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author:("Li, tingling")
1.  Oxidative Stress Activates SIRT2 to Deacetylate and Stimulate Phosphoglycerate Mutase 
Cancer research  2014;74(13):3630-3642.
Glycolytic enzyme phosphoglycerate mutase (PGAM) plays an important role in coordinating energy production with generation of reducing power and the biosynthesis of nucleotide precursors and amino acids. Inhibition of PGAM by small RNAi or small molecule attenuates cell proliferation and tumor growth. PGAM activity is commonly upregulated in tumor cells, but how PGAM activity is regulated in vivo remains poorly understood. Here we report that PGAM is acetylated at lysine 100 (K100), an active site residue that is invariably conserved from bacteria, to yeast, plant, and mammals. K100 acetylation is detected in fly, mouse, and human cells and in multiple tissues and decreases PGAM2 activity. The cytosolic protein deacetylase sirtuin 2 (SIRT2) deacetylates and activates PGAM2. Increased levels of reactive oxygen species stimulate PGAM2 deacetylation and activity by promoting its interaction with SIRT2. Substitution of endogenous PGAM2 with an acetylation mimetic mutant K100Q reduces cellular NADPH production and inhibits cell proliferation and tumor growth. These results reveal a mechanism of PGAM2 regulation and NADPH homeostasis in response to oxidative stress that impacts cell proliferation and tumor growth.
doi:10.1158/0008-5472.CAN-13-3615
PMCID: PMC4303242  PMID: 24786789
2.  Multifunctional Core/Shell Nanoparticles Cross-linked Polyetherimide-folic Acid as Efficient Notch-1 siRNA Carrier for Targeted Killing of Breast Cancer 
Scientific Reports  2014;4:7072.
In gene therapy, how genetic therapeutics can be efficiently and safely delivered into target tissues/cells remains a major obstacle to overcome. To address this issue, nanoparticles consisting of non-covalently coupled polyethyleneimine (PEI) and folic acid (FA) to the magnetic and fluorescent core/shell of Fe3O4@SiO2(FITC) was tested for their ability to deliver Notch-1 shRNA. Our results showed that Fe3O4@SiO2(FITC)/PEI-FA/Notch-1 shRNA nanoparticles are 64 nm in diameter with well dispersed and superparamagnetic. These nanoparticles with on significant cytotoxicity are capable of delivering Notch-1 shRNA into human breast cancer MDA-MB-231 cells with high efficiency while effectively protected shRNA from degradation by exogenous DNaseI and nucleases. Magnetic resonance (MR) imaging and fluorescence microscopy showed significant preferential uptake of Fe3O4@SiO2(FITC)/PEI-FA/Notch-1 shRNA nanocomplex by MDA-MB-231 cells. Transfected MDA-MB-231 cells exhibited significantly decreased expression of Notch-1, inhibited cell proliferation, and increased cell apoptosis, leading to the killing of MDA-MB-231 cells. In light of the magnetic targeting capabilities of Fe3O4@SiO2(FITC)/PEI-FA, our results show that by complexing with a second molecular targeting therapeutic, such as Notch-1 shRNA in this report, Fe3O4@SiO2(FITC)/PEI-FA can be exploited as a novel, non-viral, and concurrent targeting delivery system for targeted gene therapy as well as for MR imaging in cancer diagnosis.
doi:10.1038/srep07072
PMCID: PMC4233336  PMID: 25400232
3.  Agonist-bound structure of the human P2Y12 receptor 
Nature  2014;509(7498):119-122.
The P2Y12 receptor (P2Y12R), one of eight members of the P2YR family expressed in humans, has been identified as one of the most prominent clinical drug targets for inhibition of platelet aggregation. Consequently, extensive mutagenesis and modeling studies of the P2Y12R have revealed many aspects of agonist/antagonist binding1-4. However, the details of agonist and antagonist recognition and function at the P2Y12R remain poorly understood at the molecular level. Here, we report the structures of the human P2Y12R in complex with a full agonist 2-methylthio-adenosine-5′-diphosphate (2MeSADP, a close analogue of endogenous agonist ADP) at 2.5 Å resolution, and the corresponding ATP derivative 2-methylthio-adenosine-5′-triphosphate (2MeSATP) at 3.1 Å resolution. Analysis of these structures, together with the structure of the P2Y12R with antagonist ethyl 6-(4-((benzylsulfonyl)carbamoyl)piperidin-1-yl)-5-cyano-2-methylnicotinate (AZD1283)5, reveals dramatic conformational changes between nucleotide and non-nucleotide ligand complexes in the extracellular regions, providing the first insight into a different ligand binding landscape in the δ-group of class A G protein-coupled receptors (GPCRs). Agonist and non-nucleotide antagonist adopt different orientations in the P2Y12R, with only partially overlapped binding pockets. The agonist-bound P2Y12R structure answers long-standing ambiguities surrounding P2Y12R-agonist recognition, and reveals interactions with several residues that had not been reported to be involved in agonist binding. As a first example of a GPCR where agonist access to the binding pocket requires large scale rearrangements in the highly malleable extracellular region, the structural studies therefore will provide invaluable insight into the pharmacology and mechanisms of action of agonists and different classes of antagonists for the P2Y12R and potentially for other closely related P2YRs.
doi:10.1038/nature13288
PMCID: PMC4128917  PMID: 24784220
4.  Salicylic Acid Alleviates the Adverse Effects of Salt Stress in Torreya grandis cv. Merrillii Seedlings by Activating Photosynthesis and Enhancing Antioxidant Systems 
PLoS ONE  2014;9(10):e109492.
Background
Salt stress is a major factor limiting plant growth and productivity. Salicylic acid (SA) has been shown to ameliorate the adverse effects of environmental stress on plants. To investigate the protective role of SA in ameliorating salt stress on Torreya grandis (T. grandis) trees, a pot experiment was conducted to analyze the biomass, relative water content (RWC), chlorophyll content, net photosynthesis (Pn), gas exchange parameters, relative leakage conductivity (REC), malondialdehyde (MDA) content, and activities of superoxide dismutase (SOD) and peroxidase (POD) of T. grandis under 0.2% and 0.4% NaCl conditions with and without SA.
Methodology/Principal Findings
The exposure of T. grandis seedlings to salt conditions resulted in reduced growth rates, which were associated with decreases in RWC and Pn and increases in REC and MDA content. The foliar application of SA effectively increased the chlorophyll (chl (a+b)) content, RWC, net CO2 assimilation rates (Pn), and proline content, enhanced the activities of SOD, CAT and POD, and minimized the increases in the REC and MDA content. These changes increased the capacity of T. grandis in acclimating to salt stress and thus increased the shoot and root dry matter. However, when the plants were under 0% and 0.2% NaCl stress, the dry mass of the shoots and roots did not differ significantly between SA-treated plants and control plants.
Conclusions
SA induced the salt tolerance and increased the biomass of T. grandis cv. by enhancing the chlorophyll content and activity of antioxidative enzymes, activating the photosynthetic process, and alleviating membrane injury. A better understanding about the effect of salt stress in T. grandis is vital, in order gain knowledge over expanding the plantations to various regions and also for the recovery of T. grandis species in the future.
doi:10.1371/journal.pone.0109492
PMCID: PMC4193794  PMID: 25302987
5.  Induction of D-xylose uptake and expression of NAD(P)H-linked xylose reductase and NADP + -linked xylitol dehydrogenase in the oleaginous microalga Chlorella sorokiniana 
Biotechnology for Biofuels  2014;7(1):125.
Background
The heterotrophic and mixotrophic culture of oleaginous microalgae is a promising process to produce biofuel feedstock due to the advantage of fast growth. Various organic carbons have been explored for this application. However, despite being one of the most abundant and economical sugar resources in nature, D-xylose has never been demonstrated as a carbon source for wild-type microalgae. The purpose of the present work was to identify the feasibility of D-xylose utilization by the oleaginous microalga Chlorella sorokiniana.
Results
The sugar uptake kinetic analysis was performed with 14C-labeled sugars and the data showed that the D-glucose induced algal cells (the alga was heterotrophically grown on D-glucose and then harvested as D-glucose induced cells) exhibited a remarkably increased D-xylose uptake rate. The maximum D-xylose transport rate was 3.8 nmol min−1 mg−1 dry cell weight (DCW) with Km value of 6.8 mM. D-xylose uptake was suppressed in the presence of D-glucose, D-galactose and D-fructose but not L-arabinose and D-ribose. The uptake of D-xylose activated the related metabolic pathway, and the activities of a NAD(P)H-linked xylose reductase (XR) and a unique NADP+-linked xylitol dehydrogenase (XDH) were detected in C. sorokiniana. Compared with the culture in the dark, the consumption of D-xylose increased 2 fold under light but decreased to the same level with addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), indicating that extra chemical energy from the light-dependent reaction contributed the catabolism of D-xylose for C. sorokiniana.
Conclusions
An inducible D-xylose transportation system and a related metabolic pathway were discovered for microalga for the first time. The transportation of D-xylose across the cell membrane of C. sorokiniana could be realized by an inducible hexose symporter. The uptake of D-xylose subsequently activated the expression of key catalytic enzymes that enabled D-xylose entering central metabolism. Results of this research are useful to better understand the D-xylose metabolic pathway in the microalga C. sorokiniana and provide a target for genetic engineering to improve D-xylose utilization for microalgal lipid production.
doi:10.1186/s13068-014-0125-7
PMCID: PMC4195881  PMID: 25342968
Chlorella sorokiniana; Microalgae; D-xylose; Xylose reductase; Xylitol dehydrogenase
6.  Acetylation Stabilizes ATP-Citrate Lyase to Promote Lipid Biosynthesis and Tumor Growth 
Molecular cell  2013;51(4):506-518.
SUMMARY
Increased fatty acid synthesis is required to meet the demand for membrane expansion of rapidly growing cells. ATP-citrate lyase (ACLY) is upregulated or activated in several types of cancer, and inhibition of ACLY arrests proliferation of cancer cells. Here we show that ACLY is acetylated at lysine residues 540, 546, and 554 (3K). Acetylation at these three lysine residues is stimulated by P300/calcium-binding protein (CBP)-associated factor (PCAF) acetyltransferase under high glucose and increases ACLY stability by blocking its ubiquitylation and degradation. Conversely, the protein deacetylase sirtuin 2 (SIRT2) deacetylates and destabilizes ACLY. Substitution of 3K abolishes ACLY ubiquitylation and promotes de novo lipid synthesis, cell proliferation, and tumor growth. Importantly, 3K acetylation of ACLY is increased in human lung cancers. Our study reveals a crosstalk between acetylation and ubiquitylation by competing for the same lysine residues in the regulation of fatty acid synthesis and cell growth in response to glucose.
doi:10.1016/j.molcel.2013.07.002
PMCID: PMC4180208  PMID: 23932781
7.  Structure of the CCR5 chemokine receptor – HIV entry inhibitor Maraviroc complex 
Science (New York, N.Y.)  2013;341(6152):10.1126/science.1241475.
The CCR5 chemokine receptor acts as a co-receptor for HIV-1 viral entry. Here we report the 2.7 Å resolution crystal structure of human CCR5 bound to the marketed HIV drug Maraviroc. The structure reveals a ligand binding site that is distinct from the proposed major recognition sites for chemokines and the viral glycoprotein gp120, providing insights into the mechanism of allosteric inhibition of chemokine signaling and viral entry. A comparison between CCR5 and CXCR4 crystal structures, along with models of co-receptor/gp120-V3 complexes, suggests that different charge distributions and steric hindrances caused by residue substitutions may be major determinants of HIV-1 co-receptor selectivity. These high-resolution insights into CCR5 can enable structure-based drug discovery for the treatment of HIV-1 infection.
doi:10.1126/science.1241475
PMCID: PMC3819204  PMID: 24030490
8.  Methylation Patterns of the IFN-γ Gene in Cervical Cancer Tissues 
Scientific Reports  2014;4:6331.
Objective: To explore the relationship between methylation of interferon gamma (IFN-γ) gene and tumorigenesis in cervical cancer tissues, the biopsy specimens of cervical cancer and cervical intraepithelial neoplasia (CIN) (I-III) patients as well as normal controls were collected and analyzed. Methods: The methylation of the IFN-γ gene was verified by using methylation-specific PCR and DNA sequencing analysis, and the expression levels of IFN-γ mRNA were detected using quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR). Results: The methylation rates of the IFN-γ gene were significantly higher in cervical cancer tissues (15/43, 34.9%) than those in CIN (3/23, 13.0% of CIN I; 6/39, 15.4% of CIN II/III) and normal cervical tissues (2/43, 4.7%) (P < 0.01). Furthermore, the mRNA expression of IFN-γ in cervical tumors with methylation (0.71 ± 0.13, n = 8) was lower than that in those without methylation (1.58 ± 0.32, n = 27) (P < 0.05). Likewise, the IFN-γ expression levels in CIN II/III tissues with methylation (0.87 ± 0.16, n = 5) were significantly (P < 0.01) lower compared to those without methylation (2.12 ± 0.27, n = 32). Conclusion: The hypermethylation of IFN-γ gene may be related with tumorigenesis of cervical cancer.
doi:10.1038/srep06331
PMCID: PMC4160705  PMID: 25208560
9.  Characterisation of a Plancitoxin-1-Like DNase II Gene in Trichinella spiralis 
Background
Deoxyribonuclease II (DNase II) is a well-known acidic endonuclease that catalyses the degradation of DNA into oligonucleotides. Only one or a few genes encoding DNase II have been observed in the genomes of many species. 125 DNase II-like protein family genes were predicted in the Trichinella spiralis (T. spiralis) genome; however, none have been confirmed. DNase II is a monomeric nuclease that contains two copies of a variant HKD motif in the N- and C-termini. Of these 125 genes, only plancitoxin-1 (1095 bp, GenBank accession no. XM_003370715.1) contains the HKD motif in its C-terminus domain.
Methodology/Principal Findings
In this study, we cloned and characterised the plancitoxin-1 gene. However, the sequences of plancitoxin-1 cloned from T. spiralis were shorter than the predicted sequences in GenBank. Intriguingly, there were two HKD motifs in the N- and C-termini in the cloned sequences. Therefore, the gene with shorter sequences was named after plancitoxin-1-like (Ts-Pt, 885 bp) and has been deposited in GenBank under accession number KF984291. The recombinant protein (rTs-Pt) was expressed in a prokaryotic expression system and purified by nickel affinity chromatography. Western blot analysis showed that rTs-Pt was recognised by serum from T. spiralis-infected mice; the anti-rTs-Pt serum recognised crude antigens but not ES antigens. The Ts-Pt gene was examined at all T. spiralis developmental stages by real-time quantitative PCR. Immunolocalisation analysis showed that Ts-Pt was distributed throughout newborn larvae (NBL), the tegument of adults (Ad) and muscle larvae (ML). As demonstrated by DNase zymography, the expressed proteins displayed cation-independent DNase activity. rTs-Pt had a narrow optimum pH range in slightly acidic conditions (pH 4 and pH 5), and its optimum temperature was 25°C, 30°C, and 37°C.
Conclusions
This study indicated that Ts-Pt was classified as a somatic protein in different T. spiralis developmental stages, and demonstrated for the first time that an expressed DNase II protein from T. spiralis had nuclease activity.
Author Summary
Deoxyribonuclease II (DNase II) is classified into a unique family of nucleases and mediates the degradation of DNA associated with apoptosis. Although DNase II activity was first observed in 1947, and has been studied biochemically and enzymatically since the 1960s, only recently has genetic information on the enzyme been reported. Compared with enzymes from other species, including C. elegans, the DNase II-like protein family of the parasitic nematode T. spiralis has expanded remarkably, with an estimated 125 genes found in the draft genome of T. spiralis. However, none of these proteins have been confirmed by biochemical studies. This study describes Ts-Pt, a DNase II protein that is expressed in different T. spiralis developmental stages. The recombinant protein purified via a prokaryotic expression system displayed in vitro nuclease activity, as determined by DNase zymography. The exact function and mechanisms of Ts-Pt should be further explored in vivo.
doi:10.1371/journal.pntd.0003097
PMCID: PMC4148230  PMID: 25165857
10.  Notch-1 Signaling Promotes the Malignant Features of Human Breast Cancer through NF-κB Activation 
PLoS ONE  2014;9(4):e95912.
The aberrant activation of Notch-1 signaling pathway has been proven to be associated with the development and progression of cancers. However, the specific roles and the underlying mechanisms of Notch-1 signaling pathway on the malignant behaviors of breast cancer are poorly understood. In this study, using multiple cellular and molecular approaches, we demonstrated that activation of Notch-1 signaling pathway promoted the malignant behaviors of MDA-MB-231 cells such as increased cell proliferation, colony formation, adhesion, migration, and invasion, and inhibited apoptosis; whereas deactivation of this signaling pathway led to the reversal of the aforementioned malignant cellular behaviors. Furthermore, we found that activation of Notch-1 signaling pathway triggered the activation of NF-κB signaling pathway and up-regulated the expression of NF-κB target genes including MMP-2/-9, VEGF, Survivin, Bcl-xL, and Cyclin D1. These results suggest that Notch-1 signaling pathway play important roles in promoting the malignant phenotype of breast cancer, which may be mediated partly through the activation of NF-κB signaling pathway. Our results further suggest that targeting Notch-1 signaling pathway may become a newer approach to halt the progression of breast cancer.
doi:10.1371/journal.pone.0095912
PMCID: PMC3997497  PMID: 24760075
11.  Significance of elevated ERK expression and its positive correlation with EGFR in Kazakh patients with esophageal squamous cell carcinoma 
Extracellular signal-regulated kinases (ERKs) are activated by the MAPK pathway. ERKs are downstream effectors of the epidermal growth factor receptor (EGFR), which belongs to the receptor tyrosine kinases family. Studies on the activation of the EGFR-ERK pathway in Kazakh patients with esophageal squamous cell carcinoma (ESCC) have not been reported. Using immunohistochemical staining on tissue microarrays, we investigated the protein expression of EGFR and ERK in 90 ethnic Kazakh patients with ESCC and 48 adjacent normal esophageal tissues (NETs). EGFR and ERK1 expression was localized in the cytoplasm, whereas ERK2 expression was localized in the nucleus. Both were more highly expression in the ESCC tissues than in the NETs, and the difference was considered significant (P = 0.003, 0.002, and 0.005, respectively). ERK1 and EGFR expression was positively correlated with lymph nodes metastasis (P = 0.011 and 0.013, respectively). ERK1 staining was also significantly associated with tumor-node-metastases stage of ESCC (P = 0.044). ERK2 staining was significantly associated with Histological grade (P = 0.012). Furthermore, ERK1 and EGFR expression in the ESCC tissues were positively correlated (r = 0.413, P < 0.001); EGFR was more highly expressed in the ESCC tissues with high ERK1 expression than in the ESCC tissues with low ERK1 expression (4.95 ± 0.57 vs. 3.21 ± 0.35, P = 0.01). This study is thus far the first to demonstrate the correlation between EGFR overexpression and ERK overexpression in Kazakh patients with ESCC. This correlation suggests that the EGFR-ERK signaling pathway participates in ESCC progression and can thus be used as a prognostic marker.
PMCID: PMC4069965  PMID: 24966948
Esophageal squamous cell carcinoma; Kazakh; epidermal growth factor receptor; extracellular signal-regulated kinase
12.  Impacts of Agricultural Management and Climate Change on Future Soil Organic Carbon Dynamics in North China Plain 
PLoS ONE  2014;9(4):e94827.
Dynamics of cropland soil organic carbon (SOC) in response to different management practices and environmental conditions across North China Plain (NCP) were studied using a modeling approach. We identified the key variables driving SOC changes at a high spatial resolution (10 km×10 km) and long time scale (90 years). The model used future climatic data from the FGOALS model based on four future greenhouse gas (GHG) concentration scenarios. Agricultural practices included different rates of nitrogen (N) fertilization, manure application, and stubble retention. We found that SOC change was significantly influenced by the management practices of stubble retention (linearly positive), manure application (linearly positive) and nitrogen fertilization (nonlinearly positive) – and the edaphic variable of initial SOC content (linearly negative). Temperature had weakly positive effects, while precipitation had negligible impacts on SOC dynamics under current irrigation management. The effects of increased N fertilization on SOC changes were most significant between the rates of 0 and 300 kg ha−1 yr−1. With a moderate rate of manure application (i.e., 2000 kg ha−1 yr−1), stubble retention (i.e., 50%), and an optimal rate of nitrogen fertilization (i.e., 300 kg ha−1 yr−1), more than 60% of the study area showed an increase in SOC, and the average SOC density across NCP was relatively steady during the study period. If the rates of manure application and stubble retention doubled (i.e., manure application rate of 4000 kg ha−1 yr−1 and stubble retention rate of 100%), soils across more than 90% of the study area would act as a net C sink, and the average SOC density kept increasing from 40 Mg ha−1 during 2010s to the current worldwide average of ∼55 Mg ha−1 during 2060s. The results can help target agricultural management practices for effectively mitigating climate change through soil C sequestration.
doi:10.1371/journal.pone.0094827
PMCID: PMC3983264  PMID: 24722689
13.  Telomere Length in Peripheral Blood Leukocytes Is Associated with Risk of Colorectal Cancer in Chinese Population 
PLoS ONE  2014;9(2):e88135.
Background
Human telomeres, tandem repeats of TTAGGG nucleotides at the ends of chromosomes, are essential for maintaining genomic integrity and stability. Results of previous epidemiologic studies about the association of telomere length with risk of colorectal cancer (CRC) have been conflicting.
Methods
A case-control study was conducted in a Han population in Wuhan, central China. The relative telomere length (RTL) was measured in peripheral blood leukocytes (PBLs) using quantitative real-time polymerase chain reaction (PCR) in 628 CRC cases and 1,256 age and sex frequency matched cancer-free controls. Odds ratios (OR) and 95% confidence intervals (95% CI) were calculated using unconditional logistic regression models to evaluate the association between RTL and CRC risk.
Results
Using median RTL in the controls as the cutoff, individuals with shorter RTL were associated with a significantly increased risk of CRC (adjusted OR = 1.27, 95%CI: 1.05–1.55). When participants were further categorized into 3 and 4 groups according to the tertile and quartile RTL values of controls, significant relationships were still observed between shorter RTL and increased CRC risk (OR per tertile = 1.13, 95%CI: 1.00–1.28, Ptrend = 0.045; OR per quartile = 1.12, 95%CI: 1.03–1.23, Ptrend = 0.012). In stratified analyses, significant association between shorter RTL and increased CRC risk was found in females, individuals younger than 60 years old, never smokers and never drinkers.
Conclusions
This study suggested that short telomere length in PBLs was significantly associated with an increased risk of CRC in Chinese Han population. Further validation in large prospective studies and investigation of the biologic mechanisms are warranted.
doi:10.1371/journal.pone.0088135
PMCID: PMC3912164  PMID: 24498432
14.  A Synthetic dl-Nordihydroguaiaretic acid (Nordy), Inhibits Angiogenesis, Invasion and Proliferation of Glioma Stem Cells within a Zebrafish Xenotransplantation Model 
PLoS ONE  2014;9(1):e85759.
The zebrafish (Danio rerio) and their transparent embryos represent a promising model system in cancer research. Compared with other vertebrate model systems, we had previously shown that the zebrafish model provides many advantages over mouse or chicken models to study tumor invasion, angiogenesis, and tumorigenesis. In this study, we systematically investigated the biological features of glioma stem cells (GSCs) in a zebrafish model, such as tumor angiogenesis, invasion, and proliferation. We demonstrated that several verified anti-angiogenic agents inhibited angiogenesis that was induced by xenografted-GSCs. We next evaluated the effects of a synthetic dl-nordihydroguaiaretic acid compound (dl-NDGA or “Nordy”), which revealed anti-tumor activity against human GSCs in vitro by establishing parameters through studying its ability to suppress angiogenesis, tumor invasion, and proliferation. Furthermore, our results indicated that Nordy might inhibit GSCs invasion and proliferation through regulation of the arachidonate 5-lipoxygenase (Alox-5) pathway. Moreover, the combination of Nordy and a VEGF inhibitor exhibited an enhanced ability to suppress angiogenesis that was induced by GSCs. By contrast, even following treatment with 50 µM Nordy, there was no discernible effect on zebrafish embryonic development. Together, these results suggested efficacy and safety of using Nordy in vivo, and further demonstrated that this model should be suitable for studying GSCs and anti-GSC drug evaluation.
doi:10.1371/journal.pone.0085759
PMCID: PMC3893259  PMID: 24454929
15.  A Rapid, Highly Efficient and Economical Method of Agrobacterium-Mediated In planta Transient Transformation in Living Onion Epidermis 
PLoS ONE  2014;9(1):e83556.
Transient transformation is simpler, more efficient and economical in analyzing protein subcellular localization than stable transformation. Fluorescent fusion proteins were often used in transient transformation to follow the in vivo behavior of proteins. Onion epidermis, which has large, living and transparent cells in a monolayer, is suitable to visualize fluorescent fusion proteins. The often used transient transformation methods included particle bombardment, protoplast transfection and Agrobacterium-mediated transformation. Particle bombardment in onion epidermis was successfully established, however, it was expensive, biolistic equipment dependent and with low transformation efficiency. We developed a highly efficient in planta transient transformation method in onion epidermis by using a special agroinfiltration method, which could be fulfilled within 5 days from the pretreatment of onion bulb to the best time-point for analyzing gene expression. The transformation conditions were optimized to achieve 43.87% transformation efficiency in living onion epidermis. The developed method has advantages in cost, time-consuming, equipment dependency and transformation efficiency in contrast with those methods of particle bombardment in onion epidermal cells, protoplast transfection and Agrobacterium-mediated transient transformation in leaf epidermal cells of other plants. It will facilitate the analysis of protein subcellular localization on a large scale.
doi:10.1371/journal.pone.0083556
PMCID: PMC3885512  PMID: 24416168
16.  Increasing Threat of Brucellosis to Low-Risk Persons in Urban Settings, China 
Emerging Infectious Diseases  2014;20(1):126-130.
Cases of brucellosis were diagnosed in 3-month-old twins and their mother. An epidemiologic survey suggested that raw sheep or goat meat might be the source of Brucella melitensis infection. This finding implies that the increasing threat of brucellosis might affect low-risk persons in urban settings in China.
doi:10.3201/eid2001.130324
PMCID: PMC3884711  PMID: 24377827
brucellosis; bacteria; human infection; public health; urban setting; risk; China
17.  ERK1/2/COX-2/PGE2 signaling pathway mediates GPR91-dependent VEGF release in streptozotocin-induced diabetes 
Molecular Vision  2014;20:1109-1121.
Purpose
Retinal vascular dysfunction caused by vascular endothelial growth factor (VEGF) is the major pathological change that occurs in diabetic retinopathy (DR). It has recently been demonstrated that G protein-coupled receptor 91 (GPR91) plays a major role in both vasculature development and retinal angiogenesis. In this study, we examined the signaling pathways involved in GPR91-dependent VEGF release during the early stages of retinal vascular change in streptozotocin-induced diabetes.
Methods
Diabetic rats were assigned randomly to receive intravitreal injections of shRNA lentiviral particles targeting GPR91 (LV.shGPR91) or control particles (LV.shScrambled). Accumulation of succinate was assessed by gas chromatography-mass spectrometry (GC-MS). At 14 weeks, the ultrastructure and function of the retinal vessels of diabetic retinas with or without shRNA treatment were assessed using hematoxylin and eosin (HE) staining, transmission electron microscopy (TEM), and Evans blue dye permeability. The expression of GPR91, extracellular signal-regulated kinases 1 and 2 (ERK1/2) and cyclooxygenase-2 (COX-2) were measured using immunofluorescence and western blotting. COX-2 and VEGF mRNA were determined by quantitative RT–PCR. Prostaglandin E2 (PGE2) and VEGF secretion were detected using an enzyme-linked immunosorbent assay.
Results
Succinate exhibited abundant accumulation in diabetic rat retinas. The retinal telangiectatic vessels, basement membrane thickness, and Evans blue dye permeability were attenuated by treatment with GPR91 shRNA. In diabetic rats, knockdown of GPR91 inhibited the activities of ERK1/2 and COX-2 as well as the expression of PGE2 and VEGF. Meanwhile, COX-2, PGE2, and VEGF expression was inhibited by ERK1/2 inhibitor U0126 and COX-2 inhibitor NS-398.
Conclusions
Our data suggest that hyperglycemia causes succinate accumulation and GPR91 activity in retinal ganglion cells, which mediate VEGF-induced retinal vascular change via the ERK1/2/COX-2/PGE2 pathway. This study highlights the signaling pathway as a potential target for intervention in DR.
PMCID: PMC4119234  PMID: 25324681
18.  Shellfish Toxins Targeting Voltage-Gated Sodium Channels 
Marine Drugs  2013;11(12):4698-4723.
Voltage-gated sodium channels (VGSCs) play a central role in the generation and propagation of action potentials in excitable neurons and other cells and are targeted by commonly used local anesthetics, antiarrhythmics, and anticonvulsants. They are also common targets of neurotoxins including shellfish toxins. Shellfish toxins are a variety of toxic secondary metabolites produced by prokaryotic cyanobacteria and eukaryotic dinoflagellates in both marine and fresh water systems, which can accumulate in marine animals via the food chain. Consumption of shellfish toxin-contaminated seafood may result in potentially fatal human shellfish poisoning. This article provides an overview of the structure, bioactivity, and pharmacology of shellfish toxins that act on VGSCs, along with a brief discussion on their pharmaceutical potential for pain management.
doi:10.3390/md11124698
PMCID: PMC3877881  PMID: 24287955
VGSCs; shellfish toxins; structure; bioactivity; pharmaceutical potential
19.  Down-Regulation of Gab1 Inhibits Cell Proliferation and Migration in Hilar Cholangiocarcinoma 
PLoS ONE  2013;8(11):e81347.
Hilar cholangiocarcinoma is a highly aggressive malignancy originating from the hilar biliary duct epithelium. Due to few effective comprehensive treatments, the prognosis of hilar cholangiocarcinoma is poor. In this study, immunohistochemistry was first used to detect and analyze the expression of Gab1, VEGFR-2, and MMP-9 in hilar cholangiocarcinoma solid tumors and the relationships to the clinical pathological features. Furthermore, Gab1 and VEGFR-2 siRNA were used to interfere the hilar cholangiocarcinoma cell line ICBD-1 and then detect the PI3K/Akt signaling pathway, MMP-9 levels and malignant biological behaviors of tumor cells. The data showed that 1. Gab1, VEGFR-2, and MMP-9 were highly expressed and positively correlated with each other in hilar cholangiocarcinoma tissues, which were related to lymph node metastasis and differentiation. 2. After Gab1 or VEGFR-2 siRNA interference, PI3K/Akt pathway activity and MMP-9 levels were decreased in ICBD-1 cells. At the same time, cell proliferation decreased, cell cycle arrested in G1 phase, apoptosis increased and invasion decreased. These results suggest that the expression of Gab1, VEGFR-2, and MMP-9 are significantly related to the malignant biological behavior of hilar cholangiocarcinoma. Gab1 regulates growth, apoptosis and invasion through the VEGFR-2/Gab1/PI3K/Akt signaling pathway in hilar cholangiocarcinoma cells and influences the invasion of tumor cells via MMP-9.
doi:10.1371/journal.pone.0081347
PMCID: PMC3842939  PMID: 24312291
20.  Computational prediction of associations between long non-coding RNAs and proteins 
BMC Genomics  2013;14:651.
Background
Though most of the transcripts are long non-coding RNAs (lncRNAs), little is known about their functions. lncRNAs usually function through interactions with proteins, which implies the importance of identifying the binding proteins of lncRNAs in understanding the molecular mechanisms underlying the functions of lncRNAs. Only a few approaches are available for predicting interactions between lncRNAs and proteins. In this study, we introduce a new method lncPro.
Results
By encoding RNA and protein sequences into numeric vectors, we used matrix multiplication to score each RNA–protein pair. This score can be used to measure the interactions between an RNA–protein pair. This method effectively discriminates interacting and non-interacting RNA–protein pairs and predicts RNA–protein interactions within a given complex. Applying this method on all human proteins, we found that the long non-coding RNAs we collected tend to interact with nuclear proteins and RNA-binding proteins.
Conclusions
Compared with the existing approaches, our method shortens the time for training matrix and obtains optimal results based on the model being used. The ability of predicting the associations between lncRNAs and proteins has also been enhanced. Our method provides an idea on how to integrate different information into the prediction process.
doi:10.1186/1471-2164-14-651
PMCID: PMC3827931  PMID: 24063787
Long non-coding RNA; RNA–protein interaction; Computation
21.  Transformation of common wheat (Triticum aestivum L.) with avenin-like b gene improves flour mixing properties 
Molecular Breeding  2013;32(4):853-865.
Avenin-like b proteins may contribute to the viscoelastic properties of wheat dough via inter-chain disulphide bonds, due to their rich cysteine residues. In order to clarify the effect of the avenin-like b proteins on the functional properties of wheat flour, the functional and biochemical properties of wheat flour were analyzed in three transgenic wheat lines overexpressing the avenin-like b gene using the sodium dodecyl sulfate sedimentation (SDSS) test, Mixograph and size exclusion-high performance liquid chromatography (SE-HPLC) analysis. The results of the SDSS test and Mixograph analysis demonstrated that the overexpression of avenin-like b proteins in transgenic lines led to significantly increased SDSS volume and improved flour mixing properties. The results of SE-HPLC analysis of the gluten proteins in wheat flour demonstrated that the improvement in transgenic line flour properties was associated with the increased proportion of large polymeric proteins due to the incorporation of overexpressed avenin-like b proteins into the glutenin polymers. These results could help to understand the influence and mechanism of avenin-like b proteins on the functional properties of wheat flour.
Electronic supplementary material
The online version of this article (doi:10.1007/s11032-013-9913-1) contains supplementary material, which is available to authorized users.
doi:10.1007/s11032-013-9913-1
PMCID: PMC3830129  PMID: 24288453
Transgenic wheat; Avenin-like b protein; Mixing properties; Glutenin polymers
22.  Significance of Monoclonal Antibodies against the Conserved Epitopes within Non-Structural Protein 3 Helicase of Hepatitis C Virus 
PLoS ONE  2013;8(7):e70214.
Nonstructural protein 3 (NS3) of hepatitis C virus (HCV), codes for protease and helicase carrying NTPase enzymatic activities, plays a crucial role in viral replication and an ideal target for diagnosis, antiviral therapy and vaccine development. In this study, monoclonal antibodies (mAbs) to NS3 helicase were characterized by epitope mapping and biological function test. A total of 29 monoclonal antibodies were produced to the truncated NS3 helicase of HCV-1b (T1b-rNS3, aa1192–1459). Six mAbs recognized 8/29 16mer peptides, which contributed to identify 5 linear and 1 discontinuous putative epitope sequences. Seven mAbs reacted with HCV-2a JFH-1 infected Huh-7.5.1 cells by immunofluorescent staining, of which 2E12 and 3E5 strongly bound to the exposed linear epitope 1231PTGSGKSTK1239 (EP05) or core motif 1373IPFYGKAI1380 (EP21), respectively. Five other mAbs recognized semi-conformational or conformational epitopes of HCV helicase. MAb 2E12 binds to epitope EP05 at the ATP binding site of motif I in domain 1, while mAb 3E5 reacts with epitope EP21 close to helicase nucleotide binding region of domain 2. Epitope EP05 is totally conserved and EP21 highly conserved across HCV genotypes. These two epitope peptides reacted strongly with 59–79% chronic and weakly with 30–58% resolved HCV infected blood donors, suggesting that these epitopes were dominant in HCV infection. MAb 2E12 inhibited 50% of unwinding activity of NS3 helicase in vitro. Novel monoclonal antibodies recognize highly conserved epitopes at crucial functional sites within NS3 helicase, which may become important antibodies for diagnosis and antiviral therapy in chronic HCV infection.
doi:10.1371/journal.pone.0070214
PMCID: PMC3722154  PMID: 23894620
23.  Overexpression of Avenin-Like b Proteins in Bread Wheat (Triticum aestivum L.) Improves Dough Mixing Properties by Their Incorporation into Glutenin Polymers 
PLoS ONE  2013;8(7):e66758.
Avenin-like b proteins are a small family of wheat storage proteins, each containing 18 or 19 cysteine residues. The role of these proteins, with high numbers of cysteine residues, in determining the functional properties of wheat flour is unclear. In the present study, two transgenic lines of the bread wheat overexpressing avenin-like b gene were generated to investigate the effects of Avenin-like b proteins on dough mixing properties. Sodium dodecyl sulfate sedimentation (SDSS) test and Mixograph analysis of these lines demonstrated that overexpression of Avenin-like b proteins in both transgenic wheat lines significantly increased SDSS volume and improved dough elasticity, mixing tolerance and resistance to extension. These changes were associated with the increased proportion of polymeric proteins due to the incorporation of overexpressed Avenin-like b proteins into the glutenin polymers. The results of this study were critical to confirm the hypothesis that Avenin-like b proteins could be integrated into glutenin polymers by inter-chain disulphide bonds, which could help understand the mechanism behind strengthening wheat dough strength.
doi:10.1371/journal.pone.0066758
PMCID: PMC3699606  PMID: 23843964
24.  pKa determination of oxysophocarpine by reversed - phase high performance liquid chromatography 
SpringerPlus  2013;2:270.
Background
In this study, a RP - HPLC method was applied for determination of pKa value by using the dependence of the capacity factor (k) on the pH of the mobile phase for oxysophocarpine (OSP).
Findings
The effect of the mobile phase composition on the ionization constant was studied by measuring the pKa value at different MeOH concentrations, ranging from 10 to 20% (v/v). Based on all pH - k curves plotted and pH values at inflection point calculated, experimental pKa value obtained for oxysophocarpine was 6.5.
Conclusion
This method was successfully applied to realize low sample consumption, rapid sample throughput, high sensitivity and precision.
doi:10.1186/2193-1801-2-270
PMCID: PMC3701144  PMID: 23853749
Oxysophocarpine; pKa value; HPLC
25.  The Genetic Variant on Chromosome 10p14 Is Associated with Risk of Colorectal Cancer: Results from a Case-Control Study and a Meta-Analysis 
PLoS ONE  2013;8(5):e64310.
Background
A common single nucleotide polymorphism (SNP), rs10795668, located at 10p14, was first identified to be significantly associated with risk of colorectal cancer (CRC) by a genome-wide association study (GWAS) in 2008; however, another GWAS and following replication studies yielded conflicting results.
Methods
We conducted a case-control study of 470 cases and 475 controls in a Chinese population and then performed a meta-analysis, integrating the current study and 9 publications to evaluate the association between rs10795668 and CRC risk. Heterogeneity among studies and publication bias were assessed by the χ2-based Q statistic test and Egger's test, respectively.
Results
In the case-control study, significant association between the SNP and CRC risk was observed, with per-A-allele OR of 0.71 (95%CI: 0.54–0.94, P = 0.017). The following meta-analysis further confirmed the significant association, with per-A-allele OR of 0.91 (95%CI: 0.89–0.93, Pheterogeneity>0.05) in European population and 0.86 (95%CI: 0.78–0.96, Pheterogeneity <0.05) in Asian population. Besides, sensitivity analyses and publication bias assessment indicated the robust stability and reliability of the results.
Conclusions
Results from our case-control study and the followed meta-analysis confirmed the significant association of rs10795668 with CRC risk.
doi:10.1371/journal.pone.0064310
PMCID: PMC3661459  PMID: 23717594

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