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1.  High Fidelity Copy Number Analysis of Formalin-Fixed and Paraffin-Embedded Tissues Using Affymetrix Cytoscan HD Chip 
PLoS ONE  2014;9(4):e92820.
Detection of human genome copy number variation (CNV) is one of the most important analyses in diagnosing human malignancies. Genome CNV detection in formalin-fixed and paraffin-embedded (FFPE) tissues remains challenging due to suboptimal DNA quality and failure to use appropriate baseline controls for such tissues. Here, we report a modified method in analyzing CNV in FFPE tissues using microarray with Affymetrix Cytoscan HD chips. Gel purification was applied to select DNA with good quality and data of fresh frozen and FFPE tissues from healthy individuals were included as baseline controls in our data analysis. Our analysis showed a 91% overlap between CNV detection by microarray with FFPE tissues and chromosomal abnormality detection by karyotyping with fresh tissues on 8 cases of lymphoma samples. The CNV overlap between matched frozen and FFPE tissues reached 93.8%. When the analyses were restricted to regions containing genes, 87.1% concordance between FFPE and fresh frozen tissues was found. The analysis was further validated by Fluorescence In Situ Hybridization on these samples using probes specific for BRAF and CITED2. The results suggested that the modified method using Affymetrix Cytoscan HD chip gave rise to a significant improvement over most of the previous methods in terms of accuracy in detecting CNV in FFPE tissues. This FFPE microarray methodology may hold promise for broad application of CNV analysis on clinical samples.
doi:10.1371/journal.pone.0092820
PMCID: PMC3974686  PMID: 24699316
2.  PTEN Regulates BCRP/ABCG2 and the Side Population through the PI3K/Akt Pathway in Chronic Myeloid Leukemia 
PLoS ONE  2014;9(3):e88298.
A small population of cancer stem cells named the “side population” (SP) has been demonstrated to be responsible for the persistence of many solid tumors. However, the role of the SP in leukemic pathogenesis remains controversial. The resistance of leukemic stem cells to targeted therapies, such as tyrosine kinase inhibitors (TKIs), results in therapeutic failure or refractory/relapsed disease in chronic myeloid leukemia (CML). The drug pump, ATP-binding cassette sub-family G member 2 (ABCG2), is well known as a specific marker of the SP and could be controlled by several pathways, including the PI3K/Akt pathway. Our data demonstrated that compared with wild-type K562 cells, the higher percentage of ABCG2+ cells corresponded to the higher SP fraction in K562/ABCG2 (ABCG2 overexpressing) and K562/IMR (resistance to imatinib) cells, which exhibited enhanced drug resistance along with downregulated phosphatase and tensin homologue deleted on chromosome -10 (PTEN) and activated phosphorylated-Akt (p-Akt). PTEN and p-Akt downregulation could be abrogated by both the PI3K inhibitor LY294002 and the mTOR inhibitor rapamycin. Moreover, in CML patients in the accelerated phase/blastic phase (AP/BP), increased SP phenotype rather than ABCG2 expression was accompanied by the loss of PTEN protein and the up-regulation of p-Akt expression. These results suggested that the expression of ABCG2 and the SP may be regulated by PTEN through the PI3K/Akt pathway, which would be a potentially effective strategy for targeting CML stem cells.
doi:10.1371/journal.pone.0088298
PMCID: PMC3945754  PMID: 24603487
3.  The Fusion Protein of CTP-HBcAg18-27-Tapasin Mediates the Apoptosis of CD8+T Cells and CD8+ T Cell Response in HLA-A2 Transgenic Mice 
Hepatitis Monthly  2014;14(2):e16161.
Background:
HBV-specific cytotoxic T lymphocyte (CTL) activity is believed to play a critical role in controlling HBV infection. The phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway manipulates cell fate decisions in many different cell types by regulating the activity of downstream effectors. We have previously testified that the fusion protein of CTP-HBcAg18-27--Tapasin could enter the cytoplasm of dendritic cells and efficiently induce robust specific CTL response in vitro.
Objectives:
In the present study, we evaluated specific CTL response and the level of apoptosis of CD8+ T cells induced by CTP-HBcAg18-27-Tapasin in HLA-A2 transgenic mice (H-2Kb). Meanwhile, we preliminary investigated PI3K, phosphorylation level of Akt, and mammalian target of rapamycin (mTOR) as positive regulator of the magnitude and effector function of the hepatitis B virus-specific cytotoxic T lymphocytes in HLA-A2 transgenic mice.
Materials and Methods:
HLA-A2 transgenic mice were immunized by intramuscular injection in the hind legs three times at one-week intervals with PBS, CTP-HBcAg18-27-Tapasin (50 μg), CTP-HBcAg18-27 (50 μg), HBcAg18-27-Tapasin (50 μg), and HBcAg18-27 (50 μg). One week after the last immunization, mice were sacrificed and splenocytes were harvested in strile condition. The specific CTL response was analyzed by flow cytometry and enzyme linked immunosorbent assay (ELISA); the expression of (PI3K)/Akt signaling was detected by RT-PCR and western blot.
Results:
The results showed that CTP-HBcAg18-27-Tapasin significantly increased the percentages of IFN-γ+ CD8α+ T cells, the numbers of these polyfunctional triple-cytokine-producing (IFN-γ, TNF-α, and IL-2) CD8+T cells, the secretion of cytokine IFN-γ, IL-2, and TNF-α, while in comparison to control group, it significantly decreased the percentage of apoptotic CD8+ T cells in HLA-A2 transgenic mice. Moreover, the expression of PI3K, P-Akt, and P-mTOR was significantly upregulated in CTP-HBcAg18-27-Tapasin group compared with control groups.
Conclusions:
In conclusion, CTP-HBcAg18-27-Tapasin could reduce apoptosis of CD8+ T cells, increase the percentages of IFN-γ+ CD8α+ T cells, and elicit cell-mediated immunity in HLA-A2 transgenic mice; these processes were associated with activation of the PI3K/Akt signaling pathway.
doi:10.5812/hepatmon.16161
PMCID: PMC3950630
Tapasin; Mice, Transgenic; T-Lymphocytes, Cytotoxic; PI3K/Akt
4.  PKC α regulates netrin-1/UNC5B-mediated survival pathway in bladder cancer 
BMC Cancer  2014;14:93.
Background
Netrin-1 and its receptor UNC5B play important roles in angiogenesis, embryonic development, cancer and inflammation. However, their expression patttern and biological roles in bladder cancer have not been well characterized. The present study aims to investigating the clinical significance of PKC α, netrin-1 and UNC5B in bladder cancer as well as their association with malignant biological behavior of cancer cells.
Methods
Netrin-1 and UNC5B expression was examined in 120 bladder cancer specimens using immunohistochemistry and in 40 fresh cancer tissues by western blot. Immunofluorescence was performed in cancer cell lines. PKC α agonist PMA and PKC siRNA was employed in bladder cancer cells. CCK-8, wound healing assays and flow cytometry analysis were used to examine cell proliferation, migration and cell cycle, respectively.
Results
Netrin-1 expression was positively correlated with histological grade, T stage, metastasis and poor prognosis in bladder cancer tissues. Immunofluorescence showed elevated netrin-1 and decreased UNC5B expression in bladder cancer cells compared with normal bladder cell line. Furthermore, cell proliferation, migration and cell cycle progression were promoted with PMA treatment while inhibited by calphostin C. In addition, PMA treatment could induce while calphostin C reduce netrin-1 expression in bladder cancer cells.
Conclusions
The present study identified netrin-1/UNC5B, which could be regulated by PKC signaling, was important mediators of bladder cancer progression.
doi:10.1186/1471-2407-14-93
PMCID: PMC3937025  PMID: 24528886
PKC α; Netrin-1; UNC5B; Bladder cancer
5.  Caregiver burden and its determinants among family members of patients with chronic viral hepatitis in Shanghai, China: a community-based survey 
Background
In China, caregivers of chronic viral hepatitis patients experience considerable burdens, stress and disruption of their own well-being and social activities. Measurement of the effect on caregivers is an under-researched area. The Family Burden Interview Schedule (FBIS) was primarily devised for the caregivers of schizophrenia patients, and the adverse effect of the disease was similar to the effect of chronic viral hepatitis on family caregivers. In this study, we prospectively evaluated the psychometric properties of FBIS in the field of chronic viral hepatitis and used it to determine the factors affecting the caregiver burden on the family members of chronic viral hepatitis patients in Shanghai, China.
Methods
A representative sample of patients (n = 1478) and caregivers (n = 1478) was randomly obtained through a multi-stage cluster sampling in Shanghai, China. Reliability and validity tests were used to verify the psychometric properties of the instrument. The two-level random intercept model was applied to determine the factors of the caregiver burden between the household and the community level.
Results
Cronbach’s alpha coefficient was 0.90 for the overall instrument with statistical significance. Factor analysis suggested a three-factor model for the FBIS and confirmed that the adjusted unidimensional model and the second-order multidimensional model had better fit statistics. The average score of the caregiver burden in Shanghai was 12.62 ± 10.74, and financial burden constituted the major effect. The two-level random intercept model demonstrated that the risk factors were hospitalisation (β 1.69, 95%CI 0.48 to 2.90), elevated serum alanine aminotransferase levels (β 1.05, 95%CI 0.15 to 1.95), HCV infection (β 4.49, 95%CI 1.22 to 7.77), and acceptance of the hepatitis B vaccine (β 2.20, 95%CI 0.56 to 3.85), whereas the protective factors were no consumption of alcohol (β -2.69, 95%CI −5.19 to −0.19), average monthly costs for patients less than or equal to 100 US dollars (β -2.96, 95%CI −5.83 to −0.09), and good health status of family caregivers (β -9.91, 95%CI −12.76 to −7.05).
Conclusions
FBIS can accurately measure the caregiver burden for chronic hepatitis. Targeting interventions toward the conditions associated with the caregiver burden is of great importance.
doi:10.1186/1471-2334-14-82
PMCID: PMC3927630  PMID: 24521097
Chronic viral hepatitis; Family caregiver; The family burden interview schedule; Reliability and validity
6.  The Strain at Bone-Implant Interface Determines the Effect of Spinopelvic Reconstruction following Total Sacrectomy: A Strain Gauge Analysis in Various Spinopelvic Constructs 
PLoS ONE  2014;9(1):e85298.
Purpose
There is still some controversy regarding the optimal biomechanical concept for spinopelvic stabilization following total sacrectomy for malignancy. Strains at specific anatomical sites at pelvis/sacrum and implants interfaces have been poorly investigated. Herein, we compared and analyzed the strains applied at key points at the bone-implant interface in four different spinopelvic constructs following total sacrectomy; consequently, we defined a balanced architecture for spinopelvic fusion in that situation.
Methods
Six human cadaveric specimens, from second lumbar vertebra to proximal femur, were used to compare the partial strains at specific sites in a total sacrectomy model. Test constructs included: (1) intact pelvis (control), (2) sacral-rod reconstruction (SRR), (3) bilateral fibular flap reconstruction (BFFR), (4) four-rods reconstruction (FRR), and (5) improved compound reconstruction (ICR). Strains were measured by bonded strain gauges onto the surface of three specific sites (pubic rami, arcuate lines, and posterior spinal rods) under a 500 N axial load.
Results
ICR caused lower strains at specific sites and, moreover, on stress distribution and symmetry, compared to the other three constructs. Strains at pubic rami and arcuate lines following BFFR were lower than those following SRR, but higher at the posterior spinal rod construct. The different modes of strain distribution reflected different patient’s parameter-related conditions. FRR model showed the highest strains at all sites because of the lack of an anterior bracing frame.
Conclusions
The findings of this investigation suggest that both anterior bracing frame and the four-rods load dispersion provide significant load sharing. Additionally, these two constructs decrease the peak strains at bone-implant interface, thus determining the theoretical surgical technique to achieve optimal stress dispersion and balance for spinopelvic reconstruction in early postoperative period following total sacrectomy.
doi:10.1371/journal.pone.0085298
PMCID: PMC3891848  PMID: 24454839
7.  Reduction of Intimal Hyperplasia in Injured Rat Arteries Promoted by Catheter Balloons Coated with Polyelectrolyte Multilayers that Contain Plasmid DNA Encoding PKCδ 
Biomaterials  2012;34(1):226-236.
New therapeutic approaches that eliminate or reduce the occurrence of intimal hyperplasia following balloon angioplasty could improve the efficacy of vascular interventions and improve the quality of life of patients suffering from vascular diseases. Here, we report that treatment of arteries using catheter balloons coated with thin polyelectrolyte-based films (‘polyelectrolyte multilayers’, PEMs) can substantially reduce intimal hyperplasia in an in vivo rat model of vascular injury. We used a layer-by-layer (LbL) process to coat the surfaces of inflatable catheter balloons with PEMs composed of nanolayers of a cationic poly(β-amino ester) (polymer 1) and plasmid DNA (pPKCδ) encoding the δ isoform of protein kinase C (PKCδ), a regulator of apoptosis and other cell processes that has been demonstrated to reduce intimal hyperplasia in injured arterial tissue when administered via perfusion using viral vectors. Insertion of balloons coated with polymer 1/pPKCδ multilayers into injured arteries for 20 min resulted in local transfer of DNA and elevated levels of PKCδ expression in the media of treated tissue 3 days after delivery. IFC and IHC analysis revealed these levels of expression to promote downstream cellular processes associated with up-regulation of apoptosis. Analysis of arterial tissue 14 days after treatment revealed polymer 1/pPKCδ-coated balloons to reduce the occurrence of intimal hyperplasia by ~60% compared to balloons coated with films containing empty plasmid vectors. Our results demonstrate the potential therapeutic value of this nanotechnology-based approach to local gene delivery in the clinically important context of balloon-mediated vascular interventions. These PEM-based methods could also prove useful for other in vivo applications that require short-term, surface-mediated transfer of plasmid DNA.
doi:10.1016/j.biomaterials.2012.09.010
PMCID: PMC3483441  PMID: 23069712
Layer-by-Layer; Polyelectrolyte Multilayers; Thin Films; Gene Delivery; Intimal Hyperplasia
8.  MMP9 regulates the cellular response to inflammation after skeletal injury 
Bone  2012;52(1):111-119.
Like other tissue injuries, bone fracture triggers an inflammatory response, which plays an important role in skeletal repair. Inflammation is believed to have both positive and negative effects on bone repair, but the underlying cellular mechanisms are not well understood. To assess the role of inflammation on skeletal cell differentiation, we used mouse models of fracture repair that stimulate either intramembranous or endochondral ossification. In the first model, fractures are rigidly stabilized leading to direct bone formation, while in the second model, fracture instability causes cartilage and bone formation. We compared the inflammatory response in these two mechanical environments and found changes in the expression patterns of inflammatory genes and in the recruitment of inflammatory cells and osteoclasts. These results suggested that the inflammatory response could influence skeletal cell differentiation after fracture. We then exploited matrix metalloproteinase 9 (MMP9) that is expressed in inflammatory cells and osteoclasts, and which we previously showed is a potential regulator of cell fate decisions during fracture repair. Mmp9-/- mice heal stabilized fractures via endochondral ossification, while wild type mice heal via intramembranous ossification. In parallel, we observed increases in macrophages and T cells in the callus of Mmp9-/- compared to wild type mice. To assess the link between the profile of inflammatory cells and skeletal cell fate functionally, we transplanted Mmp9-/- mice with wild type bone marrow, to reconstitute a wild type hematopoietic lineage in interaction with the Mmp9-/- stroma and periosteum. Following transplantation, Mmp9-/- mice healed stabilized fractures via intramembranous ossification and exhibited a normal profile of inflammatory cells. Moreover, Mmp9-/- periosteal grafts healed via intramembranous ossification in wild type hosts, but healed via endochondral ossification in Mmp9-/- hosts. We observed that macrophages accumulated at the periosteal surface in Mmp9-/- mice, suggesting that cell differentiation in the periosteum is influenced by factors such as BMP2 that are produced locally by inflammatory cells. Taken together, these results show that MMP9 mediates indirect effects on skeletal cell differentiation by regulating the inflammatory response and the distribution of inflammatory cells, leading to the local regulation of periosteal cell differentiation.
doi:10.1016/j.bone.2012.09.018
PMCID: PMC3513654  PMID: 23010105
9.  An efficient expression and purification strategy for the production of S100 proteins in Escherichia coli 
Bioengineered  2013;4(1):55-58.
S100 proteins belong to a family of small, acidic, EF-hand Ca2+-binding proteins and have been found to exert both intracellular and extracellular functions in regulation of Ca2+ homeostasis, cytoskeletal dynamics, cell cycle, motility and differentiation. As a result, they have been widely investigated for their association with diseases, such as, neurological diseases, cardiomyopathy, neoplasias and inflammatory diseases. To facilitate further studies of S100 proteins, we reported a simple and efficient method for the expression and purification of human S100A4 and S100A11 proteins in Escherichia coli. Since S100 proteins share many common physical and chemical characteristics, we expect that this approach can be extended to the production of most S100 proteins.
doi:10.4161/bioe.22172
PMCID: PMC3566023  PMID: 22990588
Protein expression; Protein purification; S100 protein; S100A11; S100A4
10.  Chymase Activities and Survival in Endotoxin-Induced Human Chymase Transgenic Mice 
We examined the effects of overexpressed human chymase on survival and activity in lipopolysaccharide (LPS)-treated mice. Human chymase transgenic (Tg) and wild-type C57BL/6 (WT) mice were treated with LPS (0.03, 0.1 and 0.3 mg/day; intraperitoneal) for 2 weeks. Treatment with 0.03 mg LPS did not affect survival in either WT or Tg mice. WT mice were not affected by 0.1 mg/day of LPS, whereas 25% of Tg mice died. Survival of mice treated with 0.3 mg/day of LPS was 87.5% and 0% in WT and Tg, respectively. LPS-induced increases in chymase activity in the heart and skin were significantly greater in Tg than WT mice. These data suggest a possible contribution of human chymase activation to LPS-induced mortality.
doi:10.7150/ijms.7382
PMCID: PMC3917108  PMID: 24516344
human chymase transgenic mice; chymase activity and lipopolysaccharide; endotoxemia
11.  Unmarried male migrants and sexual risk behavior: a cross-sectional study in Shanghai, China 
BMC Public Health  2013;13:1152.
Background
In China, there is increasing concern because of the rapid increase in HIV infection recorded over recent years. Migrant workers are recognized as one of the groups most affected. In this study, HIV/AIDS-related knowledge, attitudes, and behavior among unmarried migrant workers in Shanghai are investigated, with the aim of providing critical information for policy makers and sex educators to reinforce sexual health services and sex health education targeting the behavior and sexual health of unmarried male migrants.
Methods
A cross-sectional survey was conducted among unmarried male migrant workers in Shanghai, China’s largest city and housing the most migrants. A self-administered, anonymous questionnaire was used to collect information on knowledge, attitudes, and behavior associated with increased risk of HIV/AIDS.
Results
A total of 2254 subjects were questioned, with a response rate of 91.3%. Among those interviewed, 63.5% reported sexual activities. Misconceptions regarding HIV transmission, poor perception of HIV infection, and low use of condoms were not uncommon. Among those who had sexual intercourse, 73.7% had not used condoms in their last sexual intercourse, and 28.6% reported having engaged in sexual risk behavior (defined as having at least one non-regular partner). Multivariate logistic regression analyses identified several indicators of sexual risk behavior, including younger age at first sexual intercourse (OR: 0.67, 95% CI: 0.31–0.91 for older age at first sexual intercourse), more cities of migration (OR: 2.91, 95% CI: 2.17–3.81 for high level; OR: 1.15, 95% CI: 1.06–1.29 for medium level), poor perception of acquiring HIV/AIDS (OR: 1.52, 95% CI: 1.33–1.96 for unlikely; OR: 2.38, 95% CI: 1.61–3.70 for impossible), frequent exposure to pornography (OR: 0.33, 95% CI: 0.11–0.43 for never; OR: 0.69, 95% CI: 0.60–1.81 for less frequently), not knowing someone who had or had died of HIV/AIDS and related diseases (OR: 2.13, 95% CI: 1.70–2.53 for no), and having peers who engaged in sex with a non-regular sex partner (OR: 4.40, 95% CI: 3.37–5.56 for yes).
Conclusions
Today, it is necessary to reinforce sex health education among unmarried migrants and sexual health services should target vulnerable migrant young people.
doi:10.1186/1471-2458-13-1152
PMCID: PMC3903055  PMID: 24321180
12.  MiR-223 Regulates Human Embryonic Stem Cell Differentiation by Targeting the IGF-1R/Akt Signaling Pathway 
PLoS ONE  2013;8(11):e78769.
Currently, there are difficulties associated with the culturing of pluripotent human embryonic stem cells (hESCs), and knowledge regarding their regulatory mechanisms is limited. MicroRNAs (miRNAs) regulate gene expression and have critical functions in stem cell self-renewal and differentiation. Moreover, fibroblast growth factor (FGF) and the insulin-like growth factor receptor (IGF-1R) are key activators of signaling in hESCs. Based on the identification of complementary binding sites in miR-223 and IGF-1R mRNA, it is proposed that miR-223 acts as a local regulator of IGF-1R. Therefore, levels of miR-223 were detected in differentiated versus undifferentiated hESCs. In addition, proliferation, apoptosis, and differentiation were assayed in these two hESC populations and were compared in the presence of exogenous miR-223 and miR-223 inhibitor. Inhibition of miR-223 was found to maintain the undifferentiated state of hESCs, while addition of miR-223 induced differentiation. Furthermore, these effects were found to be likely dependent on IGF-1R/Akt signaling.
doi:10.1371/journal.pone.0078769
PMCID: PMC3826748  PMID: 24250812
13.  Can Antidepressants Prevent Pegylated Interferon-α/Ribavirin-Associated Depression in Patients with Chronic Hepatitis C: Meta-Analysis of Randomized, Double-Blind, Placebo-Controlled Trials? 
PLoS ONE  2013;8(10):e76799.
Background
Antidepressants are effective in treating interferon-α/ribavirin (IFN-α/RBV)-associated depression during or after treatment of chronic hepatitis C (CHC). Whether antidepressant prophylaxis is necessary in this population remains under debate.
Methods
Comprehensive searches were performed in Medline, Embase, Cochrane Controlled Trials Register and PubMed. Reference lists were searched manually. The methodology was in accordance with the 2009 PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analysis) Statement.
Results
We identified six randomized, double-blind, placebo-controlled trials involving 522 CHC patients treated with pegylated (PEG)-IFN-α plus RBV. The antidepressants used were escitalopram, citalopram, and paroxetine, which are selective serotonin reuptake inhibitors (SSRIs). The rates of depression (17.9% vs. 31.0%, P = 0.0005), and rescue therapy (27.4% vs. 42.7%, P<0.0001) in the SSRI group were significantly lower than those in the placebo group. The rate of sustained virological response (SVR) (56.8% vs. 50.0%, P = 0.60) and drug discontinuation (18.7% vs. 21.1%, P = 0.63) in the SSRI group did not differ significantly to those in the placebo group. In terms of safety, the incidence of muscle and joint pain (40.8% vs. 52.4%, P = 0.03) and respiratory problems (29.3% vs. 40.1%, P = 0.03) were lower, but the incidence of dizziness was significantly higher (22.3% vs. 10.2%, P = 0.001) in the SSRI group.
Conclusion
Prophylactic SSRI antidepressants can significantly reduce the incidence of PEG-IFN-α/RBV-associated depression in patients with CHC, with good safety and tolerability, without reduction of SVR.
doi:10.1371/journal.pone.0076799
PMCID: PMC3813681  PMID: 24204676
14.  Correction: Structure-Guided Modification of Rhizomucor miehei Lipase for Production of Structured Lipids 
PLoS ONE  2013;8(10):10.1371/annotation/e4b16300-84fa-4c3b-97fe-4eb8fe881d00.
doi:10.1371/annotation/e4b16300-84fa-4c3b-97fe-4eb8fe881d00
PMCID: PMC3810512  PMID: 24204512
15.  MicroRNA-20b and ERK1/2 pathway independently regulate the expression of tissue factor in hematopoietic and trophoblastic differentiation of human embryonic stem cells 
Introduction
Tissue factor (TF) is expressed in various types of cells. TF expression is essential for many biological processes, such as blood coagulation and embryonic development, while its high expression in stem cells often leads to failure of transplantation. In this study, we used the human embryonic stem cell (hESC) culture system to understand the molecular mechanisms by which TF expression is regulated in hESC-derived hematopoietic and trophoblastic cells.
Methods
hESCs were induced in vitro to differentiate into hematopoietic and trophoblastic cells. TF expression in various types of cells during these differentiation processes was examined by quantitative real-time polymerase chain reaction analysis and western blot analysis. The regulatory mechanisms of TF expression were investigated by miRNA expression analysis, luciferase report assay, TF mRNA and protein analysis, and pathway phosphorylation analysis.
Results
We first found that TF was expressed only in trophoblasts and granulocyte–monocyte (G-M) cells differentiated from hESCs; and then demonstrated that miR-20b downregulated and Erk1/2 signaling pathway upregulated the TF expression in trophoblasts and G-M cells. Finally, we found that miR-20b downregulated the TF expression independently of the Erk1/2 signaling pathway.
Conclusions
The miR-20b and Erk1/2 pathway independently regulate expression of TF in trophoblasts and G-M cells differentiated from hESCs. These findings will open an avenue to further illustrate the functions of TF in various biological processes.
doi:10.1186/scrt332
PMCID: PMC3854777  PMID: 24405935
16.  Dosimetric Verification Using Monte Carlo Calculations for Tissue Heterogeneity-corrected Conformal Treatment Plans Following RTOG 0813 Dosimetric Criteria for Lung Cancer Stereotactic Body Radiotherapy 
Purpose
The recently activated RTOG studies on stereotactic body radiation therapy (SBRT) of non-small cell lung cancer (NSCLC) require tissue density heterogeneity correction, where the high and intermediate dose compliance criteria were established based on superposition-algorithm dose calculations. The study was aimed to compare superposition-algorithm dose calculations with Monte Carlo (MC) dose calculations for SBRT NSCLC treatment and to evaluate whether compliance criteria need to be adjusted for MC dose calculations.
Materials and Methods
Fifteen RTOG 0236 study sets were used. The PTV volumes range from 10.7 to 117.1 cm3. SBRT conformal treatment plans were generated using CMS XiO treatment planning software with superposition algorithm to meet the dosimetric high and intermediate compliance criteria recommended by the RTOG 0813 protocol. The plans were recalculated using the MC algorithm of a CMS Monaco treatment planning system. Tissue density heterogeneity correction was applied in both calculations.
Results
Overall, the dosimetric quantities of the MC calculations have larger magnitudes than those of the superposition calculations. On average, R100% (ratio of prescription isodose volume to PTV), R50% (ratio of 50% prescription isodose volume to PTV), D2cm (maximal dose 2 cm from PTV in any direction in percentage of prescription dose), and V20 (percentage of lung receiving dose equal to or larger than 20 Gy) increased by 9%, 12%, 7%, and 18%, respectively. In the superposition plans, 3 cases did not meet the criteria for R50% or D2cm. In the MC recalculated plans, 8 cases did not meet the criteria for R100%, R50%, or D2cm. After re-optimization with MC calculations, 5 cases did not meet the criteria for R50% or D2cm.
Conclusions
The results indicate that the dosimetric criteria, e.g., the criteria for R50%, recommended by RTOG 0813 protocol, may need to be adjusted when MC dose calculation algorithm is employed.
doi:10.1016/j.ijrobp.2011.12.005
PMCID: PMC3368093  PMID: 22365630
RTOG; stereotactic body radiation therapy; non-small-cell lung cancer; heterogeneity correction; Monte Carlo
17.  Neural mismatch indices of vowel discrimination in monolingually and bilingually exposed infants: Does attention matter? 
Neuroscience letters  2012;526(1):10-14.
Neurophysiological studies of infant speech suggest that mismatch responses (MMRs) have predictive value for later language. Their value, however, is diminished because unexplained differences in the MMR patterns are seen across studies. The current study aimed to identify the functional nature of infant MMRs by recording event-related-potentials (ERPs) to an infrequent English vowel change in internal or final positions of a sequence of ten vowels in six-month-old monolingually- and bilingually-exposed infants. Increased negativity of the MMR (infrequent minus frequent) was found in final compared to internal positions and correlated with an index of increased attention to the final position. This pattern helps explain the overall greater negativity to the speech sounds in the bilingually-exposed female infants. These findings substantially advance our understanding of neural indices of speech perception development and show promise for furthering our understanding of bilingual language development.
doi:10.1016/j.neulet.2012.07.064
PMCID: PMC3454447  PMID: 22897876
infant; attention; ERP; speech perception; neural mismatch; responses; bilingualism
18.  Room temperature rubbing for few-layer two-dimensional thin flakes directly on flexible polymer substrates 
Scientific Reports  2013;3:2697.
The functional layers of few-layer two-dimensional (2-D) thin flakes on flexible polymers for stretchable applications have attracted much interest. However, most fabrication methods are “indirect” processes that require transfer steps. Moreover, previously reported “transfer-free” methods are only suitable for graphene and not for other few-layer 2-D thin flakes. Here, a friction based room temperature rubbing method is proposed for fabricating different types of few-layer 2-D thin flakes (graphene, hexagonal boron nitride (h-BN), molybdenum disulphide (MoS2), and tungsten disulphide (WS2)) on flexible polymer substrates. Commercial 2-D raw materials (graphite, h-BN, MoS2, and WS2) that contain thousands of atom layers were used. After several minutes, different types of few-layer 2-D thin flakes were fabricated directly on the flexible polymer substrates by rubbing procedures at room temperature and without any transfer step. These few-layer 2-D thin flakes strongly adhere to the flexible polymer substrates. This strong adhesion is beneficial for future applications.
doi:10.1038/srep02697
PMCID: PMC3776208  PMID: 24045289
19.  The development of a combined mathematical model to forecast the incidence of hepatitis E in Shanghai, China 
BMC Infectious Diseases  2013;13:421.
Background
Sporadic hepatitis E has become an important public health concern in China. Accurate forecasting of the incidence of hepatitis E is needed to better plan future medical needs. Few mathematical models can be used because hepatitis E morbidity data has both linear and nonlinear patterns. We developed a combined mathematical model using an autoregressive integrated moving average model (ARIMA) and a back propagation neural network (BPNN) to forecast the incidence of hepatitis E.
Methods
The morbidity data of hepatitis E in Shanghai from 2000 to 2012 were retrieved from the China Information System for Disease Control and Prevention. The ARIMA-BPNN combined model was trained with 144 months of morbidity data from January 2000 to December 2011, validated with 12 months of data January 2012 to December 2012, and then employed to forecast hepatitis E incidence January 2013 to December 2013 in Shanghai. Residual analysis, Root Mean Square Error (RMSE), normalized Bayesian Information Criterion (BIC), and stationary R square methods were used to compare the goodness-of-fit among ARIMA models. The Bayesian regularization back-propagation algorithm was used to train the network. The mean error rate (MER) was used to assess the validity of the combined model.
Results
A total of 7,489 hepatitis E cases was reported in Shanghai from 2000 to 2012. Goodness-of-fit (stationary R2=0.531, BIC= −4.768, Ljung-Box Q statistics=15.59, P=0.482) and parameter estimates were used to determine the best-fitting model as ARIMA (0,1,1)×(0,1,1)12. Predicted morbidity values in 2012 from best-fitting ARIMA model and actual morbidity data from 2000 to 2011 were used to further construct the combined model. The MER of the ARIMA model and the ARIMA-BPNN combined model were 0.250 and 0.176, respectively. The forecasted incidence of hepatitis E in 2013 was 0.095 to 0.372 per 100,000 population. There was a seasonal variation with a peak during January-March and a nadir during August-October.
Conclusions
Time series analysis suggested a seasonal pattern of hepatitis E morbidity in Shanghai, China. An ARIMA-BPNN combined model was used to fit the linear and nonlinear patterns of time series data, and accurately forecast hepatitis E infections.
doi:10.1186/1471-2334-13-421
PMCID: PMC3847129  PMID: 24010871
Hepatitis E; Combined mathematical model; Forecast
20.  Cloning and characterization of endo-β-1,4-glucanase genes in the common wheat line three pistils 
Genetics and Molecular Biology  2013;36(3):400-407.
In this work, we report the cloning and characterization of endo-β-1,4-glucanase (EGase) genes (TaEG) in the common wheat line three pistils. Three TaEG homoeologous genes (TaEG-4A, TaEG-4B and TaEG-4D) were isolated and found to be located on chromosomes 4AL, 4BS and 4DS, respectively. The three genes showed high conservation of their coding nucleotide sequences and 3 untranslated region. The putative TaEG protein had a molecular mass of 69 kDa, a theoretical pI of 9.39 and a transmembrane domain of 74–96 amino acids in the N-terminus that anchored the protein to the membrane. The genome sequences of TaEG-4A, TaEG-4B and TaEG-4D contained six exons and five introns. All of the introns, except for intron IV, varied in length and sequence composition. Phylogenetic analysis revealed that TaEG was most closely related to rice (Oryza sativa) OsGLU1. The TaEG transcript levels increased significantly during the subsidiary pistil primordium differentiation phase (spike size ∼7–10 mm) in Chuanmai 28 TP (CM28TP). These data provide a basis for future research into the function of TaEG and offer insights into the molecular mechanism of the three pistils mutation in wheat.
doi:10.1590/S1415-47572013000300015
PMCID: PMC3795180  PMID: 24130448
cloning; endo-β-1,4-glucanase; three pistils line; wheat
21.  Correlation between virus persistent infection and cardic function in patients with dilated cardiomyopathy*  
In our study, 50 patients with dilated cardiomyopathy (DCM) were selected to investigate the correlation between virus persistent infection and cardic function. We found that 44% of patients with DCM were coxsackie virus B-RNA (CVB-RNA) positive, significantly different from that (20%) of the normal control group (P<0.05). The expression levels of coxsackie adenovirus receptor (CAR) in patients with DCM were significantly higher than those in the normal control group (P<0.01). In CVB-RNA-positive patients, expression levels of CAR were significantly higher than those in CVB-RNA-negative patients (P<0.01). There was a positive correlation between CAR expression and brain natriuretic peptide (BNP) level in patients with DCM, but no significant correlations between the CAR expression level and left ventricular ejection fraction (LVEF) or left ventricular end diastolic diameter (LVEDd). These results showed that expression levels of CAR on the surface of white cells can be used as an indicator for detecting persistent virus infection. We found that expression levels of CAR and heart function in patients with DCM were highly correlated.
doi:10.1631/jzus.BQICC708
PMCID: PMC3735976  PMID: 23897795
22.  Structure-Guided Modification of Rhizomucor miehei Lipase for Production of Structured Lipids 
PLoS ONE  2013;8(7):e67892.
To improve the performance of yeast surface-displayed Rhizomucor miehei lipase (RML) in the production of human milk fat substitute (HMFS), we mutated amino acids in the lipase substrate-binding pocket based on protein hydrophobicity, to improve esterification activity. Five mutants: Asn87Ile, Asn87Ile/Asp91Val, His108Leu/Lys109Ile, Asp256Ile/His257Leu, and His108Leu/Lys109Ile/Asp256Ile/His257Leu were obtained and their hydrolytic and esterification activities were assayed. Using Discovery Studio 3.1 to build models and calculate the binding energy between lipase and substrates, compared to wild-type, the mutant Asp256Ile/His257Leu was found to have significantly lower energy when oleic acid (3.97 KJ/mol decrease) and tripalmitin (7.55 KJ/mol decrease) were substrates. This result was in accordance with the esterification activity of Asp256Ile/His257Leu (2.37-fold of wild-type). The four mutants were also evaluated for the production of HMFS in organic solvent and in a solvent-free system. Asp256Ile/His257Leu had an oleic acid incorporation of 28.27% for catalyzing tripalmitin and oleic acid, and 53.18% for the reaction of palm oil with oleic acid. The efficiency of Asp256Ile/His257Leu was 1.82-fold and 1.65-fold that of the wild-type enzyme for the two reactions. The oleic acid incorporation of Asp256Ile/His257Leu was similar to commercial Lipozyme RM IM for palm oil acidolysis with oleic acid. Yeast surface-displayed RML mutant Asp256Ile/His257Leu is a potential, economically feasible catalyst for the production of structured lipids.
doi:10.1371/journal.pone.0067892
PMCID: PMC3700896  PMID: 23844120
23.  Apoptosis and G2/M arrest induced by Allium ursinum (ramson) watery extract in an AGS gastric cancer cell line 
OncoTargets and therapy  2013;6:779-783.
Background
The present study was designed to determine whether Allium ursinum L (ramson) could inhibit the proliferation of human AGS gastric cancer cells. Furthermore, we attempted to determine whether this inhibition could occur by targeting regulatory elements of the cell cycle.
Methods
Flow cytometry was used to observe apoptosis and the cell cycle in AGS cell lines treated or not treated with ramson watery extract. Proteins related to the cell cycle were detected by Western blotting. Caspase activity was measured using a colorimetric assay kit according to the manufacturer’s instructions.
Results
Ramson watery extract induced apoptosis and G2/M phase arrest in AGS cells. Western blotting showed that cyclin B was inhibited by ramson watery extract. However, G1 phase-related proteins remain unchanged after treatment.
Conclusion
Our results indicate that ramson effectively sup pressed proliferation and induced apoptosis and G2/M arrest in AGS cells by regulating elements of the cell cycle.
doi:10.2147/OTT.S45865
PMCID: PMC3699351  PMID: 23836991
ramson; G2/M phase arrest; apoptosis; Allium; gastric cancer
24.  Risk factors associated with severe manifestations of 2009 pandemic influenza A (H1N1) infection in China: a case–control study 
Virology Journal  2013;10:149.
Background
No studies on the risk factors of 2009 pandemic influenza A (H1N1) in China have been reported. We aimed to investigate the risk factors for severe manifestations of 2009 pandemic H1N1 influenza in China
Methods
A case–control study with 343 severe hospitalized patients and 343 randomly selected mild controls was conducted. The diagnosis was established by assessment of clinical symptoms and confirmed by the real-time reverse-transcriptase-polymerase chain reaction assay. Severe or mild patients were classified by uniform criteria issued by the Ministry of Health in China.
Results
The multivariable logistic regression analysis showed that the overweight or obese subjects admitted to hospital with H1N1 influenza were more likely to experience severe manifestations. The ORs were 3.70 (95% CI: 2.04-6.72) and 35.61 (95% CI: 7.96-159.21) respectively. Subjects at age less than 5 years or older than 60 years had an increased risk of severe manifestations (OR = 21.14, 95% CI: 7.79-57.33). We also observed increased risk among subjects with longer time interval from symptom onset to hospital admission (OR = 3.26, 95% CI: 2.08-5.11) or peasants (OR = 9.79, 95% CI: 5.11-18.78). Those with chronic disorders had increased risk of severe manifestations of H1N1 influenza.
Conclusion
We provide evidence on the risk factors associated with severe manifestations of 2009 pandemic H1N1 influenza in a study of hospitalized subjects in China.
doi:10.1186/1743-422X-10-149
PMCID: PMC3656780  PMID: 23672278
Severe manifestation; Novel influenza A; Risk factor
25.  Inhibitor Discovery of Full-Length New Delhi Metallo-β-Lactamase-1 (NDM-1) 
PLoS ONE  2013;8(5):e62955.
New Delhi metallo-β-lactmase-1 (NDM-1) has recently attracted extensive attention for its biological activities to catalyze the hydrolysis of almost all of β-lactam antibiotics. To study the catalytic property of NDM-1, the steady-kinetic parameters of NDM-1 toward several kinds of β-lactam antibiotics have been detected. It could effectively hydrolyze most β-lactams (kcat/Km ratios between 0.03 to 1.28 µmol−1.s−1), except aztreonam. We also found that thiophene-carboxylic acid derivatives could inhibit NDM-1 and have shown synergistic antibacterial activity in combination with meropenem. Flexible docking and quantum mechanics (QM) study revealed electrostatic interactions between the sulfur atom of thiophene-carboxylic acid derivatives and the zinc ion of NDM-1, along with hydrogen bond between inhibitor and His189 of NDM-1. The interaction models proposed here can be used in rational design of NDM-1 inhibitors.
doi:10.1371/journal.pone.0062955
PMCID: PMC3652859  PMID: 23675445

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