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1.  Network-Constrained Group Lasso for High-Dimensional Multinomial Classification with Application to Cancer Subtype Prediction 
Cancer Informatics  2015;13(Suppl 6):25-33.
Classic multinomial logit model, commonly used in multiclass regression problem, is restricted to few predictors and does not take into account the relationship among variables. It has limited use for genomic data, where the number of genomic features far exceeds the sample size. Genomic features such as gene expressions are usually related by an underlying biological network. Efficient use of the network information is important to improve classification performance as well as the biological interpretability. We proposed a multinomial logit model that is capable of addressing both the high dimensionality of predictors and the underlying network information. Group lasso was used to induce model sparsity, and a network-constraint was imposed to induce the smoothness of the coefficients with respect to the underlying network structure. To deal with the non-smoothness of the objective function in optimization, we developed a proximal gradient algorithm for efficient computation. The proposed model was compared to models with no prior structure information in both simulations and a problem of cancer subtype prediction with real TCGA (the cancer genome atlas) gene expression data. The network-constrained mode outperformed the traditional ones in both cases.
doi:10.4137/CIN.S17686
PMCID: PMC4295837  PMID: 25635165
cancer subtype prediction; multinomial logit model; group lasso; network-constraint; proximal gradient algorithm
2.  Efficient Inhibition of Human Glioma Development by RNA Interference-Mediated Silencing of PAK5 
Glioma is the most common type of primary intracranial tumor and is highly lethal due to its pathogenetic location, high invasiveness, and poor prognosis. Even combined surgery and chemoradiotherapy do not effectively rescue glioma patients. Molecular target therapy is considered a safe and promising therapy for glioma. The identification of a novel, effective target protein in gliomas is of great interest. We found that PAK5 was highly expressed in the tumor tissues of glioma patients and human glioma cell lines. We then used a lentivirus-delivered short hairpin RNA to stably silence PAK5 expression in glioma cells and explore its influence. The results showed that the inhibition of PAK5 reduced cell viability and delayed the cell cycle at the G0/G1 phase in the glioma cells with PAK5 high expression. In addition, silencing PAK5 expression in U87 cells weakened their colony formation ability and in vivo tumorigenesis ability. Further studies demonstrated that PAK5 inhibition led to an increase in cleaved caspase 3 and a decrease in β-catenin. In conclusion, our results suggest that the inhibition of PAK5 by RNA interference might efficiently suppress tumor development of glioma cells with PAK5 high expression. This finding provides a novel, promising therapeutic target for glioma treatment.
doi:10.7150/ijbs.9193
PMCID: PMC4308408  PMID: 25632266
glioma; PAK5; tumor development; RNA interference; cell cycle arrest
3.  The Inhibitory Effects of Npas4 on Seizures in Pilocarpine-Induced Epileptic Rats 
PLoS ONE  2014;9(12):e115801.
To explore the effects of neuronal Per-Arnt-Sim domain protein 4 (Npas4) on seizures in pilocarpine-induced epileptic rats, Npas4 expression was detected by double-label immunofluorescence, immunohistochemistry, and Western blotting in the brains of pilocarpine-induced epileptic model rats at 6 h, 24 h, 72 h, 7 d, 14 d, 30 d, and 60 d after status epilepticus. Npas4 was localized primarily in the nucleus and in the cytoplasm of neurons. The Npas4 protein levels increased in the acute phase of seizures (between 6 h and 72 h) and decreased in the chronic phases (between 7 d and 60 d) in the rat model. Npas4 expression was knocked down by specific siRNA interference. Then, the animals were treated with pilocarpine, and the effects on seizures were evaluated on the 7th day. The onset latencies of pilocarpine-induced seizures were decreased, while the seizure frequency, duration and attack rate increased in these rats. Our study indicates that Npas4 inhibits seizure attacks in pilocarpine-induced epileptic rats.
doi:10.1371/journal.pone.0115801
PMCID: PMC4275263  PMID: 25536221
4.  GEE-based SNP Set Association Test for Continuous and Discrete Traits in Family Based Association Studies 
Genetic epidemiology  2013;37(8):778-786.
Family-based genetic association studies of related individuals provide opportunities to detect genetic variants that complement studies of unrelated individuals. Most statistical methods for family association studies for common variants are single-marker-based, which test one SNP a time. In this paper, we consider testing the effect of a SNP set, e.g., SNPs in a gene, in family studies, for both continuous and discrete traits. Specifically, we propose a Generalized Estimating Equations (GEE)-based kernel association test, a variance component-based testing method, to test for the association between a phenotype and multiple variants in a SNP set jointly using family samples. The proposed approach allows for both continuous and discrete traits, where the correlation among family members is taken into account through the use of an empirical covariance estimator. We derive the theoretical distribution of the proposed statistic under the null and develop analytical methods to calculate the p-values. We also propose an efficient resampling method for correcting for small sample size bias in family studies. The proposed method allows for easily incorporating covariates and SNP-SNP interactions. Simulation studies show that the proposed method properly controls for type-I error rates under both random and ascertained sampling schemes in family studies. We demonstrate through simulation studies that our approach has superior performance for association mapping compared to the single marker based minimum p-value GEE test for a SNP set effect over a range of scenarios. We illustrate the application of the proposed method using data from the Cleveland Family GWAS Study.
doi:10.1002/gepi.21763
PMCID: PMC4007511  PMID: 24166731
Family-based association; Generalized estimation equations; Kernel machine regression; Marginal models; Score test; Variance component
5.  Association study of polymorphism of FXIIIVal34Leu gene and polycystic ovary syndrome 
Objective: To study the distribution of polymorphism of FXIIIVal34Leu gene in patients with polycystic ovary syndrome and to explore its relationship with polycystic ovary syndrome. Methods: FXIIIVal34Leu genotype of 120 patients with polycystic ovary syndrome (PCOS) and 90 controls was detected, and the endocrine and metabolic conditions of PCOS patients and the control group were analyzed. Results: Body mass index (BMI) of PCOS patients (26.45±5.81) kg/m2 was higher than the control group (22.33±5.58) kg/m2, (p=0.00); the androgen level of PCOS group was (0.67±0.85) ng/mL higher than the control group (0.42±0.22) ng/mL, (p=0.02), and the luteinizing hormone (LH) (16.8±3.61 IU/L) level is higher than the control group (9.23±4.67) IU/L, (p=0.01). Frequency of Val34Leu allele in PCOS group was 1.25%, which was not statistically significant compared to the control group (P=0.56). BMI of patients with the genotype of FXIIIVal34Leu was relatively higher (with the average 34±1 kg/m2), and serum HDL level was significantly lower (0.23±0.11 mmol/L). Polymorphism of FXIIIVal34Leu gene had no correlation with susceptibility of PCOS. Conclusion: Polymorphism of FXIIIA1 gene may be related to metabolism of PCOS patients; the mutation of FXIIIAVal34Leu is not at the common site for PCOS; polymorphism of FXIIIVal34Leu gene may not have correlation with PCOS.
PMCID: PMC4276228  PMID: 25550970
Coagulation factor XIII; polycystic ovary syndrome; polymorphism of Val34Leu gene
6.  Decreased expression of microRNA-21 correlates with the imbalance of Th17 and Treg cells in patients with rheumatoid arthritis 
The imbalance of Th17/Treg cell populations has been suggested to be involved in the regulation of rheumatoid arthritis (RA) pathogenesis; however, the mechanism behind this phenomenon remains unclear. Recent studies have shown how microRNAs (miRNAs) are important regulators of immune responses and are involved in the development of a variety of inflammatory diseases, including RA. In this study, we demonstrated that the frequencies of CD3+CD4+IL-17+Th17 cells were significantly higher, and CD4+CD25+FOXP3+ Treg cells significantly lower in peripheral blood mononuclear cells from RA patients. Detection of cytokines from RA patients revealed an elevated panel of pro-inflammatory cytokines, including IL-17, IL-6, IL-1β, TNF-α and IL-22, which carry the inflammatory signature of RA and are crucial in the differentiation and maintenance of pathogenic Th17 cells and dysfunction of Treg cells. However, the level of miR-21 was significantly lower in RA patients, accompanied by the increase in STAT3 expression and activation, and decrease in STAT5/pSTAT5 protein and Foxp3 mRNA levels. Furthermore, lipopolysaccharide stimulation up-regulated miR-21 expression from healthy controls, but down-regulated miR-21 expression from RA patients. Therefore, we speculate that miR-21 may be part of a negative feedback loop in the normal setting. However, miR-21 levels decrease significantly in RA patients, suggesting that this feedback loop is dysregulated and may contribute to the imbalance of Th17 and Treg cells. MiR-21 may thus serve as a novel regulator in T-cell differentiation and homoeostasis, and provides a new therapeutic target for the treatment of RA.
doi:10.1111/jcmm.12353
PMCID: PMC4224555  PMID: 25164131
rheumatoid arthritis; Treg; Th17; miRNA; pro-inflammatory cytokine
7.  Interleukin-1β-31C/T and -511T/C Polymorphisms Were Associated with Preeclampsia in Chinese Han Population 
PLoS ONE  2014;9(9):e106919.
Objective
The purpose of our study is to investigate the relationship between IL-1β -31C/T (rs1143627) and -511T/C (rs16944) polymorphisms and the preeclampsia (PE), and analyze the Linkage disequilibrium (LD) and haplotype frequency of the two polymorphism loci.
Methods
Polymorphisms at -31C/T and -511T/C of IL-1β were genotyped with the method of polymerase chain reaction-restriction fragment length polymorphism (PCR- RFLP) in 232 PE and 447 control subjects. Genotype and allele frequencies between case-control groups were compared by chi-square(X2) tests. Two-point LD and haplotype frequency analyses were done with the software Haploview4.2.
Results
Significant statistical differences were found between PE and control groups regarding genotype and allele frequencies of the two polymorphisms of IL-1β (For IL-1β -31C/T: X2 = 11.478, P = 0.003; For IL-1β-511T/C: X2 = 9.687, P = 0.008). LD analysis revealed that the IL-1β -31C/T SNP was in high LD with the IL-1β-511C/T SNP(D′ = 0.92, r2 = 0.79). Both CT and TC haplotypes showed significant differences between case and control groups. Only the plasma level of Prothrombin Time had a significantly statistical difference among TT, CT and CC groups of the preeclamptic two polymorphisms of IL-1β-31C/T and -511T/C (for IL-1β-31C/T, F = 1.644, P = 0.01; F = 1.587, P = 0.016).
Conclusion
Our results revealed IL-1β was associated with the PE in Chinese Han population. The CT haplotype may increase the risk of PE, while haplotype TC could be considered as a protective haplotype of PE.
doi:10.1371/journal.pone.0106919
PMCID: PMC4164436  PMID: 25222025
8.  Comparative transcriptome profiling of a desert evergreen shrub, Ammopiptanthus mongolicus, in response to drought and cold stresses 
BMC Genomics  2014;15(1):671.
Background
The molecular mechanisms involved in plant tolerance to either drought or cold have been extensively studied in many plant species. However, few studies have focused on their comparisons especially using non-model plants with strong tolerance to both stresses. Ammopiptanthus mongolicus (Maxim. ex Kom.) Cheng f. is the only evergreen broadleaf shrub grown in the central Asian desert and it has very strong cold and drought tolerance. To provide further insights into plant tolerance, the transcriptome profiles of drought- and cold-treated A. mongolicus seedlings were analyzed using Illumina technology and differentially expressed genes (DEGs) were compared.
Results
A comprehensive transcriptome of A. mongolicus was sequenced using pooled mRNA extracted from drought-, cold-stressed and unstressed seedlings as well as leaves from naturally grown shrub. These sequences were assembled into 86058 unigenes, of which 51014 unigenes had an annotated function and 2440 encoded transcription factors (TFs). Transcriptome profiles were analyzed in A. mongolicus seedlings after drought and cold treatments at three time points (2, 8 and 24 h). Between 3917 and 6102 unigenes were identified as DEGs at a single time point in both stresses. Among these DEGs 2028 and 2026 DEGs were common across the three time points of drought and cold treatments respectively, and 971 DEGs were co-regulated by both stresses. Functional enrichment analyses identified many common or specific biological processes and gene sets in response to drought and cold stresses. The most pronounced findings are that flavonoid biosynthesis genes were enriched in the DEGs co-up-regulated by both stresses; while membrane protein genes and genes related to chloroplast were abundant in the DEGs specifically up-regulated by drought or cold, respectively. Furthermore, the DREB, ERF, NAC and WRKY TFs were predominantly co-up-regulated by both stresses.
Conclusions
The present study provides the most comprehensive transcriptome resource and the first dynamic transcriptome profiles of A. mongolicus under drought and cold stresses. This information will deepen our understanding of plant tolerance to drought and cold. The up-regulated DEGs will be valuable for further investigations of key genes and molecular mechanisms involved in the adaptation of A. mongolicus to harsh environments.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-671) contains supplementary material, which is available to authorized users.
doi:10.1186/1471-2164-15-671
PMCID: PMC4143566  PMID: 25108399
Ammopiptanthus mongolicus; Drought; Cold; Transcriptome profiling; Illumina sequencing
9.  The missense Thr211Pro mutation in the factor X activation peptide of a bleeding patient causes molecular defect in the clotting cascade 
Thrombosis and haemostasis  2013;110(1):53-61.
Summary
Factor X (FX) is a vitamin K-dependent coagulation zymogen, which upon activation to factor Xa assembles into the prothrombinase complex to activate prothrombin to thrombin. FX can be activated by either factor VIIa-tissue factor or factor IXa-factor VIIIa in extrinsic and intrinsic pathways, respectively. In this study, we identified a bleeding patient with moderate FX deficiency who exhibits a clotting defect only in the intrinsic pathway. Exome sequencing revealed that the patient carries a novel homozygous missense mutation that results in substitution of Thr211 with Pro in the activation peptide of FX. Thr211 is the site of an O-linked glycosylation in the activation peptide of FX. We postulated that the lack of this post-translational modification specifically impacts the activation of FX by intrinsic Xase, thereby impairing thrombin generation in the subject. To test this hypothesis, we expressed both wild-type FX and FX containing this mutation in mammalian cells and following the purification of the zymogens to homogeneity characterized their properties in both purified and plasma-based assay systems. Analysis of the results suggests that Thr211 to Pro substitution renders the FX mutant a poor substrate for both physiological activators, however, at physiological concentration of the substrate, the clotting defect manifest itself only in the intrinsic pathway, thus explaining the bleeding phenotype for the patient carrying this mutation.
doi:10.1160/TH13-03-0184
PMCID: PMC3700658  PMID: 23677006
10.  Firth logistic regression for rare variant association tests 
Frontiers in Genetics  2014;5:187.
doi:10.3389/fgene.2014.00187
PMCID: PMC4063169  PMID: 24995013
rare variants; association test; firth logistic regression; penalized likelihood; GEE
11.  Prostate specific antigen detection in patient sera by fluorescence-free BioCD protein array 
Biosensors & bioelectronics  2010;26(5):1871-1875.
Fluorescence-free biosensor arrays for protein detection directly measure the protein surface density, and do not require a fluorophore or enzyme label, and provide quantitative and consistent signals. However, few fluorescence-free biosensor protein arrays have demonstrated successful application in high-background samples, such as serum, due to non-specific binding. We tested the BioCD as a fluorescence-free biosensor based on optical interferometry, and used it to detect prostate specific antigen (PSA, a biomarker of prostate cancer) in patient sera in a 96-well anti-PSA microarray. We have attained a 4 ng/ml detection limit in serum and have measured PSA concentrations in patient sera. The measured concentrations correlated well with enzyme-linked immunosorbent assay (ELISA) results. The measurement of PSA concentrations in high-level protein backgrounds suggests that the BioCD has a potential for clinical applications by removing the restriction of fluorescence-free biosensors from high-background applications.
doi:10.1016/j.bios.2010.02.009
PMCID: PMC4030240  PMID: 20236816
BioCD; Label-free; Interferometry; Antibody microarray; Prostate specific antigen; Cancer biomarker; Serum; Optical biosensor
12.  Molecular Layer Detection on a Diffractive Optical Balance 
Optics letters  2012;37(19):4098-4100.
Diffraction-based molecular detection is achieved by etching optical gratings into thermal oxide on silicon. The gratings perform as a stable common-path diffractive optical balance (DOB) designed to operate around a missing diffraction order. The biosensor is operated in an off-null condition with a phase bias to produce a high-contrast responsivity that is linear in accumulated molecules, but with a low background. The DOB linear responsivity is a factor of 20 larger than the reflectometric responsivity of planar thermal oxide.
PMCID: PMC4028713  PMID: 23027291
13.  Structural and biochemical insights into the V/I505T mutation found in the EIAV gp45 vaccine strain 
Retrovirology  2014;11:26.
Background
The equine infectious anemia virus (EIAV) is a lentivirus of the Retrovirus family, which causes persistent infection in horses often characterized by recurrent episodes of high fever. It has a similar morphology and life cycle to the human immunodeficiency virus (HIV). Its transmembrane glycoprotein, gp45 (analogous to gp41 in HIV), mediates membrane fusion during the infection. However, the post-fusion conformation of EIAV gp45 has not yet been determined. EIAV is the first member of the lentiviruses for which an effective vaccine has been successfully developed. The attenuated vaccine strain, FDDV, has been produced from a pathogenic strain by a series of passages in donkey dermal cells. We have previously reported that a V/I505T mutation in gp45, in combination with other mutations in gp90, may potentially contribute to the success of the vaccine strain. To this end, we now report on our structural and biochemical studies of the gp45 protein from both wide type and vaccine strain, providing a valuable structural model for the advancement of the EIAV vaccine.
Results
We resolved crystal structures of the ecto-domain of gp45 from both the wild-type EIAV and the vaccine strain FDDV. We found that the V/I505T mutation in gp45 was located in a highly conserved d position within the heptad repeat, which protruded into a 3-fold symmetry axis within the six-helix bundle. Our crystal structure analyses revealed a shift of a hydrophobic to hydrophilic interaction due to this specific mutation, and further biochemical and virological studies confirmed that the mutation reduced the overall stability of the six-helix bundle in post-fusion conformation. Moreover, we found that altering the temperatures drastically affected the viral infectivity.
Conclusions
Our high-resolution crystal structures of gp45 exhibited high conservation between the gp45/gp41 structures of lentiviruses. In addition, a hydrophobic to hydrophilic interaction change in the EIAV vaccine strain was found to modulate the stability and thermal-sensitivity of the overall gp45 structure. Our observations suggest that lowering the stability of the six-helix bundle (post-fusion), which may stabilizes the pre-fusion conformation, might be one of the reasons of acquired dominance for FDDV in viral attenuation.
doi:10.1186/1742-4690-11-26
PMCID: PMC3997929  PMID: 24656154
EIAV; gp45; Crystal structure; Stability; Vaccine strain; Heptad repeat; Pre-fusion conformation; Replication
14.  Detection and Characterization of Miniature Inverted-Repeat Transposable Elements in “Candidatus Liberibacter asiaticus” 
Journal of Bacteriology  2013;195(17):3979-3986.
Miniature inverted-repeat transposable elements (MITEs) are nonautonomous transposons (devoid of the transposase gene tps) that affect gene functions through insertion/deletion events. No transposon has yet been reported to occur in “Candidatus Liberibacter asiaticus,” an alphaproteobacterium associated with citrus Huanglongbing (HLB, yellow shoot disease). In this study, two MITEs, MCLas-A and MCLas-B, in “Ca. Liberibacter asiaticus” were detected, and the genome was characterized using 326 isolates collected in China and Florida. MCLas-A had three variants, ranging from 237 to 325 bp, and was inserted into a TTTAGG site of a prophage region. MCLas-A had a pair of 54-bp terminal inverted repeats (TIRs), which contained three tandem repeats of TGGTAACCAC. Both “filled” (with MITE) and “empty” (without MITE) states were detected, suggesting the MITE mobility. The empty sites of all bacterial isolates had TIR tandem repeat remnants (TRR). Frequencies of TRR types varied according to geographical origins. MCLas-B had four variants, ranging from 238 to 250 bp, and was inserted into a TA site of another “Ca. Liberibacter” prophage. The MITE, MCLas-B, had a pair of 23-bp TIRs containing no tandem repeats. No evidence of MCLas-B mobility was found. An identical open reading frame was found upstream of MCLas-A (229 bp) and MCLas-B (232 bp) and was predicted to be a putative tps, suggesting an in cis tps-MITE configuration. MCLas-A and MCLas-B were predominantly copresent in Florida isolates, whereas MCLas-A alone or MCLas-B alone was found in Chinese isolates.
doi:10.1128/JB.00413-13
PMCID: PMC3754606  PMID: 23813735
15.  Altered Spontaneous Brain Activity in Patients with Parkinson’s Disease Accompanied by Depressive Symptoms, as Revealed by Regional Homogeneity and Functional Connectivity in the Prefrontal-Limbic System 
PLoS ONE  2014;9(1):e84705.
As patients with Parkinson’s disease (PD) are at high risk for comorbid depression, it is hypothesized that these two diseases are sharing common pathogenic pathways. Using regional homogeneity (ReHo) and functional connectivity approaches, we characterized human regional brain activity at resting state to examine specific brain networks in patients with PD and those with PD and depression (PDD). This study comprised 41 PD human patients and 25 normal human subjects. The patients completed the Hamilton Depression Rating Scale and were further divided into two groups: patients with depressive symptoms and non-depressed PD patients (nD-PD). Compared with the non-depressed patients, those with depressive symptoms exhibited significantly increased regional activity in the left middle frontal gyrus and right inferior frontal gyrus, and decreased ReHo in the left amygdala and bilateral lingual gyrus. Brain network connectivity analysis revealed decreased functional connectivity within the prefrontal-limbic system and increased functional connectivity in the prefrontal cortex and lingual gyrus in PDD compared with the nD-PD group. In summary, the findings showed regional brain activity alterations and disruption of the mood regulation network in PDD patients. The pathogenesis of PDD may be attributed to abnormal neural activity in multiple brain regions.
doi:10.1371/journal.pone.0084705
PMCID: PMC3880326  PMID: 24404185
16.  Defining Single Molecular Forces Required to Activate Integrin and Notch Signaling 
Science (New York, N.Y.)  2013;340(6135):991-994.
Cell-cell and cell-matrix mechanical interactions through membrane receptors direct a wide range of cellular functions and orchestrate the development of multicellular organisms. To define the single molecular forces required to activate signaling through a ligand-receptor bond, we developed the Tension Gauge Tether (TGT) approach in which the ligand is immobilized to a surface through a rupturable tether before receptor engagement. TGT serves as an autonomous gauge to restrict the receptor-ligand tension. Using a range of tethers with tunable tension tolerances, we show that cells apply a universal peak tension of ~40 pN to single integrin-ligand bonds during initial adhesion. We find that less than 12 pN is required to activate Notch receptors. TGT can also provide a defined molecular mechanical cue to regulate cellular functions.
doi:10.1126/science.1231041
PMCID: PMC3710701  PMID: 23704575
17.  Abnormal Regional Homogeneity in Patients with Essential Tremor Revealed by Resting-State Functional MRI 
PLoS ONE  2013;8(7):e69199.
Essential tremor (ET) is one of the most common movement disorders in human adults. It can be characterized as a progressive neurological disorder of which the most recognizable feature is a tremor of the arms or hands that is apparent during voluntary movements such as eating and writing. The pathology of ET remains unclear. Resting-state fMRI (RS-fMRI), as a non-invasive imaging technique, was employed to investigate abnormalities of functional connectivity in ET in the brain. Regional homogeneity (ReHo) was used as a metric of RS-fMRI to assess the local functional connectivity abnormality in ET with 20 ET patients and 20 age- and gender-matched healthy controls (HC). The ET group showed decreased ReHo in the anterior and posterior bilateral cerebellar lobes, the bilateral thalamus and the insular lobe, and increased ReHo in the bilateral prefrontal and parietal cortices, the left primary motor cortex and left supplementary motor area. The abnormal ReHo value of ET patients in the bilateral anterior cerebellar lobes and the right posterior cerebellar lobe were negatively correlated with the tremor severity score, while positively correlated with that in the left primary motor cortex. These findings suggest that the abnormality in cerebello-thalamo-cortical motor pathway is involved in tremor generation and propagation, which may be related to motor-related symptoms in ET patients. Meanwhile, the abnormality in the prefrontal and parietal regions may be associated with non-motor symptoms in ET. These findings suggest that the ReHo could be utilized for investigations of functional-pathological mechanism of ET.
doi:10.1371/journal.pone.0069199
PMCID: PMC3711903  PMID: 23869236
18.  Power of Single- vs. Multi-Marker Tests of Association 
Genetic epidemiology  2012;36(5):480-487.
Current genome-wide association studies still heavily rely on a single-marker strategy, in which each single nucleotide polymorphism (SNP) is tested individually for association with a phenotype. Although methods and software packages that consider multimarker models have become available, they have been slow to become widely adopted and their efficacy in real data analysis is often questioned. Based on conducting extensive simulations, here we endeavor to provide more insights into the performance of simple multimarker association tests as compared to single-marker tests. The results reveal the power advantage as well as disadvantage of the two- vs. the single-marker test. Power differentials depend on the correlation structure among tag SNPs, as well as that between tag SNPs and causal variants. A two-marker test has relatively better performance than single-marker tests when the correlation of the two adjacent markers is high. However, using HapMap data, two-marker tests tended to have a greater chance of being less powerful than single-marker tests, due to constraints on the number of actual possible haplotypes in the HapMap data. Yet, the average power difference was small whenever the one-marker test is more powerful, while there were many situations where the two-marker test can be much more powerful. These findings can be useful to guide analyses of future studies.
doi:10.1002/gepi.21642
PMCID: PMC3708310  PMID: 22648939
Asymptotic power; single-marker test; two-marker test; genome-wide association
19.  Combined TLR7/8 and TLR9 Ligands Potentiate the Activity of a Schistosoma japonicum DNA Vaccine 
Background
Toll-like receptor (TLR) ligands have been explored as vaccine adjuvants for tumor and virus immunotherapy, but few TLR ligands affecting schistosoma vaccines have been characterized. Previously, we developed a partially protective DNA vaccine encoding the 26-kDa glutathione S-transferase of Schistosoma japonicum (pVAX1-Sj26GST).
Methodology/Principal Findings
In this study, we evaluated a TLR7/8 ligand (R848) and a TLR9 ligand (CpG oligodeoxynucleotides, or CpG) as adjuvants for pVAX1-Sj26GST and assessed their effects on the immune system and protection against S. japonicum. We show that combining CpG and R848 with pVAX1-Sj26GST immunization significantly increases splenocyte proliferation and IgG and IgG2a levels, decreases CD4+CD25+Foxp3+ regulatory T cells (Treg) frequency in vivo, and enhances protection against S. japonicum. CpG and R848 inhibited Treg-mediated immunosuppression, upregulated the production of interferon (IFN)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-4, IL-10, IL-2, and IL-6, and decreased Foxp3 expression in vitro, which may contribute to prevent Treg suppression and conversion during vaccination and allow expansion of antigen-specific T cells against pathogens.
Conclusions
Our data shows that selective TLR ligands can increase the protective efficacy of DNA vaccines against schistosomiasis, potentially through combined antagonism of Treg-mediated immunosuppression and conversion.
Author Summary
There is evidence that TLR activation can block Treg cell responses and thereby break tolerance to self-antigens. It is expected that the use of TLR ligands as vaccine adjuvants will induce potent anti-pathogen immune responses and simultaneously overcome immune inhibition mediated by Tregs. However, the impact of TLR ligands on schistosomiasis vaccines is unclear. Here, we demonstrate that the use of a TLR7/8 ligand (R848) and a TLR9 ligand (CpG) as adjuvants in combination with the S. japonicum vaccine pVAX1-Sj26GST improves disease protection. The combination of CpG and R848 administered after vaccination causes an immune response marked by an upregulation of splenocyte proliferation and IgG and IgG2a levels that also coincides with a decreased proportion of CD4+CD25+ Tregs in mice. We also show that combined adjuvant use of CpG and R848 may impair Treg development and function by promoting the secretion of proinflammatory cytokines and reducing Foxp3 expression. Our findings suggest that in combination with the vaccine, TLR ligands may protect the effector response from Treg-mediated suppression, thereby eliciting the appropriate immune response to improve vaccine efficacy. Immunization combined with the TLR ligands CpG and R848 thus represents a promising new approach for the design of schistosoma vaccines.
doi:10.1371/journal.pntd.0002164
PMCID: PMC3617091  PMID: 23593527
20.  A variance component based multi-marker association test using family and unrelated data 
BMC Genetics  2013;14:17.
Background
Incorporating family data in genetic association studies has become increasingly appreciated, especially for its potential value in testing rare variants. We introduce here a variance-component based association test that can test multiple common or rare variants jointly using both family and unrelated samples.
Results
The proposed approach implemented in our R package aggregates or collapses the information across a region based on genetic similarity instead of genotype scores, which avoids the power loss when the effects are in different directions or have different association strengths. The method is also able to effectively leverage the LD information in a region and it can produce a test statistic with an adaptively estimated number of degrees of freedom. Our method can readily allow for the adjustment of non-genetic contributions to the familial similarity, as well as multiple covariates.
Conclusions
We demonstrate through simulations that the proposed method achieves good performance in terms of Type I error control and statistical power. The method is implemented in the R package “fassoc”, which provides a useful tool for data analysis and exploration.
doi:10.1186/1471-2156-14-17
PMCID: PMC3614458  PMID: 23497289
Association studies; Family data; Score test; Multi-marker test
21.  Outcomes of Sustained-Release Formulation of Valproate and Topiramate Monotherapy in Patients with Epilepsy: A Multi-Centre, Cohort Study 
PLoS ONE  2012;7(12):e47982.
Background
New-generation antiepileptic drugs (AEDs) tend to replace traditional AEDs as the first-line choice for epilepsy. However, whether this change results in better outcome, especially in China, remains unknown.
Methodology/Principal Findings
Two broad spectrum AEDs, the traditional drug of sustained-release formulation of valproate (SRVPA) and the new-generation drug of topiramate, were compared in patients with epilepsy as monotherapy in this multi-centre, observational cohort study from 2000 to 2011. The primary outcome was time to treatment failure. The secondary outcomes included time to first seizure, time to 12-month remission, and time to 24-month remission. Drug tolerability was assessed. Cox proportional hazard models (95% confidence interval [CI]) were used to analyse the relative risks expressed as hazard ratios (HR).
Of the 1008 recruited patients, 519 received SRVPA and 489 received topiramate. SRVPA was better than topiramate (28.3% vs. 41.5%; HR = 0.62, [95% CI 0.49–0.77]; p<0.0001) in primary outcome, and in time to first seizure (56.1% vs. 69.3%; HR = 0.73, [95% CI 0.62–0.86]; p = 0.0002). No significant difference was observed between two groups in time to 12-month remission (52.6% vs. 42.5%; HR = 1.01, [95% CI 0.84–1.23]; p = 0.88) and time to 24-month remission (34.7% vs. 25.2%; HR = 1.11, [95% CI 0.88–1.42]; p = 0.38). 36 patients (6.9%) in SRVPA group and 37 patients (7.6%) in topiramate group presented treatment failure associated with intolerable adverse events, there was no significant difference between the two groups (p = 0.70).
Conclusions
The SRVPA is more suitable than topiramate for Chinese epileptic patients, and our results support the viewpoint that traditional AEDs should be the first-line choice for epilepsy rather than new-generation AEDs.
doi:10.1371/journal.pone.0047982
PMCID: PMC3519782  PMID: 23239963
22.  A Unique Feature of Iron Loss via Close Adhesion of Helicobacter pylori to Host Erythrocytes 
PLoS ONE  2012;7(11):e50314.
Iron deficiency anemia is an extra-stomach disease experienced in H. pylori carriers. Individuals with type A blood are more prone to suffering from H. pylori infection than other individuals. To clarify the molecular mechanisms underlying H. pylori-associated anemia, we collected erythrocytes from A, B, O, and AB blood donors and analyzed morphology, the number of erythrocytes with H. pylori colonies attached to them, and iron contents in erythrocytes and H. pylori (NCTC11637 and SS1 strains) by means of optical microscopy, scanning electron microscopy, and synchrotron radiation soft X-ray imaging. The number of type A erythrocytes with H. pylori attached to them was significantly higher than that of other erythrocytes (P<0.05). Far more iron distribution was observed in H. pylori bacteria using dual energy analysis near the iron L2, 3 edges by soft X-ray imaging. Iron content was significantly reduced in host erythrocytes after 4 hours of exposure to H. pylori. H. pylori are able to adhere more strongly to type A erythrocytes, and this is related to iron shift from the host to the bacteria. This may explain the reasons for refractory iron deficiency anemia and elevated susceptibility to H. pylori infection in individuals with type A blood.
doi:10.1371/journal.pone.0050314
PMCID: PMC3503993  PMID: 23185604
23.  Hypothermia Induced by Adenosine 5′-Monophosphate Attenuates Acute Lung Injury Induced by LPS in Rats 
Mediators of Inflammation  2012;2012:459617.
We have built a rat's model to investigate whether the hypothermia induced by adenosine 5′-monophosphate (5′-AMP) (AIH) could attenuate acute lung injury induced by LPS in rats. We detected the inflammatory cytokine levels in the plasma and bronchoalveolar lavage fluid samples, and we analyzed the pathological changes in the lungs. We have found that AIH can effectively inhibit acute inflammatory reactions and protect the lung from acute injury induced by LPS in rats.
doi:10.1155/2012/459617
PMCID: PMC3449152  PMID: 23024464
24.  Two-dimensional universal conductance fluctuations and the electron-phonon interaction of surface states in Bi2Te2Se microflakes 
Scientific Reports  2012;2:595.
The universal conductance fluctuations (UCFs), one of the most important manifestations of mesoscopic electronic interference, have not yet been demonstrated for the two-dimensional surface state of topological insulators (TIs). Even if one delicately suppresses the bulk conductance by improving the quality of TI crystals, the fluctuation of the bulk conductance still keeps competitive and difficult to be separated from the desired UCFs of surface carriers. Here we report on the experimental evidence of the UCFs of the two-dimensional surface state in the bulk insulating Bi2Te2Se microflakes. The solely-B⊥-dependent UCF is achieved and its temperature dependence is investigated. The surface transport is further revealed by weak antilocalizations. Such survived UCFs of the surface states result from the limited dephasing length of the bulk carriers in ternary crystals. The electron-phonon interaction is addressed as a secondary source of the surface state dephasing based on the temperature-dependent scaling behavior.
doi:10.1038/srep00595
PMCID: PMC3424525  PMID: 22916331
25.  Partial Regulatory T Cell Depletion Prior to Schistosomiasis Vaccination Does Not Enhance the Protection 
PLoS ONE  2012;7(7):e40359.
CD4+CD25+ regulatory T cells (Tregs) do not only influence self-antigen specific immune responses, but also dampen the protective effect induced by a number of vaccines. The impact of CD4+CD25+ Tregs on vaccines against schistosomiasis, a neglected tropical disease that is a major public health concern, however, has not been examined. In this study, a DNA vaccine encoding a 26 kDa glutathione S-transferase of Schistosoma japonicum (pVAX1-Sj26GST) was constructed and its potential effects were evaluated by depleting CD25+ cells prior to pVAX1-Sj26GST immunization. This work shows that removal of CD25+ cells prior to immunization with the pVAX1-Sj26GST schistosomiasis DNA vaccine significantly increases the proliferation of splenocytes and IgG levels. However, CD25+ cell-depleted mice immunized with pVAX1-Sj26GST show no improved protection against S. japonicum. Furthermore, depletion of CD25+ cells causes an increase in both pro-inflammatory cytokines (e.g. IFN-γ, GM-CSF and IL-4) and an anti-inflammatory cytokine (e.g. IL-10), with CD4+CD25- T cells being one of the major sources of both IFN-γ and IL-10. These findings indicate that partial CD25+ cell depletion fails to enhance the effectiveness of the schistosome vaccine, possibly due to IL-10 production by CD4+CD25- T cells, or other cell types, after CD25+ cell depletion during vaccination.
doi:10.1371/journal.pone.0040359
PMCID: PMC3389001  PMID: 22802961

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