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1.  Engineering a Cell-surface Aptamer Circuit for Targeted and Amplified Photodynamic Cancer Therapy 
ACS nano  2013;7(3):2312-2319.
Photodynamic therapy (PDT) is one of the most promising and noninvasive methods for clinical treatment of different malignant diseases. Here, we present a novel strategy of designing an aptamer-based DNA nanocircuit capable of the selective recognition of cancer cells, controllable activation of photosensitizer and amplification of photodynamic therapeutic effect. The aptamers can selectively recognize target cancer cells and bind to the specific proteins on cell membranes. Then the overhanging catalyst sequence on aptamer can trigger a toehold-mediated catalytic strand displacement to activate photosensitizer and achieve amplified therapeutic effect. The specific binding-induced activation allows the DNA circuit to distinguish diseased cells from healthy cells, reducing damage to nearby healthy cells. Moreover, the catalytic amplification reaction will only take place close to the target cancer cells, resulting in a high local concentration of singlet oxygen to selectively kill the target cells. The principle employed in this study demonstrated the feasibility of assembling a DNA circuit on cell membranes and could further broaden the utility of DNA circuits for applications in biology, biotechnology, and biomedicine.
doi:10.1021/nn305484p
PMCID: PMC3609929  PMID: 23397942
DNA nanocircuit; aptamer; photosensitizer; cancer therapy
2.  HMGB1-Promoted and TLR2/4-Dependent NK Cell Maturation and Activation Take Part in Rotavirus-Induced Murine Biliary Atresia 
PLoS Pathogens  2014;10(3):e1004011.
Recent studies show that NK cells play important roles in murine biliary atresia (BA), and a temporary immunological gap exists in this disease. In this study, we found high-mobility group box-1 (HMGB1) and TLRs were overexpressed in human and rotavirus-induced murine BA. The overexpressed HMGB1 released from the nuclei of rotavirus-infected cholangiocytes, as well as macrophages, activated hepatic NK cells via HMGB1-TLRs-MAPK signaling pathways. Immature NK cells had low cytotoxicity on rotavirus-injured cholangiocytes due to low expression of TLRs, which caused persistent rotavirus infection in bile ducts. HMGB1 up-regulated the levels of TLRs of NK cells and promoted NK cell activation in an age-dependent fashion. As NK cells gained increasing activation as mice aged, they gained increasing cytotoxicity on rotavirus-infected cholangiocytes, which finally caused BA. Adult NK cells eliminated rotavirus-infected cholangiocytes shortly after infection, which prevented persistent rotavirus infection in bile ducts. Moreover, adoptive transfer of mature NK cells prior to rotavirus infection decreased the incidence of BA in newborn mice. Thus, the dysfunction of newborn NK cells may, in part, participate in the immunological gap in the development of rotavirus induced murine BA.
Author Summary
Biliary atresia (BA) is the most common precipitating factor for liver transplantation in infants. BA is caused by the obstruction of hepatic bile ducts, leading to progressive obstructive jaundice and liver fibrosis. A well-recognized theory is that rotavirus injures biliary epithelia in a mouse model of BA, followed by attack of immunocytes, such as NK cells. We performed this research to investigate whether maturation and activation of NK cells take part in the development of BA. We identified that rotavirus induced HMGB1 release from injured bile ducts. HMGB1 induced NK cell activation in an age-dependent fashion via HMGB1-TLRs-MAPK signaling pathways. Newborn NK cells were unable to eliminate rotavirus-infected cholangiocytes, which caused persistent biliary infection; maturated NK cells were activated gradually and caused persistent biliary injury, which finally led to BA. We identify HMGB1 as an important pro-inflammatory initiator and a critical inducer for maturation of NK cells in the development of BA. HMGB1-induced activation of NK cells may, in part, plays crucial roles in the development of murine BA. Novel therapies targeting HMGB1 or TLRs in patients with BA may be applied in the future to decrease the activity of NK cells in order to inhibit the progression of BA.
doi:10.1371/journal.ppat.1004011
PMCID: PMC3961347  PMID: 24651485
3.  Increased Plasma DPP4 Activity Is an Independent Predictor of the Onset of Metabolic Syndrome in Chinese over 4 Years: Result from the China National Diabetes and Metabolic Disorders Study 
PLoS ONE  2014;9(3):e92222.
Aims
To determine whether fasting plasma Dipeptidyl Peptidase 4 (DPP4) activity and active Glucagon-Like Peptide-1 (GLP-1) were predictive of the onset of metabolic syndrome.
Methods
A prospective cohort study was conducted of 2042 adults (863 men and 1,179 women) aged 18-70 years without metabolic syndrome examined in 2007(baseline) and 2011(follow-up). Baseline plasma DPP4 activity was determined as the rate of cleavage of 7-amino-4- methylcoumarin (AMC) from the synthetic substrate H-glycyl-prolyl-AMC and active GLP-1 was determined by enzymoimmunoassay.
Results
During an average of 4 years of follow-up, 131 men (15.2%) and 174 women (14.8%) developed metabolic syndrome. In multiple linear regression analysis, baseline DPP4 activity was an independent predictor of an increase in insulin resistance over a 4-year period (P<0.01). In multivariable-adjusted models, the odds ratio (OR) for incident metabolic syndrome comparing the highest with the lowest quartiles of DPP4 activity and active GLP-1 were 2.82, 0.45 for men and 2.48, 0.36 for women respectively. Furthermore, plasma DPP4 activity significantly improved the area under the ROC curve for predicting new-onset metabolic syndrome based on information from metabolic syndrome components (Both P<0.01).
Conclusions
DPP4 activity is an important predictor of the onset of insulin resistance and metabolic syndrome in apparently healthy Chinese men and women. This finding may have important implications for understanding the aetiology of metabolic syndrome.
Trial Registration
#TR-CCH-Chi CTR-CCH-00000361
doi:10.1371/journal.pone.0092222
PMCID: PMC3960228  PMID: 24647445
4.  A novel mycobacterial Hsp70-containing fusion protein targeting mesothelin augments antitumor immunity and prolongs survival in murine models of ovarian cancer and mesothelioma 
Background
Although dendritic cell (DC) vaccines are considered to be promising treatments for advanced cancer, their production and administration is costly and labor-intensive. We developed a novel immunotherapeutic agent that links a single-chain antibody variable fragment (scFv) targeting mesothelin (MSLN), which is overexpressed on ovarian cancer and mesothelioma cells, to Mycobacterium tuberculosis (MTB) heat shock protein 70 (Hsp70), which is a potent immune activator that stimulates monocytes and DCs, enhances DC aggregation and maturation and improves cross-priming of T cells mediated by DCs.
Methods
Binding of this fusion protein with MSLN on the surface of tumor cells was measured by flow cytometry and fluorescence microscopy. The therapeutic efficacy of this fusion protein was evaluated in syngeneic and orthotopic mouse models of papillary ovarian cancer and malignant mesothelioma. Mice received 4 intraperitoneal (i.p.) treatments with experimental or control proteins post i.p. injection of tumor cells. Ascites-free and overall survival time was measured. For the investigation of anti-tumor T-cell responses, a time-matched study was performed. Splenocytes were stimulated with peptides, and IFNγ- or Granzyme B- generating CD3+CD8+ T cells were detected by flow cytometry. To examine the role of CD8+ T cells in the antitumor effect, we performed in vivo CD8+ cell depletion. We further determined if the fusion protein increases DC maturation and improves antigen presentation as well as cross-presentation by DCs.
Results
We demonstrated in vitro that the scFvMTBHsp70 fusion protein bound to the tumor cells used in this study through the interaction of scFv with MSLN on the surface of these cells, and induced maturation of bone marrow-derived DCs. Use of this bifunctional fusion protein in both mouse models significantly enhanced survival and slowed tumor growth while augmenting tumor-specific CD8+ T-cell dependent immune responses. We also demonstrated in vitro and in vivo that the fusion protein enhanced antigen presentation and cross-presentation by targeting tumor antigens towards DCs.
Conclusions
This new cancer immunotherapy has the potential to be cost-effective and broadly applicable to tumors that overexpress mesothelin.
doi:10.1186/1756-8722-7-15
PMCID: PMC3943805  PMID: 24565018
Mycobacterial Hsp70; Mesothelin; Single chain variable fragment; Cancer immunotherapy; Murine tumor model
5.  Full Genome Sequence of a Recombinant H1N2 Avian Influenza Virus Isolated from Wild Waterfowl in the East Dongting Lake Wetland 
Genome Announcements  2014;2(1):e00023-14.
Here, we report the full genome sequence of an H1N2 avian influenza virus (AIV) isolated from wild waterfowl in Dongting Lake. Phylogenetic analysis showed that it was a novel recombinant AIV between domestic ducks and wild waterfowl. Investigation of this virus is helpful for our understanding of the ecology of AIV in this region.
doi:10.1128/genomeA.00023-14
PMCID: PMC3931351  PMID: 24558230
6.  Few-Layer MoSe2 Possessing High Catalytic Activity towards Iodide/Tri-iodide Redox Shuttles 
Scientific Reports  2014;4:4063.
Due to the two-dimensional confinement of electrons, single- and few-layer MoSe2 nanostructures exhibit unusual optical and electrical properties and have found wide applications in catalytic hydrogen evolution reaction, field effect transistor, electrochemical intercalation, and so on. Here we present a new application in dye-sensitized solar cell as catalyst for the reduction of I3− to I− at the counter electrode. The few-layer MoSe2 is fabricated by surface selenization of Mo-coated soda-lime glass. Our results show that the few-layer MoSe2 displays high catalytic efficiency for the regeneration of I− species, which in turn yields a photovoltaic energy conversion efficiency of 9.00%, while the identical photoanode coupling with “champion” electrode based on Pt nanoparticles on FTO glass generates efficiency only 8.68%. Thus, a Pt- and FTO-free counter electrode outperforming the best conventional combination is obtained. In this electrode, Mo film is found to significantly decrease the sheet resistance of the counter electrode, contributing to the excellent device performance. Since all of the elements in the electrode are of high abundance ratios, this type of electrode is promising for the fabrication of large area devices at low materials cost.
doi:10.1038/srep04063
PMCID: PMC3924216  PMID: 24525919
7.  DNA Micelle Flares for Intracellular mRNA Imaging and Gene Therapy 
Angewandte Chemie (International ed. in English)  2013;52(7):10.1002/anie.201209440.
Multifunctional DNA micelles: Molecular beacon micelle flares (MBMFs), based on diacyllipid-molecular beacon conjugate (L-MB) self-assembly, have been developed for combined mRNA detection and gene therapy. The advantages of these micelle flares include easy probe synthesis, efficient cellular uptake, enhanced enzymatic stability, high signal-to-background ratio, excellent target selectivity, and superior biocompatibility. In addition, these probes possess a hydrophobic cavity that can be used for additional hydrophobic agents, holding great promise for constructing an all-in-one nucleic acid probe.
doi:10.1002/anie.201209440
PMCID: PMC3830797  PMID: 23319350
DNA micelle; self-assembly; mRNA imaging; gene therapy
8.  Construction of functional tissue-engineered bone using cell sheet technology in a canine model 
The aim of the present study was to construct functional tissue-engineered bone with cell sheet technology and compare the efficacy of this method with that of traditional bone tissue engineering techniques. Canine bone mesenchymal stem cells (BMSCs) were isolated using density gradient centrifugation and then cultured. The BMSCs were induced to differentiate into osteoblasts and cultured in temperature-responsive culture dishes. The BMSCs detached automatically from the temperature-responsive culture dishes when the temperature was reduced to 20°C, forming an intact cell sheet. Demineralized bone matrix (DBM) and platelet-rich plasma (PRP) were prepared and used to construct a DBM/PRP/BMSC cell sheet/BMSC complex, which was implanted under the left latissimus dorsi muscle in a dog model. A DBM/PRP/BMSC complex was used as a control and implanted under the right latissimus dorsi muscle in the dog model. Immunoblot assays were performed to detect the levels of growth factors. Osteogenesis was observed to be induced significantly more effectively in the DBM/PRP/BMSC cell sheet/BMSC implants than in the DBM/PRP/BMSC implants. Immunoblot assay results indicated that the levels of the growth factors platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) in the experimental group were 3.2- and 2.5-fold higher compared with those in the control group, respectively. These results indicated that the BMSC cell sheets were functional and more effective than the control cell complex. Therefore, cell sheet technology may be used for the effective construction of functional tissue-engineered bone with ideal properties.
doi:10.3892/etm.2014.1514
PMCID: PMC3964934  PMID: 24669258
mesenchymal stem cells; cell sheet technology; tissue-engineered bone
9.  Aptamer Conjugated Multifunctional Nanoflowers as a Platform for Targeting, Capture and Detection in Laser Desorption Ionization Mass Spectrometry 
ACS nano  2012;7(1):417-427.
Although many different nanomaterials have been tested as substrates for laser desorption and ionization mass spectrometry (LDI-MS), this emerging field still requires more efficient multifuncional nanomaterials for targeting, enrichment and detection. Here, we report the use of gold-manganese oxide (Au@MnO) hybrid nanoflowers as an efficient matrix for LDI–MS. The nanoflowers were also functionalized with two different aptamers to target cancer cells and capture adenosine triphosphate (ATP), respectively. These nanoflowers were successfully used for metabolite extraction from cancer cell lysates. Thus, in one system, our multifunctional nanoflowers can 1) act as an ionization substrate for mass spectrometry, 2) target cancer cells, and 3) detect and analyze metabolites from cancer cells.
doi:10.1021/nn304458m
PMCID: PMC3568519  PMID: 23211039
hybrid nanoparticles; nanoflower; aptamer; ATP; cancer cell; mass spectrometry
10.  Potential biomarkers for sensitivity of gallbladder cancer cells to gemcitabine 
Gemcitabine (Gem)-based chemotherapies are the main therapeutic regimens for patients with unresectable advanced or metastatic gallbladder cancer (GBC). However, the modest ORR and mild benefit on survival demonstrates the need for finding biomarkers for sensitivity to Gem and hence improving the therapy. In present work, two GBC cell lines with vast difference in sensitivity to Gem were subjected to DNA microarray analysis. Dramatic expression difference was found in protein kinase A signaling, P2Y purigenic receptor signaling, ErbB signaling and p70S6K signaling. Predicted low expression of KRAS and inactivation of AKT/ERK signaling in Gem-resistant GBC cells was validated by quantitative PCR and immunoblotting, respectively. However, p70S6K, p38MAPK and NF-κB signaling was probably activated in Gem-resistant GBC cells, which deserves further investigation in more GBC cell lines and tissues. Our work provides potential pathway signatures for Gem sensitivity of GBC patients.
PMCID: PMC3925896  PMID: 24551272
Gallbladder cancer; gemcitabine; DNA microarray analysis; KRAS; AKT signaling; ERK signaling; p38MAPK; p70S6K; NF-κB
11.  High-performance transparent and stretchable all-solid supercapacitors based on highly aligned carbon nanotube sheets 
Scientific Reports  2014;4:3612.
By using highly aligned carbon nanotube (CNT) sheets of excellent optical transmittance and mechanical stretchability as both the current collector and active electrode, high-performance transparent and stretchable all-solid supercapacitors with a good stability were developed. A transmittance up to 75% at the wavelength of 550 nm was achieved for a supercapacitor made from a cross-over assembly of two single-layer CNT sheets. The transparent supercapacitor has a specific capacitance of 7.3 F g−1 and can be biaxially stretched up to 30% strain without any obvious change in electrochemical performance even over hundreds stretching cycles.
doi:10.1038/srep03612
PMCID: PMC3885879  PMID: 24402400
12.  Keap1-Nrf2 Signaling: A Target for Cancer Prevention by Sulforaphane 
Topics in current chemistry  2013;329:163-177.
Sulforaphane is a promising agent under preclinical evaluation in many models of disease prevention. This bioactive phytochemical affects many molecular targets in cellular and animal models; however, amongst the most sensitive is Keap1, a key sensor for the adaptive stress response system regulated through the transcription factor Nrf2. Keap1 is a sulfhydryl-rich protein that represses Nrf2 signaling by facilitating the poly ubiquitination of Nrf2 thereby enabling its subsequent proteasomal degradation. Interaction of sulforaphane with Keap1 disrupts this function and allows for nuclear accumulation of Nrf2 and activation of its transcriptional program. Enhanced transcription of Nrf2 target genes provokes a strong cytoprotective response that enhances resistance to carcinogenesis and other diseases mediated by exposures to electrophiles and oxidants. Clinical evaluation of sulforaphane has been largely conducted by utilizing preparations of broccoli or broccoli sprouts rich in either sulforaphane or its precursor form in plants, a stable β-thioglucose conjugate termed glucoraphanin. We have conducted a series of clinical trials in Qidong, China, a region where exposures to food- and air-borne carcinogens has been considerable, to evaluate the suitability of broccoli sprout beverages, rich in either glucoraphanin (GRR) or sulforaphane SFR or both for their bioavailability, tolerability and pharmacodynamic action in population-based interventions. Results from these clinical trials indicate that interventions with well characterized preparations of broccoli sprouts may enhance the detoxication of aflatoxins and air-borne toxins, which may in turn attenuate their associated health risks, including cancer, in exposed individuals.
doi:10.1007/128_2012_339
PMCID: PMC3553557  PMID: 22752583
13.  Improving aqueous solubility and antitumor effects by nanosized gambogic acid-mPEG2000 micelles 
The clinical application of gambogic acid, a natural component with promising antitumor activity, is limited due to its extremely poor aqueous solubility, short half-life in blood, and severe systemic toxicity. To solve these problems, an amphiphilic polymer-drug conjugate was prepared by attachment of low molecular weight (ie, 2 kDa) methoxy poly(ethylene glycol) methyl ether (mPEG) to gambogic acid (GA-mPEG2000) through an ester linkage and characterized by 1H nuclear magnetic resonance. The GA-mPEG2000 conjugates self-assembled to form nanosized micelles, with mean diameters of less than 50 nm, and a very narrow particle size distribution. The properties of the GA-mPEG2000 micelles, including morphology, stability, molecular modeling, and drug release profile, were evaluated. MTT (3-(4,5-dimethylthiazo l-2-yl)-2,5 diphenyl tetrazolium bromide) tests demonstrated that the GA-mPEG2000 micelle formulation had obvious cytotoxicity to tumor cells and human umbilical vein endothelial cells. Further, GA-mPEG2000 micelles were effective in inhibiting tumor growth and prolonged survival in subcutaneous B16-F10 and C26 tumor models. Our findings suggest that GA-mPEG2000 micelles may have promising applications in tumor therapy.
doi:10.2147/IJN.S54050
PMCID: PMC3883552  PMID: 24403830
gambogic acid; poly(ethylene glycol)-drug conjugate; micelle; antitumor; toxicity
14.  An Effective Hybrid Self-Adapting Differential Evolution Algorithm for the Joint Replenishment and Location-Inventory Problem in a Three-Level Supply Chain 
The Scientific World Journal  2013;2013:270249.
The integration with different decisions in the supply chain is a trend, since it can avoid the suboptimal decisions. In this paper, we provide an effective intelligent algorithm for a modified joint replenishment and location-inventory problem (JR-LIP). The problem of the JR-LIP is to determine the reasonable number and location of distribution centers (DCs), the assignment policy of customers, and the replenishment policy of DCs such that the overall cost is minimized. However, due to the JR-LIP's difficult mathematical properties, simple and effective solutions for this NP-hard problem have eluded researchers. To find an effective approach for the JR-LIP, a hybrid self-adapting differential evolution algorithm (HSDE) is designed. To verify the effectiveness of the HSDE, two intelligent algorithms that have been proven to be effective algorithms for the similar problems named genetic algorithm (GA) and hybrid DE (HDE) are chosen to compare with it. Comparative results of benchmark functions and randomly generated JR-LIPs show that HSDE outperforms GA and HDE. Moreover, a sensitive analysis of cost parameters reveals the useful managerial insight. All comparative results show that HSDE is more stable and robust in handling this complex problem especially for the large-scale problem.
doi:10.1155/2013/270249
PMCID: PMC3878286  PMID: 24453822
15.  Near-Infrared Laser Adjuvant for Influenza Vaccine 
PLoS ONE  2013;8(12):e82899.
Safe and effective immunologic adjuvants are often essential for vaccines. However, the choice of adjuvant for licensed vaccines is limited, especially for those that are administered intradermally. We show that non-tissue damaging, near-infrared (NIR) laser light given in short exposures to small areas of skin, without the use of additional chemical or biological agents, significantly increases immune responses to intradermal influenza vaccination without augmenting IgE. The NIR laser-adjuvanted vaccine confers increased protection in a murine influenza lethal challenge model as compared to unadjuvanted vaccine. We show that NIR laser treatment induces the expression of specific chemokines in the skin resulting in recruitment and activation of dendritic cells and is safe to use in both mice and humans. The NIR laser adjuvant technology provides a novel, safe, low-cost, simple-to-use, potentially broadly applicable and clinically feasible approach to enhancing vaccine efficacy as an alternative to chemical and biological adjuvants.
doi:10.1371/journal.pone.0082899
PMCID: PMC3859633  PMID: 24349390
16.  Establishment of a Markerless Mutation Delivery System in Bacillus subtilis Stimulated by a Double-Strand Break in the Chromosome 
PLoS ONE  2013;8(11):e81370.
Bacillus subtilis has been a model for gram-positive bacteria and it has long been exploited for industrial and biotechnological applications. However, the availability of facile genetic tools for physiological analysis has generally lagged substantially behind traditional genetic models such as Escherichia coli and Saccharomyces cerevisiae. In this work, we have developed an efficient, precise and scarless method for rapid multiple genetic modifications without altering the chromosome of B. subtilis. This method employs upp gene as a counter-selectable marker, double-strand break (DSB) repair caused by exogenous endonuclease I-SceI and comK overexpression for fast preparation of competent cell. Foreign dsDNA can be simply and efficiently integrated into the chromosome by double-crossover homologous recombination. The DSB repair is a potent inducement for stimulating the second intramolecular homologous recombination, which not only enhances the frequency of resolution by one to two orders of magnitude, but also selects for the resolved product. This method has been successfully and reiteratively used in B. subtilis to deliver point mutations, to generate in-frame deletions, and to construct large-scale deletions. Experimental results proved that it allowed repeated use of the selectable marker gene for multiple modifications and could be a useful technique for B. subtilis.
doi:10.1371/journal.pone.0081370
PMCID: PMC3839881  PMID: 24282588
17.  Stages based molecular mechanisms for generating cholangiocytes from liver stem/progenitor cells 
Except for the most organized mature hepatocytes, liver stem/progenitor cells (LSPCs) can differentiate into many other types of cells in the liver including cholangiocytes. In addition, LSPCs are demonstrated to be able to give birth to other kinds of extra-hepatic cell types such as insulin-producing cells. Even more, under some bad conditions, these LSPCs could generate liver cancer stem like cells (LCSCs) through malignant transformation. In this review, we mainly concentrate on the molecular mechanisms for controlling cell fates of LSPCs, especially differentiation of cholangiocytes, insulin-producing cells and LCSCs. First of all, to certificate the cell fates of LSPCs, the following three features need to be taken into account to perform accurate phenotyping: (1) morphological properties; (2) specific markers; and (3) functional assessment including in vivo transplantation. Secondly, to promote LSPCs differentiation, systematical attention should be paid to inductive materials (such as growth factors and chemical stimulators), progressive materials including intracellular and extracellular signaling pathways, and implementary materials (such as liver enriched transcriptive factors). Accordingly, some recommendations were proposed to standardize, optimize, and enrich the effective production of cholangiocyte-like cells out of LSPCs. At the end, the potential regulating mechanisms for generation of cholangiocytes by LSPCs were carefully analyzed. The differentiation of LSPCs is a gradually progressing process, which consists of three main steps: initiation, progression and accomplishment. It’s the unbalanced distribution of affecting materials in each step decides the cell fates of LSPCs.
doi:10.3748/wjg.v19.i41.7032
PMCID: PMC3819537  PMID: 24222945
Liver stem/progenitor cells; Cholangiocytes; Biliary differentiation; Unbalanced distribution of materials; Cell therapy
18.  Use of National Pneumonia Surveillance to Describe Influenza A(H7N9) Virus Epidemiology, China, 2004–2013 
Emerging Infectious Diseases  2013;19(11):1784-1790.
In mainland China, most avian influenza A(H7N9) cases in the spring of 2013 were reported through the pneumonia of unknown etiology (PUE) surveillance system. To understand the role of possible underreporting and surveillance bias in assessing the epidemiology of subtype H7N9 cases and the effect of live-poultry market closures, we examined all PUE cases reported from 2004 through May 3, 2013. Historically, the PUE system was underused, reporting was inconsistent, and PUE reporting was biased toward A(H7N9)-affected provinces, with sparse data from unaffected provinces; however, we found no evidence that the older ages of persons with A(H7N9) resulted from surveillance bias. The absolute number and the proportion of PUE cases confirmed to be A(H7N9) declined after live-poultry market closures (p<0.001), indicating that market closures might have positively affected outbreak control. In China, PUE surveillance needs to be improved.
doi:10.3201/eid1911.130865
PMCID: PMC3837642  PMID: 24206646
pneumonia surveillance; influenza A(H7N9) virus; influenza virus; viruses; influenza; epidemiology; China
19.  One-Step Facile Surface Engineering of Hydrophobic Nanocrystals with Designer Molecular Recognition 
Journal of the American Chemical Society  2012;134(32):13164-13167.
High quality nanocrystals have demonstrated substantial potential for biomedical applications. However, being generally hydrophobic, their use has been greatly limited by complicated and inefficient surface engineering that often fails to yield biocompatible nanocrystals with minimal aggregation in biological fluids and active targeting toward specific biomolecules. Using chimeric DNA molecules, we developed a one-step facile surface engineering method for hydrophobic Nanocrystals. The procedure is simple and versatile, generating individual nanocrystals with multiple ligands. In addition, the resulting nanocrystals can actively and specifically target various molecular addresses, varying from nucleic acids to cancer cells. Together, the strategy developed here holds great promise in generating critical technologies needed for biomedical applications of nanocrystals.
doi:10.1021/ja304115q
PMCID: PMC3806045  PMID: 22793667
20.  DNA-capped mesoporous silica nanoparticles as an ion-responsive release system to determine the presence of mercury in aqueous solutions 
Analytical chemistry  2012;84(4):1956-1962.
We have developed DNA-functionalized silica nanoparticles for the rapid, sensitive, and selective detection of mercuric ion (Hg2+) in aqueous solution. Two DNA strands were designed to cap the pore of dye-trapped silica nanoparticles. In the presence of ppb level Hg2+, the two DNA strands are dehybridized to uncap the pore, releasing the dye cargo with detectable enhancements of fluorescence signal. This method enables rapid (less than 20min) and sensitive (LOD 4ppb) detection, and it was also able to discriminate Hg2+ from twelve other environmentally relevant metal ions. The superior properties of the as-designed DNA-functionalized silica nanoparticles can be attributed to the large loading capacity and highly ordered pore structure of mesoporous silica nanoparticles, as well as the selective binding of thymine-rich DNA with Hg2+ . Our design serves as a new prototype for metal-ion sensing systems and it also has promising potential for detection of various targets in stimulus-release systems.
doi:10.1021/ac202993p
PMCID: PMC3806059  PMID: 22240146
Mercury; DNA; Mesoporous Silica Nanoparticle; Detection
21.  A KH-Domain RNA-Binding Protein Interacts with FIERY2/CTD Phosphatase-Like 1 and Splicing Factors and Is Important for Pre-mRNA Splicing in Arabidopsis 
PLoS Genetics  2013;9(10):e1003875.
Eukaryotic genomes encode hundreds of RNA-binding proteins, yet the functions of most of these proteins are unknown. In a genetic study of stress signal transduction in Arabidopsis, we identified a K homology (KH)-domain RNA-binding protein, HOS5 (High Osmotic Stress Gene Expression 5), as required for stress gene regulation and stress tolerance. HOS5 was found to interact with FIERY2/RNA polymerase II (RNAP II) carboxyl terminal domain (CTD) phosphatase-like 1 (FRY2/CPL1) both in vitro and in vivo. This interaction is mediated by the first double-stranded RNA-binding domain of FRY2/CPL1 and the KH domains of HOS5. Interestingly, both HOS5 and FRY2/CPL1 also interact with two novel serine-arginine (SR)-rich splicing factors, RS40 and RS41, in nuclear speckles. Importantly, FRY2/CPL1 is required for the recruitment of HOS5. In fry2 mutants, HOS5 failed to be localized in nuclear speckles but was found mainly in the nucleoplasm. hos5 mutants were impaired in mRNA export and accumulated a significant amount of mRNA in the nuclei, particularly under salt stress conditions. Arabidopsis mutants of all these genes exhibit similar stress-sensitive phenotypes. RNA-seq analyses of these mutants detected significant intron retention in many stress-related genes under salt stress but not under normal conditions. Our study not only identified several novel regulators of pre-mRNA processing as important for plant stress response but also suggested that, in addition to RNAP II CTD that is a well-recognized platform for the recruitment of mRNA processing factors, FRY2/CPL1 may also recruit specific factors to regulate the co-transcriptional processing of certain transcripts to deal with environmental challenges.
Author Summary
Pre-mRNA processing, including 5′ capping, splicing, and 3′ polyadenylation, is critical for gene expression and is closely coupled with transcription. Phosphorylated carboxyl terminal domain (CTD) of RNA Polymerase II (RNAP II) serves as a platform for the recruitment of pre-mRNA processing factors, yet other components involved in the recruitment are less known. In a genetic study of stress signal transduction in Arabidopsis, we isolated a KH-domain RNA-binding protein HOS5 that plays important roles in stress gene regulation and stress tolerance. We found that HOS5 interacts with FIERY2/CTD phosphatase-like 1 (FRY2/CPL1) and they both also interact with two novel splicing factors, RS40 and RS41, in nuclear speckles. In fry2 mutants, HOS5 was unable to be recruited to nuclear speckles but rather was mainly localized in the nucleoplasm. Mutants in these genes have similar stress-sensitive phenotypes. Transcriptome analyses identified significant intron retention in many stress-related genes in these mutants under salt stress conditions. Our study reveals that, in addition to RNAP II, the CTD phosphatase may also recruit specific splicing factors and RNA binding proteins to regulate the co-transcriptional processing of certain transcripts to deal with environmental stresses.
doi:10.1371/journal.pgen.1003875
PMCID: PMC3798263  PMID: 24146632
22.  Semi quantification of ATP in Live-Cells using Nonspecific Desorption of DNA from Graphene Oxide as the Internal Reference 
Analytical chemistry  2012;84(20):8622-8627.
In this paper, we have developed an interesting imaging method for intracellular ATP molecules with semi-quantitation. While there has been a lot of work in understanding intracellular events, very few can come close to quantitation or semi-quantitation in living cells. In this work, we made an effective use of nanomaterials, graphene oxides, both as a quencher and a carrier for intracellular delivery. In addition, this graphene oxide also serves as the carrier for reference probes for fluorescent imaging. An ATP aptamer molecular beacon (AAMB) is adsorbed on graphene oxide (GO) to form a double quenching platform. The AAMB/GO spontaneously enters cells, and then AAMB is released and opened by intracellular ATP. The resulting fluorescence recovery is used to perform ATP live-cell imaging with greatly improved background and signaling. Moreover, a control ssDNA, which is released non-specifically from GO by non-target cellular proteins, can serve as an internal reference for ATP semi-quantification inside living cells using the intensity ratio of the AAMB and control. This approach can serve as a way for intracellular delivery and quantitative analysis.
doi:10.1021/ac301657f
PMCID: PMC3544202  PMID: 22978721
nonspecific desorption; graphene oxide; aptamer molecular beacon; internal reference; ATP; semi-quantification
23.  MicroRNA-155 Promotes Autophagy to Eliminate Intracellular Mycobacteria by Targeting Rheb 
PLoS Pathogens  2013;9(10):e1003697.
Mycobacterium tuberculosis is a hard-to-eradicate intracellular pathogen that infects one-third of the global population. It can live within macrophages owning to its ability to arrest phagolysosome biogenesis. Autophagy has recently been identified as an effective way to control the intracellular mycobacteria by enhancing phagosome maturation. In the present study, we demonstrate a novel role of miR-155 in regulating the autophagy-mediated anti-mycobacterial response. Both in vivo and in vitro studies showed that miR-155 expression was significantly enhanced after mycobacterial infection. Forced expression of miR-155 accelerated the autophagic response in macrophages, thus promoting the maturation of mycobacterial phagosomes and decreasing the survival rate of intracellular mycobacteria, while transfection with miR-155 inhibitor increased mycobacterial survival. However, macrophage-mediated mycobacterial phagocytosis was not affected after miR-155 overexpression or inhibition. Furthermore, blocking autophagy with specific inhibitor 3-methyladenine or silencing of autophagy related gene 7 (Atg7) reduced the ability of miR-155 to promote autophagy and mycobacterial elimination. More importantly, our study demonstrated that miR-155 bound to the 3′-untranslated region of Ras homologue enriched in brain (Rheb), a negative regulator of autophagy, accelerated the process of autophagy and sequential killing of intracellular mycobacteria by suppressing Rheb expression. Our results reveal a novel role of miR-155 in regulating autophagy-mediated mycobacterial elimination by targeting Rheb, and provide potential targets for clinical treatment.
Author Summary
microRNA-155 (miR-155) plays an essential role in regulating the host immune response by post-transcriptionally repressing the expression of target genes. However, little is known regarding its activity in modulating autophagy, an important host defense mechanism against intracellular bacterial infection. Mycobacterium tuberculosis is a hard-to-eradicate intracellular pathogen that infects approximately one-third of the global population, and causes 1.5 million deaths annually. The present study explores a novel role of miR-155 in the host response against mycobacterial infection. Our data demonstrates that mycobacterial infection triggers the expression of miR-155, and the induction of miR-155 in turn activates autophagy by targeting Rheb, a negative regulator of autophagy. miR-155-promoted autophagy accelerates the maturation of the mycobacterial phagosome, thus decreasing the survival of intracellular mycobacteria in macrophages. These findings contribute to a better understanding of the host defense mechanisms against mycobacterial infection, providing useful information for development of potential therapeutic interventions against tuberculosis.
doi:10.1371/journal.ppat.1003697
PMCID: PMC3795043  PMID: 24130493
24.  Molecular Beacon Aptamers for Direct and Universal Quantitation of Recombinant Proteins from Cell Lysates 
Analytical chemistry  2012;84(19):8272-8276.
Western blot, ELISA and fluorescent fusion proteins are currently the most common methods for detecting recombinant proteins. However, the former two are cumbersome and time-consuming, and the latter method may interfere with the trafficking and function of the fused recombinant proteins. We report here a rapid, inexpensive and simple approach to detect and quantify recombinant proteins using an anti-His-tag molecular beacon aptamer (HMBA). We demonstrated the technique by detection and quantitation of expressed recombinant proteins directly from E. coli cell lysate. The amount of expressed P78-His was determined to be 1.49 μg from the 20 μg cell lysate proteins. To the best of our knowledge, this is the first example directly measuring the concentration and expression yield of recombinant proteins from cell lysate, and the entire procedure required only 5 minutes.
doi:10.1021/ac301764q
PMCID: PMC3544214  PMID: 22916953
molecular beacon aptamer; His tag; cell lysates; quantification
25.  A Study on the Mechanism of Cinobufagin in the Treatment of Paw Cancer Pain by Modulating Local β-Endorphin Expression In Vivo 
Background. Cinobufagin has been widely used in the treatment of carcinoma and plays an important role in the relief of cancer pain. But the involved mechanism remains unknown. Aim. To investigate the changes in thermal and mechanical hyperalgesia in paw cancer pain in mice and the action mechanism of cinobufagin using a paw cancer pain model. Methods. 60 female mice were randomly divided into 5 groups: control group, model group, cinobufagin group, cinobufagin +NAL-M group, and morphine group; except ones in control group, mice were inoculated with H22 hepatoma cells in the right hind paw. From the 9th day after inoculation, mice were administrated drug once daily lasting for 8 days. The pain behavior was determined on the 2nd, 4th, 6th, and 8th days before and after administration. On the last day, they were sacrificed. The levels of β-END, CRF, and IL-1β were analyzed by ELISA; immunohistochemistry was performed to detect the expressions of β-END, POMC, and μ-OR in the tumor and adjacent tissue. Results. The thresholds of thermal pain and mechanical pain were significantly increased by cinobufagin. Moreover, the expressions of β-END, CRF, POMC, and μ-OR were significantly upregulated by cinobufagin. The analgesic effect of cinobufagin was blocked by the peripheral opioid receptor antagonist NAL-M. Conclusions. Cinobufagin significantly relieved cancer pain in mice and raised their pain threshold, mainly upregulating the expression levels of β-END and μ-OR in the hind paw tumor and adjacent tissue.
doi:10.1155/2013/851256
PMCID: PMC3800629  PMID: 24187573

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