agent of anthrax disease, has gained much attention since the anthrax spore bioterrorism attacks in the United States in 2001. In this work, a DNA probe functionalized quartz crystal microbalance (QCM) biosensor was developed to detect B. anthracis based on the recognition of its specific DNA sequences, i.e., the 168 bp fragment of the Ba813 gene in chromosomes and the 340 bp fragment of the pag gene in plasmid pXO1. A thiol DNA probe was immobilized onto the QCM gold surface through self-assembly via Au-S bond formation to hybridize with the target ss-DNA sequence obtained by asymmetric PCR. Hybridization between the target DNA and the DNA probe resulted in an increase in mass and a decrease in the resonance frequency of the QCM biosensor. Moreover, to amplify the signal, a thiol-DNA fragment complementary to the other end of the target DNA was functionalized with gold nanoparticles. The results indicate that the DNA probe functionalized QCM biosensor could specifically recognize the target DNA fragment of B. anthracis from that of its closest species, such as Bacillus thuringiensis, and that the limit of detection (LOD) reached 3.5 × 10(2)CFU/ml of B. anthracis vegetative cells just after asymmetric PCR amplification, but without culture enrichment. The DNA probe functionalized QCM biosensor demonstrated stable, pollution-free, real-time sensing, and could find application in the rapid detection of B. anthracis.Biosensors & bioelectronics 01/2011; 26(8):3398-404. · 5.43 Impact Factor -
Article: Poliovirus 2A(pro) induces the nucleic translocation of poliovirus 3CD and 3C' proteins.
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ABSTRACT: Poliovirus genomic RNA replication, protein translation, and virion assembly are performed in the cytoplasm of host cells. However, this does not mean that there is no relationship between poliovirus infection and the cellular nucleus. In this study, recombinant fluorescence-tagged poliovirus 3CD and 3C' proteins were shown to be expressed mainly in the cytoplasm of Vero cells in the absence of other viral proteins. However, upon poliovirus infection, many of these proteins redistributed to the nucleus, as well as to the cytoplasm. A series of transfection experiments revealed that the poliovirus 2A(pro) was responsible for the same redistribution of 3CD and 3C' proteins to the nucleus. Furthermore, a mutant 2A(pro) protein lacking protease activity abrogated this effect. The poliovirus 2A(pro) protein was also found to co-localize with the Nup153 protein, a component of the nuclear pore complexes on the nuclear envelope. These data provide further evidence that there are intrinsic interactions between poliovirus proteins and the cell nucleus, despite that many processes in the poliovirus replication cycle occur in the cytoplasm.Acta Biochimica et Biophysica Sinica 01/2011; 43(1):38-44. · 1.38 Impact Factor -
Article: Colorimetric detection of melamine in complex matrices based on cysteamine-modified gold nanoparticles.
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ABSTRACT: A sensitive assay for melamine in complex matrices is built using cysteamine-modified gold nanoparticles (AuNPs) and an effective sample pretreatment protocol. Citrate-stabilized AuNPs were modified by cysteamine in order to weaken the electrostatic repulsion force between the gold nanoparticles. Detection sensitivity gained through this modification increased about 100 fold compared with the result using the unmodified AuNPs. Direct colorimetric visualizations of melamine in milk products, eggs and feeds was successfully demonstrated within the linear ranges of 1-200 mg L(-1) and detection limits below 1 mg L(-1). The proposed scheme could be an alternative means for onsite detection of melamine without costly instruments.The Analyst 09/2010; 136(1):179-83. · 4.23 Impact Factor -
Article: Gold nanoparticle enhanced immuno-PCR for ultrasensitive detection of Hantaan virus nucleocapsid protein.
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ABSTRACT: A functionalized gold nanoparticle (GNP) enhanced ultrasensitive immuno-PCR assay (GNP-IPCR on ELISA plate), which was modified from the recent developed bio-barcode assay (BCA) technique, was developed to detect Hantaan virus nucleocapsid protein (HNP). During the assay, the target antigen HNP was captured by a polyclonal antibody coated on ELISA microplate wells, followed by adding GNP dually modified with oligonucleotides and a HNP specific monoclonal antibody L13 (mAb L13) to form a sandwich immuno-complex. The oligonucleotides on the GNP contained two strands: one as capture DNA immobilized on the surface of the GNP through Au-S bond and the other as signal amplification DNA, which was partially complementary with the capture DNA. After the immuno-complex was formed, the signal DNA was released by heating, and consequently characterized by PCR/gel electrophoresis and SYBR-Green real time PCR. The detection limit of this method could reach down to 10 fg/mL for detecting purified HNP in buffers as well as in human serum, which was approximately 7 orders of magnitude more sensitive than that of conventional ELISA. The current assay format might be adopted for other proteins that need ultra-high sensitive detection.Journal of immunological methods 06/2009; 346(1-2):64-70. · 2.35 Impact Factor -
Article: Rapid detection of Bacillus anthracis using monoclonal antibody functionalized QCM sensor.
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ABSTRACT: Since the anthrax spore bioterrorism attacks in America in 2001, the early detection of Bacillus anthracis spores and vegetative cells has gained significant interest. At present, many polyclonal antibody-based quartz crystal microbalance (QCM) sensors have been developed to detect B. anthracis simulates. To achieve a simultaneous rapid detection of B. anthracis spores and vegetative cells, this paper presents a biosensor that utilizes an anti-B. anthracis monoclonal antibody designated to 8G3 (mAb 8G3, IgG) functionalized QCM sensor. Having compared four kinds of antibody immobilizations on Au surface, an optimized mAb 8G3 was immobilized onto the Au electrode with protein A on a mixed self-assembled monolayer (SAM) of 11-mercaptoundecanoic acid (11-MUA) and 6-mercaptohexan-1-ol (6-MHO) as adhesive layer. The detection of B. anthracis was investigated under three conditions: dip-and-dry, static addition and flow through procedure. The results indicated that the sensor yielded a distinct response to B. anthracis spores or vegetative cells but had no significant response to Bacillus thuringiensis species. The functionalized sensor recognized B. anthracis spores and vegetative cells specifically from its homophylic ones, and the limit of detection (LOD) reached 10(3)CFU or spores/ml of B. anthracis in less than 30 min. Cyclic voltammogram (CV) and scanning electronic microscopy (SEM) were performed to characterize the surface of the sensor in variable steps during the modification and after the detection. The mAb functionalized QCM biosensor will be helpful in the fabrication of a similar biosensor that may be available in anti-bioterrorism in the future.Biosensors & bioelectronics 09/2008; 24(5):1330-5. · 5.43 Impact Factor -
Article: Glycerol-salt Mediated Stacking of Nucleic Acids in CZE
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ABSTRACT: Nucleic acid samples with high concentrations of salt could be stacked and well separated during capillary zone electrophoresis (CZE) by adding glycerol into the samples and using a Tris-Borate-EDTA (TBE) buffer (pH 8.3) as the separation medium. The so-called glycerol-salt mediated stacking was found applicable to different types of nucleic acids. Three nucleic acids: a 16s rRNA (1,542nt), a double stranded DNA (1.6kbp), and a single stranded DNA (30nt), were tested as demos in the experiments. When the sample matrix contained 50mM KCl and 50% (w/v) glycerol, the 16s rRNA sample could be stacked as high as 30 times compared with the sample without KCl. All the nucleic acids could be stacked effectively when high concentrations of glycerol (>50%) and salt (more than 50mM) were present in the sample matrix, while the dsDNA could be stacked with high concentrations of glycerol (>50%) alone.Chromatographia 02/2008; 67(5):491-494. · 1.20 Impact Factor -
Article: Visualization of the dynamic multimerization of human Cytomegalovirus pp65 in punctuate nuclear foci
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ABSTRACT: The phosphorylated protein pp65 of human Cytomegalovirus (HCMV) is the predominant virion protein and the major tegument constituent. It plays important roles in HCMV infection and virion assembly. Live cell imaging and fluorescence recovery after photobleaching (FRAP) analysis showed that HCMV pp65 accumulated dynamically in punctuate nuclear foci when transiently expressed in mammalian cells. Fluorescence resonance energy transfer (FRET) imaging disclosed that pp65 can self-interact in its localization foci. Yeast two-hybrid assay verified that pp65 is a self-associating protein, and the N-terminal amino acids 14–22 were determined to be essential for pp65 self-association. However, these amino acids were not related to pp65 localization in the specific nuclear foci. The interaction of pp65 and ppUL97 was also studied by FRET microscopy, and the result suggested that there is another signal sequence in pp65, being the ppUL97 phosphorylation site, that is responsible for localization of pp65 in nuclear foci. These results help to understand the function of pp65 in HCMV infection and virion morphogenesis.Virology. -
Article: The tegument protein UL94 of human cytomegalovirus as a binding partner for tegument protein pp28 identified by intracellular imaging
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ABSTRACT: The tegument protein pp28 of human cytomegalovirus (HCMV) is essential for the assembly of infectious HCMV virions, but how it functions during the process of HCMV tegumentation and envelopment remains unclear. By using live cell fluorescence resonance energy transfer (FRET) microscopy and yeast two-hybrid assays, we found that another HCMV tegument protein, UL94, was a specific binding partner for pp28. The interaction between pp28 and UL94 was imaged in a punctuate, juxtanuclear compartment, previously designated as the virus assembly compartment (AC). Amino acids 22–43 of pp28 were identified as being responsible for its binding with UL94, while no linear binding site could be found within UL94. The interaction between pp28 and UL94 may serve as a link in the sequential processes of HCMV capsidation, tegumentation and envelopment. This study provides a foundation for further studies into how the HCMV tegument proteins act in the assembly of HCMV virions.Virology.
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2008–2011
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Wuhan Institute Of Virology
Wuhan, Hubei, China
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