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1.  Transcription Factor ZBED6 Mediates IGF2 Gene Expression by Regulating Promoter Activity and DNA Methylation in Myoblasts 
Scientific Reports  2014;4:4570.
Zinc finger, BED-type containing 6 (ZBED6) is an important transcription factor in placental mammals, affecting development, cell proliferation and growth. In this study, we found that the expression of the ZBED6 and IGF2 were upregulated during C2C12 differentiation. The IGF2 expression levels were negatively associated with the methylation status in beef cattle (P < 0.05). A luciferase assay for the IGF2 intron 3 and P3 promoter showed that the mutant-type 439 A-SNP-pGL3 in driving reporter gene transcription is significantly higher than that of the wild-type 439 G-SNP-pGL3 construct (P < 0.05). An over-expression assay revealed that ZBED6 regulate IGF2 expression and promote myoblast differentiation. Furthermore, knockdown of ZBED6 led to IGF2 expression change in vitro. Taken together, these results suggest that ZBED6 inhibits IGF2 activity and expression via a G to A transition disrupts the interaction. Thus, we propose that ZBED6 plays a critical role in myogenic differentiation.
doi:10.1038/srep04570
PMCID: PMC3972505
2.  Network Models Predict that Reduced Excitatory Fluctuations Can Give Rise to Hippocampal Network Hyper-Excitability in MeCP2-Null Mice 
PLoS ONE  2014;9(3):e91148.
Rett syndrome is a severe pediatric neurological disorder caused by loss of function mutations within the gene encoding methyl CpG-binding protein 2 (MeCP2). Although MeCP2 is expressed near ubiquitously, the primary pathophysiology of Rett syndrome stems from impairments of nervous system function. One alteration within different regions of the MeCP2-deficient brain is the presence of hyper-excitable network responses. In the hippocampus, such responses exist despite there being an overall decrease in spontaneous excitatory drive within the network. In this study, we generated and used mathematical, neuronal network models to resolve this apparent paradox. We did this by taking advantage of previous mathematical modelling insights that indicated that decreased excitatory fluctuations, but not mean excitatory drive, more critically explain observed changes in hippocampal network oscillations from MeCP2-null mouse slices. Importantly, reduced excitatory fluctuations could also bring about hyper-excitable responses in our network models. Therefore, these results indicate that diminished excitatory fluctuations may be responsible for the hyper-excitable state of MeCP2-deficient hippocampal circuitry.
doi:10.1371/journal.pone.0091148
PMCID: PMC3958347  PMID: 24642514
3.  Enhancement of Antitumor Immunity Using a DNA-Based Replicon Vaccine Derived from Semliki Forest Virus 
PLoS ONE  2014;9(3):e90551.
A DNA-based replicon vaccine derived from Semliki Forest virus, PSVK-shFcG-GM/B7.1 (Fig. 1a) was designed for tumor immunotherapy as previously constructed. The expression of the fusion tumor antigen (survivin and hCGβ-CTP37) and adjuvant molecular protein (Granulocyte-Macrophage Colony-Stimulating Factor/ GM-CSF/B7.1) genes was confirmed by Immunofluorescence assay in vitro, and immunohistochemistry assay in vivo. In this paper, the immunological effect of this vaccine was determined using immunological assays as well as animal models. The results showed that this DNA vaccine induced both humoral and cellular immune responses in C57BL/6 mice after immunization, as evaluated by the ratio of CD4+/CD8+ cells and the release of IFN-γ. Furthermore, the vaccination of C57BL/6 mice with PSVK-shFcG-GM/B7.1 significantly delayed the in vivo growth of tumors in animal models (survivin+ and hCGβ+ murine melanoma, B16) when compared to vaccination with the empty vector or the other control constructs (Fig. 1b). These data indicate that this type of replicative DNA vaccine could be developed as a promising approach for tumor immunotherapy. Meanwhile, these results provide a basis for further study in vaccine pharmacodynamics and pharmacology, and lay a solid foundation for clinical application.
doi:10.1371/journal.pone.0090551
PMCID: PMC3946523  PMID: 24608380
4.  CXCR4/CXCL12 mediate autocrine cell cycle progression in NF1-associated malignant peripheral nerve sheath tumors 
Cell  2013;152(5):1077-1090.
Malignant Peripheral Nerve Sheath Tumors (MPNSTs) are soft tissue sarcomas that arise in connective tissue surrounding peripheral nerves. They occur sporadically in a subset of patients with Neurofibromatosis type-1 (NF1). MPNSTs are highly aggressive, therapeutically resistant, and typically fatal. Using comparative transcriptome analysis, we identified CXCR4, a G protein-coupled receptor, as highly expressed in mouse models of NF1-deficient MPNSTs, but not in non-transformed precursor cells. The chemokine receptor CXCR4 and its ligand, CXCL12, promote MPNST growth by stimulating cyclin D1 expression and cell cycle progression through PI3-Kinase (PI3K) and β-catenin signaling. Suppression of CXCR4 activity, either by shRNA or pharmacological inhibition decreases MPNST cell growth in culture and inhibits tumorigenesis in allografts and in spontaneous genetic mouse models of MPNST. We further demonstrate conservation of these activated molecular pathways in human MPNSTs. Our findings indicate a role for CXCR4 in NF1-associated MPNST development, and identify a novel therapeutic target.
doi:10.1016/j.cell.2013.01.053
PMCID: PMC3594500  PMID: 23434321
5.  The Prevalence and Molecular Spectrum of α- and β-Globin Gene Mutations in 14,332 Families of Guangdong Province, China 
PLoS ONE  2014;9(2):e89855.
Objective
To reveal the familial prevalence and molecular variation of α- and β-globin gene mutations in Guangdong Province.
Methods
A total of 40,808 blood samples from 14,332 families were obtained and analyzed for both hematological and molecular parameters.
Results
A high prevalence of α- and β-globin gene mutations was found. Overall, 17.70% of pregnant women, 15.94% of their husbands, 16.03% of neonates, and 16.83% of couples (pregnant women and their husbands) were heterozygous carriers of α- or β-thalassemia. The regions with the highest prevalence were the mountainous and western regions, followed by the Pearl River Delta; the region with the lowest prevalence was Chaoshan. The total familial carrier rate (both spouses were α- or β-thalassemia carriers) was 1.87%, and the individual carrier rates of α- and β-thalassemia were 1.68% and 0.20%, respectively. The total rate of moderate-to-severe fetal thalassemia was 12.78% among couples in which both parents were carriers.
Conclusions
There was a high prevalence of α- and β-thalassemia in Guangdong Province. This study will contribute to the development of thalassemia prevention and control strategies in Guangdong Province.
doi:10.1371/journal.pone.0089855
PMCID: PMC3937408  PMID: 24587075
6.  Genome-wide association study in Han Chinese identifies four new susceptibility loci for coronary artery disease 
Nature genetics  2012;44(8):890-894.
We performed a meta-analysis of 2 genome-wide association studies of coronary artery disease comprising 1,515 cases with coronary artery disease and 5,019 controls, followed by de novo replication studies in 15,460 cases and 11,472 controls, all of Chinese Han descent. We successfully identified four new loci for coronary artery disease reaching genome-wide significance (P < 5 × 10−8), which mapped in or near TTC32-WDR35, GUCY1A3, C6orf10-BTNL2 and ATP2B1. We also replicated four loci previously identified in European populations (PHACTR1, TCF21, CDKN2A/B and C12orf51). These findings provide new insights into biological pathways for the susceptibility of coronary artery disease in Chinese Han population.
doi:10.1038/ng.2337
PMCID: PMC3927410  PMID: 22751097
7.  A new technique combining virtual simulation and methylene blue staining for the localization of small peripheral pulmonary lesions 
BMC Cancer  2014;14:79.
Background
Quickly and accurately localizing small peripheral pulmonary lesions can avoid prolonged operative time and unplanned open thoracotomy. In this study, we aimed to introduce and evaluate a new technique combining virtual simulation and methylene blue staining for the localization of small peripheral pulmonary lesions.
Methods
Seventy four (74) patients with 80 peripheral pulmonary lesions <20 mm in size on computer tomography (CT) were virtually punctured using a radiotherapy planning simulator on the day before operation. Under general anaesthesia, methylene blue dye was injected to the virtually identified point according to the surface point, angle and depth previously determined by the simulator. The wedge resection of the marked lesion was performed under video-assisted thoracoscopic surgery (VATS) and the specimens were sent for immediate pathologic examination. According to pathology results, appropriate surgical procedures were decided and undertaken.
Results
The average lesion size was 10.4±3.5 mm (range: 4-17 mm) and the average distance to the pleural surface was 9.4±4.9 mm. Our preoperative localization procedure was successful in 75 of 80 (94%) lesions. Histological examination showed 28 benign lesions and 52 lung cancers. The shortest distance between the edges of the stain and lesion was 5.1±3.1 mm. Localization time was 17.4±2.3 min. All patients with malignant lesions subsequently underwent lobectomy and systematic lymph node dissection. No complications were observed in all participants.
Conclusions
The novel technique combining the preoperative virtual simulation and methylene blue staining techniques has a high success rate for localizing small peripheral pulmonary lesions, particularly for those tiny lesions which are difficult to visualise and palpate during VATS.
doi:10.1186/1471-2407-14-79
PMCID: PMC3926337  PMID: 24512571
Lung cancer; Pulmonary lesions; Thoracoscopy; Localization; Simulation; Methylene blue
8.  Induction of Specific Humoral and Cellular Immune Responses in a Mouse Model following Gene Fusion of HSP70C and Hantaan Virus Gn and S0.7 in an Adenoviral Vector 
PLoS ONE  2014;9(2):e88183.
Heat shock proteins (HSPs) display adjuvant functions when given as fusion proteins to enhance vaccination efficiency. To evaluate enhanced potency of Hantaan virus (HTNV) glycoprotein (GP) and nucleocapsid protein (NP) immunogenicity by heat shock protein 70 (HSP70), a recombinant adenovirus rAd-GnS0.7-pCAG-HSP70C expression vector was developed by genetically linking the HSP70 C-terminal gene (HSP70 359–610 aa, HSP70C) to the Gn and 0.7 kb fragment of the NP (aa1–274-S0.7). C57BL/6 mice were immunized with these recombinant adenoviral vectors. A series of immunological assays determined the immunogenicity of the recombinant adenoviral vectors. The results showed that rAd-GnS0.7-pCAG-HSP70C induced a stronger humoral and cellular immune response than other recombinant adenoviruses (rAd-GnS0.7-pCAG and rAd-GnS0.7) and the HFRS vaccine control. Animal protection experiments showed that rAd-GnS0.7-pCAG-HSP70C was effective at protecting C57BL/6 mice from HTNV infection. The results of the immunological experiments showed that HSP70C lead to enhanced vaccine potency, and suggested significant potential in the development of genetically engineered vaccines against HTNV.
doi:10.1371/journal.pone.0088183
PMCID: PMC3913774  PMID: 24505421
9.  Induction of Apoptosis in Human Multiple Myeloma Cell Lines by Ebselen via Enhancing the Endogenous Reactive Oxygen Species Production 
BioMed Research International  2014;2014:696107.
Ebselen a selenoorganic compound showing glutathione peroxidase like activity is an anti-inflammatory and antioxidative agent. Its cytoprotective activity has been investigated in recent years. However, experimental evidence also shows that ebselen causes cell death in several cancer cell types whose mechanism has not yet been elucidated. In this study, we examined the effect of ebselen on multiple myeloma (MM) cell lines in vitro. The results showed that ebselen significantly enhanced the production of reactive oxygen species (ROS) accompanied by cell viability decrease and apoptosis rate increase. Further studies revealed that ebselen can induce Bax redistribution from the cytosol to mitochondria leading to mitochondrial membrane potential ΔΨm changes and cytochrome C release from the mitochondria to cytosol. Furtherly, we found that exogenous addition of N-acetyl cysteine (NAC) completely diminished the cell damage induced by ebselen. This result suggests that relatively high concentration of ebselen can induce MM cells apoptosis in culture by enhancing the production of endogenous ROS and triggering mitochondria mediated apoptotic pathway.
doi:10.1155/2014/696107
PMCID: PMC3921973  PMID: 24587987
10.  Quantitative Assessment of the Influence of TP63 Gene Polymorphisms and Lung Cancer Risk: Evidence Based on 93,751 Subjects 
PLoS ONE  2014;9(1):e87004.
Background
Several genome-wide association studies on lung cancer (LC) have reported similar findings of a new susceptibility locus, 3q28. After that, a number of studies reported that the rs10937405, and rs4488809 polymorphism in chromosome 3q28 has been implicated in LC risk. However, the studies have yielded contradictory results.
Methods
PubMed, ISI web of science, EMBASE and the Chinese National Knowledge Infrastructure databases were systematically searched to identify relevant studies. Data were abstracted independently by two reviewers. A meta-analysis was performed to examine the association between rs10937405, rs4488809 polymorphism at 3q28 and susceptibility to LC. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated. Heterogeneity and publication bias were also tested.
Results
A total of 9 studies including 35,961 LC cases and 57,790 controls were involved in this meta-analysis. An overall random-effects per-allele OR of1.19 (95% CI: 1.14–1.25; P<10−5) and 1.19 (95% CI: 1.13–1.25; P<10−5) was found for the rs10937405 and rs4488809 polymorphism respectively. Similar results were also observed using dominant or recessive genetic model. After stratified by ethnicity, significant associations were found among East Asians (per-allele OR = 1.22, 95% CI: 1.17–1.27; P<10−5); whereas no significant associations were found among Caucasians for rs10937405. In the sub-group analysis by sample size, significantly increased risks were found for these polymorphisms in all genetic models. When analyzed according to histological type, the effects of rs10937405, and rs4488809 at 3q28 on the risk of lung cancer were significant mostly for lung adenocarcinoma.
Conclusions
Our findings demonstrated that rs10937405-G allele and rs4488809-G allele might be risk-conferring factors for the development of lung cancer, especially for East Asian populations.
doi:10.1371/journal.pone.0087004
PMCID: PMC3900682  PMID: 24466311
11.  Functional analysis of the involvement of apurinic/apyrimidinic endonuclease 1 in the resistance to melphalan in multiple myeloma 
BMC Cancer  2014;14:11.
Background
Melphalan resistance has been considered one of the major obstacles to improve outcomes in multiple myeloma (MM) therapy; unfortunately, the mechanistic details of this resistance remain unclear. Melphalan is a highly effective alkylating agent which causes many types of DNA lesions, including DNA base alkylation damage that is repaired by base excision repair (BER). We postulated that human apurinic/apyrimidinic endonuclease 1 (APE1), an essential BER enzyme, plays a vital role in acquired melphalan resistance. However, because APE1 is a multifunctional protein with redox activity and acetylation modification in addition to its major repair activity, the particular APE1 function that may play a more important role in melphalan resistance is unknown.
Methods
Two MM cell lines, RPMI-8226 and U266 were used to measure the difference in APE1 levels in melphalan-resistant and sensitive derivatives. APE1 functional mutants for DNA repair, redox and acetylation were employed to investigate the roles of individual APE1 activities in acquired melphalan resistance.
Results
Our results indicate that APE1 is overexpressed in both MM melphalan-resistant cells. Knocking down APE1 sensitizes the melphalan resistant MM cells to melphalan treatment. The exogenous expression of DNA repair mutant H309N and acetylation mutant K6R/K7R of APE1 failed to restore the melphalan resistance of the APE1 knockdown RPMI-8226 cells. The AP endonuclease activity and multidrug resistance protein 1 (MDR1) regulatory activity may play roles in the melphalan resistance of MM cells.
Conclusions
The present study has identified that the DNA repair functions and the acetylation modification of APE1 are involved in melphalan resistance of MM cells and has also shed light on future therapeutic strategies targeting specific APE1 functions by small molecule inhibitors.
doi:10.1186/1471-2407-14-11
PMCID: PMC3900260  PMID: 24400589
Acquired melphalan resistance; Multiple myeloma; Human apurinic/apyrimidinic endonuclease 1; DNA repair; Base excision repair
12.  Severe Hypoglycemia in a Juvenile Diabetic Rat Model: Presence and Severity of Seizures Are Associated with Mortality 
PLoS ONE  2013;8(12):e83168.
It is well accepted that insulin-induced hypoglycemia can result in seizures. However, the effects of the seizures, as well as possible treatment strategies, have yet to be elucidated, particularly in juvenile or insulin-dependent diabetes mellitus (IDDM). Here we establish a model of diabetes in young rats, to examine the consequences of severe hypoglycemia in this age group; particularly seizures and mortality. Diabetes was induced in post-weaned 22-day-old Sprague-Dawley rats by streptozotocin (STZ) administered intraperitoneally (IP). Insulin IP (15 U/kg), in rats fasted (14–16 hours), induced hypoglycemia, defined as <3.5 mM blood glucose (BG), in 68% of diabetic (STZ) and 86% of control rats (CON). Seizures occurred in 86% of STZ and all CON rats that reached hypoglycemic levels with mortality only occurring post-seizure. The fasting BG levels were significantly higher in STZ (12.4±1.3 mM) than in CON rodents (6.3±0.3 mM), resulting in earlier onset of hypoglycemia and seizures in the CON group. However, the BG at seizure onset was statistically similar between STZ (1.8±0.2 mM) and CON animals (1.6±0.1 mM) as well as between those that survived (S+S) and those that died (S+M) post-seizure. Despite this, the S+M group underwent a significantly greater number of seizure events than the S+S group. 25% glucose administered at seizure onset and repeated with recurrent seizures was not sufficient to mitigate these continued convulsions. Combining glucose with diazepam and phenytoin significantly decreased post-treatment seizures, but not mortality. Intracranial electroencephalograms (EEGs) were recorded in 10 CON and 9 STZ animals. Predictive EEG changes were not observed in these animals that underwent seizures. Fluorojade staining revealed damaged cells in non-seizing STZ animals and in STZ and CON animals post-seizure. In summary, this model of hypoglycemia and seizures in juvenile diabetic rats provides a paradigm for further study of underlying mechanisms. Our data demonstrate that severe hypoglycemia (<2.0 mM) is a necessary precondition for seizures, and the increased frequency of these seizures is associated with mortality.
doi:10.1371/journal.pone.0083168
PMCID: PMC3875447  PMID: 24386156
13.  Long-term prehypertension treatment with losartan effectively prevents brain damage and stroke in stroke-prone spontaneously hypertensive rats 
Prehypertension has been associated with adverse cerebrovascular events and brain damage. The aims of this study were to investigate i) whether short- and long-term treatments with losartan or amlodipine for prehypertension were able to prevent blood pressure (BP)-linked brain damage, and ii) whether there is a difference in the effectiveness of treatment with losartan and amlodipine in protecting BP-linked brain damage. In the present study, prehypertensive treatment with losartan and amlodipine (6 and 16 weeks treatment with each drug) was performed on 4-week-old stroke-prone spontaneously hypertensive rats (SHRSP). The results showed that long-term (16 weeks) treatment with losartan is the most effective in lowering systolic blood pressure in the long term (up to 40 weeks follow-up). Additionally, compared with the amlodipine treatment groups, the short- and long-term losartan treatments protected SHRSP from stroke and improved their brains structurally and functionally more effectively, with the long-term treatment having more benefits. Mechanistically, the short- and long-term treatments with losartan reduced the activity of the local renin-angiotensin-aldosterone system (RAAS) in a time-dependent manner and more effectively than their respective counterpart amlodipine treatment group mainly by decreasing AT1R levels and increasing AT2R levels in the cerebral cortex. By contrast, the amlodipine treatment groups inhibited brain cell apoptosis more effectively as compared with the losartan treatment groups mainly through the suppression of local oxidative stress. Taken together, the results suggest that long-term losartan treatment for prehypertension effectively protects SHRSP from stroke-induced brain damage, and this protection is associated with reduced local RAAS activity than with brain cell apoptosis. Thus, the AT1R receptor blocker losartan is a good candidate drug that may be used in the clinic for long-term treatment on prehypertensive populations in order to prevent BP-linked brain damage.
doi:10.3892/ijmm.2013.1583
PMCID: PMC3896471  PMID: 24337406
prehypertension; losartan; amlodipine; stroke-prone spontaneously hypertensive rats
14.  A Brief Analysis of Traditional Chinese Medical Elongated Needle Therapy on Acute Spinal Cord Injury and Its Mechanism 
Acute spinal cord injury is one of the most common and complicated diseases among human spinal injury. We aimed to explore the effect of point-through-point acupuncture therapy with elongated needles on acute spinal cord injury in rabbits and its possible mechanism. Adult rabbits were randomly divided into a model group, elongated needle therapy group, and blank group. Immunohistochemical staining showed that the protein levels of Fas and caspase-3 in the model group were significantly higher than those in the blank group at each time point (P < 0.05) and significantly lower than those in the elongated needle therapy group on the 3rd and 5th days after operation (P < 0.05). RT-PCR showed that Fas and caspase-3 mRNA levels in the model group and elongated needle therapy group were significantly higher than those in the blank group (P < 0.05, 0.01). The mRNA levels of Fas and caspase-3 in the elongated needle therapy group were significantly lower than those in model group on the 3rd day (P < 0.05, 0.01). Therefore, we confirmed that elongated needle therapy has an obvious effect on acute spinal cord injury in rabbits. Its mechanism is made possible by inhibiting the expression of the Fas→caspase-3 cascade, thereby inhibiting cell apoptosis after spinal cord injury.
doi:10.1155/2013/828754
PMCID: PMC3857841  PMID: 24348723
15.  Phosphoglycerate mutase 1 coordinates glycolysis and biosynthesis to promote tumor growth 
Cancer cell  2012;22(5):585-600.
SUMMARY
It remains unclear how cancer cells coordinate glycolysis and biosynthesis to support rapidly growing tumors. We found that glycolytic enzyme phosphoglycerate mutase 1 (PGAM1), commonly upregulated in human cancers due to loss of TP53, contributes to biosynthesis regulation in part by controlling intracellular levels of its substrate 3-phosphoglycerate (3-PG) and product 2-phosphoglycerate (2-PG). 3-PG binds to and inhibits 6-phosphogluconate dehydrogenase in the oxidative pentose phosphate pathway (PPP), while 2-PG activates 3-phosphoglycerate dehydrogenase to provide feedback control of 3-PG levels. Inhibition of PGAM1 by shRNA or a small molecule inhibitor PGMI-004A results in increased 3-PG and decreased 2-PG levels in cancer cells, leading to significantly decreased glycolysis, PPP flux and biosynthesis, as well as attenuated cell proliferation and tumor growth.
doi:10.1016/j.ccr.2012.09.020
PMCID: PMC3500524  PMID: 23153533
16.  Development of a Specific Monoclonal Antibody-Based ELISA to Measure the Artemether Content of Antimalarial Drugs 
PLoS ONE  2013;8(11):e79154.
Artemether is one of the artemisinin derivatives that are active ingredients in antimalarial drugs. Counterfeit and substandard antimalarial drugs have become a serious problem, which demands reliable analytical tools and implementation of strict regulation of drug quality. Structural similarity among artemisinin analogs is a challenge to develop immunoassays that are specific to artemisinin derivatives. To produce specific antibodies to artemether, we used microbial fermentation of artemether to obtain 9-hydroxyartemether, which was subsequently used to prepare a 9-O-succinylartemether hapten for conjugation with ovalbumin as the immunogen. A monoclonal antibody (mAb), designated as 2G12E1, was produced with high specificity to artemether. 2G12E1 showed low cross reactivities to dihydroartemisinin, artemisinin, artesunate and other major antimalarial drugs. An indirect competitive enzyme linked immunosorbent assay (icELISA) developed showed a concentration causing 50% of inhibition for artemether as 3.7 ng mL−1 and a working range of 0.7–19 ng mL−1. The icELISA was applied for determination of artemether content in different commercial drugs and the results were comparable to those determined by high-performance liquid chromatography analysis. In comparison with reported broad cross activity of anti-artemisinin mAbs, the most notable advantage of the 2G12E1-based ELISA is its high specificity to artemether only.
doi:10.1371/journal.pone.0079154
PMCID: PMC3827310  PMID: 24236102
17.  Rbf1 degron dysfunction enhances cellular DNA replication 
Cell Cycle  2012;11(20):3731-3738.
The E2F family of transcription factors contributes to oncogenesis through activation of multiple genes involved in cellular proliferation, a process that is opposed by the Retinoblastoma tumor suppressor protein (RB). RB also increases E2F1 stability by inhibiting its proteasome-mediated degradation, but the consequences of this post-translational regulation of E2F1 remain unknown. To better understand the mechanism of E2F stabilization and its physiological relevance, we examined the streamlined Rbf1-dE2F1 network in Drosophila. During embryonic development, Rbf1 is insulated from ubiquitin-mediated turnover by the COP9 signalosome, a multi-protein complex that modulates E3 ubiquitin ligase activity. Here, we report that the COP9 signalosome also protects the Cullin4-E3 ligase that is responsible for dE2F1 proteasome-mediated destruction. This dual role of the COP9 signalosome may serve to buffer E2F levels, enhancing its turnover via Cul4 protection and its stabilization through protection of Rbf1. We further show that Rbf1-mediated stabilization of dE2F1 and repression of dE2F1 cell cycle-target genes are distinct properties. Removal of an evolutionarily conserved Rbf1 C terminal degron disabled Rbf1 repression without affecting dE2F1 stabilization. This mutant form of Rbf1 also enhanced G1-to-S phase progression when expressed in Rbf1-containing S2 embryonic cells, suggesting that such mutations may generate gain-of-function properties relevant to cellular transformation. Consistent with this idea, several studies have identified mutations in the homologous C terminal domains of RB and p130 in human cancer.
doi:10.4161/cc.21665
PMCID: PMC3495815  PMID: 22895052
retinoblastoma; tumor suppressor; E2F1; repression; proteasome; ubiquitin; degron
18.  Reversible insulator-metal transition of LaAlO3/SrTiO3 interface for nonvolatile memory 
Scientific Reports  2013;3:2870.
We report a new type of memory device based on insulating LaAlO3/SrTiO3 (LAO/STO) hetero-interface. The microstructures of the LAO/STO interface are characterized by Cs-corrected scanning transmission electron microscopy, which reveals the element intermixing at the interface. The inhomogeneous element distribution may result in carrier localization, which is responsible for the insulating state. The insulating state of such interface can be converted to metallic state by light illumination and the metallic state maintains after light off due to giant persistent photoconductivity (PPC) effect. The on/off ratio between the PPC and the initial dark conductance is as large as 105. The metallic state also can be converted back to insulating state by applying gate voltage. Reversible and reproducible resistive switching makes LAO/STO interface promising as a nonvolatile memory. Our results deepen the understanding of PPC phenomenon in LAO/STO, and pave the way for the development of all-oxide electronics integrating information storage devices.
doi:10.1038/srep02870
PMCID: PMC3792417  PMID: 24100438
19.  Opposite Effects of Single-Dose and Multidose Administration of the Ethanol Extract of Danshen on CYP3A in Healthy Volunteers 
The aim of this study was to investigate the effect of single- and multidose administration of the ethanol extract of danshen on in vivo CYP3A activity in healthy volunteers. A sequential, open-label, and three-period pharmacokinetic interaction study design was used based on 12 healthy male individuals. The plasma concentrations of midazolam and its metabolite 1-hydroxymidazolam were measured. Treatment with single dose of the extract caused the mean Cmax of midazolam to increase by 87% compared with control. After 10 days of the danshen extract intake, the mean AUC0–12, Cmax, and t1/2 of midazolam were decreased by 79.9%, 66.6%, and 43.8%, respectively. The mean clearance of midazolam was increased by 501.6% compared with control. The in vitro study showed that dihydrotanshinone I in the extract could inhibit CYP3A, while tanshinone IIA and cryptotanshinone could induce CYP3A. In conclusion, a single-dose administration of the danshen extract can inhibit intestinal CYP3A, but multidose administration can induce intestinal and hepatic CYP3A.
doi:10.1155/2013/730734
PMCID: PMC3816049  PMID: 24223062
20.  A recombinant pseudotyped lentivirus expressing the envelope glycoprotein of Hantaan virus induced protective immunity in mice 
Virology Journal  2013;10:301.
Background
Hantaviruses cause acute hemorrhagic fever with renal syndrome (HFRS). Currently, several types of inactivated HFRS vaccines are widely used, however the limited ability of these immunogen to elicit neutralizing antibodies restricts vaccine efficacy. Development of an effective vaccine to overcome this weakness is must.
Methods
In the present study, a recombinant pseudotyped lentivirus bearing the hantaan virus (HTNV) envelope glycoproteins (GP), rLV-M, was constructed. C57BL/6 mice were immunized with the rLV-M and a series of immunological assays were conducted to determine the immunogenicity of the recombinant pseudotyped lentivirus. The humoral and cell-mediated immune responses induced by rLV-M were compared with those of the inactivated HFRS vaccine.
Results
Indirect immunofluorescence assay (IFA) showed the rLV-M expressed target proteins in HEK-293cells. In mice, the rLV-M efficiently induced GP-specific humoral responses and protection against HTNV infection. Furthermore, the rLV-M induced higher neutralizing antibody titers than the inactivated HFRS vaccine control.
Conclusions
The results indicated the potential of using a pseudotyped lentivirus as a delivery vector for a hantavirus vaccine immunogen.
doi:10.1186/1743-422X-10-301
PMCID: PMC3851560  PMID: 24093752
Recombinant pseudotyped lentivirus; Hantaan virus; Glycoprotein; Protective immunity
21.  An easy operating pathogen microarray (EOPM) platform for rapid screening of vertebrate pathogens 
BMC Infectious Diseases  2013;13:437.
Background
Infectious diseases emerge frequently in China, partly because of its large and highly mobile population. Therefore, a rapid and cost-effective pathogen screening method with broad coverage is required for prevention and control of infectious diseases. The availability of a large number of microbial genome sequences generated by conventional Sanger sequencing and next generation sequencing has enabled the development of a high-throughput high-density microarray platform for rapid large-scale screening of vertebrate pathogens.
Methods
An easy operating pathogen microarray (EOPM) was designed to detect almost all known pathogens and related species based on their genomic sequences. For effective identification of pathogens from EOPM data, a statistical enrichment algorithm has been proposed, and further implemented in a user-friendly web-based interface.
Results
Using multiple probes designed to specifically detect a microbial genus or species, EOPM can correctly identify known pathogens at the species or genus level in blinded testing. Despite a lower sensitivity than PCR, EOPM is sufficiently sensitive to detect the predominant pathogens causing clinical symptoms. During application in two recent clinical infectious disease outbreaks in China, EOPM successfully identified the responsible pathogens.
Conclusions
EOPM is an effective surveillance platform for infectious diseases, and can play an important role in infectious disease control.
doi:10.1186/1471-2334-13-437
PMCID: PMC3848773  PMID: 24053492
22.  “Dot COM”, a Nuclear Transit Center for the Primary piRNA Pathway in Drosophila 
PLoS ONE  2013;8(9):e72752.
The piRNA pathway protects genomes by silencing mobile elements. Despite advances in understanding the processing events that generate piRNAs for silencing, little is known about how primary transcripts are transported from their genomic clusters to their processing centers. Using a model of the Drosophila COM/flamenco locus in ovarian somatic cells, we identified a prominent nuclear structure called Dot COM, which is enriched in long transcripts from piRNA clusters but located far from their transcription sites. Remarkably, transcripts from multiple clusters accumulate at Dot COM, which is often juxtaposed with Yb-bodies, the cytoplasmic processing centers for cluster transcripts. Genetic evidence suggests that the accumulation of precursor transcripts at Dot COM represents one of the most upstream events in the piRNA pathway. Our results provide new insights into the initial steps of the piRNA pathway, and open up a new research area important for a complete understanding of this conserved pathway.
doi:10.1371/journal.pone.0072752
PMCID: PMC3767702  PMID: 24039799
23.  Association between 5p12 Genomic Markers and Breast Cancer Susceptibility: Evidence from 19 Case-Control Studies 
PLoS ONE  2013;8(9):e73611.
Background
The association between polymorphisms on 5p12 and breast cancer (BC) has been widely evaluated since it was first identified through genome-wide association approach; however, the studies have yielded contradictory results. We sought to investigate this inconsistency by performing a comprehensive meta-analysis on two wildly studied polymorphisms (rs10941679 and rs4415084) on 5p12.
Methods
Databases including Pubmed, EMBASE, Web of Science, EBSCO, and Cochrane Library databases were searched to find relevant studies. Odds ratios (ORs) with 95% confidence intervals (CIs) were used to assess the strength of association. The random-effects model was applied, addressing heterogeneity and publication bias.
Results
A total of 19 articles involving 100,083 cases and 163,894 controls were included. An overall random-effects per-allele OR of 1.09 (95% CI: 1.06–1.12; P = 4.5×10−8) and 1.09 (95% CI: 1.05–1.12; P = 4.2×10−7) was found for the rs10941679 and rs4415084 polymorphism respectively. Significant results were found in Asians and Caucasians when stratified by ethnicity; whereas no significant associations were found among Africans/African-Americans. Similar results were also observed using dominant or recessive genetic models. In addition, we find both rs4415084 and rs10941679 conferred significantly greater risks of ER-positive breast cancer than of ER-negative tumors.
Conclusions
Our findings demonstrated that rs10941679-G allele and rs4415084-T allele might be risk-conferring factors for the development of breast cancer, especially in Caucasians and East-Asians.
doi:10.1371/journal.pone.0073611
PMCID: PMC3765311  PMID: 24039999
24.  Rescue of behavioral and EEG deficits in male and female Mecp2-deficient mice by delayed Mecp2 gene reactivation 
Human Molecular Genetics  2013;23(2):303-318.
Mutations of the X-linked gene encoding methyl CpG binding protein type 2 (MECP2) are the predominant cause of Rett syndrome, a severe neurodevelopmental condition that affects primarily females. Previous studies have shown that major phenotypic deficits arising from MeCP2-deficiency may be reversible, as the delayed reactivation of the Mecp2 gene in Mecp2-deficient mice improved aspects of their Rett-like phenotype. While encouraging for prospective gene replacement treatments, it remains unclear whether additional Rett syndrome co-morbidities recapitulated in Mecp2-deficient mice will be similarly responsive to the delayed reintroduction of functional Mecp2. Here, we show that the delayed reactivation of Mecp2 in both male and female Mecp2-deficient mice rescues established deficits in motor and anxiety-like behavior, epileptiform activity, cortical and hippocampal electroencephalogram patterning and thermoregulation. These findings indicate that neural circuitry deficits arising from the deficiency in Mecp2 are not engrained, and provide further evidence that delayed restoration of Mecp2 function can improve a wide spectrum of the Rett-like deficits recapitulated by Mecp2-deficient mice.
doi:10.1093/hmg/ddt421
PMCID: PMC3869352  PMID: 24009314
25.  Further evidence of endogenous hydrogen sulphide as a mediator of relaxation in human and rat bladder 
Asian Journal of Andrology  2013;15(5):692-696.
We investigated the expression of hydrogen sulphide (H2S) in human and rat lower urinary tract (including bladder, prostate and urethra) tissues, and we sought to determine whether H2S induces relaxation of human and Sprague–Dawley (SD) rat bladder strips. Human normal lower urinary tract tissue was obtained for the evaluation of endogenous H2S productivity using a sulphide-sensitive electrode and for the analysis of the expression levels of all three synthases of endogenous H2S, cystathionine β-synthase (CBS), cystathionine γ lyase (CSE) and 3-mercaptopyruvate sulphur transferase (MPST, as known as 3-MST) by Western blot assay. CBS, CSE and MPST were located in human sample slides by immunohistochemistry. Human and male adult SD rat bladder strips were tested for H2S function with a transducer and recorded. All experiments were repeated six times. The endogenous H2S productivity and the H2S synthases had various distributions in the human and rat lower urinary tract tissues and were located in both epithelial and stromal sections. L-cysteine (L-Cys, a substrate of CBS, CSE and MPST) elicited relaxation in a dose-dependent manner on human bladder strips pre-contracted by acetylcholine chloride. This effect could be diminished by the ATP-sensitive potassium ion (KATP) channel blocker glibenclamide (GLB), the CSE inhibitor DL-propargylglycine (PPG) and the CBS inhibitor hydroxylamine (HA). H2S and its three synthases were present in the human and rat lower urinary tract tissues and relaxed human and rat bladder strips, which implied that endogenous H2S might play a role in physiological function and pathological disorders of the lower urinary tract symptoms (LUTS) or overactive bladder (OAB).
doi:10.1038/aja.2013.32
PMCID: PMC3881648  PMID: 23728586
cystathionine β-synthase; cystathionine γ lyase; detrusor; hydrogen sulphide; lower urinary tract symptoms; 3-mercaptopyruvate sulphur transferase

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