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2.  Porcine Epidemic Diarrhea Virus Nucleocapsid Protein Antagonizes Beta Interferon Production by Sequestering the Interaction between IRF3 and TBK1 
Journal of Virology  2014;88(16):8936-8945.
ABSTRACT
Porcine epidemic diarrhea virus (PEDV), a porcine enteropathogenic coronavirus, causes lethal watery diarrhea in piglets and results in large economic losses in many Asian and European countries. A large-scale outbreak of porcine epidemic diarrhea occurred in China in 2010, and the virus emerged in the United States in 2013 and spread rapidly, posing significant economic and public health concerns. Previous studies have shown that PEDV infection inhibits the synthesis of type I interferon (IFN), and viral papain-like protease 2 has been identified as an IFN antagonist. In this study, we found that the PEDV-encoded nucleocapsid (N) protein also inhibits Sendai virus-induced IFN-β production, IFN-stimulated gene expression, and activation of the transcription factors IFN regulatory factor 3 (IRF3) and NF-κB. We also found that N protein significantly impedes the activation of the IFN-β promoter stimulated by TBK1 or its upstream molecules (RIG-I, MDA5, IPS-1, and TRAF3) but does not counteract its activation by IRF3. A detailed analysis revealed that the PEDV N protein targets TBK1 by direct interaction and that this binding sequesters the association between TBK1 and IRF3, which in turn inhibits both IRF3 activation and type I IFN production. Together, our findings demonstrate a new mechanism evolved by PEDV to circumvent the host's antiviral immunity.
IMPORTANCE PEDV has received increasing attention since the emergence of a PEDV variant in China and the United States. Here, we identify nucleocapsid (N) protein as a novel PEDV-encoded interferon (IFN) antagonist and demonstrate that N protein antagonizes IFN production by sequestering the interaction between IRF3 and TBK1, a critical step in type I IFN signaling. This adds another layer of complexity to the immune evasion strategies evolved by this economically important viral pathogen. An understanding of its immune evasion mechanism may direct us to novel therapeutic targets and more effective vaccines against PEDV infection.
doi:10.1128/JVI.00700-14
PMCID: PMC4136253  PMID: 24872591
3.  Detrended Partial-Cross-Correlation Analysis: A New Method for Analyzing Correlations in Complex System 
Scientific Reports  2015;5:8143.
In this paper, a new method, detrended partial-cross-correlation analysis (DPCCA), is proposed. Based on detrended cross-correlation analysis (DCCA), this method is improved by including partial-correlation technique, which can be applied to quantify the relations of two non-stationary signals (with influences of other signals removed) on different time scales. We illustrate the advantages of this method by performing two numerical tests. Test I shows the advantages of DPCCA in handling non-stationary signals, while Test II reveals the “intrinsic” relations between two considered time series with potential influences of other unconsidered signals removed. To further show the utility of DPCCA in natural complex systems, we provide new evidence on the winter-time Pacific Decadal Oscillation (PDO) and the winter-time Nino3 Sea Surface Temperature Anomaly (Nino3-SSTA) affecting the Summer Rainfall over the middle-lower reaches of the Yangtze River (SRYR). By applying DPCCA, better significant correlations between SRYR and Nino3-SSTA on time scales of 6 ~ 8 years are found over the period 1951 ~ 2012, while significant correlations between SRYR and PDO on time scales of 35 years arise. With these physically explainable results, we have confidence that DPCCA is an useful method in addressing complex systems.
doi:10.1038/srep08143
PMCID: PMC4311241  PMID: 25634341
4.  Brain regions involved in moxibustion-induced analgesia in irritable bowel syndrome with diarrhea: a functional magnetic resonance imaging study 
Background
Moxibustion is one of the most commonly used therapies in acupuncture practice, and is demonstrated to be beneficial for patients with diarrhea from irritable bowel syndrome (D-IBS). But its mechanism remains unclear. Because visceral hypersensitivity in IBS patients has been documented by evaluation of perceived stimulations through functional magnetic resonance imaging (fMRI) studies, we focused on observing brain imaging changes in D-IBS patients during rectal balloon distention before and after moxibustion in order to reveal its possible central mechanism and further evaluate its effect.
Methods
This clinical trial is registered under the number: ChiCTR-TRC-10000887. Eighty D-IBS patients were randomly divided into a moxibustion and sham moxibustion group (control group) for a 4-week treatment. Fifteen patients in moxibustion group and thirteen patients in control group completed two fMRI scans during a 50 and 100 ml rectal balloon distention before and after treatment. Rectal pain were obtained with a scan test. Birmingham IBS Symptom Scale and IBS Quality of Life (QOL) Scale were used to evaluate therapeutic effect.
Results
After treatment, the decrease in Birmingham IBS Symptom Scale and IBS QOL Scale scores in moxibustion group was significantly greater than that of control group (P < 0.01). The defecation urge threshold and the pain perception threshold of moxibustion group was also significantly higher after treatment than that of control group (P < 0.01). The decrease in pain score during the 100 ml rectal balloon distention in moxibustion group was significantly greater than that of control group (P < 0.05). There was no definite activated center during the 50 ml rectal distention in either group before treatment. After treatment, the prefrontal cortex (PFC) was affected in moxibustion group, while the PFC and the anterior cingulated cortex (ACC) were affected in control group. During the 100 ml distention before treatment in both groups, the PFC and ACC were activated. After treatment, they disappeared in moxibustion group but remained in control group.
Conclusions
Moxibustion can improve symptoms and quality of life in D-IBS patients. It can also decrease rectal sensitivity. The activation of PFC and ACC during a 100 ml rectal distention disappeared after moxibustion treatment.
doi:10.1186/1472-6882-14-500
PMCID: PMC4301658  PMID: 25516481
Moxibustion; fMRI; D-IBS
5.  Light-Promoted Rhodopsin Expression and Starvation Survival in the Marine Dinoflagellate Oxyrrhis marina 
PLoS ONE  2014;9(12):e114941.
The discovery of microbial rhodopsins in marine proteobacteria changed the dogma that photosynthesis is the only pathway to use the solar energy for biological utilization in the marine environment. Although homologs of these rhodopsins have been identified in dinoflagellates, the diversity of the encoding genes and their physiological roles remain unexplored. As an initial step toward addressing the gap, we conducted high-throughput transcriptome sequencing on Oxyrrhis marina to retrieve rhodopsin transcripts, rapid amplification of cDNA ends to isolate full-length cDNAs of dominant representatives, and quantitative reverse-transcription PCR to investigate their expression under varying conditions. Our phylogenetic analyses showed that O. marina contained both the proton-pumping type (PR) and sensory type (SR) rhodopsins, and the transcriptome data showed that the PR type dominated over the SR type. We compared rhodopsin gene expression for cultures kept under light: dark cycle and continuous darkness in a time course of 24 days without feeding. Although both types of rhodopsin were expressed under the two conditions, the expression levels of PR were much higher than SR, consistent with the transcriptomic data. Furthermore, relative to cultures kept in the dark, rhodopsin expression levels and cell survival rate were both higher in cultures grown in the light. This is the first report of light-dependent promotion of starvation survival and concomitant promotion of PR expression in a eukaryote. While direct evidence needs to come from functional test on rhodopsins in vitro or gene knockout/knockdown experiments, our results suggest that the proton-pumping rhodopsin might be responsible for the light-enhanced survival of O. marina, as previously demonstrated in bacteria.
doi:10.1371/journal.pone.0114941
PMCID: PMC4266641  PMID: 25506945
6.  Specific deficiency of Plzf paralog, Zbtb20, in Sertoli cells does not affect spermatogenesis and fertility in mice 
Scientific Reports  2014;4:7062.
Ztbt20 is a POK family transcription factor and primarily functions through its conserved C2H2 Krüppel type zinc finger and BTB/POZ domains. The present study was designed to define the function of the Zbtb20, in vivo, during mouse spermatogenesis. Immunohistochemical studies revealed that ZBTB20 protein was localized specifically in the nuclei of Sertoli cells in seminiferous tubules. To investigate its role during spermatogenesis, we crossed Amh-Cre transgenic mice with Zbtb20 floxp mice to generate conditionally knockout mice (cKO) in which Zbtb20 was specifically deleted in Sertoli cells. The cKO mice were fertile and did not show any detectable abnormalities in spermatogenesis. Taken together, though specific deletion of transcription factor Zbtb20 in Sertoli cells has no apparent influence on spermatogenesis, its specific localization in Sertoli cells makes Zbtb20 a useful marker for the identification of Sertoli cells in seminiferous tubules.
doi:10.1038/srep07062
PMCID: PMC4231391  PMID: 25395169
7.  Stomach Virtual Non-Enhanced CT with Second-Generation, Dual-Energy CT: A Preliminary Study 
PLoS ONE  2014;9(11):e112295.
Objectives
To compare the true non-enhanced (TNE) and virtual non-enhanced (VNE) data sets in patients who underwent gastric preoperative dual-energy CT (DECT) and to evaluate potential radiation dose reduction by omitting a TNE scan.
Methods
A total of 74 patients underwent gastric DECT. The mean CT values, length, image quality and effective radiation doses for VNE and TNE images were compared.
Results
There was no statistical difference in maximal thickness of gastric tumors and maximal diameter of enlarged lymph nodes among the TNE and VNE images (P>0.05). The mean CT value differences between TNE and VNE were statistically significant for all tissue types, except for aorta attenuation measurements (P<0.05), but the absolute differences were under 10 HU. Lower noise was found for VNE images than TNE images (P<0.01). Image quality of VNE was diagnostic but lower than that of TNE (P<0.01). The dose reduction achieved by omitting the TNE acquisition was 21.40±4.44%.
Conclusion
VNE scan may potentially replace TNE as part of a multi-phase gastric preoperative staging imaging protocol with consequent saving in radiation dose.
doi:10.1371/journal.pone.0112295
PMCID: PMC4231030  PMID: 25393001
8.  Dehydroepiandrosterone improves follicular fluid bone morphogenetic protein-15 and accumulated embryo score of infertility patients with diminished ovarian reserve undergoing in vitro fertilization: a randomized controlled trial 
Objective
To evaluate the effect of dehydroepiandrosterone (DHEA) on infertility patients with diminished ovarian reserve undergoing in vitro fertilization.
Methods
This is a prospective study. Ninety-five patients with diminished ovarian reserve were included in this study. Of them, 42 patients were randomly allocated to the DHEA group, who received DHEA 75 mg daily for three consecutive menstrual cycles prior to IVF cycles, and 53 patients were allocated to the control group, who entered IVF cycles directly. All patients were treated with the same ovarian stimulation protocol. Follicular fluid samples from both groups were collected for bone morphogenetic protein-15 (BMP-15) and growth differentiation factor-9 (GDF-9). Fluid from the first aspirated follicle without any visible blood contamination was carefully collected. In addition, day 3 Blood samples were collected pre- and post-treatment of DHEA for serum anti-Mullerian hormone (AMH), follicle stimulating hormone (FSH) and estradiol (E2) in the DHEA group.
Results
The level of BMP-15 in follicular fluid samples from the DHEA group was significantly higher than that of the control samples (P=.000). Patients after DHEA treatment demonstrated a significantly higher level of AMH and a significantly lower level of FSH, E2 compared to themselves prior to DHEA therapy (P=.015; P=.036; P=.002; respectively). Moreover, the accumulated score of embryos was significantly higher in the DHEA group (P=.033).
Conclusions
These observations confirm the beneficial effect of DHEA for infertility patients with diminished ovarian reserve.
Trial registration
ChiCTR-TRC-14005002
doi:10.1186/s13048-014-0093-3
PMCID: PMC4210503  PMID: 25330837
Dehydroepiandrosterone; Diminished ovarian reserve; BMP-15; GDF-9; AMH
9.  Modules, networks and systems medicine for understanding disease and aiding diagnosis 
Genome Medicine  2014;6(10):82.
Many common diseases, such as asthma, diabetes or obesity, involve altered interactions between thousands of genes. High-throughput techniques (omics) allow identification of such genes and their products, but functional understanding is a formidable challenge. Network-based analyses of omics data have identified modules of disease-associated genes that have been used to obtain both a systems level and a molecular understanding of disease mechanisms. For example, in allergy a module was used to find a novel candidate gene that was validated by functional and clinical studies. Such analyses play important roles in systems medicine. This is an emerging discipline that aims to gain a translational understanding of the complex mechanisms underlying common diseases. In this review, we will explain and provide examples of how network-based analyses of omics data, in combination with functional and clinical studies, are aiding our understanding of disease, as well as helping to prioritize diagnostic markers or therapeutic candidate genes. Such analyses involve significant problems and limitations, which will be discussed. We also highlight the steps needed for clinical implementation.
doi:10.1186/s13073-014-0082-6
PMCID: PMC4254417  PMID: 25473422
10.  Equilibrium spin current in graphene with Rashba spin-orbit coupling 
Scientific Reports  2014;4:6464.
The existence of a background spin current under thermodynamic equilibrium is an interesting phenomenon in the two-dimensional electron gas with Rashba spin-orbit coupling (RSOC). Here we study the equilibrium spin current (ESC) in graphene with RSOC. For an infinite graphene with uniform RSOC, we found that the ESC is proportional to λ2 with λ the Rashba strength and mainly comes from the energy window [−λ, λ] near Dirac points. In the regime of energy far away from Dirac points, the λ3 dependence as that in a normal two-dimensional electron gas is recovered. In a system with a normal graphene strip inserted between two Rashba graphene sheets, we found that the ESC can penetrate through the normal graphene layer (perpendicular to the interface). This unique effect can be understood by considering the spin-filtered scattering from the normal region to the RSOC region. The finding of the ESC through the normal region without RSOC advances the understanding of ESC and provides a new way to generate a pure spin current in graphene. For an experimentally accessible strength of Rashba spin-orbit coupling, the ESC remains over room temperature.
doi:10.1038/srep06464
PMCID: PMC4173034  PMID: 25249215
11.  A Survey of Overlooked Viral Infections in Biological Experiment Systems 
PLoS ONE  2014;9(8):e105348.
It is commonly accepted that there are many unknown viruses on the planet. For the known viruses, do we know their prevalence, even in our experimental systems? Here we report a virus survey using recently published small (s)RNA sequencing datasets. The sRNA reads were assembled and contigs were screened for virus homologues against the NCBI nucleotide (nt) database using the BLASTn program. To our surprise, approximately 30% (28 out of 94) of publications had highly scored viral sequences in their datasets. Among them, only two publications reported virus infections. Though viral vectors were used in some of the publications, virus sequences without any identifiable source appeared in more than 20 publications. By determining the distributions of viral reads and the antiviral RNA interference (RNAi) pathways using the sRNA profiles, we showed evidence that many of the viruses identified were indeed infecting and generated host RNAi responses. As virus infections affect many aspects of host molecular biology and metabolism, the presence and impact of viruses needs to be actively investigated in experimental systems.
doi:10.1371/journal.pone.0105348
PMCID: PMC4140767  PMID: 25144530
12.  Mucin 5B Promoter Polymorphism Is Associated with Susceptibility to Interstitial Lung Diseases in Chinese Males 
PLoS ONE  2014;9(8):e104919.
The variation of G>T in the MUC5B promoter (rs35705950) has been associated with idiopathic pulmonary fibrosis (IPF) and familial interstitial pneumonia (FIP) in Caucasians, but no information is available regarding this variant in the Chinese population. We recruited 405 patients with interstitial lung diseases (ILD), including 165 IPF patients and 2043 healthy controls, for genotyping the MUC5B gene in the Chinese population. One hundred three patients with pneumonia and 360 patients with autoimmune diseases (ADs) were recruited as disease controls. Our results indicated that the prevalence of the minor allele (T) of the polymorphism rs35705950 in healthy Chinese subjects was approximately 0.66%, which was lower than that described in the Caucasian population. The frequencies of the T allele were 3.33% and 2.22% in IPF and ILD patients, respectively, and these values were significantly higher than those of healthy controls (P = 0.001, OR = 4.332 for IPF, and P = 0.002, OR = 2.855 for ILD). A stratified analysis showed that this variant in MUC5B associated with the risk for ILD mainly in older male Chinese subjects. No difference was observed between patients with pneumonia, AD patients, and healthy controls.
doi:10.1371/journal.pone.0104919
PMCID: PMC4133265  PMID: 25121989
13.  Prediction of clinical pregnancy in vitrified-warmed single blastocyst transfer cycles by pre-freeze morphology 
Background: The selection of blastocyst warmed for transfer is based on pre-freeze morphology in vitrified-warmed single blastocyst transfer cycles. But, it is controversial which parameter of blastocyst morphology most closely related to the clinical outcomes.
Objective: To estimate the effect of blastocoele expansion, trophectoderm (TE) morphology grade, and inner cell mass (ICM) morphology grade on clinical pregnancy in vitrified-warmed single blastocyst transfers.
Materials and Methods: There were 172 vitrified-warmed single blastocyst transfer cycles during the year 2012 included in this analysis. Comparison of clinical results between pregnancy and no pregnancy group based on patient and blastocyst morphology characteristics was done. Then stepwise logistic regression analysis was used to select the best morphological predictor for clinical pregnancy. Last, comparison of patient characteristics and clinical outcomes separated by the best independent morphological predictor was done.
Results: Comparison of clinical results between pregnancy and no pregnancy group and logistic regression showed the clinical pregnancy rate was affected by ICM. Comparison of patient characteristics separated by ICM grade, ICM grade A cycles got higher clinical pregnancy rate than ICM grade B cycles (54.3% vs. 35.0% respectively, p=0.037).
Conclusion: Blastocyst with good ICM morphology could increase clinical pregnancy rate in vitrified-warmed single blastocyst transfer cycles.
PMCID: PMC4233316  PMID: 25408707
In vitro fertilization; Innercellmass; Blastocyst; Vitrification; Morphology
14.  Draft Sequencing and Analysis of the Genome of Pufferfish Takifugu flavidus 
The pufferfish Takifugu flavidus is an important economic species due to its outstanding flavour and high market value. It has been regarded as an excellent model of genetic study for decades as well. In the present study, three mate-pair libraries of T. flavidus genome were sequenced by the SOLiD 4 next-generation sequencing platform, and the draft genome was constructed with the short reads using an assisted assembly strategy. The draft consists of 50,947 scaffolds with an N50 value of 305.7 kb, and the average GC content was 45.2%. The combined length of repetitive sequences was 26.5 Mb, which accounted for 6.87% of the genome, indicating that the compactness of T. flavidus genome was approximative with that of T. rubripes genome. A total of 1,253 non-coding RNA genes and 30,285 protein-encoding genes were assigned to the genome. There were 132,775 and 394 presumptive genes playing roles in the colour pattern variation, the relatively slow growth and the lipid metabolism, respectively. Among them, genes involved in the microtubule-dependent transport system, angiogenesis, decapentaplegic pathway and lipid mobilization were significantly expanded in the T. flavidus genome. This draft genome provides a valuable resource for understanding and improving both fundamental and applied research with pufferfish in the future.
doi:10.1093/dnares/dsu025
PMCID: PMC4263296  PMID: 25053628
Takifugu flavidus; draft genome; NGS
15.  The Polymorphism in the Promoter of HSP70 Gene Is Associated with Heat Tolerance of Two Congener Endemic Bay Scallops (Argopecten irradians irradians and A. i. concentricus) 
PLoS ONE  2014;9(7):e102332.
Background
The heat shock protein 70 (HSP70) is one kind of molecular chaperones, which plays a key role in protein metabolism under normal and stress conditions.
Methodology
In the present study, the mRNA expressions of HSP70 under normal physiological condition and after acute heat stress were investigated in gills of two bay scallop populations (Argopecten irradians irradians and A. i. concentricus). The heat resistant scallops A. i. concentricus showed significantly lower basal level and higher induction of HSP70 compared with that of the heat sensitive scallops A. i. irradians. The promoter sequence of HSP70 gene from bay scallop (AiHSP70) was cloned and the polymorphisms within this region were investigated to analyze their association with heat tolerance. Totally 11 single nucleotide polymorphisms (SNPs) were identified, and four of them (−967, −480, −408 and −83) were associated with heat tolerance after HWE analysis and association analysis. Based on the result of linkage disequilibrium analysis, the in vitro transcriptional activities of AiHSP70 promoters with different genotype were further determined, and the results showed that promoter from A. i. concentricus exhibited higher transcriptional activity than that from A. i. irradians (P<0.05).
Conclusions
The results provided insights into the molecular mechanisms underlying the thermal adaptation of different congener endemic bay scallops, which suggested that the increased heat tolerance of A. i. concentricus (compared with A. i. irradians) was associated with the higher expression of AiHSP70. Meanwhile, the −967 GG, −480 AA, −408 TT and −83 AG genotypes could be potential markers for scallop selection breeding with higher heat tolerance.
doi:10.1371/journal.pone.0102332
PMCID: PMC4100766  PMID: 25028964
16.  Charge-to-mass Ratio of Saltating Particles in Wind-Blown Sand 
Scientific Reports  2014;4:5590.
The electrification of sand particles plays an important role in aeolian events. In this paper, the charge-to-mass ratio vertical profiles of saltating particles in wind-blown sand were measured by a field experiments. By combining the results of field measurements with our previous wind-tunnel measurements, we discussed the factors affecting the charge-to-mass ratio of saltating particles. It reveals that the magnitude of the charge-to-mass ratio increases exponentially with height above the surface. In addition, the charge polarity of saltating particles depends on the relative size between saltating and creeping particles, and the magnitude of charge-to-mass ratio is determined by wind velocity and the relative size difference ratio between saltating and creeping particles.
doi:10.1038/srep05590
PMCID: PMC4083261  PMID: 24998641
17.  De Novo Transcriptomes of a Mixotrophic and a Heterotrophic Ciliate from Marine Plankton 
PLoS ONE  2014;9(7):e101418.
Studying non-model organisms is crucial in the context of the current development of genomics and transcriptomics for both physiological experimentation and environmental characterization. We investigated the transcriptomes of two marine planktonic ciliates, the mixotrophic oligotrich Strombidium rassoulzadegani and the heterotrophic choreotrich Strombidinopsis sp., and their respective algal food using Illumina RNAseq. Our aim was to characterize the transcriptomes of these contrasting ciliates and to identify genes potentially involved in mixotrophy. We detected approximately 10,000 and 7,600 amino acid sequences for S. rassoulzadegani and Strombidinopsis sp., respectively. About half of these transcripts had significant BLASTP hits (E-value <10−6) against previously-characterized sequences, mostly from the model ciliate Oxytricha trifallax. Transcriptomes from both the mixotroph and the heterotroph species provided similar annotations for GO terms and KEGG pathways. Most of the identified genes were related to housekeeping activity and pathways such as the metabolism of carbohydrates, lipids, amino acids, nucleotides, and vitamins. Although S. rassoulzadegani can keep and use chloroplasts from its prey, we did not find genes clearly linked to chloroplast maintenance and functioning in the transcriptome of this ciliate. While chloroplasts are known sources of reactive oxygen species (ROS), we found the same complement of antioxidant pathways in both ciliates, except for one enzyme possibly linked to ascorbic acid recycling found exclusively in the mixotroph. Contrary to our expectations, we did not find qualitative differences in genes potentially related to mixotrophy. However, these transcriptomes will help to establish a basis for the evaluation of differential gene expression in oligotrichs and choreotrichs and experimental investigation of the costs and benefits of mixotrophy.
doi:10.1371/journal.pone.0101418
PMCID: PMC4077812  PMID: 24983246
18.  The Direct Inhibitory Effect of Dutasteride or Finasteride on Androgen Receptor Activity is Cell Line Specific 
The Prostate  2013;73(14):1483-1494.
BACKGROUND
Finasteride and dutasteride were developed originally as 5α-reductase inhibitors to block the conversion of testosterone to dihydrotestosterone (DHT). These drugs may possess off-target effects on the androgen receptor (AR) due to their structural similarity to DHT.
METHODS
A total of 4 human prostate cancer cell models were examined: LNCaP (T877A mutant AR), 22Rv1 (H874Y mutant AR), LAPC4 (wild type AR) and VCaP (wild type AR). Cells were cultured in 10% charcoal-stripped fetal bovine serum, either with or without DHT added to the medium. AR activity was evaluated using the ARE-luciferase assay or the expression of AR regulated genes.
RESULTS
Dutasteride was more potent than finasteride in interfering with DHT-stimulated AR signaling. Disruption of AR function was accompanied by decreased cell growth. Cells that rely on DHT for protection against death were particularly vulnerable to dutasteride. Different prostate cancer cell models exhibited different sensitivities to dutasteride and finasteride. LNCaP was most sensitive, LAPC4 and VCaP were intermediate, while 22Rv1 was least sensitive. Regardless of the AR genotype, if AR was transfected into drug-sensitive cells, AR was inhibited by drug treatment; and if AR was transfected into drug-resistant cells, AR was not inhibited.
CONCLUSIONS
The direct inhibitory effect of dutasteride or finasteride on AR signaling is cell line specific. Mutations in the ligand binding domain of AR do not appear to play a significant role in influencing the AR antagonistic effect of these drugs. Subcellular constituent is an important factor in determining the drug effect on AR function.
doi:10.1002/pros.22696
PMCID: PMC3992475  PMID: 23813737
finasteride; dutasteride; antiandrogen; androgen receptor signaling; prostate cancer
19.  Quantitative analysis of real-time tissue elastography for evaluation of liver fibrosis 
The present study aimed to investigate the feasibility of quantitative analysis of liver fibrosis using real-time tissue elastography (RTE) and its pathological and molecule biological basis. Methods: Fifty-four New Zealand rabbits were subcutaneously injected with thioacetamide (TAA) to induce liver fibrosis as the model group, and another eight New Zealand rabbits served as the normal control group. Four rabbits were randomly taken every two weeks for real-time tissue elastography (RTE) and quantitative analysis of tissue diffusion. The obtained twelve characteristic quantities included relative mean value (MEAN), standard deviation (SD), blue area % (% AREA), complexity (COMP), kurtosis (KURT), skewness (SKEW), contrast (CONT), entropy (ENT), inverse different moment (IDM), angular secon moment (ASM), correlation (CORR) and liver fibrosis index (LF Index). Rabbits were executed and liver tissues were taken for pathological staging of liver fibrosis (grouped by pathological stage into S0 group, S1 group, S2 group, S3 group and S4 group). In addition, the collagen I (Col I) and collagen III (Col III) expression levels in liver tissue were detected by Western blot. Results: Except for KURT, there were significant differences among the other eleven characteristic quantities (P < 0.05). LF Index, Col I and Col III expression levels showed a rising trend with increased pathological staging of liver fibrosis, presenting a positive correlation with the pathological staging of liver fibrosis (r = 0.718, r = 0.693, r = 0.611, P < 0.05). Conclusion: RTE quantitative analysis is expected for noninvasive evaluation of the pathological staging of liver fibrosis.
PMCID: PMC4057854  PMID: 24955175
Ultrasound; elastography; liver fibrosis; non-invasive diagnosis
20.  Pattern of cerebral hyperperfusion in Alzheimer’s disease and amnestic mild cognitive impairment using voxel-based analysis of 3D arterial spin-labeling imaging: initial experience 
Purpose
A three-dimensional (3D) continuous pulse arterial spin labeling (ASL) technique was used to investigate cerebral blood flow (CBF) changes in patients with Alzheimer’s disease (AD), amnestic mild cognitive impairment (aMCI), and age- and sex-matched healthy controls.
Materials and methods
Three groups were recruited for comparison, 24 AD patients, 17 MCI patients, and 21 age- and sex-matched control subjects. Three-dimensional ASL scans covering the entire brain were acquired with a 3.0 T magnetic resonance scanner. Spatial processing was performed with statistical parametric mapping 8. A second-level one-way analysis of variance analysis (threshold at P<0.05) was performed on the preprocessed ASL data. An average whole-brain CBF for each subject was also included as group-level covariates for the perfusion data, to control for individual CBF variations.
Results
Significantly increased CBF was detected in bilateral frontal lobes and right temporal subgyral regions in aMCI compared with controls. When comparing AD with aMCI, the major hyperperfusion regions were the right limbic lobe and basal ganglia regions, including the putamen, caudate, lentiform nucleus, and thalamus, and hypoperfusion was found in the left medial frontal lobe, parietal cortex, the right middle temporo-occipital lobe, and particularly, the left anterior cingulate gyrus. We also found decreased CBF in the bilateral temporo-parieto-occipital cortices and left limbic lobe in AD patients, relative to the control group. aMCI subjects showed decreased blood flow in the left occipital lobe, bilateral inferior temporal cortex, and right middle temporal cortex.
Conclusion
Our results indicated that ASL provided useful perfusion information in AD disease and may be used as an appealing alternative for further pathologic and neuropsychological studies, especially of compensatory mechanisms for cerebral hypoperfusion.
doi:10.2147/CIA.S58879
PMCID: PMC3971940  PMID: 24707173
Alzheimer’s disease; amnestic mild cognitive impairment; perfusion image; arterial spin labeling
21.  CypA, a Gene Downstream of HIF-1α, Promotes the Development of PDAC 
PLoS ONE  2014;9(3):e92824.
Hypoxia-inducible factor-1α (HIF-1α) is a highly important transcription factor involved in cell metabolism. HIF-1α promotes glycolysis and inhibits of mitochondrial respiration in pancreatic ductal adenocarcinoma (PDAC). In response to tumor hypoxia, cyclophilin A (CypA) is over-expressed in various cancer types, and is associated with cell apoptosis, tumor invasion, metastasis, and chemoresistance in PDAC. In this study, we showed that both HIF-1α and CypA expression were significantly associated with lymph node metastasis and tumor stage. The expression of CypA was correlated with HIF-1α. Moreover, the mRNA and protein expression of CypA markedly decreased or increased following the suppression or over-expression of HIF-1α in vitro. Chromatin immunoprecipitation analysis showed that HIF-1α could directly bind to the hypoxia response element (HRE) in the CypA promoter regions and regulated CypA expression. Consistent with other studies, HIF-1α and CypA promoted PDAC cell proliferation and invasion, and suppressed apoptosis in vitro. Furthermore, we proved the combination effect of 2-methoxyestradiol and cyclosporin A both in vitro and in vivo. These results suggested that,CypA, a gene downstream of HIF-1α, could promote the development of PDAC. Thus, CypA might serve as a potential therapeutic target for PDAC.
doi:10.1371/journal.pone.0092824
PMCID: PMC3963943  PMID: 24662981
22.  Integration of partial least squares and Monte Carlo gene expression analysis in coronary artery disease 
Coronary artery disease (CAD) is the most common type of cardiovascular disease and leading cause of mortality worldwide. Microarray technology for gene expression analysis has facilitated the identification of the molecular mechanism that underlies the pathogenesis of CAD. Previous studies have primarily used variance or regression analysis, without considering array specific factors. Thus, the aim of the present study was to investigate the mechanism of CAD using partial least squares (PLS)-based analysis, which was integrated with the Monte Carlo technique. Microarray analysis was performed with a data set of 110 CAD patients and 111 controls obtained from the Gene Expression Omnibus database. A total of 390 dysregulated genes were acquired. Significantly increased representations of dysregulated genes in Gene Ontology items, including transforming growth factor β-activated receptor activity and acyl-CoA oxidase activity, were identified. Network analysis revealed three hub genes with a degree of >10, including ESR1, ITGA4 and ARRB2. The results of the present study provide novel information on the gene expression signatures of CAD patients and offer further theoretical support for future therapeutic study.
doi:10.3892/etm.2014.1610
PMCID: PMC3991499  PMID: 24940402
coronary artery disease; gene expression; partial least squares; Monte Carlo
23.  Integrated genomic and prospective clinical studies show the importance of modular pleiotropy for disease susceptibility, diagnosis and treatment 
Genome Medicine  2014;6(2):17.
Background
Translational research typically aims to identify and functionally validate individual, disease-specific genes. However, reaching this aim is complicated by the involvement of thousands of genes in common diseases, and that many of those genes are pleiotropic, that is, shared by several diseases.
Methods
We integrated genomic meta-analyses with prospective clinical studies to systematically investigate the pathogenic, diagnostic and therapeutic roles of pleiotropic genes. In a novel approach, we first used pathway analysis of all published genome-wide association studies (GWAS) to find a cell type common to many diseases.
Results
The analysis showed over-representation of the T helper cell differentiation pathway, which is expressed in T cells. This led us to focus on expression profiling of CD4+ T cells from highly diverse inflammatory and malignant diseases. We found that pleiotropic genes were highly interconnected and formed a pleiotropic module, which was enriched for inflammatory, metabolic and proliferative pathways. The general relevance of this module was supported by highly significant enrichment of genetic variants identified by all GWAS and cancer studies, as well as known diagnostic and therapeutic targets. Prospective clinical studies of multiple sclerosis and allergy showed the importance of both pleiotropic and disease specific modules for clinical stratification.
Conclusions
In summary, this translational genomics study identified a pleiotropic module, which has key pathogenic, diagnostic and therapeutic roles.
doi:10.1186/gm534
PMCID: PMC4064311  PMID: 24571673
24.  The Identification and Characteristics of Immune-Related MicroRNAs in Haemocytes of Oyster Crassostrea gigas 
PLoS ONE  2014;9(2):e88397.
Background
MicroRNAs (miRNAs) represent a class of small ncRNAs that repress gene expression on the post-transcriptional level by the degradation or translation inhibition of target mRNA.
Methodology
Three small RNA libraries from oyster haemocytes were sequenced on the Illumina platform to investigate the latent immunomodulation of miRNAs after bacteria challenge and heat stress. Totally, 10,498,663, 8,588,606 and 9,679,663 high-quality reads were obtained in the control, bacteria and bacteria+heat library, respectively, from which 199 oyster miRNAs including 71 known and 128 novel ones were identified. Among these miRNAs, 6 known and 23 novel ones were predicted to possess more than one precursor-coding region, and cgi-miR-10a, cgi-miR-184b, cgi-miR-100, cgi-miR-1984 and cgi-miR-67a were observed to be the most abundant miRNAs in the control library. The expression levels of 22 miRNAs in the bacteria library were significantly higher than those in the control library, while there were another 33 miRNAs whose expression levels were significantly lower than that in the control library. Meanwhile, the expression levels of 65 miRNAs in the bacteria+heat library changed significantly compared to those in the bacteria library. The target genes of these differentially expressed miRNAs were annotated, and they fell in immune and stress-related GO terms including antioxidant, cell killing, death, immune system process, and response to stimulus. Furthermore, there were 42 differentially expressed miRNAs detected in both control/bacteria and bacteria/bacteria+heat comparisons, among which 9 miRNAs displayed the identical pattern in the two comparisons, and the expression alterations of 8 miRNAs were confirmed using quantitative RT-PCR.
Conclusions
These results indicated collectively that immune challenge could induce the expression of immune-related miRNAs, which might modulate the immune response such as redox reaction, phagocytosis and apoptosis, and the expression of some immune-related miRNAs could be also regulated by heat stress to improve the environmental adaption of oyster.
doi:10.1371/journal.pone.0088397
PMCID: PMC3916443  PMID: 24516648
25.  DNA Methylation Changes Separate Allergic Patients from Healthy Controls and May Reflect Altered CD4+ T-Cell Population Structure 
PLoS Genetics  2014;10(1):e1004059.
Altered DNA methylation patterns in CD4+ T-cells indicate the importance of epigenetic mechanisms in inflammatory diseases. However, the identification of these alterations is complicated by the heterogeneity of most inflammatory diseases. Seasonal allergic rhinitis (SAR) is an optimal disease model for the study of DNA methylation because of its well-defined phenotype and etiology. We generated genome-wide DNA methylation (Npatients = 8, Ncontrols = 8) and gene expression (Npatients = 9, Ncontrols = 10) profiles of CD4+ T-cells from SAR patients and healthy controls using Illumina's HumanMethylation450 and HT-12 microarrays, respectively. DNA methylation profiles clearly and robustly distinguished SAR patients from controls, during and outside the pollen season. In agreement with previously published studies, gene expression profiles of the same samples failed to separate patients and controls. Separation by methylation (Npatients = 12, Ncontrols = 12), but not by gene expression (Npatients = 21, Ncontrols = 21) was also observed in an in vitro model system in which purified PBMCs from patients and healthy controls were challenged with allergen. We observed changes in the proportions of memory T-cell populations between patients (Npatients = 35) and controls (Ncontrols = 12), which could explain the observed difference in DNA methylation. Our data highlight the potential of epigenomics in the stratification of immune disease and represents the first successful molecular classification of SAR using CD4+ T cells.
Author Summary
T-cells, a type of white blood cell, are an important part of the immune-system in humans. T-cells allow us to adapt our immune-response to the various infectious agents we encounter during life. However, T-cells can also cause disease when they target the body's own cells, e.g. Psoriasis, or when they react to a harmless particle or ‘antigen’, i.e. allergy. Much evidence supports an environmental, or ‘epigenetic’, component to allergy. Surprisingly, although allergy is viewed as a T-cell disease with an epigenetic component, no studies have identified epigenetic differences between healthy individuals and allergic individuals. Using a state-of-the-art genome-wide approach, we found that we could clearly and robustly separate allergic patients from healthy controls. It is often assumed that these changes reflect changes in DNA methylation in a given type of cell; however such differences can also result from different mixtures of T-cell subtypes in the samples. Indeed, we found that allergic patients had different proportions of T-cell sub-types compared to healthy controls. These changes in T-cell proportions may explain the difference in DNA methylation profile we observed between patients and controls. Our study is the first successful molecular classification of allergy using CD4+ T cells.
doi:10.1371/journal.pgen.1004059
PMCID: PMC3879208  PMID: 24391521

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