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1.  Identification of Candidate Reference Genes in Perennial Ryegrass for Quantitative RT-PCR under Various Abiotic Stress Conditions 
PLoS ONE  2014;9(4):e93724.
Background
Quantitative real-time reverse-transcriptase PCR (qRT-PCR) is an important technique for analyzing differences in gene expression due to its sensitivity, accuracy and specificity. However, the stability of the expression of reference genes is necessary to ensure accurate qRT-PCR assessment of expression in genes of interest. Perennial ryegrass (Lolium perenne L.) is important forage and turf grass species in temperate regions, but the expression stability of its reference genes under various stresses has not been well-studied.
Methodology/Principal Findings
In this study, 11 candidate reference genes were evaluated for use as controls in qRT-PCR to quantify gene expression in perennial ryegrass under drought, high salinity, heat, waterlogging, and ABA (abscisic acid) treatments. Four approaches – Delta CT, geNorm, BestKeeper and Normfinder were used to determine the stability of expression in these reference genes. The results are consistent with the idea that the best reference genes depend on the stress treatment under investigation. Eukaryotic initiation factor 4 alpha (eIF4A), Transcription elongation factor 1 (TEF1) and Tat binding protein-1 (TBP-1) were the three most stably expressed genes under drought stress and were also the three best genes for studying salt stress. eIF4A, TBP-1, and Ubiquitin-conjugating enzyme (E2) were the most suitable reference genes to study heat stress, while eIF4A, TEF1, and E2 were the three best reference genes for studying the effects of ABA. Finally, Ubiquitin (UBQ), TEF1, and eIF4A were the three best reference genes for waterlogging treatments.
Conclusions/Significance
These results will be helpful in choosing the best reference genes for use in studies related to various abiotic stresses in perennial ryegrass. The stability of expression in these reference genes will enable better normalization and quantification of the transcript levels for studies of gene expression in such studies.
doi:10.1371/journal.pone.0093724
PMCID: PMC3974806  PMID: 24699822
2.  Effect of Naphthalene Acetic Acid on Adventitious Root Development and Associated Physiological Changes in Stem Cutting of Hemarthria compressa 
PLoS ONE  2014;9(3):e90700.
In order to find a way to induce rooting on cuttings of Hemarthria compressa cv. Ya’an under controlled conditions, a project was carried out to study the effect of naphthalene acetic acid (NAA) on rooting in stem cuttings and related physiological changes during the rooting process of Hemarthria compressa. The cuttings were treated with five concentrations of NAA (0, 100, 200 300, 400 mg/l) at three soaking durations (10, 20, 30 minutes), and cuttings without treatment were considered as control. Samples were planted immediately into pots after treatment. IAA-oxidase (IAAO) activity, peroxidase (POD) activity and polyphenol oxidase (PPO) activity were determined after planting. Results showed that NAA had positive effect on rooting at the concentration of 200 mg/l compared to other concentrations at 30 days after planting (DAP). Among the three soaking durations, 20 minutes (min) of 200 mg/l NAA resulted in higher percentages of rooting, larger numbers of adventitious roots and heavier root dry weight per cutting. The lowest IAAO activity was obtained when soaked at 200 mg/l NAA for 20 min soaking duration. This was consistent with the best rooting ability, indicating that the lower IAAO activity, the higher POD activity and PPO activity could be used as an indicator of better rooting ability for whip grass cuttings and might serve as a good marker for rooting ability in cuttings.
doi:10.1371/journal.pone.0090700
PMCID: PMC3942460  PMID: 24595064
3.  Oleanolic acid and ursolic acid inhibit proliferation in transformed rat hepatic oval cells 
AIM: To investigate H2O2-induced promotion proliferation and malignant transformation in WB-F344 cells and anti-tumor effects of ursolic acid (UA) and oleanolic acid (OA).
METHODS: WB-F344 cells were continuously exposed to 7 x 10-7 mol/L H2O2 for 21 d. Observations of cell morphology, colony formation rates, flow cytometric analysis of cell cycle changes and aneuploidy formation indicated that H2O2 was able to induce malignant transformation of WB-F344 cells. We treated malignantly transformed WB-F344 cells with 4 μmol/L OA or 8 μmol/L UA for 72 h and analyzed the cell cycle distribution by flow cytometry.
RESULTS: MTT assay showed that 7 x 10-7 mol/L H2O2 decreased G1 phase subpopulation from 73.8% to 49.6% compared with the control group, and increased S phase subpopulation from 14.5% to 31.8% (P < 0.05 vs control group). Cell morphology showed that nucleus to cytoplasm ratio increased, many mitotic cells, prokaryotes and even tumor giant cells were shown in H2O2-induced WB-F344 cells. Fluorescence activated cell sorting analysis showed that WB-F344 cell aneuploidy increased to 12% following H2O2 treatment. Flow cytometric analysis of the transformed WB-F344 cells following treatment with OA (4 μmol/L) and UA (8 μmol/L) showed that OA increased G1 subpopulation to 68.6%, compared to 49.7% in unexposed cells. UA increased G1 subpopulation to 67.4% compared to 49.7% in unexposed cells (P < 0.05 vs H2O2 model group).
CONCLUSION: H2O2 causes the malignant transformation of WB-F344 cells. OA and UA exert anti-tumor effects by inhibiting the proliferation in malignantly transformed WB-F344 cells.
doi:10.3748/wjg.v20.i5.1348
PMCID: PMC3921518  PMID: 24574810
Oxidative stress; Hepatocarcinogenesis; Malignant transformation; Oleanolic acid; Ursolic acid
4.  Endoplasmic Reticulum Stress-Unfolding Protein Response-Apoptosis Cascade Causes Chondrodysplasia in a col2a1 p.Gly1170Ser Mutated Mouse Model 
PLoS ONE  2014;9(1):e86894.
The collagen type II alpha 1 (COL2A1) mutation causes severe skeletal malformations, but the pathogenic mechanisms of how this occurs are unclear. To understand how this may happen, a col2a1 p.Gly1170Ser mutated mouse model was constructed and in homozygotes, the chondrodysplasia phenotype was observed. Misfolded procollagen was largely synthesized and retained in dilated endoplasmic reticulum and the endoplasmic reticulum stress (ERS)-unfolded protein response (UPR)-apoptosis cascade was activated. Apoptosis occurred prior to hypertrophy, prevented the formation of a hypertrophic zone, disrupted normal chondrogenic signaling pathways, and eventually caused chondrodysplasia. Heterozygotes had normal phenotypes and endoplasmic reticulum stress intensity was limited with no abnormal apoptosis detected. Our results suggest that earlier chondrocyte death was related to the ERS-UPR-apoptosis cascade and that this was the chief cause of chondrodysplaia. The col2a1 p.Gly1170Ser mutated mouse model offered a novel connection between misfolded collagen and skeletal malformation. Further investigation of this mouse mutant model can help us understand mechanisms of type II collagenopathies.
doi:10.1371/journal.pone.0086894
PMCID: PMC3903611  PMID: 24475193
5.  Premature Attraction of Pollinators to Inaccessible Figs of Ficus altissima: A Search for Ecological and Evolutionary Consequences 
PLoS ONE  2014;9(1):e86735.
Adult life spans of only one or two days characterise life cycles of the fig wasps (Agaonidae) that pollinate fig trees (Ficus spp., Moraceae). Selection is expected to favour traits that maximise the value of the timing of encounters between such mutualistic partners, and fig wasps are usually only attracted to their hosts by species- and developmental-stage specific volatiles released from figs at the time when they are ready to be entered, oviposited in and pollinated. We found that Ficus altissima is exceptional, because it has persistent tight-fitting bud covers that prevent its Eupristina altissima pollinator (and a second species of ‘cheater’ agaonid) from entering its figs for several days after they start to be attracted. We examined the consequences of delayed entry for the figs and fig wasps and tested whether delayed entry has been selected to increase adult longevity. We found that older pollinators produced fewer and smaller offspring, but seed production was more efficient. Pollinator offspring ratios also varied depending on the age of figs they entered. The two agaonids from F. altissima lived slightly longer than six congeners associated with typical figs, but this was explainable by their larger body sizes. Delayed entry generates reproductive costs, especially for the pollinator. This opens an interesting perspective on the coevolution of figs and their pollinators and on the nature of mutualistic interactions in general.
doi:10.1371/journal.pone.0086735
PMCID: PMC3899289  PMID: 24466217
6.  The NAC-like gene ANTHER INDEHISCENCE FACTOR acts as a repressor that controls anther dehiscence by regulating genes in the jasmonate biosynthesis pathway in Arabidopsis  
Journal of Experimental Botany  2013;65(2):621-639.
ANTHER INDEHISCENCE FACTOR (AIF), a NAC-like gene, was identified in Arabidopsis. In AIF:GUS flowers, β-glucuronidase (GUS) activity was detected in the anther, the upper parts of the filaments, and in the pollen of stage 7–9 young flower buds; GUS activity was reduced in mature flowers. Yellow fluorescent protein (YFP)+AIF-C fusion proteins, which lacked a transmembrane domain, accumulated in the nuclei of the Arabidopsis cells, whereas the YFP+AIF fusion proteins accumulated in the membrane and were absent in the nuclei. Further detection of a cleaved AIF protein in flowers revealed that AIF needs to be processed and released from the endoplasmic reticulum in order to function. The ectopic expression of AIF-C caused a male-sterile phenotype with indehiscent anthers throughout flower development in Arabidopsis. The presence of a repressor domain in AIF and the similar phenotype of indehiscent anthers in AIF-C+SRDX plants suggest that AIF acts as a repressor. The defect in anther dehiscence was due to the down-regulation of genes that participate in jasmonic acid (JA) biosynthesis, such as DAD1/AOS/AOC3/OPR3/OPCL1. The external application of JA rescued the anther indehiscence in AIF-C and AIF-C+SRDX flowers. In AIF-C+VP16 plants, which are transgenic dominant-negative mutants in which AIF is converted to a potent activator via fusion to a VP16-AD motif, the anther dehiscence was promoted, and the expression of DAD1/AOS/AOC3/OPR3/OPCL1 was up-regulated. Furthermore, the suppression of AIF through an antisense strategy resulted in a mutant phenotype similar to that observed in the AIF-C+VP16 flowers. The present data suggest a role for AIF in controlling anther dehiscence by suppressing the expression of JA biosynthesis genes in Arabidopsis.
doi:10.1093/jxb/ert412
PMCID: PMC3904717  PMID: 24323506
Anther dehiscence; ANTHER INDEHISCENCE FACTOR; jasmonate signalling; NAC-like gene; repressor.
7.  Pharmacogenetics and pharmacogenomics: a bridge to individualized cancer therapy 
Pharmacogenomics  2013;14(3):315-324.
In the past decade, advances in pharmacogenetics and pharmacogenomics (PGx) have gradually unveiled the genetic basis of interindividual differences in drug responses. A large portion of these advances have been made in the field of anticancer therapy. Currently, the US FDA has updated the package inserts of approximately 30 anticancer agents to include PGx information. Given the complexity of this genetic information (e.g., tumor mutation and gene overexpression, chromosomal translocation and germline variations), as well as the variable level of scientific evidence, the FDA recommendation and potential action needed varies among drugs. In this review, we have highlighted some of these PGx discoveries for their scientific values and utility in improving therapeutic efficacy and reducing side effects. Furthermore, examples are also provided for the role of PGx in new anticancer drug development by revealing novel druggable targets.
doi:10.2217/pgs.12.213
PMCID: PMC3605891  PMID: 23394393
anticancer agents; drug label; pharmacogenetics; pharmacogenomics
8.  Effects of valproate sodium on extracellular signal-regulated kinase 1/2 phosphorylation following hippocampal neuronal epileptiform discharge in rats 
The aim of the present study was to investigate the effects of valproate sodium (VPAS) on the phosphorylation extracellular signal-regulated kinase 1/2 (ERK1/2) following hippocampal neuronal epileptiform discharge in rat neurons. The study used neurons from female and male neonate Sprague-Dawley (SD) rats (at least 24 h old), which were rapidly decapitated. Following the successful development of the epileptiform discharge cell model, the neurons were divided into two groups, the VPAS group and the control group. In the concentration-response experiment, the neurons were incubated with three different concentrations of VPAS (50, 75 and 100 mg/l) 30 min prior to the epileptiform discharge. The expression of phosphorylated ERK1/2 (p-ERK1/2) was examined using an immunofluorescence technique. In the time-response experiment, the neurons were incubated with VPAS (50 mg/l) and monitored at different time-points (30 min prior to the epileptiform discharge and 0 min, 30 min, 2 h and 6 h subsequent to epileptiform discharge), and western blotting was employed to measure the changes in p-ERK1/2 protein expression. No significant differences in the expression of p-ERK1/2 among the neurons treated with different concentrations of VPAS were identified in the concentration-response experiment. However, in the time-response experiment, the expression of p-ERK1/2 30 min prior to the epileptiform discharge was significantly lower compared with that at the other time-points. Furthermore, 50 mg/l VPAS was capable of decreasing the action potential frequency of the neuronal epileptiform discharge. ERK1/2 was excessively and persistently activated following the epileptiform discharge of the neurons. In addition, a low concentration of VPAS was effective at inhibiting the phosphorylation of ERK1/2 at an earlier period of neuronal epileptiform discharge.
doi:10.3892/etm.2013.1343
PMCID: PMC3829712  PMID: 24250721
valproate sodium; extracellular signal-regulated kinase 1/2; neuron; epileptiform discharge
9.  Larger Fig Wasps Are More Careful About Which Figs to Enter – With Good Reason 
PLoS ONE  2013;8(9):e74117.
Floral longevity reflects a balance between gains in pollinator visitation and the costs of flower maintenance. Because rewards to pollinators change over time, older flowers may be less attractive, reducing the value of extended longevity. Un-pollinated figs, the inflorescences of Ficus species, can remain receptive for long periods, but figs that are older when entered by their host-specific fig wasp pollinators produce fewer seeds and fig wasp offspring. Our field experiments with Ficushispida, a dioecious fig tree, examined how the length of time that receptive figs have remained un-pollinated influences the behaviour and reproductive success of its short-lived fig wasp pollinator, Ceratosolensolmsi marchali. The results were consistent in three different seasons, and on male and female trees, although receptivity was greatly extended during colder months. Pollinators took longer to find the ostioles of older figs, and longer to penetrate them. They also became increasingly unwilling to enter figs as they aged, and increasing numbers of the wasps became trapped in the ostiolar bracts. Larger individuals were particularly unwilling to enter older figs, resulting in older figs being pollinated by smaller wasps. On female trees, where figs produce only seeds, seed production declined rapidly with fig age. On male trees, the numbers and size of fig wasp offspring declined, and a higher proportion were male. Older male figs are harder to enter, especially for larger individuals, and offer poorer quality oviposition opportunities. This study opens an interesting new perspective on the coevolution of figs and their pollinators, especially factors influencing pollinator body size and emphasises the subtleties of interactions between mutualists.
doi:10.1371/journal.pone.0074117
PMCID: PMC3781092  PMID: 24086315
11.  C3-Heteroaroyl cannabinoids as photolabeling ligands for the CB2 cannabinoid receptor 
A series of tricyclic cannabinoids incorporating a heteroaroyl group at C3 were prepared as probes to explore the binding site(s) of the CB1 and CB2 receptors. This relatively unexplored structural motif is shown to be CB2 selective with Ki values at low nanomolar concentrations when the heteroaromatic group is 3-benzothiophenyl (41) or 3-indolyl (50). When photoactivated, the lead compound 41 was shown to successfully label the CB2 receptor through covalent attachment at the active site while 50 failed to label. The benzothiophenone moiety may be a photoactivatable moiety suitable for selective labeling.
doi:10.1016/j.bmcl.2012.06.013
PMCID: PMC3445631  PMID: 22796181
CB2; Cannabinoid; Ligand-assisted protein structure; Photolabeling; Photoactivatable group
12.  A Biodegradable, Sustained-Released, Prednisolone Acetate Microfilm Drug Delivery System Effectively Prolongs Corneal Allograft Survival in the Rat Keratoplasty Model 
PLoS ONE  2013;8(8):e70419.
Frequent and long-term use of topical corticosteroids after corneal transplantation is necessary to prevent graft rejection. However, it relies heavily on patient compliance, and sustained therapeutic drug levels are often not achieved with administration of topical eye drops. A biodegradable drug delivery system with a controlled and sustained drug release may circumvent these limitations. In this study, we investigated the efficacy of a prednisolone acetate (PA)-loaded poly (d,l-lactide-co-ε-caprolactone) (PLC) microfilm drug delivery system on promoting the survival of allogeneic grafts after penetrating keratoplasty (PK) using a rat model. The drug release profiles of the microfilms were characterized (group 1). Subsequently, forty-eight PK were performed in four experimental groups: syngeneic control grafts (group 2), allogeneic control grafts (group 3), allogeneic grafts with subconjunctivally-implanted PA microfilm (group 4), and allogeneic grafts with PA eye drops (group 5; n = 12 in each). PA-loaded microfilm achieved a sustained and steady release at a rate of 0.006–0.009 mg/day, with a consistent aqueous drug concentration of 207–209 ng/ml. The mean survival days was >28 days in group 2, 9.9±0.8 days in group 3, 26.8±2.7 days in group 4, and 26.4±3.4 days in group 5 (P = 0.023 and P = 0.027 compared with group 3). Statistically significant decrease in CD4+, CD163+, CD 25+, and CD54+ cell infiltration was observed in group 4 and group 5 compared with group 3 (P<0.001). There was no significant difference in the mean survival and immunohistochemical analysis between group 4 and group 5. These results showed that sustained PA-loaded microfilm effectively prolongs corneal allograft survival. It is as effective as conventional PA eye drops, providing a promising clinically applicable alternative for patients undergoing corneal transplantation.
doi:10.1371/journal.pone.0070419
PMCID: PMC3734265  PMID: 23940573
13.  Threonine Affects Intestinal Function, Protein Synthesis and Gene Expression of TOR in Jian Carp (Cyprinus carpio var. Jian) 
PLoS ONE  2013;8(7):e69974.
This study aimed to investigate the effects of threonine (Thr) on the digestive and absorptive ability, proliferation and differentiation of enterocytes, and gene expression of juvenile Jian carp (Cyprinus carpio var. Jian). First, seven isonitrogenous diets containing graded levels of Thr (7.4–25.2 g/kg diet) were fed to the fishes for 60 days. Second, enterocyte proliferation and differentiation were assayed by culturing enterocytes with graded levels of Thr (0–275 mg/l) in vitro. Finally, enterocytes were cultured with 0 and 205 mg/l Thr to determine protein synthesis. The percent weight gain (PWG), specific growth rate, feed intake, feed efficiency, protein retention value, activities of trypsin, lipase and amylase, weights and protein contents of hepatopancreas and intestine, folds heights, activities of alkaline phosphatase (AKP), γ- glutamyl transpeptidase and Na+/K+-ATPase in all intestinal segments, glutamate-oxaloacetate transaminase (GOT) and glutamate-pyruvate transaminase (GPT) activities in hepatopancreas, and 4E-BP2 gene expression in muscle, hepatopancreas and intestinal segments were significantly enhanced by Thr (p<0.05). However, the plasma ammonia concentration and TOR gene expression decreased (p<0.05). In vitro, Thr supplement significantly increased cell numbers, protein content, the activities of GOT, GPT, AKP and Na+/K+-ATPase, and protein synthesis rate of enterocytes, and decreased LDH activity and ammonia content in cell medium (p<0.05). In conclusion, Thr improved growth, digestive and absorptive capacity, enterocyte proliferation and differentiation, and protein synthesis and regulated TOR and 4E-BP2 gene expression in juvenile Jian carp. The dietary Thr requirement of juvenile Jian carp was 16.25 g/kg diet (51.3 g/kg protein) based on quadratic regression analysis of PWG.
doi:10.1371/journal.pone.0069974
PMCID: PMC3724917  PMID: 23922879
14.  Synergistic effect of clinically used antibiotics and peptide antibiotics against Gram-positive and Gram-negative bacteria 
Ribosomally synthesized (natural) peptides demonstrate antimicrobial potency and may represent a novel therapeutic approach for the treatment of infections. The aim of the present study was to investigate the interaction between polycationic peptides and clinically used antimicrobial agents in the treatment of clinical isolates of Gram-positive and Gram-negative aerobic bacteria in vitro, using the microbroth dilution method. The combination studies demonstrated synergies between ranalexin and polymyxin E, doxycycline and clarithromycin. Similarly, magainin II was demonstrated to be synergistic with ceftriaxone, amoxicillin clavulanate, ceftazidime, meropenem, piperacillin and β-lactam antibiotics. Buforin II, cecropin P1 and indolicidin were not observed to be synergistic with the clinically used antibiotics, but demonstrated additive effects with them. Notably, no antagonistic effects were identified in all the combinations examined.
doi:10.3892/etm.2013.1231
PMCID: PMC3797290  PMID: 24137305
Gram-positive aerobic bacteria; Gram-negative aerobic bacteria; synergy; peptide antibiotics
15.  Curcumin Protects against 1-Methyl-4-phenylpyridinium Ion- and Lipopolysaccharide-Induced Cytotoxicities in the Mouse Mesencephalic Astrocyte via Inhibiting the Cytochrome P450 2E1 
Curcumin is extracted from the rhizomes of the ginger family plant Curcuma longa L., which has a good protection for liver, kidney, and immune system. However, there is little information about its contribution in protection of astrocytes recently. The present study was undertaken to elucidate the protective effect of curcumin, an herbal antioxidant, on 1-methyl-4-phenylpyridinium ion- (MPP+-) and lipopolysaccharide- (LPS-) induced cytotoxicities, as well as the underlying mechanisms by using primary mouse mesencephalic astrocytes. The results showed that curcumin protected the mesencephalic astrocytes from MPP+- and LPS-induced toxicities along with reducing reactive oxygen species (P < 0.05) and maleic dialdehyde (P < 0.05) sufficiently. Moreover, curcumin significantly inhibited the cytochrome P450 2E1 (CYP2E1) expression (P < 0.01 at mRNA level, P < 0.05 at protein level) and its activity (P < 0.05) sufficiently induced by MPP+ and LPS in the mouse mesencephalic astrocytes. And curcumin as well as diallyl sulphide, a CYP2E1 positive inhibitor, ameliorated MPP+- and LPS-induced mouse mesencephalic astrocytes damage. Accordingly, curcumin protects against MPP+- and LPS-induced cytotoxicities in the mouse mesencephalic astrocyte via inhibiting the CYP2E1 expression and activity.
doi:10.1155/2013/523484
PMCID: PMC3703411  PMID: 23843878
16.  Synthesis, Characterization, and Interaction with Biomolecules of Platinum(II) Complexes with Shikimic Acid-Based Ligands 
Starting from the active ingredient shikimic acid (SA) of traditional Chinese medicine and NH2(CH2)nOH, (n = 2–6), we have synthesized a series of new water-soluble Pt(II) complexes PtLa–eCl2, where La–e are chelating diamine ligands with carbon chain covalently attached to SA (La–e = SA-NH(CH2)nNHCH2CH2NH2; La, n = 2; Lb, n = 3; Lc, n = 4; Ld, n = 5; Le, n = 6). The results of the elemental analysis, LC-MS, capillary electrophoresis, and 1H, 13C NMR indicated that there was only one product (isomer) formed under the present experimental conditions, in which the coordinate mode of PtLa–eCl2 was two-amine bidentate. Their in vitro cytotoxic activities were evaluated by MTT method, where these compounds only exhibited low cytotoxicity towards BEL7404, which should correlate their low lipophilicity. The interactions of the five Pt(II) complexes with DNA were investigated by agarose gel electrophoresis, which suggests that the Pt(II) complexes could induce DNA alteration. We also studied the interactions of the Pt(II) complexes with 5′-GMP with ESI-MS and 1H NMR and found that PtLbCl2, PtLcCl2, and PtLdCl2 could react with 5′-GMP to form mono-GMP and bis-GMP adducts. Furthermore, the cell-cycle analysis revealed that PtLbCl2, PtLcCl2 cause cell G2-phase arrest after incubation for 72 h. Overall, these water-soluble Pt(II) complexes interact with DNA mainly through covalent binding, which blocks the DNA synthesis and replication and thus induces cytotoxicity that weakens as the length of carbon chain increases.
doi:10.1155/2013/565032
PMCID: PMC3603162  PMID: 23533373
17.  Genomic polymorphisms of G-Protein Estrogen Receptor 1 are associated with severity of adolescent idiopathic scoliosis 
International Orthopaedics  2011;36(3):671-677.
Purpose
Adolescent idiopathic scoliosis (AIS) is reported to be associated with the two traditional estrogen receptor genes, ESR1 and ESR2. Yet, the novel estrogen receptor G protein-coupled estrogen receptor 1 (GPER) has not been studied. To investigate the association of GPER gene polymorphisms with the onset and deterioration of AIS, we performed a case-control study.
Methods
Clinical information was recorded, blood samples were taken and genomic DNA was extracted. After resequencing the gene in 45 cases and 45 controls who were randomly selected, 16 tag single nucleotide polymorphisms (SNPs) were selected. Then the association study was extended by an additional 344 patients and 293 controls with direct sequencing and a TaqMan-based genotyping assay. The chi-square test and logistic regression were used to analyse the genotypic and allelic association. One-way analysis of variance was used to compare the mean maximum Cobb angles and ages with different genotypes in the case-only data set.
Results
No association was observed between the polymorphisms of the GPER gene and susceptibility to AIS. However, heterozygotes in three SNPs of the gene (rs3808351, rs10269151 and rs426655s3) were related significantly with the curve severity in AIS patients (P = 0.004, 0.048 and 0.028, respectively).
Conclusions
Our results demonstrate that GPER gene polymorphisms are associated with the severity of curvature in AIS; deficits of GPER may contribute to the deterioration of spine deformity.
doi:10.1007/s00264-011-1374-8
PMCID: PMC3291774  PMID: 22002330
18.  Clinical study of isosorbide mononitrate treatment for angina pectoris in coronary heart disease 
The aim of this study was to observe and investigate the clinical efficacy of an intravenous drip of isosorbide mononitrate (ISMN) for the treatment of angina pectoris in coronary heart disease. A total of 102 patients with angina pectoris in coronary heart disease were divided into two groups. For the treatment group (n=51), 20 mg ISMN was added to 250 ml 0.9% normal saline and administered by intravenous drip for 14 consecutive days, twice daily. For the control group (n=51), 10 mg glyceryl trinitrate was added to 250 ml 0.9% normal saline and administered by intravenous drip for 14 consecutive days, twice daily. The clinical efficacy and adverse reactions were compared between the two groups. The disease symptoms of the two groups were improved. Compared with the control group, the clinical efficacy and electrocardiogram examination results of the treatment group were significantly improved (P<0.05). The intravenous formulation of ISMN is an effective treatment for angina pectoris in coronary heart disease and it has the advantages of fewer adverse reactions and higher safety.
doi:10.3892/etm.2013.958
PMCID: PMC3628368  PMID: 23596481
isosorbide mononitrate; intravenous drip; angina pectoris; coronary heart disease
19.  Association between Common Variants near LBX1 and Adolescent Idiopathic Scoliosis Replicated in the Chinese Han Population 
PLoS ONE  2013;8(1):e53234.
Background
Adolescent idiopathic scoliosis (AIS) is one of the most common spinal deformities found in adolescent populations. Recently, a genome-wide association study (GWAS) in a Japanese population indicated that three single nucleotide polymorphisms (SNPs), rs11190870, rs625039 and rs11598564, all located near the LBX1 gene, may be associated with AIS susceptibility [1]. This study suggests a novel AIS predisposition candidate gene and supports the hypothesis that somatosensory functional disorders could contribute to the pathogenesis of AIS. These findings warrant replication in other populations.
Methodology/Principal Findings
First, we conducted a case-control study consisting of 953 Chinese Han individuals from southern China (513 patients and 440 healthy controls), and the three SNPs were all found to be associated with AIS predisposition. The ORs were observed as 1.49 (95% CI 1.23–1.80, P = 5.09E-5), 1.70 (95% CI 1.42–2.04, P = 1.17E-8) and 1.52 (95% CI 1.27–1.83, P = 5.54E-6) for rs625039, rs11190870 and rs11598564, respectively. Second, a case-only study including a subgroup of AIS patients (N = 234) was performed to determine the effects of these variants on the severity of the condition. However, we did not find any association between these variants and the severity of curvature.
Conclusion
This study shows that the genetic variants near the LBX1 gene are associated with AIS susceptibility in Chinese Han population. It successfully replicates the results of the GWAS, which was performed in a Japanese population.
doi:10.1371/journal.pone.0053234
PMCID: PMC3537668  PMID: 23308168
20.  Use of computed tomographic reconstruction to establish the ideal entry point for pedicle screws in idiopathic scoliosis 
European Spine Journal  2011;21(1):23-30.
Objective
To determine the ideal entry point for individual pedicle screw in the surgical treatment of idiopathic scoliosis using computed tomographic (CT) three-dimensional (3D) reconstruction.
Methods
Twenty patients with moderate or severe idiopathic scoliosis from two groups received surgical treatment using “Free Hand technique” and “Assisted Free Hand technique”. Computed tomographic scanning with 3D reconstruction of the thoracic and lumbar spine was conducted to determine the entry point and to evaluate the placement accuracy.
Results
The accuracy of placement was 88.2% in convexity and 84.5% in concavity for the “Free Hand” group, and 94.1% in convexity and 94% in concavity for the “Assisted Free Hand” group. Evidence showed that “Assisted Free Hand” group had higher accuracy when screws were placed in the thoracic spine (P = 0.02), while no obvious difference was seen in the lumbar spine placement (P = 0.47).
Conclusions
We conclude that in the surgical treatment of severe scoliosis, individual screw placement guided by entry points determined by CT reconstruction can result in improved accuracy and ease of the procedure.
doi:10.1007/s00586-011-1962-8
PMCID: PMC3252450  PMID: 21842236
Pedicle screw; Computed tomography; Entry point; Scoliosis
21.  Nutritional and Chemical Composition and Antiviral Activity of Cultivated Seaweed Sargassum naozhouense Tseng et Lu 
Marine Drugs  2012;11(1):20-32.
Sargassum naozhouense is a brown seaweed used in folk medicine and applied for thousands of years in Zhanjiang, Guangdong province, China. This study is the first time to investigate its chemical composition and antiviral activity. On the dry weight basis, this seaweed was constituted of ca. 35.18% ash, 11.20% protein, 1.06% lipid and 47.73% total carbohydrate, and the main carbohydrate was water-soluble polysaccharide. The protein analysis indicated the presence of essential amino acids, which accounted for 36.35% of the protein. The most abundant fatty acids were C14:0, C16:0, C18:1 and C20:4. The ash fraction analysis indicated that essential minerals and trace elements, such as Fe, Zn and Cu, were present in the seaweed. IR analysis revealed that polysaccharides from cultivated S. naozhouense may be alginates and fucoidan. The polysaccharides possessed strong antiviral activity against HSV-1 in vitro with EC50 of 8.92 μg/mL. These results demonstrated cultivated S. naozhouense has a potential for its use in functional foods and antiviral new drugs.
doi:10.3390/md11010020
PMCID: PMC3564154  PMID: 23271422
Sargassum naozhouense; seaweed; chemical composition; antiviral activity
22.  Expression Profile in Rice Panicle: Insights into Heat Response Mechanism at Reproductive Stage 
PLoS ONE  2012;7(11):e49652.
Rice at reproductive stage is more sensitive to environmental changes, and little is known about the mechanism of heat response in rice panicle. Here, using rice microarray, we provided a time course gene expression profile of rice panicle at anther developmental stage 8 after 40°C treatment for 0 min, 20 min, 60 min, 2 h, 4 h, and 8 h. The identified differentially expressed genes were mainly involved in transcriptional regulation, transport, cellular homeostasis, and stress response. The predominant transcription factor gene families responsive to heat stress were Hsf, NAC, AP2/ERF, WRKY, MYB, and C2H2. KMC analysis discovered the time-dependent gene expression pattern under heat stress. The motif co-occurrence analysis on the promoters of genes from an early up-regulated cluster showed the important roles of GCC box, HSE, ABRE, and CE3 in response to heat stress. The regulation model central to ROS combined with transcriptome and ROS quantification data in rice panicle indicated the great importance to maintain ROS balance and the existence of wide cross-talk in heat response. The present study increased our understanding of the heat response in rice panicle and provided good candidate genes for crop improvement.
doi:10.1371/journal.pone.0049652
PMCID: PMC3498232  PMID: 23155476
24.  An Extreme Case of Plant–Insect Codiversification: Figs and Fig-Pollinating Wasps 
Systematic Biology  2012;61(6):1029-1047.
It is thought that speciation in phytophagous insects is often due to colonization of novel host plants, because radiations of plant and insect lineages are typically asynchronous. Recent phylogenetic comparisons have supported this model of diversification for both insect herbivores and specialized pollinators. An exceptional case where contemporaneous plant–insect diversification might be expected is the obligate mutualism between fig trees (Ficus species, Moraceae) and their pollinating wasps (Agaonidae, Hymenoptera). The ubiquity and ecological significance of this mutualism in tropical and subtropical ecosystems has long intrigued biologists, but the systematic challenge posed by >750 interacting species pairs has hindered progress toward understanding its evolutionary history. In particular, taxon sampling and analytical tools have been insufficient for large-scale cophylogenetic analyses. Here, we sampled nearly 200 interacting pairs of fig and wasp species from across the globe. Two supermatrices were assembled: on an average, wasps had sequences from 77% of 6 genes (5.6 kb), figs had sequences from 60% of 5 genes (5.5 kb), and overall 850 new DNA sequences were generated for this study. We also developed a new analytical tool, Jane 2, for event-based phylogenetic reconciliation analysis of very large data sets. Separate Bayesian phylogenetic analyses for figs and fig wasps under relaxed molecular clock assumptions indicate Cretaceous diversification of crown groups and contemporaneous divergence for nearly half of all fig and pollinator lineages. Event-based cophylogenetic analyses further support the codiversification hypothesis. Biogeographic analyses indicate that the present-day distribution of fig and pollinator lineages is consistent with a Eurasian origin and subsequent dispersal, rather than with Gondwanan vicariance. Overall, our findings indicate that the fig-pollinator mutualism represents an extreme case among plant–insect interactions of coordinated dispersal and long-term codiversification. [Biogeography; coevolution; cospeciation; host switching; long-branch attraction; phylogeny.]
doi:10.1093/sysbio/sys068
PMCID: PMC3478567  PMID: 22848088
25.  New Cembrane Diterpenoids from a Hainan Soft Coral Sinularia sp 
Marine Drugs  2012;10(9):2023-2032.
Five new cembrane diterpenoids, named sinuflexibilins A–E (1–5), along with nine other known diterpenoids (6–14), have been isolated from the organic extract of a Hainan soft coral Sinularia sp. Their structures were determined on the basis of extensive spectroscopic analyses and by comparison of their spectral data with those of related metabolites. Compound 13, flexibilide, exhibited significant inhibitory activity of NF-κB activation using the cell-based HEK293 NF-κB luciferase reporter gene assay.
doi:10.3390/md10092023
PMCID: PMC3475270  PMID: 23118718
Sinularia sp.; cembrane diterpenoids; NF-κB inhibitor

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