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1.  Effects of Gray-Scale Ultrasonography Immediate Post-Contrast on Characterization of Focal Liver Lesions 
BioMed Research International  2015;2015:193178.
This study compared the imaging features of conventional gray scale ultrasound (US) before and after contrast-enhanced ultrasound (CEUS) for focal liver lesions and 22 evaluated the role of US post-CEUS in characterizing liver lesions. 126 patients with 158 focal liver lesions underwent CEUS and US post-CEUS examination and entered this study. There were 74 hepatocellular carcinomas (HCC), 43 hepatic metastases, and 41 hemangiomas. Imaging features of US pre-CEUS and US post-CEUS were analyzed offsite by two blinded experienced radiologists to evaluate size, boundary, echogenicity, internal texture, posterior acoustic enhancement, spatial resolution, and contrast resolution. In the end with pathological and clinical evidence, the diagnostic accuracy rate of US pre-CEUS was 53.8% (85/158 lesions), lower than that of CEUS (88.0%, 139/158 lesions); with the complementation of US post-CEUS the rate rose to 93.0% (147/158 lesions). US post-CEUS could improve the visibility of typical structures of focal liver lesions and might provide important complementary information for CEUS diagnosis. It also increases the visibility of small liver lesions compared with US pre-CEUS and helps to guide local interventional procedure.
doi:10.1155/2015/193178
PMCID: PMC4450236  PMID: 26090387
2.  Hospital Variation in Non-Invasive Positive Pressure Ventilation for Acute Decompensated Heart Failure 
Circulation. Heart failure  2014;7(3):427-433.
Background
Although non-invasive positive pressure ventilation (NIPPV) for patients with acute decompensated heart failure (ADHF) was introduced almost 20 years ago, the variation in its use among hospitals remains unknown. We sought to define hospital practice patterns of NIPPV use for ADHF and their relationship with intubation and mortality.
Methods and Results
We conducted a cross-sectional study using a database maintained by Premier, Inc., that includes a date-stamped log of all billed items for hospitalizations at over 400 hospitals. We examined hospitalizations for ADHF in this database from 2005–2010 and included hospitals with annual average volume of greater than 25 such hospitalizations. We identified 384 hospitals that encompassed 524,430 hospitalizations (median annual average volume: 206). We used hierarchical logistic regression models to calculate hospital-level outcomes: risk-standardized NIPPV rate (RS-NIPPV), risk-standardized intubation rate (RSIR), and in-hospital risk-standardized mortality rate (RSMR). We grouped hospitals into quartiles by RS-NIPPV and compared RSMRs and RSIRs across quartiles. Median RS-NIPPV was 6.2% (interquartile range, 2.8–9.3%; 5th percentile, 0.2%; 95th percentile, 14.8%). There was no clear pattern of RSMRs across quartiles. The bottom quartile of hospitals had higher RSIR (11.4%) than each of the other quartiles (9.0%, 9.7%, and 9.1%; P<0.02 for all comparisons).
Conclusion
Substantial variation exists among hospitals in the use of NIPPV for ADHF without evidence for differences in mortality. There may be a threshold effect in relation to intubation rates, with the lowest utilizers of NIPPV having higher intubation rates.
doi:10.1161/CIRCHEARTFAILURE.113.000698
PMCID: PMC4386575  PMID: 24633829
heart failure; mortality; ventilation
3.  Identifying differential transcription factor binding in ChIP-seq 
Frontiers in Genetics  2015;6:169.
ChIP seq is a widely used assay to measure genome-wide protein binding. The decrease in costs associated with sequencing has led to a rise in the number of studies that investigate protein binding across treatment conditions or cell lines. In addition to the identification of binding sites, new studies evaluate the variation in protein binding between conditions. A number of approaches to study differential transcription factor binding have recently been developed. Several of these methods build upon established methods from RNA-seq to quantify differences in read counts. We compare how these new approaches perform on different data sets from the ENCODE project to illustrate the impact of data processing pipelines under different study designs. The performance of normalization methods for differential ChIP-seq depends strongly on the variation in total amount of protein bound between conditions, with total read count outperforming effective library size, or variants thereof, when a large variation in binding was studied. Use of input subtraction to correct for non-specific binding showed a relatively modest impact on the number of differential peaks found and the fold change accuracy to biological validation, however a larger impact might be expected for samples with more extreme copy number variations between them. Still, it did identify a small subset of novel differential regions while excluding some differential peaks in regions with high background signal. These results highlight proper scaling for between-sample data normalization as critical for differential transcription factor binding analysis and suggest bioinformaticians need to know about the variation in level of total protein binding between conditions to select the best analysis method. At the same time, validation using fold-change estimates from qRT-PCR suggests there is still room for further method improvement.
doi:10.3389/fgene.2015.00169
PMCID: PMC4413818  PMID: 25972895
ChIP-seq; differential binding; methods comparison; normalization; validation
4.  Antigenic Relatedness of Norovirus GII.4 Variants Determined by Human Challenge Sera 
PLoS ONE  2015;10(4):e0124945.
The GII.4 noroviruses (NoVs) are a single genotype that is responsible for over 50% of NoV gastroenteritis epidemics worldwide. However, GII.4 NoVs have been found to undergo antigenic drifts, likely selected by host herd immunity, which raises an issue for vaccine strategies against NoVs. We previously characterized GII.4 NoV antigenic variations and found significant levels of antigenic relatedness among different GII.4 variants. Further characterization of the genetic and antigenic relatedness of recent GII.4 variants (2008b and 2010 cluster) was performed in this study. The amino acid sequences of the receptor binding interfaces were highly conserved among all GII.4 variants from the past two decades. Using serum samples from patients enrolled in a GII.4 virus challenge study, significant cross-reactivity between major GII.4 variants from 1998 to 2012 was observed using enzyme-linked immunosorbent assays and HBGA receptor blocking assays. The overall abilities of GII.4 NoVs to bind to the A/B/H HBGAs were maintained while their binding affinities to individual ABH antigens varied. These results highlight the importance of human HBGAs in NoV evolution and how conserved antigenic types impact vaccine development against GII.4 variants.
doi:10.1371/journal.pone.0124945
PMCID: PMC4411064  PMID: 25915764
5.  Mitochondrial proteomics with siRNA knockdown to reveal ACAT1 and MDH2 in the development of doxorubicin-resistant uterine cancer 
Mitochondria are key organelles in mammary cells in responsible for a number of cellular functions including cell survival and energy metabolism. Moreover, mitochondria are one of the major targets under doxorubicin treatment. In this study, low-abundant mitochondrial proteins were enriched for proteomic analysis with the state-of-the-art two-dimensional differential gel electrophoresis (2D-DIGE) and matrix-assistant laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) strategy to compare and identify the mitochondrial protein profiling changes in response to the development of doxorubicin resistance in human uterine cancer cells. The mitochondrial proteomic results demonstrate more than fifteen hundred protein features were resolved from the equal amount pooled of three purified mitochondrial proteins and 101 differentially expressed spots were identified. In which, 39 out of these 101 identified proteins belong to mitochondrial proteins. Mitochondrial proteins such as acetyl-CoA acetyltransferase (ACAT1) and malate dehydrogenase (MDH2) have not been reported with the roles on the formation of doxorubicin resistance in our knowledge. Further studies have used RNA interference and cell viability analysis to evidence the essential roles of ACAT1 and MDH2 on their potency in the formation of doxorubicin resistance through increased cell viability and decreased cell apoptosis during doxorubicin treatment. To sum up, our current mitochondrial proteomic approaches allowed us to identify numerous proteins, including ACAT1 and MDH2, involved in various drug-resistance-forming mechanisms. Our results provide potential diagnostic markers and therapeutic candidates for the treatment of doxorubicin-resistant uterine cancer.
doi:10.1111/jcmm.12388
PMCID: PMC4395189  PMID: 25639359
mitochondrial; DIGE; doxorubicin; resistance; uterine cancer
6.  Synthesis and characterization of ZnS with controlled amount of S vacancies for photocatalytic H2 production under visible light 
Scientific Reports  2015;5:8544.
Controlling amount of intrinsic S vacancies was achieved in ZnS spheres which were synthesized by a hydrothermal method using Zn and S powders in concentrated NaOH solution with NaBH4 added as reducing agent. These S vacancies efficiently extend absorption spectra of ZnS to visible region. Their photocatalytic activities for H2 production under visible light were evaluated by gas chromatograph, and the midgap states of ZnS introduced by S vacancies were examined by density functional calculations. Our study reveals that the concentration of S vacancies in the ZnS samples can be controlled by varying the amount of the reducing agent NaBH4 in the synthesis, and the prepared ZnS samples exhibit photocatalytic activity for H2 production under visible-light irradiation without loading noble metal. This photocatalytic activity of ZnS increases steadily with increasing the concentration of S vacancies until the latter reaches an optimum value. Our density functional calculations show that S vacancies generate midgap defect states in ZnS, which lead to visible-light absorption and responded.
doi:10.1038/srep08544
PMCID: PMC4339798  PMID: 25712901
7.  Global analysis of the rat and human platelet proteome – the molecular blueprint for illustrating multi-functional platelets and cross-species function evolution 
Proteomics  2010;10(13):2444-2457.
Emerging evidences indicate that blood platelets function in multiple biological processes including immune response, bone metastasis and liver regeneration in addition to their known roles in hemostasis and thrombosis. Global elucidation of platelet proteome will provide the molecular base of these platelet functions. Here, we set up a high throughput platform for maximum exploration of the rat/human platelet proteome using integrated proteomics technologies, and then applied to identify the largest number of the proteins expressed in both rat and human platelets. After stringent statistical filtration, a total of 837 unique proteins matched with at least two unique peptides were precisely identified, making it the first comprehensive protein database so far for rat platelets. Meanwhile, quantitative analyses of the thrombin-stimulated platelets offered great insights into the biological functions of platelet proteins and therefore confirmed our global profiling data. A comparative proteomic analysis between rat and human platelets was also conducted, which revealed not only a significant similarity, but also an across-species evolutionary link that the orthologous proteins representing ‘core proteome’, and the ‘evolutionary proteome’ is actually a relatively static proteome.
doi:10.1002/pmic.200900271
PMCID: PMC4302335  PMID: 20443191
Blood platelets; multidimensional separation; mass spectrometry; global profiling; cross-species comparison
8.  Identification of inflammatory mediators in patients with rhegmatogenous retinal detachment associated with choroidal detachment 
Molecular Vision  2015;21:417-427.
Purpose
To investigate the expression profile of intravitreous cytokines, chemokines, and growth factors in patients with rhegmatogenous retinal detachment associated with choroidal detachment (RRDCD) in comparison with patients with only rhegmatogenous retinal detachment (RRD).
Methods
Twenty RRDCD patients and 30 RRD patients were included in this case-control study. A multiplex bead-based immunoassay was performed to determine the expression of a wide range of 29 inflammatory mediators in undiluted vitreous from the patients. Data were analyzed using the Mann–Whitney U-test for nonparametric values and multivariate logistic regression analysis.
Results
Compared with the patients with RRD, intravitreous inflammatory mediators, including migration inhibitor factor (MIF), interleukin-6 (IL-6), CCL4, CCL11, CCL17, CCL19, CCL22, CXCL9, CXCL8, soluble inter-cellular adhesion molecule 1 (sICAM-1), transforming growth factor β3 (TGF-β3), and platelet-derived growth factor AA (PDGF-AA), were upregulated in patients with RRDCD. After calibrating the factors duration of detachment, preoperative proliferative vitreoretinopathy grade, and presence or absence of macular hole, the PDGF-AA concentrations were not significantly different according to the multivariate logistic regression analysis. MIF and sICAM-1 markers were significantly different between the two groups and represented a forward stepwise logistic regression trend.
Conclusions
This is the first report to use multiplex bead analysis to investigate inflammatory mediators related to RRDCD. We proposed that the upregulated expression of these mediators may be involved in the inflammation process of RRDCD and that regulation of their expression may be potentially therapeutic by altering local inflammation.
PMCID: PMC4443581  PMID: 26015767
9.  Strain-induced quantum spin Hall effect in methyl-substituted germanane GeCH3 
Scientific Reports  2014;4:7297.
Quantum spin Hall (QSH) insulators exhibit a bulk insulting gap and metallic edge states characterized by nontrivial topology. We investigated the electronic structure of an isolated layer of methyl substituted germanane GeCH3 by density functional calculations (DFT), and its dynamic stability by phonon dispersion calculations. Our results show that an isolated GeCH3 layer has no dynamic instability, and is a QSH insulator under reasonable strain. This QSH insulator has a large enough band gap (up to 108 meV) at 12% strain. The advantageous features of this QSH insulator for practical room-temperature applications are discussed.
doi:10.1038/srep07297
PMCID: PMC4252893  PMID: 25465887
10.  Distinct, genome-wide, gene-specific selectivity patterns of four glucocorticoid receptor coregulators 
Glucocorticoids are a class of steroid hormones that bind to and activate the glucocorticoid receptor (GR), which then positively or negatively regulates transcription of many genes that govern multiple important physiological pathways such as inflammation and metabolism of glucose, fat and bone. The remodeling of chromatin and regulated assembly or disassembly of active transcription complexes by GR and other DNA-binding transcription factors is mediated and modulated by several hundred transcriptional coregulator proteins. Previous studies focusing on single coregulators demonstrated that each coregulator is required for regulation of only a subset of all the genes regulated by a steroid hormone. We hypothesized that the gene-specific patterns of coregulators may correspond to specific physiological pathways such that different coregulators modulate the pathway-specificity of hormone action, thereby providing a mechanism for fine tuning of the hormone response. We tested this by direct comparison of multiple coregulators, using siRNA to deplete the products of four steroid hormone receptor coregulator genes (CCAR1, CCAR2, CALCOCO1 and ZNF282). Global analysis of glucocorticoid-regulated gene expression after siRNA mediated depletion of coregulators confirmed that each coregulator acted in a selective and gene-specific manner and demonstrated both positive and negative effects on glucocorticoid-regulated expression of different genes. We identified several classes of hormone-regulated genes based on the effects of coregulator depletion. Each coregulator supported hormonal regulation of some genes and opposed hormonal regulation of other genes (coregulator-modulated genes), blocked hormonal regulation of a second class of genes (coregulator-blocked genes), and had no effect on hormonal regulation of a third gene class (coregulator-independent genes). In spite of previously demonstrated physical and functional interactions among these four coregulators, the majority of the several hundred modulated and blocked genes for each of the four coregulators tested were unique to that coregulator. Finally, pathway analysis on coregulator-modulated genes supported the hypothesis that individual coregulators may regulate only a subset of the many physiological pathways controlled by glucocorticoids. We conclude that gene-specific actions of coregulators correspond to specific physiological pathways, suggesting that coregulators provide a potential mechanism for physiological fine tuning in vivo and may thus represent attractive targets for therapeutic intervention.
doi:10.1621/nrs.12002
PMCID: PMC4242289  PMID: 25422592
coregulators; gene expression; microarray; glucocorticoid receptor; gene regulation
11.  Identification of Chemokines and Growth Factors in Proliferative Diabetic Retinopathy Vitreous 
BioMed Research International  2014;2014:486386.
Associations were investigated between levels of chemokines and growth factors in the vitreous and proliferative diabetic retinopathy (PDR). Enrolled were 58 patients (58 eyes) requiring pars plana vitrectomy (PPV), with PDR (n = 32, none with traction retinal detachment) or not (non-PDR). In the latter, 16 had macular hole (MH) and 10 had epiretinal membrane (ERM). With a multiplex bead immunoassay, levels of 11 chemokines and growth factors were measured from the undiluted vitreous sample from each patient. In the non-PDR eyes, the levels of the 11 chemokines and growth factors tested were similar between patients with MH and those with ERM. However, the levels of all 11 were significantly higher in the PDR eyes relative to the non-PDR; CCL17, CCL19, and TGFβ3 were markedly upregulated and have not been investigated in PDR previously. The significantly higher levels of CCL4 and CCL11 in PDR contradict the results of previous reports. Based on Spearman's nonparametric test, moderate-to-strong correlations were found between VEGF and other mediators. Our results indicate that these chemokines and growth factors could be candidates for research into targeted therapies applied either singly or in combination with anti-VEGF drugs for the treatment of PDR.
doi:10.1155/2014/486386
PMCID: PMC4226181  PMID: 25401103
12.  Non-Genetic Direct Reprogramming and Biomimetic Platforms in a Preliminary Study for Adipose-Derived Stem Cells into Corneal Endothelia-Like Cells 
PLoS ONE  2014;9(10):e109856.
Cell fate and function can be regulated and reprogrammed by intrinsic genetic program, extrinsic factors and niche microenvironment. Direct reprogramming has shown many advantages in the field of cellular reprogramming. Here we tried the possibility to generate corneal endothelia (CE) -like cells from human adipose-derived stem cells (ADSCs) by the non-genetic direct reprogramming of recombinant cell-penetrating proteins Oct4/Klf4/Sox2 (PTD-OKS) and small molecules (purmorphamine, RG108 and other reprogramming chemical reagents), as well as biomimetic platforms of simulate microgravity (SMG) bioreactor. Co-cultured with corneal cells and decellularized corneal ECM, Reprogrammed ADSCs revealed spherical growth and positively expressing Nanog for RT-PCR analysis and CD34 for immunofluorescence staining after 7 days-treatment of both purmorphamine and PTD-OKS (P-OKS) and in SMG culture. ADSCs changed to CEC polygonal morphology from spindle shape after the sequential non-genetic direct reprogramming and biomimetic platforms. At the same time, induced cells converted to weakly express CD31, AQP-1 and ZO-1. These findings demonstrated that the treatments were able to promote the stem-cell reprogramming for human ADSCs. Our study also indicates for the first time that SMG rotary cell culture system can be used as a non-genetic means to promote direct reprogramming. Our methods of reprogramming provide an alternative strategy for engineering patient-specific multipotent cells for cellular plasticity research and future autologous CEC replacement therapy that avoids complications associated with the use of human pluripotent stem cells.
doi:10.1371/journal.pone.0109856
PMCID: PMC4198143  PMID: 25333522
13.  Behavioral and metabolic characterization of heterozygous and homozygous POLG mutator mice 
Mitochondrion  2013;13(4):282-291.
The mitochondrial DNA (mtDNA) polymerase γ (POLG) mutator mice provide the first experimental evidence that high levels of somatic mtDNA mutations can be functionally significant. Here we report that older homozygous, but not heterozygous, POLG mice show significant reductions in striatal dopaminergic terminals as well as deficits in motor function. However, resting oxygen consumption, heat production, mtDNA content and mitochondrial electron transport chain activities are significantly decreased at older ages in both homozygous and heterozygous mice. These results indicate that high levels of somatic mtDNA mutations can contribute to dopaminergic dysfunction and to behavioral and metabolic deficits.
doi:10.1016/j.mito.2013.03.006
PMCID: PMC3682692  PMID: 23542163
mtDNA polymerase γ; mtDNA mutation; Parkinson’s disease; dopamine; behavioral deficits; metabolic deficits
14.  A Dual Chicken IgY Against Rotavirus and Norovirus 
Antiviral research  2012;97(3):293-300.
Rotavirus (RV) and norovirus (NoV) are the two most important causes of viral gastroenteritis. While vaccine remains an effective prophylactic strategy, development of other approaches, such as passive immunization to control and treat clinical infection and illness of the two pathogens, is necessary. Previously we demonstrated that high titers of NoV-specific IgY were readily developed by immunization of chickens with the NoV P particles. In this study, we developed a dual IgY against both RV and NoV through immunization of chickens with a divalent vaccine comprising neutralizing antigens of both RV and NoV. This divalent vaccine, named P-VP8* particle, is made of the NoV P particle as a carrier with the RV spike protein VP8* as a surface insertion. Approximately 45 mg of IgY were readily obtained from each yolk with high titers of anti-P particle and anti-VP8* antibodies detected by ELISA, Western blot, HBGA blocking (NoV and RV) and neutralization (RV) assays. Reductions of RV replication were observed with viruses treated with the IgY before and after inoculation into cells, suggesting an application of the IgY as both prophylactic and a therapeutic treatment. Collectively, our data suggested that the P-VP8* based IgY could serve as a practical approach against both NoV and RV.
doi:10.1016/j.antiviral.2012.12.011
PMCID: PMC3995418  PMID: 23267830
rotavirus; norovirus; diarrhea; immunoglobulin Y (IgY); passive immunization
15.  Noble-metal-free plasmonic photocatalyst: hydrogen doped semiconductors 
Scientific Reports  2014;4:3986.
The unique capacity of localized surface plasmon resonance (LSPR) offers a new opportunity to overcome the limited efficiency of semiconductor photocatalyst. Here we unravel that LSPR, which usually occurs in noble metal nanoparticles, can be realized by hydrogen doping in noble-metal-free semiconductor using TiO2 as a model photocatalyst. Moreover, its LSPR is located in infrared region, which supplements that of noble metal whose LSPR is generally in the visible region, making it possible to extend the light response of photocatalyst to infrared region. The near field enhancement is shown to be comparable with that of noble-metal nanoparticles, indicating that highly enhanced light absorption rate can be expected. The present work can provide a key guideline for the creation of highly efficient noble-metal-free plasmonic photocatalysts and have a much wider impact in infrared bioimaging and spectroscopy where infrared LSPR is essential.
doi:10.1038/srep03986
PMCID: PMC3913932  PMID: 24496400
16.  Evaluation of anti-norovirus IgY from egg yolk of chickens immunized with norovirus P particles 
Journal of virological methods  2012;186(1-2):126-131.
Noroviruses (NoVs) are a leading cause of epidemic acute gastroenteritis affecting millions of people worldwide. Understanding of NoV remains limited due to the lack of a cell culture system and small animal models. Currently, there are no available vaccines or antivirals against NoVs. In this study, an approach for large-scale production of anti-NoV antibodies for use as a potential treatment for NoV disease using passive immunization was evaluated. NoV-specific immunoglobulins (IgY) were produced by immunizing chickens with NoV P particles. The birds continuously produced high titers of antibodies in their eggs for at least 3 months, in which NoV-specific antibody levels reached 4.7-9.2 mg/egg yolk. The egg yolk antibodies strongly reacted with NoV P particles by both ELISA and Western blot and blocked NoV virus-like particle (VLP) and P particle binding to the histo-blood group antigen (HBGA) receptors with a BT50 of about 1:800. The blocking activity of the chicken IgY remained after an incubation at 70°C for 30 min or treatment at pH 4 to 9 for 3 h. These data suggested that chicken IgY could be a practical strategy for large-scale production of anti-NoV antibodies for potential use as passive immunization against NoV infection, as well as for diagnostic purposes.
doi:10.1016/j.jviromet.2012.07.002
PMCID: PMC3496071  PMID: 22867844
Norovirus; Immunoglobulin; IgY; Chicken; Norovirus P particle; Diarrhea
17.  Identification of Breast Cancer Prognosis Markers via Integrative Analysis 
Summary
In breast cancer research, it is of great interest to identify genomic markers associated with prognosis. Multiple gene profiling studies have been conducted for such a purpose. Genomic markers identified from the analysis of single datasets often do not have satisfactory reproducibility. Among the multiple possible reasons, the most important one is the small sample sizes of individual studies. A cost-effective solution is to pool data from multiple comparable studies and conduct integrative analysis. In this study, we collect four breast cancer prognosis studies with gene expression measurements. We describe the relationship between prognosis and gene expressions using the accelerated failure time (AFT) models. We adopt a 2-norm group bridge penalization approach for marker identification. This integrative analysis approach can effectively identify markers with consistent effects across multiple datasets and naturally accommodate the heterogeneity among studies. Statistical and simulation studies demonstrate satisfactory performance of this approach. Breast cancer prognosis markers identified using this approach have sound biological implications and satisfactory prediction performance.
doi:10.1016/j.csda.2012.02.017
PMCID: PMC3389801  PMID: 22773869
Breast cancer prognosis; Gene expression; Marker identification; Integrative analysis; 2-norm group bridge
18.  Activation of the JAK-STAT pathway is necessary for desensitization of 5-HT2A receptor-stimulated phospholipase C signaling by olanzapine, clozapine and MDL100907 
We previously demonstrated that olanzapine-induced desensitization of 5-HT2A receptor-stimulated phospholipase C (PLC) activity is associated with increases in RGS7 protein levels both in vivo and in cells in culture, and the increase in RGS7 is dependent on activation of the JAK-STAT pathway in cells in culture (Muma, et al., 2007;Singh, et al., 2007). In the current study, we found that desensitization of 5-HT2A receptor-stimulated PLC activity induced by olanzapine is dependent on activation of the JAK-STAT pathway. Similar to olanzapine, clozapine-induced desensitization of 5-HT2A receptor signaling is accompanied by increases in RGS7 and activation of JAK2. Treatment with the selective 5-HT2A receptor antagonist MDL100907 also increased RGS7 protein levels and JAK2 activation. Using a JAK2 inhibitor AG490, we found that clozapine and MDL100907-induced increases in RGS7 are dependent on activation of the JAK-STAT pathway. Olanzapine, clozapine, and MDL100907 treatment increased mRNA levels of RGS7. Using a chromatin immunoprecipitation assay we found STAT3 binding to the putative RGS7 promoter region. Taken together, olanzapine-induced activation of the JAK-STAT pathway, and STAT3 binding to the RGS7 gene could underlie the increase in RGS7 mRNA which could subsequently increase protein expression. Furthermore, the increase in RGS7 protein could play a role in the desensitization of 5-HT2A receptor signaling by terminating the activated Gαq/11 proteins more rapidly. Overall, our data suggest that the complete desensitization of 5-HT2A receptor-stimulated PLC activity by olanzapine, clozapine and MDL100,907 requires activation of the JAK-STAT pathway, which in turn increases RGS7 expression likely by direct transcriptional activity of STAT3.
doi:10.1017/S1461145708009590
PMCID: PMC3733235  PMID: 18976543
JAK-STAT; 5-HT2A receptor; Clozapine; Olanzapine; Desensitization
19.  Ephexin1 is required for structural maturation and neurotransmission at the neuromuscular junction 
Neuron  2010;65(2):204-216.
Summary
The maturation of neuromuscular junctions (NMJs) requires the topological transformation of postsynaptic acetylcholine receptor (AChR)-containing structures from a simple plaque to an elaborate structure composed of pretzel-like branches. This maturation process results in the precise apposition of the pre- and postsynaptic specializations. However, little is known about the molecular mechanisms underlying the plaque-to-pretzel transition of AChR clusters. In this study, we identify an essential role for the RhoGEF ephexin1 in the maturation of AChR clusters. Adult ephexin1−/− mice exhibit severe muscle weakness and impaired synaptic transmission at the NMJ. Intriguingly, when ephexin1 expression is deficient in vivo, the NMJ fails to differentiate into the pretzel-like shape, and such abnormalities can be rescued by re-expression of ephexin1. We further demonstrate that ephexin1 regulates the stability of AChR clusters in a RhoA-dependent manner. Taken together, our findings reveal an indispensible role for ephexin1 in regulating the structural maturation and neurotransmission of NMJs.
doi:10.1016/j.neuron.2010.01.012
PMCID: PMC3718047  PMID: 20152127
synapse; ephrin; EphA4; Rho GTPase; AChR
20.  Inhibition of Histo-blood Group Antigen Binding as a Novel Strategy to Block Norovirus Infections 
PLoS ONE  2013;8(7):e69379.
Noroviruses (NoVs) are the most important viral pathogens that cause epidemic acute gastroenteritis. NoVs recognize human histo-blood group antigens (HBGAs) as receptors or attachment factors. The elucidation of crystal structures of the HBGA-binding interfaces of a number of human NoVs representing different HBGA binding patterns opens a new strategy for the development of antiviral compounds against NoVs through rational drug design and computer-aided virtual screening methods. In this study, docking simulations and virtual screening were used to identify hit compounds targeting the A and B antigens binding sites on the surface of the capsid P protein of a GII.4 NoV (VA387). Following validation by re-docking of the A and B ligands, these structural models and AutoDock suite of programs were used to screen a large drug-like compound library (derived from ZINC library) for inhibitors blocking GII.4 binding to HBGAs. After screening >2 million compounds using multistage protocol, 160 hit compounds with best predicted binding affinities and representing a number of distinct chemical classes have been selected for subsequent experimental validation. Twenty of the 160 compounds were found to be able to block the VA387 P dimers binding to the A and/or B HBGAs at an IC50<40.0 µM, with top 5 compounds blocking the HBGA binding at an IC50<10.0 µM in both oligosaccharide- and saliva-based blocking assays. Interestingly, 4 of the top-5 compounds shared the basic structure of cyclopenta [a] dimethyl phenanthren, indicating a promising structural template for further improvement by rational design.
doi:10.1371/journal.pone.0069379
PMCID: PMC3716607  PMID: 23894462
21.  Variable selection for semiparametric regression models with iterated penalization 
Semiparametric regression models with multiple covariates are commonly encountered. When there are covariates not associated with response variable, variable selection may lead to sparser models, more lucid interpretations and more accurate estimation. In this study, we adopt a sieve approach for the estimation of nonparametric covariate effects in semiparametric regression models. We adopt a two-step iterated penalization approach for variable selection. In the first step, a mixture of the Lasso and group Lasso penalties are employed to conduct the first-round variable selection and obtain the initial estimate. In the second step, a mixture of the weighted Lasso and weighted group Lasso penalties, with weights constructed using the initial estimate, are employed for variable selection. We show that the proposed iterated approach has the variable selection consistency property, even when number of unknown parameters diverges with sample size. Numerical studies, including simulation and analysis of a diabetes dataset, show satisfactory performance of the proposed approach.
doi:10.1080/10485252.2012.661054
PMCID: PMC3367330  PMID: 22685376
Iterated penalization; Variable selection; Semiparametric regression
22.  Expression of a Functional Recombinant Human Basic Fibroblast Growth Factor from Transgenic Rice Seeds 
Basic fibroblast growth factor (FGF-2) is an important member of the FGF gene family. It is widely used in clinical applications for scald and wound healing in order to stimulate cell proliferation. Further it is applied for inhibiting stem cell differentiation in cultures. Due to a shortage of plasma and low expression levels of recombinant rbFGF in conventional gene expression systems, we explored the production of recombinant rbFGF in rice grains (Oryza sativa bFGF, OsrbFGF). An expression level of up to 185.66 mg/kg in brown rice was obtained. A simple purification protocol was established with final recovery of 4.49% and resulting in a yield of OsrbFGF reaching up to 8.33 mg/kg OsrbFGF. The functional assay of OsrbFGF indicated that the stimulating cell proliferation activity on NIH/3T3 was the same as with commercialized rbFGF. Wound healing in vivo of OsrbFGF is equivalent to commercialized rbFGF. Our results indicate that rice endosperm is capable of expressing small molecular mass proteins, such as bFGF. This again demonstrates that rice endosperm is a promising system to express various biopharmaceutical proteins.
doi:10.3390/ijms14023556
PMCID: PMC3588058  PMID: 23434658
recombinant human bFGF; rice endosperm; protein expression and processing; cell proliferation and wound healing
23.  Realization of tunable Dirac cone and insulating bulk states in topological insulators (Bi1−xSbx)2Te3 
Scientific Reports  2012;2:976.
The bulk-insulating topological insulators with tunable surface states are necessary for applications in spintronics and quantum computation. Here we present theoretical evidence for modulating the topological surface states and achieving the insulating bulk states in solid-solution (Bi1−xSbx)2Te3. Our results reveal that the band inversion occurs in (Bi1−xSbx)2Te3, indicating the non-triviality across the entire composition range, and the Dirac point moves upwards till it lies within the bulk energy gap accompanying the increase of Sb concentration x. In addition, with increasing x, the formation of prominent native defects becomes much more difficult, resulting in the truly insulating bulk. The solid-solution system is a promising way of tuning the properties of topological insulators and designing novel topologically insulating devices.
doi:10.1038/srep00976
PMCID: PMC3521154  PMID: 23240080
24.  Characterizing the Effects of VPA, VC and RCCS on Rabbit Keratocytes onto Decellularized Bovine Cornea 
PLoS ONE  2012;7(11):e50114.
To investigate the morphological and growth characteristics of rabbit keratocytes when cultured on decellularized cornea under simulate microgravity (SMG) rotary cell culture system (RCCS) and static culture or in plastic culture supplemented with small molecules of valproic acid (VPA) and vitamin C (VC). Bovine corneas were firstly decellularized with Triton X-100 and NH4OH and through short-term freezing process. Then cell count kit-8 (CCK-8) and flow cytometry were used to test the effects of VPA and VC on the proliferation, cell cycle and apoptosis of rabbit keratocytes. Hematoxylin-eosin (H&E) staining and scanning electron microscopy (SEM) imaging showed that cells were eliminated in the decellularized bovine corneas. The proliferation of cultured keratocytes was promoted by VPA and VC in the cell proliferation assay. VPA and VC moderately decreased the number of apoptotic cells and obviously promoted cell-cycle entrance of keratocytes. Rabbit keratocytes in plastic displayed spindle shape and rare interconnected with or without VPA and VC. Cells revealed dendritic morphology and reticular cellular connections when cultured on the carriers of decellularized corneas supplemented with VPA and VC even in the presence of 10% fetal bovine serum (FBS). When cultured in RCCS supplemented with VPA, VC and 10% FBS, keratocytes displayed round shape with many prominences and were more prone to grow into the pores of carriers with aggregation. Reverse transcription-polymerase chain reaction (RT-PCR) analysis proved that the keratocytes cultured on decellularized bovine cornea under SMG with VPA and VC expressed keratocan and lumican. Keratocytes cultured on plastic expressed lumican but not keratocan. Immunofluorescence identification revealed that cells in all groups were positively immunostained for vimentin. Keratocytes on decellularized bovine cornea under SMG or in static culture were positively immunostained for keratocan and lumican. Thus, we reasonably made a conclusion that the combination of VPA, VC, RCCS and decellularized corneal carriers provide a good condition for keratocytes to well grow. Keratocytes can be manipulated to be aggregates or physiological morphological growth in vitro, which are important for the research of corneal stem cells and corneal tissue engineering.
doi:10.1371/journal.pone.0050114
PMCID: PMC3510233  PMID: 23209652
25.  Pgc-1α Overexpression Downregulates Pitx3 and Increases Susceptibility to MPTP Toxicity Associated with Decreased Bdnf 
PLoS ONE  2012;7(11):e48925.
Multiple mechanisms likely contribute to neuronal death in Parkinson’s disease (PD), including mitochondrial dysfunction and oxidative stress. Peroxisome proliferator-activated receptor gamma co-activator-1 alpha (PGC-1α) positively regulates the expression of genes required for mitochondrial biogenesis and the cell’s antioxidant responses. Also, expression of PGC-1α-regulated genes is low in substantia nigra (SN) neurons in early PD. Thus upregulation of PGC-1α is a candidate neuroprotective strategy in PD. Here, an adeno-associated virus (AAV) was used to induce unilateral overexpression of Pgc-1α, or a control gene, in the SN of wild-type C57BL/6CR mice. Three weeks after AAV administration, mice were treated with saline or MPTP. Overexpression of Pgc-1α in the SN induced expression of target genes, but unexpectedly it also greatly reduced the expression of tyrosine hydroxylase (Th) and other markers of the dopaminergic phenotype with resultant severe loss of striatal dopamine. Reduced Th expression was associated with loss of Pitx3, a transcription factor that is critical for the development and maintenance of dopaminergic cells. Expression of the neurotrophic factor Bdnf, which also is regulated by Pitx3, similarly was reduced. Overexpression of Pgc-1α also led to increased sensitivity to MPTP-induced death of Th+ neurons. Pgc-1α overexpression alone, in the absence of MPTP treatment, did not lead to cell loss in the SN or to loss of dopaminergic terminals. These data demonstrate that overexpression of Pgc-1α results in dopamine depletion associated with lower levels of Pitx3 and enhances susceptibility to MPTP. These data may have ramifications for neuroprotective strategies targeting overexpression of PGC-1α in PD.
doi:10.1371/journal.pone.0048925
PMCID: PMC3492133  PMID: 23145024

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