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1.  Proteomic analysis of ubiquitin ligase KEAP1 reveals associated proteins that inhibit NRF2 ubiquitination 
Cancer research  2013;73(7):2199-2210.
Somatic mutations in the KEAP1 ubiquitin ligase or its substrate NRF2 (NFE2L2) commonly occur in human cancer, resulting in constitutive NRF2-mediated transcription of cytoprotective genes. However, many tumors display high NRF2 activity in the absence of mutation, supporting the hypothesis that alternative mechanisms of pathway activation exist. Previously, we and others discovered that via a competitive binding mechanism, the proteins WTX (AMER1), PALB2 and SQSTM1 bind KEAP1 to activate NRF2. Proteomic analysis of the KEAP1 protein interaction network revealed a significant enrichment of associated proteins containing an ETGE amino acid motif, which matches the KEAP1 interaction motif found in NRF2. Like WTX, PALB2, and SQSTM1, we found that the dipeptidyl peptidase 3 (DPP3) protein binds KEAP1 via an ‘ETGE’ motif to displace NRF2, thus inhibiting NRF2 ubiquitination and driving NRF2-dependent transcription. Comparing the spectrum of KEAP1 interacting proteins with the genomic profile of 178 squamous cell lung carcinomas characterized by The Cancer Genome Atlas revealed amplification and mRNA over-expression of the DPP3 gene in tumors with high NRF2 activity but lacking NRF2 stabilizing mutations. We further show that tumor-derived mutations in KEAP1 are hypomorphic with respect to NRF2 inhibition and that DPP3 over-expression in the presence of these mutants further promotes NRF2 activation. Collectively, our findings further support the competition model of NRF2 activation and suggest that ‘ETGE’-containing proteins like DPP3 contribute to NRF2 activity in cancer.
PMCID: PMC3618590  PMID: 23382044
2.  Ratiometric electrochemical proximity assay for sensitive one-step protein detection 
Scientific Reports  2014;4:4360.
This work proposes the concept of ratiometric electrochemical proximity assay (REPA), which can be used for one-step, highly sensitive and selective detection of protein. The assay strategy was achieved on a sensing interface that was formed by hybridization of methylene blue (MB)-labeled antibody-DNA probe (MB-DNA1-Ab1) with ferrocene (Fc)-labeled DNA capture probe (Fc-P) modified gold electrode. On the interface the target protein could trigger the formation of immunocomplex between MB-DNA1-Ab1 and detection antibody-DNA probe (Ab2-DNA2) and subsequently the proximity hybridization of DNA1-DNA2, which led to the departure of MB-DNA1-Ab1 from the interface. The remained Fc-P could form a hairpin structure to take Fc group to electrode surface. Therefore, the recognition of target protein to Ab1 and Ab2 resulted in both the “signal-off” of MB and the “signal-on” of Fc for dual-signal electrochemical ratiometric readout. The proposed REPA could be carried out in one-step with 40-min duration and showed a wide detection range from 0.05 to 100 ng/mL with pg/mL limit of detection, displaying great potential for convenient point-of-care testing and commercial application.
PMCID: PMC3950580  PMID: 24618513
3.  Fractal Scaling of Particle Size Distribution and Relationships with Topsoil Properties Affected by Biological Soil Crusts 
PLoS ONE  2014;9(2):e88559.
Biological soil crusts are common components of desert ecosystem; they cover ground surface and interact with topsoil that contribute to desertification control and degraded land restoration in arid and semiarid regions.
Methodology/Principal Findings
To distinguish the changes in topsoil affected by biological soil crusts, we compared topsoil properties across three types of successional biological soil crusts (algae, lichens, and mosses crust), as well as the referenced sandland in the Mu Us Desert, Northern China. Relationships between fractal dimensions of soil particle size distribution and selected soil properties were discussed as well. The results indicated that biological soil crusts had significant positive effects on soil physical structure (P<0.05); and soil organic carbon and nutrients showed an upward trend across the successional stages of biological soil crusts. Fractal dimensions ranged from 2.1477 to 2.3032, and significantly linear correlated with selected soil properties (R2 = 0.494∼0.955, P<0.01).
Biological soil crusts cause an important increase in soil fertility, and are beneficial to sand fixation, although the process is rather slow. Fractal dimension proves to be a sensitive and useful index for quantifying changes in soil properties that additionally implies desertification. This study will be essential to provide a firm basis for future policy-making on optimal solutions regarding desertification control and assessment, as well as degraded ecosystem restoration in arid and semiarid regions.
PMCID: PMC3917891  PMID: 24516668
4.  A Modified Nonlinear Damage Accumulation Model for Fatigue Life Prediction Considering Load Interaction Effects 
The Scientific World Journal  2014;2014:164378.
Many structures are subjected to variable amplitude loading in engineering practice. The foundation of fatigue life prediction under variable amplitude loading is how to deal with the fatigue damage accumulation. A nonlinear fatigue damage accumulation model to consider the effects of load sequences was proposed in earlier literature, but the model cannot consider the load interaction effects, and sometimes it makes a major error. A modified nonlinear damage accumulation model is proposed in this paper to account for the load interaction effects. Experimental data of two metallic materials are used to validate the proposed model. The agreement between the model prediction and experimental data is observed, and the predictions by proposed model are more possibly in accordance with experimental data than that by primary model and Miner's rule. Comparison between the predicted cumulative damage by the proposed model and an existing model shows that the proposed model predictions can meet the accuracy requirement of the engineering project and it can be used to predict the fatigue life of welded aluminum alloy joint of Electric Multiple Units (EMU); meanwhile, the accuracy of approximation can be obtained from the proposed model though more simple computing process and less material parameters calling for extensive testing than the existing model.
PMCID: PMC3918351  PMID: 24574866
5.  Relationship between seedling and mature vegetation on the hilly-gullied Loess Plateau 
SpringerPlus  2013;2(Suppl 1):S17.
Seedling is an indispensable stage in plant cycle life, and seedling survival is important during natural vegetation restoration, especially on the Loess Plateau. In 2007, we selected 4 plots of Artemisia scoparia communities (ASC) and 4 plots of Artemisia gmelinii + Artemisia giraldii communities (AGC), examined seedling richness, diversity during the rainy season, and examined mature vegetation richness, coverage, and frequency in August. The results showed that seedlings density of ASC were 29 n m-2, 33 n m-2, 20 n m-2 and 31 n m-2 in July to October respectively, and that of AGC were 14 n m-2, 12 n m-2, 6 n m-2 and 9 n m-2 respectively; A. scoparia seedlings represented 53.2% of the total seedlings in ASC, the dominant species in AGC only account for less than 5% of the total seedlings. Most of the seedlings found were belonged to Compositae, Leguminoseae and Gramineae; 80% of seedlings in ASC were mainly comprised of A. scoparia and Lespedeza davurica, while in AGC that consisted of more than 6 species, such as L. davurica, Sophora viciifolia, Dracocephalum moldavicaand, A. gmelinii, Patrinia heterophylla, Heteropappus altaicus so on. Sørensen similarity index between monthly seedlings was approximately 0.47 in ASC and 0.35 in AGC; Sørensen similarity index between seedlings and mature vegetation ranged from 0.18 to 0.34 in ASC, and varied from 0.26 to 0.39 in AGC. These results suggested that seedling establishment would be a bottleneck for natural vegetation restoration when seed supply and seedling emergence were possible.
PMCID: PMC3973415
Seedling density; Seedling species; Diversity; Similarity
6.  Covalent Intermediate in the Catalytic Mechanism of the Radical SAM Methyl Synthase RlmN Trapped by Mutagenesis 
Journal of the American Chemical Society  2012;134(43):18074-18081.
The posttranscriptional modification of ribosomal RNA (rRNA) modulates ribosomal function and confers resistance to antibiotics targeted to the ribosome. The radical SAM (S-adenosyl-L-methionine) methyl synthases, RlmN and Cfr, both methylate A2503 within the peptidyl transferase center (PTC) of prokaryotic ribosomes, yielding 2-methyl- and 8-methyl-adenosine, respectively. The C2 and C8 positions of adenosine are unusual methylation substrates due to their electrophilicity. To accomplish this reaction, RlmN and Cfr proceed by a shared radical-mediated mechanism. However, in addition to the radical SAM CX3CX2C motif, both RlmN and Cfr contain two conserved cysteine residues required for in vivo function. These conserved cysteine residues are putatively involved in a covalent intermediate employed by RlmN and Cfr in order to achieve this challenging transformation. Currently, there is no direct evidence for this proposed covalent intermediate. We have further investigated the roles of these conserved cysteines in the mechanism of RlmN. Cysteine 118 mutants of RlmN are unable to resolve the covalent intermediate, either in vivo or in vitro, enabling us to isolate and characterize this intermediate. Additionally, tandem mass spectrometric analyses of mutant RlmN reveal a methylene-linked adenosine modification at cysteine 355. Employing deuterium-labeled SAM and RNA substrates in vitro has allowed us to further elucidate the mechanism of formation of this intermediate. Together, these experiments provide compelling evidence for the formation of a covalent intermediate species formed between RlmN and its rRNA substrate and the roles of the conserved cysteine residues in catalysis.
PMCID: PMC3499099  PMID: 23088750
7.  Structural basis of transfer between lipoproteins by cholesteryl ester transfer protein 
Nature chemical biology  2012;8(4):342-349.
Human cholesteryl ester transfer protein (CETP) mediates the net transfer of cholesteryl ester mass from atheroprotective high-density lipoproteins to atherogenic low-density lipoproteins by an unknown mechanism. Delineating this mechanism would be an important step toward the rational design of new CETP inhibitors for treating cardiovascular diseases. Using EM, single-particle image processing and molecular dynamics simulation, we discovered that CETP bridges a ternary complex with its N-terminal β-barrel domain penetrating into high-density lipoproteins and its C-terminal domain interacting with low-density lipoprotein or very-low-density lipoprotein. In our mechanistic model, the CETP lipoprotein-interacting regions, which are highly mobile, form pores that connect to a hydrophobic central cavity, thereby forming a tunnel for transfer of neutral lipids from donor to acceptor lipoproteins. These new insights into CETP transfer provide a molecular basis for analyzing mechanisms for CETP inhibition.
PMCID: PMC3792710  PMID: 22344176
8.  The sodium channel gene family is specifically expressed in hen uterus and associated with eggshell quality traits 
BMC Genetics  2013;14:90.
Eggshell quality is important for the poultry industry. During eggshell formation a mass of inorganic minerals is deposited. The Sodium Channel (SCNN1) gene family plays an essential role in cation transportation. The objective of this study was to investigate the pattern of expression of members of the SCNN1 gene family, their variation and their effects on eggshell quality.
The highest expression of SCNN1a, SCNN1b, and SCNN1g genes were in the active uterus during eggshell mineralization, while SCNN1d showed its highest expression level in the quiescent uterus (no egg present). Nineteen candidate SNPs from the four genes were genotyped in a population of 338 White Leghorn layers. Association analysis between SNPs (haplotypes/diplotypes) and eggshell traits was performed. Among seven significant SNPs, five SNPs were associated with eggshell strength, eggshell thickness, eggshell percentage or/and egg weight, while the other two SNPs within SCNN1d were only associated with eggshell percentage. These SNPs had a 0.25-6.99% contribution to phenotypic variance, depending on the trait. In haplotype analysis, SCNN1b and SCNN1d were associated with egg weight. The SCNN1b and SCNN1g were significantly associated with eggshell weight while only SCNN1g explained 2.04% of phenotypic variance. All the alleles of the members of SCNN1 gene family were associated with eggshell percentage and eggshell thickness, and others members had an association with eggshell strength except for SCNN1a. The contribution of different haplotypes of the SCNN1 gene family to eggshell phenotypic variance ranged from 0.09% to 5.74%.
Our study indicated that the SCNN1 gene family showed tissue expression specificity and was significantly associated with eggshell traits in chicken. This study provides evidence that genetic variation in members of the sodium channel can influence eggshell quality.
PMCID: PMC3851161  PMID: 24059973
Sodium channel gene family; Eggshell quality; Cation transportation; Phenotypic variance; Uterus
9.  Transgenic Overexpression of Peroxiredoxin-2 Attenuates Ischemic Neuronal Injury Via Suppression of a Redox-Sensitive Pro-Death Signaling Pathway 
Antioxidants & Redox Signaling  2012;17(5):719-732.
Aims: Peroxiredoxins (PRXs) are a newly characterized family of peroxide scavenging enzymes that not only help maintain cellular redox homeostasis but also may directly engage in a variety of intracellular signaling pathways. PRX2 is a neuronal-specific PRX believed to participate in cerebral antioxidant responses in several neurodegenerative diseases. This study investigates the potential neuroprotective effect and the underlying mechanism of PRX2 in models of ischemic neuronal injury. Results: Transgenic mice overexpressing PRX2 showed reduced brain injury and improved neurological recovery up to 3 weeks after transient focal cerebral ischemia compared to wild-type littermates. In primary cultures of cortical neurons, transfection of PRX2 but not the loss-of-catalytic-site PRX2 mutant conferred neuroprotection against cell death induced by oxygen glucose deprivation. PRX2 exhibited potent pro-survival effects in ischemic neurons by maintaining thioredoxin (Trx) in its reduced state, thereby preventing oxidative stress-mediated activation of apoptosis signal–regulating kinase 1 (ASK1) and the downstream MKK/JNK pro-death signaling pathway. PRX2 failed to provide additional neuroprotection against ischemic injury in Trx- or ASK1-knockdown neuron cultures and in mice treated with a JNK inhibitor. Innovation: This study provides evidence that neuronal overexpression of PRX2 confers prolonged neuroprotection against ischemic/reperfusion brain injury. Moreover, the results suggest a signaling pathway by which PRX2 suppresses ischemia-induced neuronal apoptosis. Conclusions: Enhanced neuronal expression and activity of PRX2 protect against ischemic neuronal injury by directly modulating the redox-sensitive Trx-ASK1 signaling complex. Antioxid. Redox Signal. 17, 719–732.
PMCID: PMC3387778  PMID: 22356734
10.  Laparoscopic splenic hilum lymph node dissection for advanced proximal gastric cancer: A modified approach for pancreas- and spleen-preserving total gastrectomy 
AIM: To investigate the feasibility and optimal approach for laparoscopic pancreas- and spleen-preserving splenic hilum lymph node dissection in advanced proximal gastric cancer.
METHODS: Between August 2009 and August 2012, 12 patients with advanced proximal gastric cancer treated in Nanfang Hospital, Southern Medical University, Guangzhou, China were enrolled and subsequently underwent laparoscopic total gastrectomy with pancreas- and spleen-preserving splenic hilum lymph node (LN) dissection. The clinicopathological characteristics, surgical outcomes, postoperative course and follow-up data of these patients were retrospectively collected and analyzed in the study.
RESULTS: Based on our anatomical understanding of peripancreatic structures, we combined the characteristics of laparoscopic surgery and developed a modified approach (combined supra- and infra-pancreatic approaches) for laparoscopic pancreas- and spleen-preserving splenic hilum LN dissection. Surgery was completed in all 12 patients laparoscopically without conversion. Only one patient experienced intraoperative bleeding when dissecting LNs along the splenic artery and was handled with laparoscopic hemostasis. The mean operating time was 268.4 min and mean number of retrieved splenic hilum LNs was 4.8. One patient had splenic hilum LN metastasis (8.3%). Neither postoperative morbidity nor mortality was observed. Peritoneal metastasis occurred in one patient and none of the other patients died or experienced recurrent disease during the follow-up period.
CONCLUSION: Laparoscopic total gastrectomy with pancreas- and spleen-preserving splenic hilum LN dissection using the modified approach for advanced proximal gastric cancer could be safely achieved.
PMCID: PMC3740431  PMID: 23946606
Proximal stomach; Stomach neoplasm; Laparoscopy; Lymph node excision; Splenic hilum
11.  FAM123A Binds Microtubules and Inhibits the Guanine Nucleotide Exchange Factor ARHGEF2 to Decrease Actomyosin Contractility*** 
Science signaling  2012;5(240):ra64.
The FAM123 gene family comprises three members, FAM123A, the tumor suppressor WTX(FAM123B) and FAM123C. WTX is required for normal development and causally contributes to human disease, in part through its regulation of β-catenin-dependent WNT signaling. The roles of FAM123A and FAM123C in signaling, cell behavior and human disease remain less understood. We defined and compared the protein-protein interaction networks for each member of the FAM123 family by affinity purification and mass spectrometry. Protein localization and functional studies suggest that the FAM123 family members have conserved and divergent cellular roles. In contrast to WTX and FAM123C, we found that microtubule-associated proteins were enriched in the FAM123A protein interaction network. FAM123A interacted with and tracked dynamic microtubules in a plus-end direction. Domain interaction experiments revealed a ‘SKIP’ amino acid motif in FAM123A that mediated interaction with the microtubule tip tracking proteins EB1 and EB3, and therefore with microtubules. Cells depleted of FAM123A showed compartment-specific effects on microtubule dynamics, increased actomyosin contractility, larger focal adhesions and decreased cell migration. These effects required binding of FAM123A to and inhibition of the guanine nucleotide exchange factor ARHGEF2, a microtubule-associated activator of RhoA. Together, these data suggest that the ‘family-unique’ SKIP motif enables FAM123A to bind EB proteins, localize to microtubules and coordinate microtubule dynamics and actomyosin contractility.
PMCID: PMC3733483  PMID: 22949735
12.  Reduction of Waste Water in Erhai Lake Based on MIKE21 Hydrodynamic and Water Quality Model 
The Scientific World Journal  2013;2013:958506.
In order to study the ecological water environment in Erhai Lake, different monitoring sections were set to research the change of hydrodynamics and water quality. According to the measured data, MIKE21 Ecolab, the water quality simulation software developed by DHI, is applied to simulate the water quality in Erhai Lake. The hydrodynamics model coupled with water quality is established by MIKE21FM software to simulate the current situation of Erhai Lake. Then through the comparison with the monitoring data, the model parameters are calibrated and the simulation results are verified. Based on this, water quality is simulated by the two-dimensional hydrodynamics and water quality coupled model. The results indicate that the level of water quality in the north and south of lake is level III, while in the center of lake, the water quality is level II. Finally, the water environment capacity and total emmision reduction of pollutants are filtered to give some guidance for the water resources management and effective utilization in the Erhai Lake.
PMCID: PMC3749603  PMID: 23997684
13.  Necrotizing myositis causes restrictive hypoventilation in a mouse model for human enterovirus 71 infection 
Virology Journal  2013;10:215.
Enterovirus 71 (EV71) infections are associated with a high prevalence of hand, foot and mouth disease (HFMD) in children and occasionally cause lethal complications. Most infections are self-limiting. However, resulting complications, including aseptic meningitis, encephalitis, poliomyelitis-like acute flaccid paralysis, and neurological pulmonary edema or hemorrhage, are responsible for the lethal symptoms of EV71 infection, the pathogenesis of which remain to be clarified.
In the present study, 2-week-old Institute of Cancer Research (ICR) mice were infected with a mouse-adapted EV71 strain. These infected mice demonstrated progressive paralysis and died within 12 days post infection (d.p.i.). EV71, which mainly replicates in skeletal muscle tissues, caused severe necrotizing myositis. Lesions in the central nervous system (CNS) and other tissues were not observed.
Necrotizing myositis of respiratory-related muscles caused severe restrictive hypoventilation and subsequent hypoxia, which could explain the fatality of EV71-infected mice. This finding suggests that, in addition to CNS injury, necrotic myositis may also be responsible for the paralysis and death observed in EV71-infected mice.
PMCID: PMC3710232  PMID: 23809248
Enterovirus 71; Skeletal muscle; Necrotizing myositis; Restrictive hypoventilation
14.  Synergy of Taxol and rhein lysinate associated with the downregulation of ERK activation in lung carcinoma cells 
Oncology Letters  2013;6(2):525-528.
In previous studies we observed that rhein lysinate (RHL), a salt of rhein and lysine that is easily dissolved in water, inhibited the growth of tumor cells in breast cancer, ovarian cancer, hepatocellular carcinoma and cervical cancer. The present study aimed to investigate the effects of RHL on H460 and A549 non-small cell lung cancer (NSCLC) cells using a combination of RHL and Taxol. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay was used to determine the growth inhibition effect of the drugs in the H460 and A549 cells. Cell apoptosis was analyzed by flow cytometry combined with fluorescein-isothiocyanate-Annexin V/propidium iodide (PI) staining. The expression levels of proteins were detected by western blotting. There was a significant reduction in the proliferation of the NSCLC cell lines treated with a combination of Taxol and RHL. The overall growth inhibition was directly correlated with apoptotic cell death. RHL potentiated Taxol-induced cell killing by reducing extracellular signal-regulated kinase (ERK) activity and increasing the levels of cleaved poly(ADP-ribose) polymerase (PARP) and caspase-3. Notably, the results for the Bcl-2 and NF-κB proteins also showed downregulation in the combined treatment group compared with the single-agent treatment and the untreated control groups. The present results showed that RHL potentiates the growth inhibition induced by Taxol in NSCLC cells and showed that this synergy may be associated with the downregulation of ERK activation.
PMCID: PMC3789082  PMID: 24137360
rhein lysate; Taxol; lung cancer; combination therapy; extracellular signal-regulated kinase
15.  Cellular Delivery of Quantum Dot-Bound Hybridization Probe for Detection of Intracellular Pre-MicroRNA Using Chitosan/Poly(γ-Glutamic Acid) Complex as a Carrier 
PLoS ONE  2013;8(6):e65540.
A quantum dot (QD)-bound hybridization probe was designed for detection of intracellular pre-miRNA using chitosan (CS)/poly(γ-glutamic acid) (γ-PGA) complex as a gene vector. The probe was prepared by assembling thiolated RNA to gold nanoparticle (Au NP) via Au-S bond and then binding 3′-end amine of the RNA to the carboxy group capped on quantum dot surface. The QD-RNA-Au NP probe was assembled on the vector by mixing with aqueous γ-PGA solution and then CS solution to construct a gene delivery system for highly effective cellular uptake and delivery. After the probe was released from CS/γ-PGA complex to the cytoplasm by electrostatic repulsion at intracellular pH, it hybridized with pre-miRNA precursor as target. The formed product was then cleaved by RNase III Dicer, leading to the separation of QDs from Au NPs and fluorescence emission of QDs, which could be detected by confocal microscopic imaging to monitor the amount of the intracellular pre-miRNA precursor. The in vitro assays revealed that the QD-RNA-Au NP was a robust, sensitive and selective probe for quantitative detection of target pre-miRNA. Using MDA-MB231 and MCF-7 breast cancer cells as models, the relative amount of pre-miRNA let-7a could be successfully compared. Since the amount of miRNA is related to the progress and prognosis of cancer, this strategy could be expected to hold promising application potential in medical research and clinical diagnostics.
PMCID: PMC3676334  PMID: 23762388
16.  Modified frontolateral partial laryngectomy operation: combined muscle-pedicle hyoid bone and thyrohyoid membrane flap in laryngeal reconstruction 
Cancer Biology & Medicine  2013;10(2):103-109.
Laryngeal reconstruction is needed to preserve laryngeal function in patients who have undergone extensive vertical or frontal partial laryngectomy. However, the procedure remains a difficult challenge. Several reconstruction techniques have been described, but these techniques pose risks of complications such as laryngeal stenosis. This study aimed to evaluate the postoperative course and functional outcomes of a new technique that combined a muscle-pedicle hyoid bone and a thyrohyoid flap during laryngeal reconstruction after tumor resection.
Four patients underwent extensive vertical partial or frontal partial laryngectomy for cancer. After tumor resection, laryngeal reconstruction was performed using the proposed technique. Postoperative recovery time, complications, and oncologic results were evaluated.
The four patients were successfully treated with the proposed technique. No dyspnea, dysphagia, or death occurred during the postoperative course. Decannulation was performed after a median of 3 days. The average postoperative hospital stay was 7 days. Short-term postoperative functional recovery was normal. No laryngeal stenosis or tumor recurrence was observed in any of the four patients after a follow-up period of more than 24 months.
The combination of the muscle-pedicle hyoid bone and the thyrohyoid flap is a reliable procedure for laryngeal reconstruction after extensive vertical partial or frontal partial laryngectomy.
PMCID: PMC3719186  PMID: 23882426
Hyoid bone; reconstruction; laryngeal cancer; flap; operation
17.  DNMT3A −448A>G polymorphism and the risk for hepatocellular carcinoma 
Biomedical Reports  2013;1(4):664-668.
DNA-methyltransferase (DNMT) 3A plays a significant role in carcinogenesis. Findings of a previous study suggested an association between the DNMT3A −448A>G single-nucleotide polymorphism (SNP) and susceptibility to gastric cancer (GC) and colorectal cancer (CRC). Hepatocellular carcinoma (HCC) is a common malignancy, with a similar expression pattern to GC. The aim of this case-control study was to determine whether there is an association between DNMT3A gene polymorphism and susceptibility to HCC. Real-time quantitive PCR (qPCR) was employed to detect DNMT3A expression in tumor and non-cancer liver tissue from 13 HCC patients. An increased expression of DNMT3A was detected, as well as −448A>G polymorphisms of DNMT3A promoter by polymerase chain reaction/restriction fragment length polymorphism (PCR-RFLP), confirmed by sequencing. The distribution of −448A>G polymorphisms was examined in 108 HCC patients and 225 healthy controls who were matched for age and gender. The association of −448A>G polymorphisms of DNMT3A and the risk of HCC was evaluated by stratified analysis according to the patient's age and gender. The allele frequency of −448A among HCC patients and the controls was 24.07 vs. 24.22%, respectively. The frequency of genotypes GG, AG, AA was 55.56 vs. 56.89%, 40.74 vs. 37.78%, 3.7 vs. 5.33%, respectively. The results indicated that −448A>G is not associated with susceptibility to HCC, although −448A>G is a functional single-nucleotide polymorphism (SNP) and increased the expression of DNMT3A in HCC cases. Findings of the present study suggested that the DNMT3A −448A>G polymorphism is an insufficient biomarker to predict the susceptibility to HCC.
PMCID: PMC3917098  PMID: 24649006
hepatocellular carcinoma; DNA-methyltransferase 3A; single-nucleotide polymorphism; susceptibility
18.  Use of immuno-dominant epitope derived from genotype 4 as a diagnostic reagent for detecting the antibodies against Hepatitis E Virus 
Virology Journal  2013;10:131.
Despite the genotype 4 has become the dominant cause of hepatitis E disease in China, none antigen derived from genotype 4 of hepatitis E virus (HEV) was used in current commercial anti-HEV immunoassay, and the serological reactivity of antigen derive from genotype 4 is not well-charactered.
We expressed and purified the 4 main immuno-dominant epitopes derived from genotype 1 and 4 including ORF2 (410-621aa) of genotype 4, ORF3 (47-114aa) of genotype 4, ORF2 (396-606aa) of genotype 1 and ORF3 (56-123aa) of genotype 4.
The ORF2 of genotype 4 displayed good diagnostics performance according to ROC analysis using in-house panel, and the immunoassays based the ORF2 of genotype 4 was then developed to detect the anti-HEV IgG antibodies and evaluated further in 530 anti-HEV IgG positive specimens and 380 negative specimens. The sensitivity and the specificity is 98.1% (520/530) and 94.7% (360/380) for immunoassay based on ORF2 of genotype 4, 96.6% (512/530) and 92.6% (352/380) for commercial immunoassay based on genotype 1. It is noted that all of the positive samples will be detected by combing two assays together. The anti-HEV immunoassays based on genotype 4 are in accordance with Chinese anti-HEV national standard,and show an good agreement of 95.8% with commercial assay (kappa=0.913, P=0.014).
The immunoassay based on ORF2G4 displays good performance, and combining assay based on genotype 1 together with genotype 4 will benefit the HEV diagnosis in large scale samples.
PMCID: PMC3655869  PMID: 23618011
HEV; ORF2; ORF3; Genotype; Immunoassay
19.  Intervention Effects of Ganoderma Lucidum Spores on Epileptiform Discharge Hippocampal Neurons and Expression of Neurotrophin-4 and N-Cadherin 
PLoS ONE  2013;8(4):e61687.
Epilepsy can cause cerebral transient dysfunctions. Ganoderma lucidum spores (GLS), a traditional Chinese medicinal herb, has shown some antiepileptic effects in our previous studies. This was the first study of the effects of GLS on cultured primary hippocampal neurons, treated with Mg2+ free medium. This in vitro model of epileptiform discharge hippocampal neurons allowed us to investigate the anti-epileptic effects and mechanism of GLS activity. Primary hippocampal neurons from <1 day old rats were cultured and their morphologies observed under fluorescence microscope. Neurons were confirmed by immunofluorescent staining of neuron specific enolase (NSE). Sterile method for GLS generation was investigated and serial dilutions of GLS were used to test the maximum non-toxic concentration of GLS on hippocampal neurons. The optimized concentration of GLS of 0.122 mg/ml was identified and used for subsequent analysis. Using the in vitro model, hippocampal neurons were divided into 4 groups for subsequent treatment i) control, ii) model (incubated with Mg2+ free medium for 3 hours), iii) GLS group I (incubated with Mg2+ free medium containing GLS for 3 hours and replaced with normal medium and incubated for 6 hours) and iv) GLS group II (neurons incubated with Mg2+ free medium for 3 hours then replaced with a normal medium containing GLS for 6 hours). Neurotrophin-4 and N-Cadherin protein expression were detected using Western blot. The results showed that the number of normal hippocampal neurons increased and the morphologies of hippocampal neurons were well preserved after GLS treatment. Furthermore, the expression of neurotrophin-4 was significantly increased while the expression of N-Cadherin was decreased in the GLS treated group compared with the model group. This data indicates that GLS may protect hippocampal neurons by promoting neurotrophin-4 expression and inhibiting N-Cadherin expression.
PMCID: PMC3634853  PMID: 23637882
20.  FCGR2B and FCRLB Gene Polymorphisms Associated with IgA Nephropathy 
PLoS ONE  2013;8(4):e61208.
IgA nephropathy (IgAN) is a complex syndrome characterized by deposition of IgA and IgA containing immune complexes (ICs) composed of IgG and complement C3 proteins in the mesangial area of glomeruli. The low-affinity receptors for the Fc region of IgG (FcγRs) are involved in autoantibody/immune complex-induced organ injury as well as ICs clearance. The aim of the study was to associate multiple polymorphisms within FCGR gene locus with IgAN in a large Chinese cohort.
Patients and Methods
60 single nucleotide polymorphisms (SNPs) spanning a 400 kb range within FCGR gene locus were analyzed in 2100 DNA samples from patients with biopsy proven IgAN and healthy age- and sex-matched controls from the same population in Chinese.
Among the 60 SNPs investigated, 15 gene polymorphisms within FCGR gene locus (25%) were associated with susceptibility to IgAN. The most significantly associated SNPs within individual genes were FCGR2B rs12118043 (p = 8.74*10−3, OR 0.76, 95% CI 0.62–0.93), and FCRLB rs4657093 (p = 2.28*10−3, OR 0.77, 95% CI 0.65–0.91). Both conditional analysis and linkage disequilibrium analysis suggested they were independent signals associated with IgAN. Associations between FCGR2B rs12118043 and proteinuria (p = 3.65×10−2) as well as gross hematuria (p = 4.53×10−2), between FCRLB rs4657093 and levels of serum creatinine (p = 2.67×10−2) as well as eGFR (p = 5.41*10−3) were also observed. Electronic cis-expression quantative trait loci analysis supported their possible functional significance, with protective genotypes correlating lower gene expressions.
Our data from genetic associations and expression associations revealed potentially pathogenic roles of Fc receptor gene polymorphisms in IgAN.
PMCID: PMC3625155  PMID: 23593433
21.  Step rate-determined walking intensity and walking recommendation in Chinese young adults: a cross-sectional study 
BMJ Open  2013;3(1):e001801.
There is lack of data on the physiological characteristics of over ground walking and walking recommendations for Chinese young adult. The purpose of the study was to measure walking-related energy expenditure during field testing, to identify step-rate cut-point associated with moderate and vigorous intensity, and to translate physical activity (PA) guidelines into walking goals for Chinese young adults.
Cross-sectional analytic study.
Two communities from Beijing and Shanghai in China.
A sample of 226 Chinese adults (117 men, 109 women) with a mean age of 21.7 (±0.2) years, volunteered to participate in the study. All Participants were recreationally active without orthopaedic limitations, free of chronic diseases, not taking any medications that affect metabolism and non-smokers.
Outcome measures
All the participants completed four 6 minincremental over ground walking at different speeds of 3.8, 4.8, 5.6 and 6.4 km/h, respectively. Indirect calorimeter was used to measure energy expenditure at each speed. Receiver operating characteristic curves were used to determine the step-rate cut-points associated with moderate and vigorous intensity activity.
At the same walking speed, step counts per minute were higher in women than in men. No significant differences were found in VO2 per weight (ml/kg/min) between women and men. Step-rate cut-point associated with walking at 3 metabolic equivalents (METs) and 6METs were 105 and 130 step/min when analysing men and women together. There were slight differences on the cut-points between women and men if data were analysed separately.
In order to meet PA guidelines, Chinese young adult should walk 30 min with at least 105 step/min or 3150 steps or 2 km with the same step-rate per day. Walking at a higher speed of 130 step/min might provide additional health benefit.
PMCID: PMC3549247  PMID: 23335555
Sports Medicine; Public Health; Statistics & Research Methods
22.  Anti-proliferative and pro-apoptotic effect of carvacrol on human hepatocellular carcinoma cell line HepG-2 
Cytotechnology  2011;64(1):43-51.
Carvacrol is one of the members of monoterpene phenol and is present in the volatile oils of Thymus vulgaris, Carum copticum, origanum and oregano. It is a safe food additive commonly used in our daily life, and few studies have indicated that carvacrol has anti-hepatocarcinogenic activities. The rationale of the study was to examine whether carvacrol affects apoptosis of human hepatoma HepG2 cells. In this study, we showed that carvacrol inhibited HepG2 cell growth by inducing apoptosis as evidenced by Hoechst 33258 stain and Flow cytometric (FCM) analysis. Incubation of HepG2 cells with carvacrol for 24 h induced apoptosis by the activation of caspase-3, cleavage of PARP and decreased Bcl-2 gene expression. These results demonstrated that a significant fraction of carvacrol treated cells died by an apoptotic pathway in HepG2 cells. Moreover, carvacrol selectively altered the phosphorylation state of members of the MAPK superfamily, decreasing phosphorylation of ERK1/2 significantly in a dose-dependent manner, and activated phosphorylation of p38 but not affecting JNK MAPK phosphorylation. These results suggest that carvacrol may induce apoptosis by direct activation of the mitochondrial pathway, and the mitogen-activated protein kinase pathway may play an important role in the antitumor effect of carvacrol. These results have identified, for the first time, the biological activity of carvacrol in HepG2 cells and should lead to further development of carvacrol for liver disease therapy.
PMCID: PMC3261448  PMID: 21938469
Carvacrol; Hepatocellular carcinoma; Apoptosis; Mitochondrial pathway; Mitogen-activated protein kinase pathway
23.  Delayed Inhibition of c-Jun N-Terminal Kinase Worsens Outcomes after Focal Cerebral Ischemia 
The Journal of Neuroscience  2012;32(24):8112-8115.
The stress-activated protein kinase c-Jun N-terminal kinase (JNK) is a central regulator in neuronal death cascades. In animal models of cerebral ischemia, acute inhibition of JNK reduces infarction and improves outcomes. Recently however, emerging data suggest that many neuronal death mediators may have biphasic properties—deleterious in the acute stage but potentially beneficial in the delayed stage. Here, we hypothesized that JNK may also have biphasic actions, so some caution may be required in the development of JNK inhibitors for stroke. Sprague Dawley rats underwent 90 min transient occlusions of the middle cerebral artery. Acute treatment (10 min poststroke) with the JNK inhibitor SP600125 reduced infarction volumes. In contrast, delayed treatment (7 d poststroke) worsened infarction volumes and neurological outcomes. Immunostaining of peri-infarct cortex showed that JNK inhibition suppressed surrogate markers of neurovascular remodeling, including matrix metalloproteinase-9 in GFAP-positive astrocytes and microvascular density. Consistent with these in vivo data, SP600125 significantly suppressed in vitro angiogenesis in rat brain endothelial cultures. Our data provide initial proof-of-concept that the neuronal death target JNK may also participate in endogenous processes of neurovascular remodeling and recovery after cerebral ischemia.
PMCID: PMC3752060  PMID: 22699892
24.  High expression levels of COX-2 and P300 are associated with unfavorable survival in laryngeal squamous cell carcinoma 
In order to provide a basis for clinical treatment decisions, we explored whether there was a correlation between the expression of COX-2 and P300 and clinical factors in a group of patients with laryngeal squamous cell carcinoma (LSCC). A retrospective analysis of clinicopathological data was conducted in 80 patients with LSCC who presented between January 1997 and December 1998. An immunohistochemistry tissue microarray was conducted of 80 surgically resected LSCC and 20 adjacent normal tissue specimens. Survival analysis and Kaplan–Meier curves were used to compare the effects of clinicopathological factors on survival. The Cox model was applied for multivariate analysis. The expression level of COX-2/P300 in LSCC tissues and adjacent normal tissues were 47.5/50.0 versus 0.0/15.0 %. The expression of COX-2 and P300 was correlated with higher T category, N category, clinical staging, histological grade and recurrence (P < 0.05). P300 expression was correlated with COX-2 expression (P < 0.05). Univariate survival analysis showed that P300, COX-2, N category, clinical staging and recurrence factors were closely correlated with unfavorable survival (P < 0.05). Multivariate analysis showed that COX-2 expression, histological grade and recurrence were independent prognostic factors for LSCC. High expression levels of COX-2 and P300 indicated poor survival outcomes for patients with LSCC.
PMCID: PMC3580132  PMID: 23179937
Laryngeal squamous cell carcinoma (LSCC); Prognosis; Survival; P300; COX-2
25.  Poly[[hexa-μ-aqua-diaqua­bis­(μ4-dihydrogen benzene-1,2,4,5-tetra­carboxyl­ato)magnesiumdisodium] dihydrate] 
The asymmetric unit of the title compound, {[MgNa2(C10H4O8)2(H2O)8]·2H2O}n, contains one octa­hedrally coordin­ated MgII atom (site symmetry 2/m), one octahedrally coordinated NaI atom (site symmetry 2) and one half of the dihydrogen benzene-1,2,4,5-tetra­carboxyl­ate (btec) ligand, the second half of the ligand being generated by a twofold rotation axis. The basic framework of the title compound features infinite (–Na–Na–Mg–)n chains along [10-1] with the metal cations bridged by the coordinating water molecules. The chains are isolated from each other by μ4-bridging btec ligands, which form inter­molecular O—H⋯O hydrogen bonds to uncoordinated water mol­ecules and the coordinated water mol­ecules of a neighbouring chain. In each btec ligand, there are also intramolecular O—H⋯O hydrogen bonds.
PMCID: PMC3393174  PMID: 22807742

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