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1.  Draft Genome Sequence of Vibrio owensii GRA50-12, Isolated from Green Algae in the Intertidal Zone of Eastern Taiwan 
Genome Announcements  2015;3(1):e01438-14.
Vibrio owensii GRA50-12 was isolated from symbiotic green algae of coral. The genome contains genes encoding toxin production, virulence regulation, stress response proteins, types II, IV, and VI secretion systems, and proteins for the metabolism of aromatic compounds, which reflects its pathogenic potential and its ecological roles in the ocean.
PMCID: PMC4299907  PMID: 25593265
2.  Systematic approach to Escherichia coli cell population control using a genetic lysis circuit 
BMC Systems Biology  2014;8(Suppl 5):S7.
Cell population control allows for the maintenance of a specific cell population density. In this study, we use lysis gene BBa_K117000 from the Registry of Standard Biological Parts, formed by MIT, to lyse Escherichia coli (E. coli). The lysis gene is regulated by a synthetic genetic lysis circuit, using an inducer-regulated promoter-RBS component. To make the design more easily, it is necessary to provide a systematic approach for a genetic lysis circuit to achieve control of cell population density.
Firstly, the lytic ability of the constructed genetic lysis circuit is described by the relationship between the promoter-RBS components and inducer concentration in a steady state model. Then, three types of promoter-RBS libraries are established. Finally, according to design specifications, a systematic design approach is proposed to provide synthetic biologists with a prescribed I/O response by selecting proper promoter-RBS component set in combination with suitable inducer concentrations, within a feasible range.
This study provides an important systematic design method for the development of next-generation synthetic gene circuits, from component library construction to genetic circuit assembly. In future, when libraries are more complete, more precise cell density control can be achieved.
PMCID: PMC4305986  PMID: 25559865
Genetic lysis circuit; Cell population control; Promoter-RBS library
3.  LRP-1: Functions, Signaling and Implications in Kidney and Other Diseases 
Low-density lipoprotein (LDL)-related protein-1 (LRP-1) is a member of LDL receptor family that is implicated in lipoprotein metabolism and in the homeostasis of proteases and protease inhibitors. Expression of LRP-1 is ubiquitous. Up-regulation of LRP-1 has been reported in numerous human diseases. In addition to its function as a scavenger receptor for various ligands, LRP-1 has been shown to transduce multiple intracellular signal pathways including mitogen-activated protein kinase (MAPK), Akt, Rho, and the integrin signaling. LRP-1 signaling plays an important role in the regulation of diverse cellular process, such as cell proliferation, survival, motility, differentiation, and transdifferentiation, and thus participates in the pathogenesis of organ dysfunction and injury. In this review, we focus on the current understanding of LRP-1 signaling and its roles in the development and progression of kidney disease. The role and signaling of LRP-1 in the nervous and cardiovascular systems, as well as in carcinogenesis, are also briefly discussed.
PMCID: PMC4284744  PMID: 25514242
LRP-1; signaling; tPA; integrin; tyrosine phosphorylation; pathophysiology
4.  Quality of Life of Taiwanese Adults with Autism Spectrum Disorder 
PLoS ONE  2014;9(10):e109567.
To date, few recent studies have investigated the quality of life of adults with autism spectrum disorder (ASD). It remains unclear how individuals with ASD view their own quality of life.
The primary purpose of this study was to compare the quality of life scores among adults with ASD with those of a non-ASD control group and the Taiwanese health population reference group.
The study comprised 41 adults with ASD (M age = 26.9, SD = 5.0), and without intellectual disabilities (IQ>70). A comparison sample of 41 adults without ASD was selected by matching the age and sex of the participants with ASD. A validated measure, the Taiwanese version of the World Health Organization Quality of Life-BREF (WHOQOL-BREF), was used. Independent t-tests were performed to examine the differences in the quality of life between groups.
The highest quality of life was scored in the environment domain, followed by the physical health and psychological health domains. The lowest quality of life score was found in the social relationship domain. Adults with ASD scored significantly lower in all domains than did the non-ASD control group. Additionally, adults with ASD scored significantly lower in the physical health, psychological health, and social relationship domains than did the Taiwanese health population reference group. Comorbid psychiatric disorders, self-rated health status, and perceived happiness were correlated with quality of life among adults with ASD.
The preliminary findings suggest that adults with ASD need more supportive social contexts and interventions to promote their quality of life. Based on our findings, social relationship must be considered in designing and applying treatment programs for adults with ASD.
PMCID: PMC4192352  PMID: 25299379
5.  Bio-Inspired Voltage-Dependent Calcium Channel Blockers 
Nature communications  2013;4:2540.
Ca2+ influx via voltage-dependent CaV1/CaV2 channels couples electrical signals to biological responses in excitable cells. CaV1/CaV2 channel blockers have broad biotechnological and therapeutic applications. Here we report a general method for developing novel genetically-encoded calcium channel blockers inspired by Rem, a small G-protein that constitutively inhibits CaV1/CaV2 channels. We show that diverse cytosolic proteins (CaVβ, 14-3-3, calmodulin, and CaMKII) that bind pore-forming α1-subunits can be converted into calcium channel blockers with tunable selectivity, kinetics, and potency, simply by anchoring them to the plasma membrane. We term this method “channel inactivation induced by membrane-tethering of an associated protein” (ChIMP). ChIMP is potentially extendable to small-molecule drug discovery, as engineering FK506-binding protein into intracellular sites within CaV1.2-α1C permits heterodimerization-initiated channel inhibition with rapamycin. The results reveal a universal method for developing novel calcium channel blockers that may be extended to develop probes for a broad cohort of unrelated ion channels.
PMCID: PMC4190111  PMID: 24096474
6.  Apelin-13 Decreases Lipid Storage in Hypertrophic Adipocytes In Vitro Through the Upregulation of AQP7 Expression by the PI3K Signaling Pathway 
Adipocyte-secreted apelin contributes to decreased adiposity and to improved insulin resistance, but the mechanisms remain unknown. The present study aimed to assess if apelin-13 is an upstream signal regulation factor of aquaporin 7 (AQP7), a water-glycerol transporter present in the plasma membrane of adipocytes that plays a key role in the regulation of lipid accumulation.
3T3-L1 pre-adipocytes were induced to fully differentiated adipocytes; hypertrophic adipocytes were then induced using palmitate. The effects of apelin-13 on AQP7 expression in hypertrophic adipocytes were investigated before and after treatment with LY249002, a PI3K inhibitor. Accumulation of cytoplasmic triglycerides (TG) in hypertrophic adipocytes was also determined.
We found that 0.1 mM of palmitate induced a model of hypertrophic adipocytes with a lower AQP7 expression (0.26±0.07 vs. 0.46±0.04, P<0.05). Apelin-13 100 nM or 1000 nM upregulated AQP7 mRNA expression (100 nM: 0.54±0.06 and 1000 nM: 0.58±0.09 vs. control: 0.33±0.04, both P<0.05), and decreased accumulation of cytoplasmic triglycerides in hypertrophic adipocytes. Pretreatment using 10 μM LY294002 prevented the increase in AQP7 expression observed when using apelin-13 alone (apelin-13 + LY49002: 0.38±0.03 vs. apelin-13: 0.54±0.06, P<0.05), as well as the decreased cytoplasmic TG accumulation (apelin-13 + LY294002: 3.79±0.04 μM per μg/ml vs. apelin-13: 3.32±0.08 μM per μg/ml, P<0.05).
Apelin-13 decreases lipid storage in hypertrophic adipocytes in vitro, possibly through the upregulation of AQP7 expression by the PI3K signaling pathway. Treatment using apelin-13 and AQP modulators might represent novel treatment strategies against obesity and its related complications.
PMCID: PMC4136933  PMID: 25080850
Adipocytes; Apelin-13; Phosphatidylinositol 3-Kinases; 3T3-L1 Cells; Aquaporins
7.  Effects of Pristane Alone or Combined with Chloroquine on Macrophage Activation, Oxidative Stress, and Th1/Th2 Skewness 
Journal of Immunology Research  2014;2014:613136.
We investigated the protective role of chloroquine against pristane-induced macrophage activation, oxidative stress, and Th1/Th2 skewness in C57BL/6J mice. Those mice were treated with pristane alone or combined with chloroquine. Hematological and biochemical parameters, macrophage phagocytic function, the oxidant/antioxidant index, cytokine for IFN-γ, TNF-α, IL-4, and IL-6, and the isotypes of IgG2a and IgG1 were determined. And the expression of T-bet/GATA-3 and IL-12/IL-10 mRNA in spleen were analyzed by real-time PCR. We found that pristane treatment for a period of 12 or 24 weeks triggered macrophage activation syndrome, characterized by hemophagocytosis in spleen and peripheral blood, enhanced lipid phagocytosis by peritoneal macrophages in vitro, erythropenia and leucopenia, increased anti-Smith, lactic dehydrogenase, triglyceride, and ferritin, as well as hypercytokinemia of IFN-γ, TNF-α, IL-4, and IL-6. In parallel, a significant increase in lipid peroxidation and a decrease in superoxide dismutase, glutathione, and catalase activity, as well as a skewed Th1/Th2 balance in spleen, were observed. However, chloroquine supplementation showed a remarkable amelioration of these abnormalities. Our data indicate that pristane administration induces macrophage activation, oxidative stress, and Th1/Th2 skewness, which can be attenuated by chloroquine.
PMCID: PMC4127244  PMID: 25136646
8.  Real-Time Light Scattering Tracking of Gold Nanoparticles- bioconjugated Respiratory Syncytial Virus Infecting HEp-2 Cells 
Scientific Reports  2014;4:4529.
Real-time tracking of virus invasion is crucial for understanding viral infection mechanism, which, however, needs simple and efficient labeling chemistry with improved signal-to-noise ratio. For that purpose, herein we investigated the invasion dynamics of respiratory syncytial virus (RSV) through dark-field microscopic imaging (iDFM) technique by using Au nanoparticles (AuNPs) as light scattering labels. RSV, a ubiquitous, non-segmented, pleiomorphic and negative-sense RNA virus, is an important human pathogen in infants, the elderly, and the immunocompromised. In order to label the enveloped virus of paramyxoviridae family, an efficient streptavidin (SA)-biotin binding chemistry was employed, wherein AuNPs and RSV particles modified with SA and biotin, respectively, allowing the AuNP-modified RSVs to maintain their virulence without affecting the native activities of RSV, making the long dynamic visualization successful for the RSV infections into human epidermis larynx carcinoma cells.
PMCID: PMC3970125  PMID: 24681709
9.  Tissue plasminogen activator and inflammation: from phenotype to signaling mechanisms 
In disease conditions, inflammatory cells, such as neutrophils, T cells, and monocytes/macrophages, are recruited in response to injury cues and express panoply of proinflammatory genes through a combination of transcription factors. Tissue plasminogen activator (tPA), a member of the serine protease family, has been shown to act as cytokine to activate profound receptor-mediated signaling events. In this review, we will discuss the role of tPA in inflammation in various models, and illuminate its signaling mechanisms underlying its modulation of inflammation.
PMCID: PMC3960759  PMID: 24660119
Tissue plasminogen activator; tPA; inflammation; phenotype; signaling mechanisms
10.  Re-sequencing Expands Our Understanding of the Phenotypic Impact of Variants at GWAS Loci 
PLoS Genetics  2014;10(1):e1004147.
Genome-wide association studies (GWAS) have identified >500 common variants associated with quantitative metabolic traits, but in aggregate such variants explain at most 20–30% of the heritable component of population variation in these traits. To further investigate the impact of genotypic variation on metabolic traits, we conducted re-sequencing studies in >6,000 members of a Finnish population cohort (The Northern Finland Birth Cohort of 1966 [NFBC]) and a type 2 diabetes case-control sample (The Finland-United States Investigation of NIDDM Genetics [FUSION] study). By sequencing the coding sequence and 5′ and 3′ untranslated regions of 78 genes at 17 GWAS loci associated with one or more of six metabolic traits (serum levels of fasting HDL-C, LDL-C, total cholesterol, triglycerides, plasma glucose, and insulin), and conducting both single-variant and gene-level association tests, we obtained a more complete understanding of phenotype-genotype associations at eight of these loci. At all eight of these loci, the identification of new associations provides significant evidence for multiple genetic signals to one or more phenotypes, and at two loci, in the genes ABCA1 and CETP, we found significant gene-level evidence of association to non-synonymous variants with MAF<1%. Additionally, two potentially deleterious variants that demonstrated significant associations (rs138726309, a missense variant in G6PC2, and rs28933094, a missense variant in LIPC) were considerably more common in these Finnish samples than in European reference populations, supporting our prior hypothesis that deleterious variants could attain high frequencies in this isolated population, likely due to the effects of population bottlenecks. Our results highlight the value of large, well-phenotyped samples for rare-variant association analysis, and the challenge of evaluating the phenotypic impact of such variants.
Author Summary
Abnormal serum levels of various metabolites, including measures relevant to cholesterol, other fats, and sugars, are known to be risk factors for cardiovascular disease and type 2 diabetes. Identification of the genes that play a role in generating such abnormalities could advance the development of new treatment and prevention strategies for these disorders. Investigations of common genetic variants carried out in large sets of research subjects have successfully pinpointed such genes within many regions of the human genome. However, these studies often have not led to the identification of the specific genetic variations affecting metabolic traits. To attempt to detect such causal variations, we sequenced genes in 17 genomic regions implicated in metabolic traits in >6,000 people from Finland. By conducting statistical analyses relating specific variations (individually and grouped by gene) to the measures for these metabolic traits observed in the study subjects, we added to our understanding of how genotypes affect these traits. Our findings support a long-held hypothesis that the unique history of the Finnish population provides important advantages for analyzing the relationship between genetic variations and biomedically important traits.
PMCID: PMC3907339  PMID: 24497850
11.  Increased Risk for Developing Major Adverse Cardiovascular Events in Stented Chinese Patients Treated with Dual Antiplatelet Therapy after Concomitant Use of the Proton Pump Inhibitor 
PLoS ONE  2014;9(1):e84985.
Some clinical studies have demonstrated that the proton pump inhibitor (PPI) could decrease clopidogrel platelet response and increase major adverse cardiovascular events (MACE) in white or black subjects. However, that remains to be determined in Chinese patients. In this study, we sought to determine whether there could be an increased risk for developing MACE after concomitant use of dual antiplatelet therapy (DAT) and a PPI in Chinese patients treated with percutaneous coronary intervention (PCI) and stenting.
This study was a 5-year, single-center, retrospective cohort analysis of eligible patients (n = 6188) who received DAT and a PPI concomitantly (defined as PPI users) before discharge and/or 12-month follow-up after discharge as compared with those who received DAT alone (also defined as non-PPI users, n = 1465). The incidence of recurrent MACE, such as myocardial infarction (MI), definite stent thromboses (ST), or cardiovascular death, was compared between the PPI users and non-users.
PPI users had a significantly higher incidence of the MACE than non-users (13.9% vs. 10.6%; adjusted HR: 1.33; 95% CI: 1.12 – 1.57, P = 0.007). Stratified analysis revealed that concurrent use of DAT and a PPI was associated with a significantly increased risk for developing ST compared with DAT alone (1% vs. 0.4%; adjusted HR: 2.66, 95% CI: 1.16 – 5.87, P = 0.012). However, there were no significant differences in the risk of MI, cardiovascular death and other adverse events, regardless of combination of clopidogrel and a PPI.
The study further suggests that concomitant use of DAT and a PPI may be associated with an increased risk for developing MACE, in particular definite ST, in Chinese PCI patients after discharge as compared with use of DAT alone.
PMCID: PMC3885647  PMID: 24416326
12.  Safety and efficacy of thoracoscopic wedge resection for elderly high-risk patients with stage I peripheral non-small-cell lung cancer 
Elderly patients with severe cardiopulmonary and other system dysfunctions are unable to tolerate pulmonary lobectomy. This study aimed to evaluate the risk and efficacy of wedge resection under video-assisted thoracoscopic surgery (VATS) on elderly high-risk patients with stage I peripheral non-small-cell lung cancer (PNSCLC).
Elderly patients (≥70 years) with suspected PNSCLC were divided into high-risk group and conventional risk group. The high-risk patients confirmed in stage I by the examination of positron emission tomography computed tomography (PET-CT) and the postoperative patients in stage I PNSCLC with negative incisal margin were treated with VATS wedge resection. The conventional risk patients were treated with VATS radical resection and systematic lymphadenectomy. The clinical and pathological data were recorded. The total survival, tumor-free survival, recurrence time and style of patients were followed up.
The operative time and blood loss of the VATS wedge resection group (69.4 ± 15.5 min, 52.1 ± 11.2 ml) were significantly less than those of the VATS radical resection group (128 ± 35.5 min, 217.9 ± 87.1 ml). Neither groups had postoperative death. The overall and tumor-free survival rate of the VATS wedge resection group within three years were 66.7% and 60.0%, and those of the VATS radical resection group were 93.8% and 94.1%, without significant difference (P > 0.05). The recurrence rates of the VATS wedge resection group and VATS radical resection group were 14.3% and 3.0%, without significant difference (P > 0.05).
It is safe, minimally invasive and meaningful to perform VATS wedge resection on the elderly high-risk patients with stage I PNSCLC.
PMCID: PMC3896765  PMID: 24359930
Elderly; Early lung cancer; Thoracoscope
13.  miR-150 Promotes Human Breast Cancer Growth and Malignant Behavior by Targeting the Pro-Apoptotic Purinergic P2X7 Receptor 
PLoS ONE  2013;8(12):e80707.
The P2X7 receptor regulates cell growth through mediation of apoptosis. Low level expression of P2X7 has been linked to cancer development because tumor cells harboring a defective P2X7 mechanism can escape P2X7 pro-apoptotic control. microRNAs (miRNAs) function as negative regulators of post-transcriptional gene expression, playing major roles in cellular differentiation, proliferation, and metastasis. In this study, we found that miR-150 was over-expressed in breast cancer cell lines and tissues. In these breast cancer cell lines, blocking the action of miR-150 with inhibitors leads to cell death, while ectopic expression of the miR-150 results in increased cell proliferation. We deploy a microRNA sponge strategy to inhibit miR-150 in vitro, and the result demonstrates that the 3′-untranslated region (3′UTR) of P2X7 receptor contains a highly conserved miR-150-binding motif and its direct interaction with miR-150 down-regulates endogenous P2X7 protein levels. Furthermore, our findings demonstrate that miR-150 over-expression promotes growth, clonogenicity and reduces apoptosis in breast cancer cells. Meanwhile, these findings can be decapitated in nude mice with breast cancer xenografts. Finally, these observations strengthen our working hypothesis that up-regulation of miR-150 in breast cancer is inversely associated with P2X7 receptor expression level. Together, these findings establish miR-150 as a novel regulator of P2X7 and a potential therapeutic target for breast cancer.
PMCID: PMC3846619  PMID: 24312495
14.  Genome Wide Analysis of Narcolepsy in China Implicates Novel Immune Loci and Reveals Changes in Association Prior to Versus After the 2009 H1N1 Influenza Pandemic 
PLoS Genetics  2013;9(10):e1003880.
Previous studies in narcolepsy, an autoimmune disorder affecting hypocretin (orexin) neurons and recently associated with H1N1 influenza, have demonstrated significant associations with five loci. Using a well-characterized Chinese cohort, we refined known associations in TRA@ and P2RY11-DNMT1 and identified new associations in the TCR beta (TRB@; rs9648789 max P = 3.7×10−9 OR 0.77), ZNF365 (rs10995245 max P = 1.2×10−11 OR 1.23), and IL10RB-IFNAR1 loci (rs2252931 max P = 2.2×10−9 OR 0.75). Variants in the Human Leukocyte Antigen (HLA)- DQ region were associated with age of onset (rs7744020 P = 7.9×10−9 beta −1.9 years) and varied significantly among cases with onset after the 2009 H1N1 influenza pandemic compared to previous years (rs9271117 P = 7.8×10−10 OR 0.57). These reflected an association of DQB1*03:01 with earlier onset and decreased DQB1*06:02 homozygosity following 2009. Our results illustrate how genetic association can change in the presence of new environmental challenges and suggest that the monitoring of genetic architecture over time may help reveal the appearance of novel triggers for autoimmune diseases.
Author Summary
Narcolepsy-hypocretin deficiency results from a highly specific autoimmune attack on hypocretin cells. Recent studies have established antigen presentation by specific class II proteins encoded by (HLA DQB1*06:02 and DQA1*01:02) to the cognate T cell receptor as the main disease pathway, with a role for H1N1 influenza in the triggering process. Here, we have used a large and well-characterized cohort of Chinese narcolepsy cases to examine genetic architecture not observed in European samples. We confirmed previously implicated susceptibility genes (T cell receptor alpha, P2RY11), and identify new loci (ZNF365, IL10RB-IFNAR1), most notably, variants at the beta chain of the T cell receptor. We found that one HLA variant, (DQB1*03:01), is associated with dramatically earlier disease onset (nearly 2 years). We also identified differences in HLA haplotype frequencies among cases with onset following the 2009 H1N1 influenza pandemic as compared to before the outbreak, with fewer HLA DQB1*06:02 homozygotes. This may be the first demonstration of such an effect, and suggests that the study of changes in GWAS signals over time could help identify environmental factors in other autoimmune diseases.
PMCID: PMC3814311  PMID: 24204295
15.  Systematic design methodology for robust genetic transistors based on I/O specifications via promoter-RBS libraries 
BMC Systems Biology  2013;7:109.
Synthetic genetic transistors are vital for signal amplification and switching in genetic circuits. However, it is still problematic to efficiently select the adequate promoters, Ribosome Binding Sides (RBSs) and inducer concentrations to construct a genetic transistor with the desired linear amplification or switching in the Input/Output (I/O) characteristics for practical applications.
Three kinds of promoter-RBS libraries, i.e., a constitutive promoter-RBS library, a repressor-regulated promoter-RBS library and an activator-regulated promoter-RBS library, are constructed for systematic genetic circuit design using the identified kinetic strengths of their promoter-RBS components.
According to the dynamic model of genetic transistors, a design methodology for genetic transistors via a Genetic Algorithm (GA)-based searching algorithm is developed to search for a set of promoter-RBS components and adequate concentrations of inducers to achieve the prescribed I/O characteristics of a genetic transistor. Furthermore, according to design specifications for different types of genetic transistors, a look-up table is built for genetic transistor design, from which we could easily select an adequate set of promoter-RBS components and adequate concentrations of external inducers for a specific genetic transistor.
This systematic design method will reduce the time spent using trial-and-error methods in the experimental procedure for a genetic transistor with a desired I/O characteristic. We demonstrate the applicability of our design methodology to genetic transistors that have desirable linear amplification or switching by employing promoter-RBS library searching.
PMCID: PMC4015965  PMID: 24160305
Genetic transistor; Input/Output (I/O) characteristics; Promoter-RBS library; Systematic design methodology; Design specifications
16.  Reduction of Decoy Receptor 3 Enhances TRAIL-Mediated Apoptosis in Pancreatic Cancer 
PLoS ONE  2013;8(10):e74272.
Most human pancreatic cancer cells are resistant to tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis. However, the mechanisms by which pancreatic cancer cells utilize their extracellular molecules to counteract the proapoptotic signaling mediated by the TNF family are largely unknown. In this study, we demonstrate for the first time that DcR3, a secreted decoy receptor that malignant pancreatic cancer cells express at a high level, acts as an extracellular antiapoptotic molecule by binding to TRAIL and counteracting its death-promoting function. The reduction of DcR3 with siRNA unmasked TRAIL and greatly enhanced TRAIL-induced apoptosis. Gemcitabine, a first-line drug for pancreatic cancer, also reduced the level of DcR3. The addition of DcR3 siRNA further enhanced gemcitabine-induced apoptosis. Notably, our in vivo study demonstrated that the therapeutic effect of gemcitabine could be enhanced via further reduction of DcR3, suggesting that downregulation of DcR3 in tumor cells could tip the balance of pancreatic cells towards apoptosis and potentially serve as a new strategy for pancreatic cancer therapy.
PMCID: PMC3808375  PMID: 24204567
17.  Clinical and polysomnographic course of childhood narcolepsy with cataplexy 
Brain  2013;136(12):3787-3795.
Our aim was to investigate the natural evolution of cataplexy and polysomnographic features in untreated children with narcolepsy with cataplexy. To this end, clinical, polysomnographic, and cataplexy-video assessments were performed at diagnosis (mean age of 10 ± 3 and disease duration of 1 ± 1 years) and after a median follow-up of 3 years from symptom onset (mean age of 12 ± 4 years) in 21 children with narcolepsy with cataplexy and hypocretin 1 deficiency (tested in 19 subjects). Video assessment was also performed in two control groups matched for age and sex at first evaluation and follow-up and was blindly scored for presence of hypotonic (negative) and active movements. Patients’ data at diagnosis and at follow-up were contrasted, compared with controls, and related with age and disease duration. At diagnosis children with narcolepsy with cataplexy showed an increase of sleep time during the 24 h; at follow-up sleep time and nocturnal sleep latency shortened, in the absence of other polysomnographic or clinical (including body mass index) changes. Hypotonic phenomena and selected facial movements decreased over time and, tested against disease duration and age, appeared as age-dependent. At onset, childhood narcolepsy with cataplexy is characterized by an abrupt increase of total sleep over the 24 h, generalized hypotonia and motor overactivity. With time, the picture of cataplexy evolves into classic presentation (i.e. brief muscle weakness episodes triggered by emotions), whereas total sleep time across the 24 h decreases, returning to more age-appropriate levels.
PMCID: PMC3859221  PMID: 24142146
children; narcolepsy; cataplexy; sleep; sleepiness
18.  Four Common Vascular Endothelial Growth Factor Polymorphisms (−2578C>A, −460C>T, +936C>T, and +405G>C) in Susceptibility to Lung Cancer: A Meta-Analysis 
PLoS ONE  2013;8(10):e75123.
Background and Objective
Vascular endothelial growth factor (VEGF) is one of the key initiators and regulators of angiogenesis and it plays a vital role in the onset and development of malignancy. The association between VEGF gene polymorphisms and lung cancer risk has been extensively studied in recent years, but currently available results remain controversial or ambiguous. The aim of this meta-analysis is to investigate the associations between four common VEGF polymorphisms (i.e., −2578C>A, −460C>T, +936C>T and +405C>G) and lung cancer risk.
A comprehensive search was conducted to identify all eligible studies to estimate the association between VEGF polymorphisms and lung cancer risk. Crude odds ratios (ORs) with 95% confidence intervals (CIs) were used to evaluate the strength of this association.
A total of 14 published case-control studies with 4,664 cases and 4,571 control subjects were identified. Our meta-analysis provides strong evidence that VEGF −2578C>A polymorphism is capable of increasing lung cancer susceptibility, especially among smokers and lung squamous cell carcinoma (SCC) patients. Additionally, for +936C>T polymorphism, increased lung cancer susceptibility was only observed among lung adenocarcinoma patients. In contrast, VEGF −460C>T polymorphism may be a protective factor among nonsmokers and SCC patients. Nevertheless, we did not find any association between +405C>G polymorphism and lung cancer risk, even when the groups were stratified by ethnicity, smoking status or histological type.
This meta-analysis recommends more investigations into the relationship between −2578C>A and −460C>T lung cancer risks. More detailed and well-designed studies should be conducted to identify the causal variants and the underlying mechanisms of the possible associations.
PMCID: PMC3788083  PMID: 24098368
19.  Role of Toll-Like Receptor 4 on Lupus Lung Injury and Atherosclerosis in LPS-Challenge ApoE−/− Mice 
To investigate the pathologic mechanisms of toll-like receptor 4 (TLR4) in lung injury and atherosclerosis, ApoE−/− or wild-type mice were intraperitoneally administered saline, lipopolysaccharides (LPS), or LPS plus TAK-242 (TLR4 inhibitor), respectively, twice a week for 4 weeks. Serum autoantibody of antinuclear antibody (ANA), anti-double-stranded DNA (anti-dsDNA), and cytokines of interferon-gamma (IFN-γ), tumor necrosis factor (TNF-α), and interleukin-1 (IL-1β) were assessed by ELISA. Hematoxylin and eosin (HE) and Perl's stains for lung pathomorphology as well as HE staining for atherosclerosis were employed. TLR4 in macrophages was detected by double immunofluorescent staining. While protein expressions of TLR4, nuclear factor-kappa B p65 (NF-κB p65), and B cell activating factor belonging to the TNF family (BAFF) were examined by immunohistochemistry. We found that serum autoantibody (ANA and anti-dsDNA), cytokines (IFN-γ, TNF-α, IL-1β), lung inflammation, and intima-media thickness in brachiocephalic artery were obviously increased after LPS challenge in both genotypes, but to a lesser extent in wild-type strains. And those alterations were alleviated by coadministration of LPS and TAK-242. Mechanistically, upregulation of TLR4, NF-κb, and BAFF was involved. We concluded that TLR4/NF-κb/BAFF in macrophages might be a possible common autoimmune pathway that caused lung injury and atherosclerosis. TLR4 signal will be a therapeutic target in atherosclerosis and immune-mediated lung injury.
PMCID: PMC3784175  PMID: 24324506
20.  The origin and evolution of mutations in Acute Myeloid Leukemia 
Cell  2012;150(2):264-278.
Most mutations in cancer genomes are thought to be acquired after the initiating event, which may cause genomic instability, driving clonal evolution. However, for acute myeloid leukemia (AML), normal karyotypes are common, and genomic instability is unusual. To better understand clonal evolution in AML, we sequenced the genomes of AML samples with a known initiating event (PML-RARA) vs. normal karyotype AML samples, and the exomes of hematopoietic stem/progenitor cells (HSPCs) from healthy people. Collectively, the data suggest that most of the mutations found in AML genomes are actually random events that occurred in HSPCs before they acquired the initiating mutation; the mutational history of that cell is “captured” as the clone expands. In many cases, only one or two additional, cooperating mutations are needed to generate the malignant founding clone. Cells from the founding clone can acquire additional cooperating mutations, yielding subclones that can contribute to disease progression and/or relapse.
PMCID: PMC3407563  PMID: 22817890
21.  Mutations in DNMT1 cause autosomal dominant cerebellar ataxia, deafness and narcolepsy 
Human Molecular Genetics  2012;21(10):2205-2210.
Autosomal dominant cerebellar ataxia, deafness and narcolepsy (ADCA-DN) is characterized by late onset (30–40 years old) cerebellar ataxia, sensory neuronal deafness, narcolepsy–cataplexy and dementia. We performed exome sequencing in five individuals from three ADCA-DN kindreds and identified DNMT1 as the only gene with mutations found in all five affected individuals. Sanger sequencing confirmed the de novo mutation p.Ala570Val in one family, and showed co-segregation of p.Val606Phe and p.Ala570Val, with the ADCA-DN phenotype, in two other kindreds. An additional ADCA-DN kindred with a p.GLY605Ala mutation was subsequently identified. Narcolepsy and deafness were the first symptoms to appear in all pedigrees, followed by ataxia. DNMT1 is a widely expressed DNA methyltransferase maintaining methylation patterns in development, and mediating transcriptional repression by direct binding to HDAC2. It is also highly expressed in immune cells and required for the differentiation of CD4+ into T regulatory cells. Mutations in exon 20 of this gene were recently reported to cause hereditary sensory neuropathy with dementia and hearing loss (HSAN1). Our mutations are all located in exon 21 and in very close spatial proximity, suggesting distinct phenotypes depending on mutation location within this gene.
PMCID: PMC3465691  PMID: 22328086
22.  Background mutations in parental cells account for most of the genetic heterogeneity of Induced Pluripotent Stem Cells 
Cell Stem Cell  2012;10(5):570-582.
To assess the genetic consequences of induced Pluripotent Stem Cell (iPSC) reprogramming, we sequenced the genomes of ten murine iPSC clones derived from three independent reprogramming experiments, and compared them to their parental cell genomes. We detected hundreds of single nucleotide variants (SNVs) in every clone, with an average of 11 in coding regions. In two experiments, all SNVs were unique for each clone and did not cluster in pathways, but in the third, all four iPSC clones contained 157 shared genetic variants, which could also be detected in rare cells (<1 in 500) within the parental MEF pool. This data suggests that most of the genetic variation in iPSC clones is not caused by reprogramming per se, but is rather a consequence of cloning individual cells, which “captures” their mutational history. These findings have implications for the development and therapeutic use of cells that are reprogrammed by any method.
PMCID: PMC3348423  PMID: 22542160
23.  DQB1*06:02 allele specific expression varies by allelic dosage, not narcolepsy status 
Human immunology  2012;73(4):405-410.
The association of narcolepsy-cataplexy, a sleep disorder caused by the loss of hypocretin/orexin neurons in the hypothalamus, with DQA1*01:02-DQB1*06:02 is one of the tightest known single allele HLA associations. In this study, we explored genome wide expression in peripheral white blood cells of 50 narcolepsy versus 47 controls (half of whom were DQB1*06:02 positive) and found the largest differences between the groups to be in the signal from HLA probes. Further studies of HLA-DQ expression (mRNA and protein in a subset) in 125 controls and 147 narcolepsy cases did not reveal any difference, a result we explain by the lack of proper control of allelic diversity in Affymetrix HLA probes. Rather, a clear effect of DQB1*06:02 allelic dosage on DQB1*06:02 mRNA levels (1.65 fold) and protein (1.59 fold) could be demonstrated independent of the disease status. These results indicate that allelic dosage is transmitted into changes in heterodimer availability, a phenomenon that may explain increased risk for narcolepsy in DQB1*06:02 homozygotes versus heterozygotes.
PMCID: PMC3501142  PMID: 22326585
Narcolepsy; Human Leukocyte Antigen; DQB1*06:02; gene expression
24.  TCRA, P2RY11, and CPT1B/CHKB associations in Chinese narcolepsy 
Sleep Medicine  2011;13(3):269-272.
Polymorphisms in the TCRA and P2RY11, two immune related genes, are associated with narcolepsy in Caucasians and Asians. In contrast, CPT1B/CHKB polymorphisms have only been shown to be associated with narcolepsy in Japanese, with replication in a small group of Koreans. Our aim was to study whether these polymorphisms are associated with narcolepsy and its clinical characteristics in Chinese patients with narcolepsy.
We collected clinical data on 510 Chinese patients presenting with narcolepsy/hypocretin deficiency. Patients were included either when hypocretin deficiency was documented (CSF hypocretin-1 ≤110 pg/ml, n=91) or on the basis of the presence of clear cataplexy and HLA-DQB1*0602 positivity (n=419). Genetic data was compared to typing obtained in 452 controls matched for geographic origin within China. Clinical evaluations included demographics, the Stanford Sleep Inventory (presence and age of onset of each symptom), and Multiple Sleep Latency Test (MSLT) data.
Chinese narcolepsy was strongly and dose dependently associated with TCRA (rs1154155C) and P2RY11 (rs2305795A) but not CPT1B/CHKB (rs5770917C) polymorphisms. CPT1B/CHKB polymorphisms were not associated with any specific clinical characteristics. TCRA rs1154155A homozygotes (58 subjects) had a later disease onset, but this was not significant when corrected for multiple comparisons, thus replication is needed. CPT1B/CHKB or P2RY11 polymorphisms were not associated with any specific clinical characteristics.
The study extends on the observation of a strong multiethnic association of polymorphisms in the TCRA and P2RY11 with narcolepsy, but does not confirm the association of CPT1B/CHKB (rs5770917) in the Chinese population.
PMCID: PMC3288279  PMID: 22177342
narcolepsy; TCR alpha; P2RY11; CPT1B/CHKB; hypocretin; orexin; MSLT; HLADQB1*0602
25.  Hepatitis D Virus Isolates with Low Replication and Epithelial-Mesenchymal Transition-Inducing Activity Are Associated with Disease Remission 
Journal of Virology  2012;86(17):9044-9054.
Clearance of hepatitis D virus (HDV) viremia leads to disease remission. Large hepatitis delta antigen (L-HDAg) has been reported to activate transforming growth factor β, which may induce epithelial-mesenchymal transition (EMT) and fibrogenesis. This study analyzed serum HDV RNA “quasispecies” in HDV-infected patients at two stages of infection: before and after alanine aminotransferase (ALT) elevations. Included in the study were four patients who went into remission after ALT elevation and three patients who did not go into remission and progressed to cirrhosis or hepatocellular carcinoma. Full-length HDV cDNA clones were obtained from the most abundant HDV RNA species at the pre- and post-ALT elevation stages. Using an in vitro model consisting of Huh-7 cells transfected with cloned HDV cDNAs, the pre- or post-ALT elevation dominant HDV RNA species were characterized for (i) their replication capacity by measuring HDV RNA and HDAg levels in transfected cells and (ii) their capacity to induce EMT by measuring the levels of the mesenchymal-cell-specific protein vimentin, the EMT regulators twist and snail, and the epithelial-cell-specific protein E-cadherin. Results show that in patients in remission, the post-ALT elevation dominant HDV RNA species had a lower replication capacity in vitro and lower EMT activity than their pre-ALT elevation counterparts. This was not true of patients who did not go into remission. The expression of L-HDAg, but not small HDAg, increased the expression of the EMT-related proteins. It is concluded that in chronically infected patients, HDV quasispecies with a low replication capacity and low EMT activity are associated with disease remission.
PMCID: PMC3416108  PMID: 22674995

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