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1.  Fibroblast growth factor receptor 4 protein expression and clinicopathological features in gastric cancer 
AIM: To investigate fibroblast growth factor receptor 4 (FGFR4) protein expression in Chinese patients with resectable gastric cancer (GC) and the association with clinicopathological characteristics and survival.
METHODS: One hundred and seventy-five GC patients who underwent curative surgical procedures were enrolled in this study. The protein expression of FGFR4 in formalin-fixed, paraffin-embedded (FFPE) GC tissues was determined by immunohistochemical (IHC) analysis. Patient clinicopathological data and survival information were also collected and χ2 statistical analysis was performed to analyze FGFR4 protein expression in the subgroups with differing clinicopathological characteristics including; gender, age, tumor location, differentiation, tumor-node-metastasis stage, macroscopic type, depth of invasion, lymph node metastases, distant metastasis, neural invasion and vascular invasion. Furthermore, some common molecular markers of GC in our cancer center, including p53, p27, topoisomerase IIα (Topo IIα) were also determined by IHC and their association with FGFR4 protein expression evaluated. The probability of survival for different subgroups with different clinicopathological characteristics was calculated using the Kaplan-Meier method and survival curves plotted using the log rank test.
RESULTS: Seventy seven cases (44%) were found to have high expression of FGFR4 protein. Significantly different FGFR4 expression was observed between gastric cancers with differing expression of Topo IIα (log rank χ2 = 9.4760, P = 0.0236). No significant differences were observed between subgroups defined by any of the other clinicopathological characteristics. The median survival time of the FGFR4 high expression (77 cases) and low expression groups (98 cases) was 27 mo and 39 mo, respectively. The five-year survival rates and median survival times of gastric cancers with high FGFR4 expression were worse than those with low expression (30.8% vs 39.2%, 27 mo vs 39 mo), respectively, however, no significant difference was observed in survival time (log rank χ2 = 1.0477, P = 0.3060). Survival analysis revealed that high expression of FGFR4 was a predictor of poor outcome in GC patients if the tumor was small (less than or equal to 3 cm in size) (log rank χ2 = 5.5033, P = 0.0190), well differentiated (log rank χ2 = 7.9757, P = 0.0047), and of T1 or T2 stage invasion depth (log rank χ2 = 4.8827, P = 0.0271).
CONCLUSION: Our results suggest that high tumor expression of FGFR4 protein is not an independent risk factor for GC cancer initiation, but is a useful prognostic marker for GC patients when the tumor is relatively small, well differentiated, or in the early stages of invasion.
PMCID: PMC4323460
Gastric cancer; Fibroblast growth factor receptor 4; Protein expression; Clinicopathological characteristics; Prognosis
2.  A novel adenoviral vector carrying an all-in-one Tet-On system with an autoregulatory loop for tight, inducible transgene expression 
BMC Biotechnology  2015;15(1):4.
One of the most commonly used vectors for gene therapy is the adenoviral vector; its ability to tightly regulate transgene expression is critical for optimizing therapeutic outcomes. The tetracycline-regulated system (especially the Tet-On system) for gene expression is one of the most valuable tools for controlling gene expression. The major problem of an adenoviral vector carrying a Tet-On system is suboptimal regulation of transgene expression.
We constructed a single adenoviral vector carrying in its E1 region a novel “all-in-one” Tet-On system with an autoregulatory loop. This system had improved Dox-inducible gene expression in terms of low basal expression, high induced expression and high responsiveness to Dox. To our knowledge, this is the first reported adenovirus-based, all-in-one Tet-On system with an autoregulatory loop inserted into a single region of adenoviral genome. This system was further tested by inducible expression of soluble tumor necrosis factor-related apoptosis-inducing ligand (sTRAIL). The adenovirus that expressed soluble TRAIL under the control of this novel Tet-On system showed tumor-derived cells inhibitory activity in SW480 cells only under induced conditions.
Our novel, single adenoviral vector carrying in its E1 region an all-in-one Tet-On system with an autoregulatory loop displayed tight regulation of transgene expression in vitro. This system has great potential for a variety of applications, including gene therapy and the study of gene function.
PMCID: PMC4331377
Adenoviral vector; Tet-On; Transgene; Inducible vector; Gene therapy
3.  A high-resolution 2D J-resolved NMR detection technique for metabolite analyses of biological samples 
Scientific Reports  2015;5:8390.
NMR spectroscopy is a commonly used technique for metabolite analyses. Due to the observed macroscopic magnetic susceptibility in biological tissues, current NMR acquisitions in measurements of biological tissues are generally performed on tissue extracts using liquid NMR or on tissues using magic-angle spinning techniques. In this study, we propose an NMR method to achieve high-resolution J-resolved information for metabolite analyses directly from intact biological samples. A dramatic improvement in spectral resolution is evident in our contrastive demonstrations on a sample of pig brain tissue. Metabolite analyses for a postmortem fish from fresh to decayed statuses are presented to further reveal the capability of the proposed method. This method is a previously-unreported high-resolution 2D J-resolved spectroscopy for biological applications without specialised hardware requirements or complicated sample pretreatments. It provides a significant contribution to metabolite analyses of biological samples, and may be potentially applicable to in vivo samples. Furthermore, this method also can be applied to measurements of semisolid and viscous samples.
PMCID: PMC4323641  PMID: 25670027
4.  Serpin Treatment Suppresses Inflammatory Vascular Lesions in Temporal Artery Implants (TAI) from Patients with Giant Cell Arteritis 
PLoS ONE  2015;10(2):e0115482.
Giant cell arteritis (GCA) and Takayasu’s disease are inflammatory vasculitic syndromes (IVS) causing sudden blindness and widespread arterial obstruction and aneurysm formation. Glucocorticoids and aspirin are mainstays of treatment, predominantly targeting T cells. Serp-1, a Myxomavirus-derived serpin, blocks macrophage and T cells in a wide range of animal models. Serp-1 also reduced markers of myocardial injury in a Phase IIa clinical trial for unstable coronary disease. In recent work, we detected improved survival and decreased arterial inflammation in a mouse Herpesvirus model of IVS. Here we examine Serp-1 treatment of human temporal artery (TA) biopsies from patients with suspected TA GCA arteritis after implant (TAI) into the aorta of immunodeficient SCID (severe combined immunodeficiency) mice. TAI positive for arteritis (GCApos) had significantly increased inflammation and plaque when compared to negative TAI (GCAneg). Serp-1 significantly reduced intimal inflammation and CD11b+ cell infiltrates in TAI, with reduced splenocyte Th1, Th17, and Treg. Splenocytes from mice with GCApos grafts had increased gene expression for interleukin-1beta (IL-1β), IL-17, and CD25 and decreased Factor II. Serp-1 decreased IL-1β expression. In conclusion, GCApos TAI xenografts in mice provide a viable disease model and have increased intimal inflammation as expected and Serp-1 significantly reduces vascular inflammatory lesions with reduced IL-1β.
PMCID: PMC4319900  PMID: 25658487
5.  The bacterial interactions in the nasopharynx of children receiving adenoidectomy 
Biomedicine  2015;5(1):6.
Staphylococcus aureus, Streptococcus pneumoniae, and Haemophilus influenzae are the common pathogens that colonize in the nasopharynx of children. Polymicrobial interactions are thought to play an important role in different sites throughout the human body. However, there are currently very few studies that investigate the interactions between S. aureus, S. pneumoniae, and H. influenzae in the nasopharynx. We retrospectively analyzed the adenoid tissue culture from 269 children who received adenoidectomy. S. aureus, S. pneumoniae, and H. influenzae constituted the major microorganisms which were cultured from these adenoidectomies, at 23.4%, 21.6%, and 18.2%, respectively. S. pneumoniae and H. influenzae were the most prevalent in the preschool-aged children (3 < age ≤ 6), whereas S. aureus was more prevalent in infants and toddlers (age ≤ 3) and school-aged children (age > 6). Bacterial interference was found between S. aureus and S. pneumoniae and between S. aureus and H. influenzae, whereas there was an association found between S. pneumoniae and H. influenzae. The synergism and antagonism among these three species are investigated in the following paper, with the possible mechanisms involved in these interactions also discussed.
PMCID: PMC4326659
Adenoid;; Bacterial interactions;; Haemophilus influenzae;; Staphylococcus aureus;; Streptococcus; pneumoniae
6.  Snail Recruits Ring1B to Mediate Transcriptional Repression and Cell Migration in Pancreatic Cancer Cells 
Cancer research  2014;74(16):4353-4363.
Transcriptional repressor Snail is a master regulator of epithelial–mesenchymal transition (EMT), yet the epigenetic mechanism governing Snail to induce EMT is not well understood. Here, we report that in pancreatic ductal adenocarcinoma (PDAC), elevated levels of the ubiquitin E3 ligase Ring1B and Snail, along with elevated monoubiquitination of H2A at K119 (H2AK119Ub1), are highly correlated with poor survival. Mechanistic investigations identified Ring1B as a Snail-interacting protein and showed that the carboxyl zinc fingers of Snail recruit Ring1B and its paralog Ring1A to repress its target promoters. Simultaneous depletion of Ring1A and Ring1B in pancreatic cancer cells decreased Snail binding to the target chromatin, abolished H2AK119Ub1 modification, and thereby compromised Snail-mediated transcriptional repression and cell migration. We found that Ring1B and the SNAG-associated chromatin modifier EZH2 formed distinct protein complexes with Snail and that EZH2 was required for Snail-Ring1A/B recruitment to the target promoter. Collectively, our results unravel an epigenetic mechanism underlying transcriptional repression by Snail, suggest Ring1A/B as a candidate therapeutic target, and identify H2AK119Ub1 as a potential biomarker for PDAC diagnosis and prognosis. Cancer Res; 74(16); 4353-63. ©2014 AACR
PMCID: PMC4285394  PMID: 24903147
7.  Precision and Agreement of Corneal Power Measurements Obtained Using a New Corneal Topographer OphthaTOP 
PLoS ONE  2015;10(1):e109414.
To evaluate repeatability and reproducibility of anterior corneal power measurements obtained with a new corneal topographer OphthaTOP (Hummel AG, Germany) and agreement with measurements by a rotating Scheimpflug camera (Pentacam HR, Oculus, Germany) and an automated keratometer (IOLMaster, Carl Zeiss Meditec, Germany).
The right eyes of 79 healthy subjects were prospectively measured three times with all three devices. Another examiner performed three additional scans with the OphthaTOP in the same session. Within one week, the first examiner repeated the measurements using the OphthaTOP. The flat simulated keratometry (Kf), steep K (Ks), mean K (Km), J0, and J45 were noted. Repeatability and reproducibility of measurements were assessed by within-subject standard deviation (Sw), repeatability (2.77 Sw), coefficient of variation (CoV), and intraclass correlation coefficient (ICC). Agreement between devices was assessed using 95% limits of agreement (LoA).
Intraobserver repeatability and interobserver and intersession reproducibility of all measured parameters showed a 2.77 Sw of 0.29 diopter or less, a CoV of less than 0.24%, and an ICC of more than 0.906. Statistically significant differences (P<0.001) were found between the parameters analyzed by the three devices, except J0 and J45. The mean differences between OphthaTOP and the other two devices were small, and the 95% LoA was narrow for all results.
The OphthaTOP showed excellent intraobserver repeatability and interobserver and intersession reproducibility of corneal power measurements. Good agreements with the other two devices in these parameters were found in healthy eyes.
PMCID: PMC4283956  PMID: 25559203
8.  Rats are the smart choice: Rationale for a renewed focus on rats in behavioral genetics 
Neuropharmacology  2013;76(0 0):10.1016/j.neuropharm.2013.05.047.
Due in part to their rich behavioral repertoire rats have been widely used in behavioral studies of drug abuse-related traits for decades. However, the mouse became the model of choice for researchers exploring the genetic underpinnings of addiction after the first mouse study was published demonstrating the capability of engineering the mouse genome through embryonic stem cell technology. The sequencing of the mouse genome and more recent re-sequencing of numerous inbred mouse strains has further cemented the status of mice as the premier mammalian organism for genetic studies. As a result, many of the behavioral paradigms initially developed and optimized for rats have been adapted to mice. However, numerous complex and interesting drug abuse-related behaviors that can be studied in rats are very difficult or impossible to adapt for use in mice, impeding the genetic dissection of those traits. Now, technological advances have removed many of the historical limitations of genetic studies in rats. For instance, the rat genome has been sequenced and many inbred rat strains are now being re-sequenced and outbred rat stocks are being used to fine-map QTLs. In addition, it is now possible to create “knockout” rats using zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALENs) and related techniques. Thus, rats can now be used to perform quantitative genetic studies of sophisticated behaviors that have been difficult or impossible to study in mice.
PMCID: PMC3823679  PMID: 23791960
QTL; GWAS; rats; behavioral genetics; addiction
9.  Comparative Genomics of Oral Isolates of Streptococcus mutans by in silico Genome Subtraction Does Not Reveal Accessory DNA Associated with Severe Early Childhood Caries 
Comparative genomics is a popular method for the identification of microbial virulence determinants, especially since the sequencing of a large number of whole bacterial genomes from pathogenic and non-pathogenic strains has become relatively inexpensive. The bioinformatics pipelines for comparative genomics usually include gene prediction and annotation and can require significant computer power. To circumvent this, we developed a rapid method for genome-scale in silico subtractive hybridization, based on blastn and independent of feature identification and annotation. Whole genome comparisons by in silico genome subtraction were performed to identify genetic loci specific to Streptococcus mutans strains associated with severe early childhood caries (S-ECC), compared to strains isolated from caries-free (CF) children.
The genome similarity of the 20 S. mutans strains included in this study, calculated by Simrank k-mer sharing, ranged from 79.5 to 90.9%, confirming this is a genetically heterogeneous group of strains. We identified strain-specific genetic elements in 19 strains, with sizes ranging from 200 bp to 39 kb. These elements contained protein-coding regions with functions mostly associated with mobile DNA. We did not, however, identify any genetic loci consistently associated with dental caries, i.e., shared by all the S-ECC strains and absent in the CF strains. Conversely, we did not identify any genetic loci specific with the healthy group. Comparison of previously published genomes from pathogenic and carriage strains of Neisseria meningitidis with our in silico genome subtraction yielded the same set of genes specific to the pathogenic strains, thus validating our method.
Our results suggest that S. mutans strains derived from caries active or caries free dentitions cannot be differentiated based on the presence or absence of specific genetic elements. Our in silico genome subtraction method is available as the Microbial Genome Comparison (MGC) tool, with a user-friendly JAVA graphical interface.
PMCID: PMC3940162  PMID: 24291226
Comparative genomics; Software; Streptococcus mutans; Dental caries; Virulence; Pathogenesis
10.  miR-150-5p Inhibits Hepatoma Cell Migration and Invasion by Targeting MMP14 
PLoS ONE  2014;9(12):e115577.
Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related mortality worldwide. Despite progress in diagnostics and treatment of HCC, its prognosis remains poor because the molecular mechanisms underlying hepatocarcinogenesis are not well understood. In the study, we focused on identifying the role of miRNAs in HCC progression. miRNA microarray was used to analyze the differentially expressed miRNAs, and the results were validated by qPCR. We found that the miR-150-5p expression is down-regulated in HCC tissues compared with pair non-tumor tissues. miR-150-5p expression is also decreased in metastatic cancer tissues compared with pair primary tissues, indicating that miR-150-5p may be involved in HCC metastasis. Functionally, miR-150-5p inhibition significantly promotes hepatoma cell migration and invasion, whereas miR-150-5p overexpression suppresses cancer cell migration and invasion in vitro. The matrix metalloproteinase 14 (MMP14) is identified as a new target gene of miR-150-5p. miR-150-5p markedly inhibits MMP14 expression in hepatoma cells, and miR-150-5p expression is negative correlation with MMP14 expression in vivo. More important, re-expression of MMP14 in hepatoma cells partially reverses the effect of miR-150-5p in inhibiting cell invasion.
PMCID: PMC4280173  PMID: 25549355
11.  Western white pine SNP discovery and high-throughput genotyping for breeding and conservation applications 
BMC Plant Biology  2014;14(1):380.
Western white pine (WWP, Pinus monticola Douglas ex D. Don) is of high interest in forest breeding and conservation because of its high susceptibility to the invasive disease white pine blister rust (WPBR, caused by the fungus Cronartium ribicola J. C. Fisch). However, WWP lacks genomic resource development and is evolutionarily far away from plants with available draft genome sequences. Here we report a single nucleotide polymorphism (SNP) study by bulked segregation-based RNA-Seq analysis.
A collection of resistance germplasm was used for construction of cDNA libraries and SNP genotyping. Approximately 36–89 million 2 × 100-bp reads were obtained per library and de-novo assembly generated the first shoot-tip reference transcriptome containing a total of 54,661 unique transcripts. Bioinformatic SNP detection identified >100,000 high quality SNPs in three expressed candidate gene groups: Pinus highly conserved genes (HCGs), differential expressed genes (DEGs) in plant defense response, and resistance gene analogs (RGAs). To estimate efficiency of in-silico SNP discovery, genotyping assay was developed by using Sequenom iPlex and it unveiled SNP success rates from 40.1% to 61.1%. SNP clustering analyses consistently revealed distinct populations, each composed of multiple full-sib seed families by parentage assignment in the WWP germplasm collection. Linkage disequilibrium (LD) analysis identified six genes in significant association with major gene (Cr2) resistance, including three RGAs (two NBS-LRR genes and one receptor-like protein kinase -RLK gene), two HCGs, and one DEG. At least one SNP locus provided an excellent marker for Cr2 selection across P. monticola populations.
The WWP shoot tip transcriptome and those validated SNP markers provide novel genomic resources for genetic, evolutionary and ecological studies. SNP loci of those candidate genes associated with resistant phenotypes can be used as positional and functional variation sites for further characterization of WWP major gene resistance against C. ribicola. Our results demonstrate that integration of RNA-seq-based transcriptome analysis and high-throughput genotyping is an effective approach for discovery of a large number of nucleotide variations and for identification of functional gene variants associated with adaptive traits in a non-model species.
Electronic supplementary material
The online version of this article (doi:10.1186/s12870-014-0380-6) contains supplementary material, which is available to authorized users.
PMCID: PMC4302426  PMID: 25547170
Five-needle pine; Genotyping array; Linkage disequilibrium; Marker-based selection; Pedigree reconstruction
12.  Carbon Disulfide Mediates Socially-Acquired Nicotine Self-Administration 
PLoS ONE  2014;9(12):e115222.
The social environment plays a critical role in smoking initiation as well as relapse. We previously reported that rats acquired nicotine self-administration with an olfactogustatory cue only when another rat consuming the same cue was present during self-administration. Because carbon disulfide (CS2) mediates social learning of food preference in rodents, we hypothesized that socially acquired nicotine self-administration is also mediated by CS2. We tested this hypothesis by placing female adolescent Sprague-Dawley rats in operant chambers equipped with two lickometers. Licking on the active spout meeting a fixed-ratio 10 schedule triggered the concurrent delivery of an i.v. infusion (saline, or 30 µg/kg nicotine, free base) and an appetitive olfactogustatory cue containing CS2 (0–500 ppm). Rats that self-administered nicotine with the olfactogustatory cue alone licked less on the active spout than on the inactive spout. Adding CS2 to the olfactogustatory cue reversed the preference for the spouts. The group that received 500 ppm CS2 and the olfactogustatory cue obtained a significantly greater number of nicotine infusions than other groups. After extinction training, the original self-administration context reinstated nicotine-seeking behavior in all nicotine groups. In addition, in rats that received the olfactogustatory cue and 500 ppm CS2 during SA, a social environment where the nicotine-associated olfactory cue is present, induced much stronger drug-seeking behavior compared to a social environment lacking the olfactogustatory cue. These data established that CS2 is a critical signal that mediates social learning of nicotine self-administration with olfactogustatory cues in rodents. Additionally, these data showed that the social context can further enhance the drug-seeking behavior induced by the drug-taking environment.
PMCID: PMC4274004  PMID: 25532105
13.  Measuring Protein-Ligand Interactions Using Liquid Sample Desorption Electrospray Ionization Mass Spectrometry 
Analytical chemistry  2013;85(24):11966-11972.
We have shown previously that liquid sample desorption electrospray ionization-mass spectrometry (DESI-MS) is able to measure large proteins and noncovalently-bound protein complexes (to 150 kDa) (Ferguson et al., Anal. Chem. 2011, 83, 6468-6473). In this study, we further investigate the application of liquid sample DESI-MS to probe protein-ligand interactions. Liquid sample DESI allows the direct formation of intact protein-ligand complex ions by spraying ligands toward separate protein sample solutions. This type of “reactive” DESI methodology can provide rapid information on binding stiochiometry, selectivity, and kinetics, as demonstrated by the binding of ribonuclease A (RNaseA, 13.7 kDa) with cytidine nucleotide ligands and the binding of lysozyme (14.3 kDa) with acetyl chitose ligands. A higher throughput method for ligand screening by liquid sample DESI was demonstrated, in which different ligands were sequentially injected as a segmented flow for DESI ionization. Furthermore, supercharging to enhance analyte charge can be integrated with liquid sample DESI-MS without interfering with the formation of protein-ligand complexes.
PMCID: PMC3901310  PMID: 24237005
Desorption electrospray ionization; mass spectrometry; noncovalent protein-ligand complexes; ligand screening; supercharging
14.  Meta-analysis of the association between CCAAT/enhancer binding protein-ε polymorphism and the risk of childhood acute lymphoblastic leukemia 
CEBPE rs2239633 polymorphism has been implicated in susceptibility to childhood acute lymphoblastic leukemia (ALL) risk. Several studies investigated the association of this polymorphism with ALL in different populations. However, the results were contradictory. A meta-analysis was conducted to assess the association between CEBPE rs2239633 polymorphism and ALL susceptibility. Databases including Pubmed, EMBASE, Chinese National Knowledge Infrastructure (CNKI) and Wangfang were searched to find relevant studies. Odds ratios (ORs) with 95% confidence intervals (CIs) were used to assess the strength of associations. A random-effects model was used. A significant association was found between CEBPE rs2239633 polymorphism and childhood ALL (OR = 1.19, 95% CI, 1.11-1.28). In the subgroup analyses by ethnicity, the significant association was found among Caucasians (OR = 1.19, 95% CI, 1.09-1.30) and Hispanics (OR = 1.39, 95% CI, 1.18-1.63), but not in Asians (OR = 1.05, 95% CI, 0.90-1.22). In the subgroup analysis by histology, B-cell ALL risk (OR = 1.29, 95% CI, 1.15-1.44) and B hyperdiploid ALL risk (OR = 1.84, 95% CI, 1.40-2.43) were increased. Our results suggested that CEBPE rs2239633 polymorphism conferred a risk factor of childhood ALL.
PMCID: PMC4307517  PMID: 25664070
Acute lymphoblastic leukemia; CEBPE; meta-analysis
15.  Purification and Functional Characterization of a Protein: Bombyx mori Human Growth Hormone Like Protein in Silkworm Pupa 
PLoS ONE  2014;9(12):e114351.
Human growth hormone (hGH) is a peptide hormone secreted by eosinophils of the human anterior pituitary, and a regulatory factor for a variety of metabolic pathways. A 30-kD protein from the pupa stage of silkworm was detected by Western blotting and confirmed by immunoprecipitation based on its ability to bind to anti-hGH antibody. This protein, named BmhGH-like protein, was purified from fresh silkworm pupas through low-temperature homogenization, filtration, and centrifugation to remove large impurity particles. The supernatants were precipitated, resuspended, and passed through a molecular sieve. Further purification by affinity chromatography and two-dimensional electrophoresis resulted in pure protein for analysis by MS MALDI-TOF-MS analysis. An alignment with predicted proteins indicated that BmhGH-like protein consisted of two lipoproteins, which we named hGH-L1 and hGH-L2. These proteins belong to the β-trefoil superfamily, with β domains similar to the spatial structure of hGH. Assays with K562 cells demonstrated that these proteins could promote cell division in vitro. To further validate the growth-promoting effects, hGH-L2 was cloned from pupa cDNA to create recombinant silkworm baculovirus vBmNPV-hGH-L2, which was used to infect silkworm BmN cells at low titer. Flow cytometric analysis demonstrated that the protein shortened the G0/G1 phase of the cells, and enabled the cells to rapidly traverse the G1/S phase transition point to enter S phase and promote cell division. Discovery of hGH-like protein in silkworm will once again arouse people’s interest in the potential medicinal value of silkworm and establish the basis for the development of new hormone drugs.
PMCID: PMC4254979  PMID: 25469649
16.  Trihelix transcription factor GT-4 mediates salt tolerance via interaction with TEM2 in Arabidopsis 
BMC Plant Biology  2014;14(1):339.
Trihelix transcription factor family is plant-specific and plays important roles in developmental processes. However, their function in abiotic stress response is largely unclear.
We studied one member GT-4 from Arabidopsis in relation to salt stress response. GT-4 expression is induced by salt stress and GT-4 protein is localized in nucleus and cytoplasm. GT-4 acts as a transcriptional activator and its C-terminal end is the activation domain. The protein can bind to the cis-elements GT-3 box, GT-3b box and MRE4. GT-4 confers enhanced salt tolerance in Arabidopsis likely through direct binding to the promoter and activation of Cor15A, in addition to possible regulation of other relevant genes. The gt-4 mutant shows salt sensitivity. TEM2, a member of AP2/ERF family was identified to interact with GT-4 in yeast two-hybrid, BiFC and Co-IP assays. Loss-of-function of TEM2 exerts no significant difference on salt tolerance or Cor15A expression in Arabidopsis. However, double mutant gt-4/tem2 shows greater sensitivity to salt stress and lower transcript level of Cor15A than gt-4 single mutant. GT-4 plus TEM2 can synergistically increase the promoter activity of Cor15A.
GT-4 interacts with TEM2 and then co-regulates the salt responsive gene Cor15A to improve salt stress tolerance.
Electronic supplementary material
The online version of this article (doi:10.1186/s12870-014-0339-7) contains supplementary material, which is available to authorized users.
PMCID: PMC4267404  PMID: 25465615
Salt stress; Trihelix transcription factor; GT-4; TEM2
17.  Epstein-Barr Virus Infection Induces Indoleamine 2,3-Dioxygenase Expression in Human Monocyte-Derived Macrophages through p38/Mitogen-Activated Protein Kinase and NF-κB Pathways: Impairment in T Cell Functions 
Journal of Virology  2014;88(12):6660-6671.
Epstein-Barr virus (EBV) infection has been observed in tumor-infiltrated macrophages, but its infection effects on macrophage immune functions are poorly understood. Here, we showed that some macrophages in the tumor stroma of nasopharyngeal carcinoma (NPC) tissue expressed the immunosuppressive protein indoleamine 2,3-dioxygenase (IDO) more strongly than did tumor cells. EBV infection induced mRNA, protein, and enzymatic activity of IDO in human monocyte-derived macrophages (MDMs). Infection increased the production of tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6), whereas the neutralizing antibodies against TNF-α and IL-6 inhibited IDO induction. EBV infection also activated the mitogen-activated protein kinase (MAPK) p38 and NF-κB, and the inhibition of these two pathways with SB202190 and SN50 almost abrogated TNF-α and IL-6 production and inhibited IDO production. Moreover, the activation of IDO in response to EBV infection of MDMs suppressed the proliferation of T cells and impaired the cytotoxic activity of CD8+ T cells, whereas the inhibition of IDO activity with 1-methyl-l-tryptophan (1-MT) did not affect T cell proliferation and function. These findings indicate that EBV-induced IDO expression in MDMs is substantially mediated by IL-6- and TNF-α-dependent mechanisms via the p38/MAPK and NF-κB pathways, suggesting that a possible role of EBV-mediated IDO expression in tumor stroma of NPC may be to create a microenvironment of suppressed T cell immune responses.
IMPORTANCE CD8+ cytotoxic T lymphocytes (CTLs) play an important role in the control of viral infections and destroy tumor cells. Activation of the tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase (IDO) in cancer tissues facilitates immune escape by the impairment of CTL functions. IDO expression was observed in some macrophages of the tumor stroma of nasopharyngeal carcinoma (NPC) tissue, and IDO could be induced in Epstein-Barr virus (EBV)-infected human monocyte-derived macrophages (MDMs). NPC cells and macrophages have been found to produce IDO in a gamma interferon (IFN-γ)-dependent manner. Instead, EBV-induced IDO expression in MDMs is substantially mediated by IL-6- and TNF-α-dependent mechanisms via the p38/MAPK and NF-κB pathways, which suppressed the proliferation of T cells and impaired the cytotoxic activity of CD8+ T cells. This finding provides a new interpretation of the mechanism of immune escape of EBV and shows the immunosuppressive role of EBV-mediated IDO expression in tumor stroma of NPC.
PMCID: PMC4054364  PMID: 24696473
18.  Laparoscopic vs open D2 gastrectomy for locally advanced gastric cancer: A meta-analysis 
World Journal of Gastroenterology : WJG  2014;20(44):16750-16764.
AIM: To conduct a meta-analysis comparing laparoscopic (LGD2) and open D2 gastrectomies (OGD2) for the treatment of advanced gastric cancer (AGC).
METHODS: Randomized controlled trials (RCTs) and non-RCTs comparing LGD2 with OGD2 for AGC treatment, published between 1 January 2000 and 12 January 2013, were identified in the PubMed, Embase, and Cochrane Library databases. Primary endpoints included operative outcomes (operative time, intraoperative blood loss, and conversion rate), postoperative outcomes (postoperative analgesic consumption, time to first ambulation, time to first flatus, time to first oral intake, postoperative hospital stay length, postoperative morbidity, incidence of reoperation, and postoperative mortality), and oncologic outcomes (the number of lymph nodes harvested, tumor recurrence and metastasis, disease-free rates, and overall survival rates). The Cochrane Collaboration tools and the modified Newcastle-Ottawa scale were used to assess the quality and risk of bias of RCTs and non-RCTs in the study. Subgroup analyses were conducted to explore the incidence rate of various postoperative morbidities as well as recurrence and metastasis patterns. A Begg’s test was used to evaluate the publication bias.
RESULTS: One RCT and 13 non-RCTs totaling 2596 patients were included in the meta-analysis. LGD2 in comparison to OGD2 showed lower intraoperative blood loss [weighted mean difference (WMD) = -137.87 mL, 95%CI: -164.41--111.33; P < 0.01], lower analgesic consumption (WMD = -1.94, 95%CI: -2.50--1.38; P < 0.01), shorter times to first ambulation (WMD = -1.03 d, 95%CI: -1.90--0.16; P < 0.05), flatus (WMD = -0.98 d, 95%CI: -1.30--0.66; P < 0.01), and oral intake (WMD = -0.85 d, 95%CI: -1.67--0.03; P < 0.05), shorter hospitalization (WMD = -3.08 d, 95%CI: -4.38--1.78; P < 0.01), and lower postoperative morbidity (odds ratio = 0.78, 95%CI: 0.61-0.99; P < 0.05). No significant differences were observed between LGD2 and OGD2 for the following criteria: reoperation incidence, postoperative mortality, number of harvested lymph nodes, tumor recurrence/metastasis, or three- or five-year disease-free and overall survival rates. However, LGD2 had longer operative times (WMD = 57.06 min, 95%CI: 41.87-72.25; P < 0.01).
CONCLUSION: Although a technically demanding and time-consuming procedure, LGD2 may be safe and effective, and offer some advantages over OGD2 for treatment of locally AGC.
PMCID: PMC4248223  PMID: 25469048
D2 lymph node dissection; Gastrectomy; Gastric cancer; Laparoscopy; Meta-analysis
19.  Integration of online digestion and electrolytic reduction with mass spectrometry for rapid disulfide-containing protein structural analysis 
Bottom-up structural analysis of disulfide-bond containing proteins usually involves time-consuming offline enzymatic digestion, chemical reduction and thiol protection prior to mass spectrometric detection, which takes many hours. This paper presents an expedited bottom-up approach, employing desorption electrospray ionization-mass spectrometry (DESI-MS) coupled with online pepsin digestion and online electrochemical reduction of disulfide bonds. Peptides are generated in high digestion yield as its precursor protein in acidic aqueous solution flows through a pepsin column, which can undergo direct electrolysis. The electrolytic behaviors of peptides, as online monitored by DESI-MS, suggest the presence or absence of disulfide bonds in the peptides, and also provide information to relate disulfide bond-containing peptide precursors to their corresponding reduced products. Furthermore, selective electrolysis simply using different reduction potentials can be adopted to generate either partially or fully reduced peptides to assist disulfide bond mapping. In addition, it turns out that DESI is suitable for ionizing peptides in water without organic solvent additives (organic solvent additives would not be compatible with the use of pepsin column). The feasibility of this method was demonstrated using insulin, a protein carrying three pairs of disulfide-bonds as an example, in which all disulfide bond linkages and most of the protein sequence were successfully determined. Strikingly, this method shortens the sample digestion, reduction and MS detection from hours to less than 7 min, which could be of high value in high-throughput proteomics research.
PMCID: PMC4240030  PMID: 25419170
Mass spectrometry; Electrochemistry; Disulfide bond mapping; Protein sequencing; Selective reduction
20.  The efficacy and safety of certolizumab pegol (CZP) in the treatment of active rheumatoid arthritis (RA): a meta-analysis from nine randomized controlled trials 
Objective: Certolizumab pegol (CZP) is a novel anti-TNF agent that is used for patients with moderate to severe active rheumatoid arthritis (RA). However, the efficacy of CZP in RA remains controversial. Thus, we performed this meta-analysis to assess the efficacy and safety of CZP in the treatment of RA patients. Methods: Eligible studies were randomized controlled trials (RCTs) that evaluated the efficacy and safe of CZP in the patients with active RA. The primary outcome was American College of Rheumatology 20% (ACR20), and secondary outcome were ACR50, ACR70, disease activity, patient-reported outcomes (PROs), and adverse events. A fixed-effect model or random-effect model was used to pool the estimates, depending on the absence or presence of heterogeneity among the included studies. Results: Nine RCTs with a total of 5228 patients were included in this meta-analysis, and all of the patients were administered CZP or placebo. The pooled results showed that CZP significantly improved the ACR20, ACR50, ACR70 response rates, and physical function. CZP was associated with a statistically significant reduction in Disease Activity Score in 28 joints-Erythrocyte sedimentation rate, arthritis pain, and fatigue. Patients who received CZP treatment did not have a higher incidence of treatment-related adverse events, no matter in any intensity. Conclusions: CZP 200 or 400mg in the treatment of active RA significantly reduced the RA signs and symptoms, and improved physical function as compared with the placebo. More large-scale RCTs are needed to evaluate the long-term efficacy and safety of CZP in the treatment of active RA.
PMCID: PMC4276153  PMID: 25550895
Certolizumab pegol; rheumatoid arthritis; meta-analysis
21.  Effect of (-)-epigallocatechin-3-gallate in preventing bone loss in ovariectomized rats and possible mechanisms 
Despite recent developments reported in studies of (-)-epigallocatechin-3-gallate (EGCG), its early preventive effect of mitigating bone loss is not well understood. We investigated the effect of EGCG in preventing bone loss in ovariectomized (OVX) female rats, and explored the possible underlying mechanisms. Twelve-week-old female Sprague-Dawley rats, were divided into 3 groups: group A received intraperitoneal EGCG for 12 consecutive weeks, begun 3 days after ovariectomy; group B received ovariectomy alone; group C, received a sham operation. At the end of the experiment, tibias and femurs were harvested for: (1) micro-CT scanning and measurement of bone mineral density (BMD) and bone morphological parameters; (2) a 3-point bending test; (3) HE staining and an immunohistological study investigating Sema4D expression. Results: The BMD and BV/TV of group A were significantly higher than for the OVX group. The trabecular separation (Tb.Sp) of group A was significantly lower than for group B. Results from the 3-point bending test showed no statistical significance among all the groups. Bone histological studies indicated that trabecular bone was denser in group C, while group B had less dense trabecular bone, and the bone morphological status of group A was intermediate between groups A and C. The immunohistological study demonstrated that Sema4D was more highly expressed as a percentage of the brown-stained area in group B than in the other 2 groups. Conclusion: EGCG had a positive effect on mitigating bone loss in ovariectomized rats, and it inhibited Sema4D expression in bone tissue. Early stage supplementation of EGCG at a dose of 10 mg/kg/day after the onset of ovariectomy did not entirely eliminate bone loss.
PMCID: PMC4276187  PMID: 25550929
EGCG; osteoporosis; bone loss; microarchitecture; BMD; Sema4D
22.  The Relationship between Visual Field Global Indices and Retinal Nerve Fiber Layer Thickness in Healthy Myopes 
Journal of Ophthalmology  2014;2014:431901.
The aim of the current study was to investigate the association between the thickness of the retinal nerve fiber layer (RNFL) and central visual field indices in otherwise healthy myopes. In total, 57 otherwise healthy subjects were cross-sectionally studied. General ophthalmic examinations, refractive measurements, RNFL thickness by spectral domain optical coherence tomography (OCT), and central visual fields were examined. Linear models were used to assess the associations. In this young and mid-aged population, the mean spherical equivalent was −4.79 (SD 1.66) and −4.59 (SD 1.88) diopters in the right and left eyes, respectively. Approximately 7% to 14% of the eyes showed the average RNFL thickness out of the normal range. The temporal RNFL was remarkably thicker, whereas the nasal RNFL was thinner. The higher the refractive error, the thinner the RNFL thickness. A thicker overall RNFL was significantly associated with decreased mean sensitivity and increased mean defect, and further adjustments for age, sex, refractive error, optic disk area, or ocular magnification did not change the association. Although nonpathologic myopia does not significantly affect central visual field global indices, its effects on the RNFL may be linked with performance on the central visual field test.
PMCID: PMC4241731  PMID: 25436141
23.  An improved independent component analysis model for 3D chromatogram separation and its solution by multi-areas genetic algorithm 
BMC Bioinformatics  2014;15(Suppl 12):S8.
The 3D chromatogram generated by High Performance Liquid Chromatography-Diode Array Detector (HPLC-DAD) has been researched widely in the field of herbal medicine, grape wine, agriculture, petroleum and so on. Currently, most of the methods used for separating a 3D chromatogram need to know the compounds' number in advance, which could be impossible especially when the compounds are complex or white noise exist. New method which extracts compounds from 3D chromatogram directly is needed.
In this paper, a new separation model named parallel Independent Component Analysis constrained by Reference Curve (pICARC) was proposed to transform the separation problem to a multi-parameter optimization issue. It was not necessary to know the number of compounds in the optimization. In order to find all the solutions, an algorithm named multi-areas Genetic Algorithm (mGA) was proposed, where multiple areas of candidate solutions were constructed according to the fitness and distances among the chromosomes.
Simulations and experiments on a real life HPLC-DAD data set were used to demonstrate our method and its effectiveness. Through simulations, it can be seen that our method can separate 3D chromatogram to chromatogram peaks and spectra successfully even when they severely overlapped. It is also shown by the experiments that our method is effective to solve real HPLC-DAD data set.
Our method can separate 3D chromatogram successfully without knowing the compounds' number in advance, which is fast and effective.
PMCID: PMC4251091  PMID: 25474487
24.  Inhibition of angiogenesis by a novel neutralizing antibody targeting human VEGFR-3 
mAbs  2013;5(6):956-961.
Vascular endothelial growth factor receptor 3 (VEGFR-3) is a receptor for the vascular endothelial growth factor C and D (VEGF-C and D) and plays a critical role in the development of embryonic vascular system and regulation of tumor lymphangiogenesis. In this report, we generated a novel panel of 17 monoclonal antibodies (mAbs) against human VEGFR-3 and determined their ability to inhibit the proliferation of human erythroleukemia (HEL) cells and angiogenesis of chick embryo chorioallantoic membrane (CAM). Among these mAbs, BDD073 was demonstrated to inhibit the interaction of soluble VEGFR-3 with VEGF-D and the proliferation of HEL cells. Furthermore, in chick embryo CAM angiogenesis experiments, the angiogenesis induced by recombinant glutathione-S-transferase-VEGF-D was decreased in the presence of antibody BDD073. These data suggest that this novel neutralizing antibody against human VEGFR-3 could be a tool for the investigations into the biology of VEGFR-3, and potentially a reagent for blocking VEGF-D-induced angiogenesis and lymphogenesis.
PMCID: PMC3896609  PMID: 23995616
VEGFR-3; VEGF-D; antibody; angiogenesis
25.  Metabolism of 2,2′,3,3′,6,6′-Hexachlorobiphenyl (PCB 136) Atropisomers in Tissue Slices from Phenobarbital or Dexamethasone-Induced Rats is Sex-Dependent 
Xenobiotica; the fate of foreign compounds in biological systems  2013;43(11):10.3109/00498254.2013.785626.
Chiral polychlorinated biphenyls (PCBs) such as PCB 136 enantioselectively sensitize the ryanodine receptor (RyR). In light of recent evidence that PCBs cause developmental neurotoxicity via RyR-dependent mechanisms, this suggests that enantioselective PCB metabolism may influence the developmental neurotoxicity of chiral PCBs. However, enantioselective disposition of PCBs has not been fully characterized.The effect of sex and cytochrome P450 (P450) enzyme induction on the enantioselective metabolism of PCB 136 was studied using liver tissue slices prepared from naïve control (CTL), phenobarbital (PB; CYP2B inducer) or dexamethasone (DEX; CYP3A inducer) pretreated adult Sprague-Dawley rats. PCB 136 metabolism was also examined in hippocampal slices derived from untreated rat pups.In liver tissue slices, hydroxylated PCB (OH-PCB) profiles depended on sex and inducer pretreatment, and OH-PCB levels followed the rank orders male > female and PB > DEX > CTL. In contrast, the enantiomeric enrichment of PCB 136 and its metabolites was independent of sex and inducer pretreatment. Only small amounts of PCB 136 partitioned into hippocampal tissue slices and no OH-PCB metabolites were detected.Our results suggest that enantioselective metabolism, sex and induction status of P450 enzymes in the liver may modulate the neurotoxic outcomes of developmental exposure to chiral PCBs.
PMCID: PMC3878182  PMID: 23581876
Sex differences; Cytochrome P450 enzymes; Atropisomeric enrichment

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