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1.  Ectopic Expression of Activated Notch or SOX2 Reveals Similar and Unique Roles in the Development of the Sensory Cell Progenitors in the Mammalian Inner Ear 
The Journal of Neuroscience  2013;33(41):16146-16157.
Hearing impairment or vestibular dysfunction in humans often results from a permanent loss of critical cell types in the sensory regions of the inner ear, including hair cells, supporting cells, or cochleovestibular neurons. These important cell types arise from a common sensory or neurosensory progenitor, although little is known about how these progenitors are specified. Studies have shown that Notch signaling and the transcription factor Sox2 are required for the development of these lineages. Previously we and others demonstrated that ectopic activation of Notch can direct nonsensory cells to adopt a sensory fate, indicating a role for Notch in early specification events. Here, we explore the relationship between Notch and SOX2 by ectopically activating these factors in nonsensory regions of the mouse cochlea, and demonstrate that, similar to Notch, SOX2 can specify sensory progenitors, consistent with a role downstream of Notch signaling. However, we also show that Notch has a unique role in promoting the proliferation of the sensory progenitors. We further demonstrate that Notch can only induce ectopic sensory regions within a certain time window of development, and that the ectopic hair cells display specialized stereocilia bundles similar to endogenous hair cells. These results demonstrate that Notch and SOX2 can both drive the sensory program in nonsensory cells, indicating these factors may be useful in cell replacement strategies in the inner ear.
doi:10.1523/JNEUROSCI.3150-12.2013
PMCID: PMC3792456  PMID: 24107947
2.  Pallidal Index as Biomarker of Manganese Brain Accumulation and Associated with Manganese Levels in Blood: A Meta-Analysis 
PLoS ONE  2014;9(4):e93900.
Objective
The current study was designed to evaluate the sensitivity, feasibility, and effectiveness of the pallidal index (PI) serving as a biomarker of brain manganese (Mn) accumulation, which would be used as an early diagnosis criteria for Mn neurotoxicity.
Methods
The weighted mean difference (WMD) of the PI between control and Mn-exposed groups was estimated by using a random-effects or fixed-effects meta-analysis with 95% confidence interval (CI) performed by STATA software version 12.1. Moreover, the R package “metacor” was used to estimate correlation coefficients between PI and blood Mn (MnB).
Results
A total of eight studies with 281 occupationally Mn-exposed workers met the inclusion criteria. Results were pooled and performed with the Meta-analysis. Our data indicated that the PI of the exposed group was significantly higher than that of the control (WMD: 7.76; 95% CI: 4.86, 10.65; I2 = 85.7%, p<0.0001). A random effects model was used to perform meta-analysis. These findings were remarkably robust in the sensitivity analysis, and publication bias was shown in the included studies. Seven out of the eight studies reported the Pearson correlation (r) values. Significantly positive correlation between PI and MnB was observed (r = 0.42; 95% CI, 0.31, 0.52).
Conclusions
PI can be considered as a sensitive, feasible, effective and semi-quantitative index in evaluating brain Mn accumulation. MnB can also augment the evaluation of brain Mn accumulation levels in the near future. However, the results should be interpreted with caution.
doi:10.1371/journal.pone.0093900
PMCID: PMC3981755  PMID: 24718592
3.  Altered cholesterol homeostasis in aged macrophages linked to neovascular macular degeneration 
Cell metabolism  2013;17(4):471-472.
Abnormal lipid metabolism has been linked to age-related macular degeneration (AMD); choroidal neovascularization in late AMD commonly causes blindness. Sene et al. now demonstrate that in aged macrophages decreased ABCA1 expression, regulated by liver X receptor and miR-33, impairs export of intracellular cholesterol which promotes neovascular AMD (Sene et al., 2013).
doi:10.1016/j.cmet.2013.03.010
PMCID: PMC3669899  PMID: 23562072
4.  Intravitreal Injection of IGFBP-3 Restores Normal Insulin Signaling in Diabetic Rat Retina 
PLoS ONE  2014;9(4):e93788.
Diabetes-induced changes in growth factor binding protein 3 (IGFBP-3) and tumor necrosis factor alpha (TNFα) have been linked to decreased insulin receptor signaling in diabetic retinopathy. Our previous studies in retinas of diabetic rats have shown that Compound 49b, a novel β-adrenergic receptor agonist, prevented diabetic changes by increasing IGFBP-3 and decreasing TNFα, thus restoring insulin signaling and protection against diabetic retinopathy. The current study was designed to determine whether boosted expression of IGFBP-3 NB (a non-IGF-1 binding form of IGFBP-3) alone is sufficient to mimic the full actions of Compound 49b in protecting against diabetic retinopathy, as well as testing whether IGFBP-3 NB is linked to a restoration of normal insulin signal transduction. Two months after initiation of streptozotocin-induced diabetes, rats received a single intravitreal injection of IGFBP-3 NB plasmid in the right eye. Four days after injection, electroretinogram (ERG) analyses were performed prior to sacrifice. Whole retinal lysates from control, diabetic, diabetic + control plasmid, and diabetic+ IGFBP-3 NB were analyzed for IGFBP-3, TNFα, suppressor of cytokine signaling 3 (SOCS3), and insulin receptor signaling partners using Western blotting or ELISA. Data show that a single intraocular injection of IGFBP-3 NB in diabetic animals significantly reduced TNFα levels, concomitant with reductions in IRS-1Ser307, SOCS3, and pro-apoptotic markers, while restoring insulin receptor phosphorylation and increasing anti-apoptotic marker levels. These cellular changes were linked to restoration of retinal function. Our findings establish IGFBP-3 as a pivotal regulator of the insulin receptor/TNFα pathway and a potential therapeutic target for diabetic retinopathy.
doi:10.1371/journal.pone.0093788
PMCID: PMC3973588  PMID: 24695399
5.  IL-17 induces expression of vascular cell adhesion molecule through signaling pathway of NF-κB, but not Akt1 and TAK1 in vascular smooth muscle cells 
Interleukin 17 (IL-17) plays an important role in several autoimmune diseases. IL-17 can induce the expression of vascular cell adhesion molecule (VCAM-1) in aortic vascular smooth muscle cells (SMCs), which is important for the development of atherosclerosis. However, the signaling pathway of IL-17-induced VCAM-1 expression remains unclear. In this study, we reported that IL-17 induced expression of VCAM-1 in SMCs is dependent on NF-κB, but independent of Akt1 and TAK1. This is because knocking down Akt1 or TAK1 by siRNA did not reduce IL-17-induced activation of NF-κB and expression of VCAM-1, whereas knocking down NF-κB by siRNA markedly inhibited IL-17-mediated upregulation of VCAM-1 expression. In addition, IL-17-induced expression of VCAM-1 is partially dependent on activation of ERK1/2. Therefore, these signaling pathways of IL-17-mediated upregulation of VCAM-1 expression might be therapeutic targets for treatment of IL-17-mediated inflammation.
doi:10.1111/sji.12030
PMCID: PMC3683581  PMID: 23421430
IL-17; NF-κB; vascular smooth muscle cells; adhesion molecule
6.  Dynamin 2 Potentiates Invasive Migration of Pancreatic Tumor Cells through Stabilization of the Rac1 GEF Vav1 
Developmental cell  2013;24(6):573-585.
Summary
The large GTPase Dynamin 2 (Dyn2) is markedly upregulated in pancreatic cancer, is a potent activator of metastatic migration, and is required for Rac1-mediated formation of lamellipodia. Here we demonstrate an unexpected mechanism of Dyn2 action in these contexts, via direct binding to the Rac1 guanine nucleotide exchange factor (GEF) Vav1. Surprisingly, disruption of the Dyn2-Vav1 interaction targets Vav1 to the lysosome for degradation, via an interaction with the cytoplasmic chaperone Hsc70, resulting in a dramatic reduction of Vav1 protein stability. Importantly, a specific mutation in Vav1 near its Dyn2-binding C-terminal SH3 domain prevents Hsc70 binding, resulting in a stabilization of Vav1 levels. Dyn2 binding regulates the interaction of Vav1 with Hsc70 to control the stability and subsequent activity of this oncogenic GEF. These findings elucidate how Dyn2 activates Rac1, lamellipod protrusion, and invasive cellular migration and provide insight into how this specific Vav is ectopically expressed in pancreatic tumors.
doi:10.1016/j.devcel.2013.02.010
PMCID: PMC3905678  PMID: 23537630
7.  Control of Nutrient Stress-Induced Metabolic Reprogramming by PKCζ in Tumorigenesis 
Cell  2013;152(3):599-611.
SUMMARY
Tumor cells have high-energetic and anabolic needs and are known to adapt their metabolism to be able to survive and keep proliferating under conditions of nutrient stress. We show that PKCζ deficiency promotes the plasticity necessary for cancer cells to reprogram their metabolism to utilize glutamine through the serine biosynthetic pathway in the absence of glucose. PKCζ represses the expression of two key enzymes of the pathway, PHGDH and PSAT1, and phosphorylates PHGDH at key residues to inhibit its enzymatic activity. Interestingly, the loss of PKCζ in mice results in enhanced intestinal tumorigenesis and increased levels of these two metabolic enzymes, whereas patients with low levels of PKCζ have a poor prognosis. Furthermore, PKCζ and caspase-3 activities are correlated with PHGDH levels in human intestinal tumors. Taken together, this demonstrates that PKCζ is a critical metabolic tumor suppressor in mouse and human cancer.
doi:10.1016/j.cell.2012.12.028
PMCID: PMC3963830  PMID: 23374352
8.  CypA, a Gene Downstream of HIF-1α, Promotes the Development of PDAC 
PLoS ONE  2014;9(3):e92824.
Hypoxia-inducible factor-1α (HIF-1α) is a highly important transcription factor involved in cell metabolism. HIF-1α promotes glycolysis and inhibits of mitochondrial respiration in pancreatic ductal adenocarcinoma (PDAC). In response to tumor hypoxia, cyclophilin A (CypA) is over-expressed in various cancer types, and is associated with cell apoptosis, tumor invasion, metastasis, and chemoresistance in PDAC. In this study, we showed that both HIF-1α and CypA expression were significantly associated with lymph node metastasis and tumor stage. The expression of CypA was correlated with HIF-1α. Moreover, the mRNA and protein expression of CypA markedly decreased or increased following the suppression or over-expression of HIF-1α in vitro. Chromatin immunoprecipitation analysis showed that HIF-1α could directly bind to the hypoxia response element (HRE) in the CypA promoter regions and regulated CypA expression. Consistent with other studies, HIF-1α and CypA promoted PDAC cell proliferation and invasion, and suppressed apoptosis in vitro. Furthermore, we proved the combination effect of 2-methoxyestradiol and cyclosporin A both in vitro and in vivo. These results suggested that,CypA, a gene downstream of HIF-1α, could promote the development of PDAC. Thus, CypA might serve as a potential therapeutic target for PDAC.
doi:10.1371/journal.pone.0092824
PMCID: PMC3963943  PMID: 24662981
9.  Variation in Genes that Regulate Blood Pressure Are Associated with Glomerular Filtration Rate in Chinese 
PLoS ONE  2014;9(3):e92468.
Chronic kidney disease (CKD) can be a consequence of diabetes, hypertension, immunologic disorders, and other exposures, as well as genetic factors that are still largely unknown. Glomerular filtration rate (GFR), which is widely used to measure kidney function, has a heritability ranging from 25% to 75%, but only 1.5% of this heritability is explained by genetic loci that have been identified to date. In this study we tested for associations between GFR and 234 SNPs in 26 genes from pathways of blood pressure regulation in 3,025 rural Chinese participants of the “Genetic Epidemiology Network of Salt Sensitivity” (GenSalt) study. We estimated GFR (eGFR) using baseline serum creatinine measurements obtained prior to dietary intervention. We identified significant associations between eGFR and 12 SNPs in 6 genes (ACE, ADD1, AGT, GRK4, HSD11B1, and SCNN1G). The cumulative effect of the protective alleles was an increase in mean eGFR of 4 mL/min per 1.73 m2, while the cumulative effect of the risk alleles was a decrease in mean eGFR of 3 mL/min per 1.73 m2. In addition, we identified a significant interaction between SNPs in CYP11B1 and ADRB2. We have identified common variants in genes from pathways that regulate blood pressure and influence kidney function as measured by eGFR, providing new insights into the genetic determinants of kidney function. Complex genetic effects on kidney function likely involve interactions among genes as we observed for CYP11B1 and ADRB2.
doi:10.1371/journal.pone.0092468
PMCID: PMC3962404  PMID: 24658007
10.  Clinical and Prognostic Significance of HIF-1α, PTEN, CD44v6, and Survivin for Gastric Cancer: A Meta-Analysis 
PLoS ONE  2014;9(3):e91842.
Purpose
This study was to quantitatively summarize published data for evaluating the clinical and prognostic significance of four proteins involved in hypoxia-inducible factor-1 (HIF-1α) regulation of the metastasis cascade.
Methods
Searches were performed using the MEDLINE, EMBASE, Cochrane Library, and Chinese Biomedicine databases without any language restrictions. Studies were pooled and either the summary risk ratio (RR) or odds ratio (OR) was calculated. Potential sources of heterogeneity were sought out via subgroup and sensitivity analyses, and publication bias was also performed.
Results
Seventeen studies evaluated HIF-1α, 20 studies evaluated phosphatase and tensin homolog (PTEN), 20 studies evaluated Survivin, and 16 studies evaluated CD44v6. Our results showed that increased HIF-1α expression was linked to a poor 5-year overall survival (RR = 1.508; 95% confidence interval (CI) 1.318–1.725; P<0.001). Decreased survival was heavily influenced by advanced tumor invasion (OR = 3.050; 95% CI 2.067–4.501; P<0.001), lymph node metastasis (1415 patients; OR = 3.486, 95% CI 2.737–4.440; P<0.001), distant metastasis (OR = 6.635; 95% CI 1.855–23.738; P = 0.004), vascular invasion (OR = 2.368; 95% CI 1.725–3.252; P<0.001), dedifferentiation (OR = 2.112; 95% CI 1.410–3.163; P<0.001), tumor size (OR = 1.921; 95% CI 1.395–2.647; P<0.001), and a higher TNM stage (OR =  2.762; 95% CI 1.941–3.942; P<0.001). Similarly, aberrant expression of PTEN, CD44v6, and Survivin were also observed in tumors that correlated with poor OS. The higher ORs of death at 5 years were 1.637 (95% CI = 1.452–1.845; P<0.001), 1.901 (95% CI = 1.432–2.525; P<0.001), and 1.627 (95% CI = 1.384–1.913; P<0.001), respectively, with an OR>2 for the main stratified meta-analyses of clinical factors.
Conclusions
Our findings indicate that HIF-1α/PTEN/CD44v6/Survivin, as measured by immunohistochemistry, can be used to predict the prognosis and potential for invasion and metastasis in Asian patients with gastric cancer. The development of strategies against this subset of proteins could lead to new therapeutic approaches.
doi:10.1371/journal.pone.0091842
PMCID: PMC3960154  PMID: 24647137
11.  Periodic Heat Shock Accelerated the Chondrogenic Differentiation of Human Mesenchymal Stem Cells in Pellet Culture 
PLoS ONE  2014;9(3):e91561.
Osteoarthritis (OA) is one of diseases that seriously affect elderly people's quality of life. Human mesenchymal stem cells (hMSCs) offer a potential promise for the joint repair in OA patients. However, chondrogenic differentiation from hMSCs in vitro takes a long time (∼6 weeks) and differentiated cells are still not as functionally mature as primary isolated chondrocytes, though chemical stimulations and mechanical loading have been intensively studied to enhance the hMSC differentiation. On the other hand, thermal stimulations of hMSC chondrogenesis have not been well explored. In this study, the direct effects of mild heat shock (HS) on the differentiation of hMSCs into chondrocytes in 3D pellet culture were investigated. Periodic HS at 41°C for 1 hr significantly increased sulfated glycosaminoglycan in 3D pellet culture at Day 10 of chondrogenesis. Immunohistochemical and Western Blot analyses revealed an increased expression of collagen type II and aggrecan in heat-shocked pellets than non heat-shocked pellets on Day 17 of chondrogenesis. In addition, HS also upregulated the expression of collagen type I and X as well as heat shock protein 70 on Day 17 and 24 of differentiation. These results demonstrate that HS accelerated the chondrogenic differentiation of hMSCs and induced an early maturation of chondrocytes differentiated from hMSCs. The results of this study will guide the design of future protocols using thermal treatments to facilitate cartilage regeneration with human mesenchymal stem cells.
doi:10.1371/journal.pone.0091561
PMCID: PMC3954764  PMID: 24632670
12.  Automated Detection and Measurement of Isolated Retinal Arterioles by a Combination of Edge Enhancement and Cost Analysis 
PLoS ONE  2014;9(3):e91791.
Pressure myography studies have played a crucial role in our understanding of vascular physiology and pathophysiology. Such studies depend upon the reliable measurement of changes in the diameter of isolated vessel segments over time. Although several software packages are available to carry out such measurements on small arteries and veins, no such software exists to study smaller vessels (<50 µm in diameter). We provide here a new, freely available open-source algorithm, MyoTracker, to measure and track changes in the diameter of small isolated retinal arterioles. The program has been developed as an ImageJ plug-in and uses a combination of cost analysis and edge enhancement to detect the vessel walls. In tests performed on a dataset of 102 images, automatic measurements were found to be comparable to those of manual ones. The program was also able to track both fast and slow constrictions and dilations during intraluminal pressure changes and following application of several drugs. Variability in automated measurements during analysis of videos and processing times were also investigated and are reported. MyoTracker is a new software to assist during pressure myography experiments on small isolated retinal arterioles. It provides fast and accurate measurements with low levels of noise and works with both individual images and videos. Although the program was developed to work with small arterioles, it is also capable of tracking the walls of other types of microvessels, including venules and capillaries. It also works well with larger arteries, and therefore may provide an alternative to other packages developed for larger vessels when its features are considered advantageous.
doi:10.1371/journal.pone.0091791
PMCID: PMC3953588  PMID: 24626349
13.  ZiBuPiYin Recipe Protects db/db Mice from Diabetes-Associated Cognitive Decline through Improving Multiple Pathological Changes 
PLoS ONE  2014;9(3):e91680.
Multiple organ systems, including the brain, which undergoes changes that may increase the risk of cognitive decline, are adversely affected by diabetes mellitus (DM). Here, we demonstrate that type 2 diabetes mellitus (T2DM) db/db mice exhibited hippocampus-dependent memory impairment, which might associate with a reduction in dendritic spine density in the pyramidal neurons of brain, Aβ1-42 deposition in the prefrontal cortex (PFC) and hippocampus, and a decreased expression of neurostructural proteins including microtubule-associated protein (MAP2), a marker of dendrites, and postsynaptic density 95 (PSD95), a marker of excitatory synapses. To investigate the effects of the ZiBuPiYin recipe (ZBPYR), a traditional Chinese medicine recipe, on diabetes-related cognitive decline (DACD), db/db mice received daily administration of ZBPYR over an experimental period of 6 weeks. We then confirmed that ZBPYR rescued learning and memory performance impairments, reversed dendritic spine loss, reduced Aβ1-42 deposition and restored the expression levels of MAP2 and PSD95. The present study also revealed that ZBPYR strengthened brain leptin and insulin signaling and inhibited GSK3β overactivity, which may be the potential mechanism or underlying targets of ZBPYR. These findings conclude that ZBPYR prevents DACD, most likely by improving dendritic spine density and attenuating brain leptin and insulin signaling pathway injury. Our findings provide further evidence for the effects of ZBPYR on DACD.
doi:10.1371/journal.pone.0091680
PMCID: PMC3948870  PMID: 24614172
14.  Test of Einstein-Podolsky-Rosen Steering Based on the All-Versus-Nothing Proof 
Scientific Reports  2014;4:4291.
In comparison with entanglement and Bell nonlocality, Einstein-Podolsky-Rosen steering is a newly emerged research topic and in its incipient stage. Although Einstein-Podolsky-Rosen steering has been explored via violations of steering inequalities both theoretically and experimentally, the known inequalities in the literatures are far from well-developed. As a result, it is not yet possible to observe Einstein-Podolsky-Rosen steering for some steerable mixed states. Recently, a simple approach was presented to identify Einstein-Podolsky-Rosen steering based on all-versus-nothing argument, offering a strong condition to witness the steerability of a family of two-qubit (pure or mixed) entangled states. In this work, we show that the all-versus-nothing proof of Einstein-Podolsky-Rosen steering can be tested by measuring the projective probabilities. Through the bound of probabilities imposed by local-hidden-state model, the proposed test shows that steering can be detected by the all-versus-nothing argument experimentally even in the presence of imprecision and errors. Our test can be implemented in many physical systems and we discuss the possible realizations of our scheme with non-Abelian anyons and trapped ions.
doi:10.1038/srep04291
PMCID: PMC3944728  PMID: 24598858
15.  Expression, purification and crystallization of an indole prenyltransferase from Aspergillus fumigatus  
Residues 38–440 of CdpNPT from A. fumigatus were overexpressed in E. coli, purified and crystallized. Diffraction data have been collected to a resolution of 2.5 Å.
CdpNPT from Aspergillus fumigatus is a dimethylallyltryptophan synthase/indole prenyltransferase that catalyzes reverse prenylation at position N1 of tryptophan-containing cyclic dipeptides. Residues 38–440 of CdpNPT were expressed in Escherichia coli and crystallized using the sitting-drop vapour-diffusion and microseeding techniques. The crystals belonged to space group P212121, with unit-cell parameters a = 84.4, b = 157.1, c = 161.8 Å, α = β = γ = 90.0°.
doi:10.1107/S1744309112004964
PMCID: PMC3310551  PMID: 22442243
prenyltransferases; alkaloids; tryptophan
16.  Identification of Salmonella enterica Serovar Pullorum Antigenic Determinants Expressed In Vivo 
Infection and Immunity  2013;81(9):3119-3127.
Salmonella enterica serovar Pullorum affecting poultry causes pullorum disease and results in severe economic loss in the poultry industry. Currently, it remains a major threat in countries with poor poultry surveillance and no efficient control measures. As S. Pullorum could induce strong humoral immune responses, we applied an immunoscreening technique, the in vivo-induced antigen technology (IVIAT), to identify immunogenic bacterial proteins expressed or upregulated during S. Pullorum infection. Convalescent-phase sera from chickens infected with S. Pullorum were pooled, adsorbed against antigens expressed in vitro, and used to screen an S. Pullorum genomic expression library. Forty-five proteins were screened out, and their functions were implicated in molecular biosynthesis and degradation, transport, metabolism, regulation, cell wall synthesis and antibiotic resistance, environmental adaptation, or putative functions. In addition, 11 of these 45 genes were assessed for their differential expression by quantitative real-time reverse transcription-PCR (RT-PCR), revealing that 9 of 11 genes were upregulated to different degrees under in vivo conditions, especially the regulator of virulence determinants, phoQ. Then, four in vivo-induced proteins (ShdA, PhoQ, Cse3, and PbpC) were tested for their immunoreactivity in 28 clinical serum samples from chickens infected with S. Pullorum. The rate of detection of antibodies against ShdA reached 82% and was the highest among these proteins. ShdA is a host colonization factor known to be upregulated in vivo and related to the persistence of S. Typhimurium in the intestine. Furthermore, these antigens identified by IVIAT warrant further evaluation for their contributions to pathogenesis, and more potential roles, such as diagnostic, therapeutic, and preventive uses, need to be developed in future studies.
doi:10.1128/IAI.00145-13
PMCID: PMC3754199  PMID: 23774596
17.  Establishment of Real Time Allele Specific Locked Nucleic Acid Quantitative PCR for Detection of HBV YIDD (ATT) Mutation and Evaluation of Its Application 
PLoS ONE  2014;9(2):e90029.
Background
Long-term use of nucleos(t)ide analogues can increase risk of HBV drug-resistance mutations. The rtM204I (ATT coding for isoleucine) is one of the most important resistance mutation sites. Establishing a simple, rapid, reliable and highly sensitive assay to detect the resistant mutants as early as possible is of great clinical significance.
Methods
Recombinant plasmids for HBV YMDD (tyrosine-methionine-aspartate-aspartate) and YIDD (tyrosine-isoleucine-aspartate-aspartate) were constructed by TA cloning. Real time allele specific locked nucleic acid quantitative PCR (RT-AS-LNA-qPCR) with SYBR Green I was established by LNA-modified primers and evaluated with standard recombinant plasmids, clinical templates (the clinical wild type and mutant HBV DNA mixture) and 102 serum samples from nucleos(t)ide analogues-experienced patients. The serum samples from a chronic hepatitis B (CHB) patient firstly received LMV mono therapy and then switched to LMV + ADV combined therapy were also dynamically analyzed for 10 times.
Results
The linear range of the assay was between 1×109 copies/μl and 1×102 copies/μl. The low detection limit was 1×101 copies/μl. Sensitivity of the assay were 10−6, 10−4 and 10−2 in the wild-type background of 1×109 copies/μl, 1×107 copies/μl and 1×105 copies/μl, respectively. The sensitivity of the assay in detection of clinical samples was 0.03%. The complete coincidence rate between RT-AS-LNA-qPCR and direct sequencing was 91.2% (93/102), partial coincidence rate was 8.8% (9/102), and no complete discordance was observed. The two assays showed a high concordance (Kappa = 0.676, P = 0.000). Minor variants can be detected 18 weeks earlier than the rebound of HBV DNA load and alanine aminotransferase level.
Conclusions
A rapid, cost-effective, high sensitive, specific and reliable method of RT-AS-LNA-qPCR with SYBR Green I for early and absolute quantification of HBV YIDD (ATT coding for isoleucine) variants was established, which can provide valuable information for clinical antiretroviral regimens.
doi:10.1371/journal.pone.0090029
PMCID: PMC3938556  PMID: 24587198
18.  Identification of Key Genes in the Response to Salmonella enterica Enteritidis, Salmonella enterica Pullorum, and Poly(I:C) in Chicken Spleen and Caecum 
BioMed Research International  2014;2014:154946.
Salmonella enterica Enteritidis (S. Enteritidis) and Salmonella enterica Pullorum (S. pullorum) are regarded as a threat to poultry production. This study's aim is to characterize the expression profiles in response to three different challenges and to identify infection-related genes in the chicken spleen and caecum. Groups of the Chinese chicken breed Langshan were challenged with either S. Enteritidis, S. pullorum, or poly(I:C). The concentrations of cytokines and antibodies and the Salmonella colonization level of the caecum and liver were detected in each group at 7 days postinfection. Expression microarray experiments were conducted using mRNA isolated from both spleen and caecum. Crucial differentially expressed genes (DEGs) associated with immunity were identified. Four DEGs were identified in spleen of all three challenge groups (RBM16, FAH, SOX5, and RBM9) and different four genes in caecum (SOUL, FCN2, ANLN, and ACSL1). Expression profiles were clearly different among the three challenged groups. Genes enriched in the spleen of birds infected with S. pullorum were enriched in lymphocyte proliferation related pathways, but the enriched genes in the caecum of the same group were primarily enriched in innate immunity or antibacterial responses. The DEGs that appear across all three challenge groups might represent global response factors for different pathogens.
doi:10.1155/2014/154946
PMCID: PMC3950952
19.  Human papillomavirus prevalence and type-distribution in women with cervical lesions: a cross-sectional study in Sri Lanka 
BMC Cancer  2014;14:116.
Background
Cervical cancer ranks second among all cancers reported in Sri Lankan women. This study assessed the prevalence and type-distribution of human papillomavirus (HPV) among Sri Lankan women with invasive cervical cancer (ICC) and pre-cancerous lesions.
Methods
114 women aged 21 years and above, hospitalized in the National Cancer Institute, Sri Lanka with a diagnosis of ICC or cervical intraepithelial neoplasia (CIN) 2/3 were prospectively enrolled between October 2009 and September 2010 (110430/NCT01221987). The cervical biopsy or excision specimens collected during routine clinical procedures were subjected to histopathological review. DNA was extracted from samples with a confirmed histological diagnosis and was amplified using polymerase chain reaction and HPV DNA was detected using Enzyme Immuno Assay. HPV positive samples were typed using reverse hybridization Line Probe Assay.
Results
Of the cervical samples collected, 93.0% (106/114) had a histologically confirmed diagnosis of either ICC (98/106) or CIN 2/3 (8/106). Among all ICC cases, squamous cell carcinoma was diagnosed in the majority of women (81.6% [80/98]). HPV prevalence among ICC cases was 84.7% (83/98). The HPV types most commonly detected in ICC cases with single HPV infection (98.8% [82/83]) were HPV-16 (67.3%) and HPV-18 (9.2%). Infection with multiple HPV types was recorded in a single case (co-infection of HPV-16 and HPV-59).
Conclusions
HPV was prevalent in most women with ICC in Sri Lanka; HPV-16 and HPV-18 were the predominantly detected HPV types. An effective prophylactic vaccine against the most prevalent HPV types may help to reduce the burden of ICC disease.
doi:10.1186/1471-2407-14-116
PMCID: PMC3936905  PMID: 24558979
Cervical cancer; Human papillomavirus; Prevalence; Sri Lanka; Type-distribution
20.  Superparamagnetic iron oxide nanoparticles mediated 131I-hVEGF siRNA inhibits hepatocellular carcinoma tumor growth in nude mice 
BMC Cancer  2014;14:114.
Background
Hepatocellular carcinoma (HCC) is a primary liver tumor and is the most difficult human malignancy to treat. In this study, we sought to develop an integrative approach in which real-time tumor monitoring, gene therapy, and internal radiotherapy can be performed simultaneously. This was achieved through targeting HCC with superparamagnetic iron oxide nanoparticles (SPIOs) carrying small interfering RNA with radiolabled iodine 131 (131I) against the human vascular endothelial growth factor (hVEGF).
Methods
hVEGF siRNA was labeled with 131I by the Bolton-Hunter method and conjugated to SilenceMag, a type of SPIOs. 131I-hVEGF siRNA/SilenceMag was then subcutaneously injected into nude mice with HCC tumors exposed to an external magnetic field (EMF). The biodistribution and cytotoxicity of 131I-hVEGF siRNA/SilenceMag was assessed by SPECT (Single-Photon Emission Computed Tomography) and MRI (Magnetic Resonance Imaging) studies and blood kinetics analysis. The body weight and tumor size of nude mice bearing HCC were measured daily for the 4-week duration of the experiment.
Results
131I-hVEGF siRNA/SilenceMag was successfully labeled; with a satisfactory radiochemical purity (>80%) and biological activity in vitro. External application of an EMF successfully attracted and retained more 131I-hVEGF siRNA/SilenceMag in HCC tumors as shown by SPECT, MRI and biodistribution studies. The tumors treated with 131I-hVEGF siRNA/SilenceMag grew nearly 50% slower in the presence of EMF than those without EMF and the control. Immunohistochemical assay confirmed that the tumor targeted by 131I-hVEGF siRNA/SilenceMag guided by an EMF had a lower VEGF protein level compared to that without EMF exposure and the control.
Conclusions
EMF-guided 131I-hVEGF siRNA/SilenceMag exhibited an antitumor effect. The synergic therapy of 131I-hVEGF siRNA/SilenceMag might be a promising future treatment option against HCC with the dual functional properties of tumor therapy and imaging.
doi:10.1186/1471-2407-14-114
PMCID: PMC3938316  PMID: 24555445
VEGF; Small interfering RNA; 131I; Hepatocellular carcinoma; SPIOs
21.  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
22.  All Solution-processed Stable White Quantum Dot Light-emitting Diodes with Hybrid ZnO@TiO2 as Blue Emitters 
Scientific Reports  2014;4:4085.
White quantum dot light-emitting diodes (QD-LEDs) have been a promising candidate for high-efficiency and color-saturated displays. However, it is challenging to integrate various QD emitters into one device and also to obtain efficient blue QDs. Here, we report a simply solution-processed white QD-LED using a hybrid ZnO@TiO2 as electron injection layer and ZnCdSeS QD emitters. The white emission is obtained by integrating the yellow emission from QD emitters and the blue emission generated from hybrid ZnO@TiO2 layer. We show that the performance of white QD-LEDs can be adjusted by controlling the driving force for hole transport and electroluminescence recombination region via varying the thickness of hole transport layer. The device is demonstrated with a maximum luminance of 730 cd/m2 and power efficiency of 1.7 lm/W, exhibiting the Commission Internationale de l'Enclairage (CIE) coordinates of (0.33, 0.33). The unencapsulated white QD-LED has a long lifetime of 96 h at its initial luminance of 730 cd/m2, primarily due to the fact that the device with hybrid ZnO@TiO2 has low leakage current and is insensitive to the oxygen and the moisture. These results indicate that hybrid ZnO@TiO2 provides an alternate and effective approach to achieve high-performance white QD-LEDs and also other optoelectronic devices.
doi:10.1038/srep04085
PMCID: PMC3923213  PMID: 24522341
23.  Light Manipulation for Organic Optoelectronics Using Bio-inspired Moth's Eye Nanostructures 
Scientific Reports  2014;4:4040.
Organic-based optoelectronic devices, including light-emitting diodes (OLEDs) and solar cells (OSCs) hold great promise as low-cost and large-area electro-optical devices and renewable energy sources. However, further improvement in efficiency remains a daunting challenge due to limited light extraction or absorption in conventional device architectures. Here we report a universal method of optical manipulation of light by integrating a dual-side bio-inspired moth's eye nanostructure with broadband anti-reflective and quasi-omnidirectional properties. Light out-coupling efficiency of OLEDs with stacked triple emission units is over 2 times that of a conventional device, resulting in drastic increase in external quantum efficiency and current efficiency to 119.7% and 366 cd A−1 without introducing spectral distortion and directionality. Similarly, the light in-coupling efficiency of OSCs is increased 20%, yielding an enhanced power conversion efficiency of 9.33%. We anticipate this method would offer a convenient and scalable way for inexpensive and high-efficiency organic optoelectronic designs.
doi:10.1038/srep04040
PMCID: PMC3918972  PMID: 24509524
24.  Vitamin D receptor expression controls proliferation of naïve CD8+ T cells and development of CD8 mediated gastrointestinal inflammation 
BMC Immunology  2014;15:6.
Background
Vitamin D receptor (VDR) deficiency contributes to the development of experimental inflammatory bowel disease (IBD) in several different models. T cells have been shown to express the VDR, and T cells are targets of vitamin D. In this article we determined the effects of VDR expression on CD8+ T cells.
Results
VDR KO CD8+ T cells, but not WT CD8+ T cells, induced colitis in Rag KO recipients. In addition, co-transfer of VDR KO CD8+ T cells with naïve CD4+ T cells accelerated colitis development. The more severe colitis was associated with rapidly proliferating naïve VDR KO CD8+ T cells and increased IFN-γ and IL-17 in the gut. VDR KO CD8+ T cells proliferated in vitro without antigen stimulation and did not downregulate CD62L and upregulate CD44 markers following proliferation that normally occurred in WT CD8+ T cells. The increased proliferation of VDR KO CD8+ cells was due in part to the higher production and response of the VDR KO cells to IL-2.
Conclusions
Our data indicate that expression of the VDR is required to prevent replication of quiescent CD8+ T cells. The inability to signal through the VDR resulted in the generation of pathogenic CD8+ T cells from rapidly proliferating cells that contributed to the development of IBD.
doi:10.1186/1471-2172-15-6
PMCID: PMC3923390  PMID: 24502291
Vitamin D receptor; CD8+ T cells; Proliferation; Inflammatory bowel disease
25.  Expression of Signaling Components in Embryonic Eyelid Epithelium 
PLoS ONE  2014;9(2):e87038.
Closure of an epithelium opening is a critical morphogenetic event for development. An excellent example for this process is the transient closure of embryonic eyelid. Eyelid closure requires shape change and migration of epithelial cells at the tip of the developing eyelids, and is dictated by numerous signaling pathways. Here we evaluated gene expression in epithelial cells isolated from the tip (leading edge, LE) and inner surface epithelium (IE) of the eyelid from E15.5 mouse fetuses by laser capture microdissection (LCM). We showed that the LE and IE cells are different at E15.5, such that IE had higher expression of muscle specific genes, while LE acquired epithelium identities. Despite their distinct destinies, these cells were overall similar in expression of signaling components for the “eyelid closure pathways”. However, while the LE cells had more abundant expression of Fgfr2, Erbb2, Shh, Ptch1 and 2, Smo and Gli2, and Jag1 and Notch1, the IE cells had more abundant expression of Bmp5 and Bmpr1a. In addition, the LE cells had more abundant expression of adenomatosis polyposis coli down-regulated 1 (Apcdd1), but the IE cells had high expression of Dkk2. Our results suggest that the functionally distinct LE and IE cells have also differential expression of signaling molecules that may contribute to the cell-specific responses to morphogenetic signals. The expression pattern suggests that the EGF, Shh and NOTCH pathways are preferentially active in LE cells, the BMP pathways are effective in IE cells, and the Wnt pathway may be repressed in LE and IE cells via different mechanisms.
doi:10.1371/journal.pone.0087038
PMCID: PMC3911929  PMID: 24498290

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