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1.  Genome-Wide Linkage, Exome Sequencing and Functional Analyses Identify ABCB6 as the Pathogenic Gene of Dyschromatosis Universalis Hereditaria 
PLoS ONE  2014;9(2):e87250.
Background
As a genetic disorder of abnormal pigmentation, the molecular basis of dyschromatosis universalis hereditaria (DUH) had remained unclear until recently when ABCB6 was reported as a causative gene of DUH.
Methodology
We performed genome-wide linkage scan using Illumina Human 660W-Quad BeadChip and exome sequencing analyses using Agilent SureSelect Human All Exon Kits in a multiplex Chinese DUH family to identify the pathogenic mutations and verified the candidate mutations using Sanger sequencing. Quantitative RT-PCR and Immunohistochemistry was performed to verify the expression of the pathogenic gene, Zebrafish was also used to confirm the functional role of ABCB6 in melanocytes and pigmentation.
Results
Genome-wide linkage (assuming autosomal dominant inheritance mode) and exome sequencing analyses identified ABCB6 as the disease candidate gene by discovering a coding mutation (c.1358C>T; p.Ala453Val) that co-segregates with the disease phenotype. Further mutation analysis of ABCB6 in four other DUH families and two sporadic cases by Sanger sequencing confirmed the mutation (c.1358C>T; p.Ala453Val) and discovered a second, co-segregating coding mutation (c.964A>C; p.Ser322Lys) in one of the four families. Both mutations were heterozygous in DUH patients and not present in the 1000 Genome Project and dbSNP database as well as 1,516 unrelated Chinese healthy controls. Expression analysis in human skin and mutagenesis interrogation in zebrafish confirmed the functional role of ABCB6 in melanocytes and pigmentation. Given the involvement of ABCB6 mutations in coloboma, we performed ophthalmological examination of the DUH carriers of ABCB6 mutations and found ocular abnormalities in them.
Conclusion
Our study has advanced our understanding of DUH pathogenesis and revealed the shared pathological mechanism between pigmentary DUH and ocular coloboma.
doi:10.1371/journal.pone.0087250
PMCID: PMC3911924  PMID: 24498303
2.  An association study of TOLL and CARD with leprosy susceptibility in Chinese population 
Human Molecular Genetics  2013;22(21):4430-4437.
Previous genome-wide association studies (GWASs) identified multiple susceptibility loci that have highlighted the important role of TLR (Toll-like receptor) and CARD (caspase recruitment domain) genes in leprosy. A large three-stage candidate gene-based association study of 30 TLR and 47 CARD genes was performed in the leprosy samples of Chinese Han. Of 4363 SNPs investigated, eight SNPs showed suggestive association (P < 0.01) in our previously published GWAS datasets (Stage 1). Of the eight SNPs, rs2735591 and rs4889841 showed significant association (P < 0.001) in an independent series of 1504 cases and 1502 controls (Stage 2), but only rs2735591 (next to BCL10) showed significant association in the second independent series of 938 cases and 5827 controls (Stage 3). Rs2735591 showed consistent association across the three stages (P > 0.05 for heterogeneity test), significant association in the combined validation samples (Pcorrected = 5.54 × 10−4 after correction for 4363 SNPs tested) and genome-wide significance in the whole GWAS and validation samples (P = 1.03 × 10−9, OR = 1.24). In addition, we demonstrated the lower expression of BCL10 in leprosy lesions than normal skins and a significant gene connection between BCL10 and the eight previously identified leprosy loci that are associated with NFκB, a major regulator of downstream inflammatory responses, which provides further biological evidence for the association. We have discovered a novel susceptibility locus on 1p22, which implicates BCL10 as a new susceptibility gene for leprosy. Our finding highlights the important role of both innate and adaptive immune responses in leprosy.
doi:10.1093/hmg/ddt286
PMCID: PMC3792694  PMID: 23784377
3.  Effects of Feedstock and Pyrolysis Temperature on Biochar Adsorption of Ammonium and Nitrate 
PLoS ONE  2014;9(12):e113888.
Biochar produced by pyrolysis of biomass can be used to counter nitrogen (N) pollution. The present study investigated the effects of feedstock and temperature on characteristics of biochars and their adsorption ability for ammonium N (NH4+-N) and nitrate N (NO3−-N). Twelve biochars were produced from wheat-straw (W-BC), corn-straw (C-BC) and peanut-shell (P-BC) at pyrolysis temperatures of 400, 500, 600 and 700°C. Biochar physical and chemical properties were determined and the biochars were used for N sorption experiments. The results showed that biochar yield and contents of N, hydrogen and oxygen decreased as pyrolysis temperature increased from 400°C to 700°C, whereas contents of ash, pH and carbon increased with greater pyrolysis temperature. All biochars could sorb substantial amounts of NH4+-N, and the sorption characteristics were well fitted to the Freundlich isotherm model. The ability of biochars to adsorb NH4+-N followed: C-BC>P-BC>W-BC, and the adsorption amount decreased with higher pyrolysis temperature. The ability of C-BC to sorb NH4+-N was the highest because it had the largest cation exchange capacity (CEC) among all biochars (e.g., C-BC400 with a CEC of 38.3 cmol kg−1 adsorbed 2.3 mg NH4+-N g−1 in solutions with 50 mg NH4+ L−1). Compared with NH4+-N, none of NO3−-N was adsorbed to biochars at different NO3− concentrations. Instead, some NO3−-N was even released from the biochar materials. We conclude that biochars can be used under conditions where NH4+-N (or NH3) pollution is a concern, but further research is needed in terms of applying biochars to reduce NO3−-N pollution.
doi:10.1371/journal.pone.0113888
PMCID: PMC4254611  PMID: 25469875
4.  Hierarchical Macro-meso-microporous ZSM-5 Zeolite Hollow Fibers With Highly Efficient Catalytic Cracking Capability 
Scientific Reports  2014;4:7276.
Zeolite fibers have attracted growing interest for a range of new applications because of their structural particularity while maintaining the intrinsic performances of the building blocks of zeolites. The fabrication of uniform zeolite fibers with tunable hierarchical porosity and further exploration of their catalytic potential are of great importance. Here, we present a versatile and facile method for the fabrication of hierarchical ZSM-5 zeolite fibers with macro-meso-microporosity by coaxial electrospinning. Due to the synergistic integration of the suitable acidity and the hierarchical porosity, high yield of propylene and excellent anti-coking stability were demonstrated on the as-prepared ZSM-5 hollow fibers in the catalytic cracking reaction of iso-butane. This work may also provide good model catalysts with uniform wall thickness and tunable porosity for studying a series of important catalytic reactions.
doi:10.1038/srep07276
PMCID: PMC4250908  PMID: 25450726
5.  Estrogen improves the proliferation and differentiation of hBMSCs derived from postmenopausal osteoporosis through notch signaling pathway 
Molecular and Cellular Biochemistry  2014;392(1-2):85-93.
Estrogen deficiency is the main reason of bone loss, leading to postmenopausal osteoporosis, and estrogen replacement therapy (ERT) has been demonstrated to protect bone loss efficiently. Notch signaling controls proliferation and differentiation of bone marrow-derived mesenchymal stem cells (BMSCs). Moreover, imperfect estrogen-responsive elements (EREs) were found in the 5′-untranslated region of Notch1 and Jagged1. Thus, we examined the molecular and biological links between estrogen and the Notch signaling in postmenopausal osteoporosis in vitro. hBMSCs were obtained from healthy women and patients with postmenopausal osteoporosis. Notch signaling molecules were quantified using real-time polymerase chain reaction (real-time PCR) and Western Blot. Luciferase reporter constructs with putative EREs were transfected into hBMSCs and analyzed. hBMSCs were transduced with lentiviral vectors containing human Notch1 intracellular domain (NICD1). We also used N-[N-(3, 5-diflurophenylacetate)-l-alanyl]-(S)-phenylglycine t-butyl ester, a γ-secretase inhibitor, to suppress the Notch signaling. We found that estrogen enhanced the Notch signaling in hBMSCs by promoting the expression of Jagged1. hBMSCs cultured with estrogen resulted in the up-regulation of Notch signaling and increased proliferation and differentiation. Enhanced Notch signaling could enhance the proliferation and differentiation of hBMSCs from patients with postmenopausal osteoporosis (OP-hBMSCs). Our results demonstrated that estrogen preserved bone mass partly by activating the Notch signaling. Because long-term ERT has been associated with several side effects, the Notch signaling could be a potential target for treating postmenopausal osteoporosis.
Electronic supplementary material
The online version of this article (doi:10.1007/s11010-014-2021-7) contains supplementary material, which is available to authorized users.
doi:10.1007/s11010-014-2021-7
PMCID: PMC4053611  PMID: 24390089
Estrogen; Postmenopausal osteoporosis; HBMSCs; Notch signaling pathway; Cell proliferation; Cell differentiation
6.  PPARδ Activation Protects Endothelial Function in Diabetic Mice 
Diabetes  2012;61(12):3285-3293.
Recent evidence highlights the therapeutic potential of peroxisome proliferator–activated receptor-δ (PPARδ) agonists to increase insulin sensitivity in diabetes. However, the role of PPARδ in regulating vascular function is incompletely characterized. We investigate whether PPARδ activation improves endothelial function in diabetic and obese mice. PPARδ knockout (KO) and wild-type (WT) mice fed with high-fat diet and db/db mice were used as diabetic mouse models, compared with PPARδ KO and WT mice on normal diet and db/m+ mice. Endothelium-dependent relaxation (EDR) was measured by wire myograph. Flow-mediated vasodilatation (FMD) was measured by pressure myograph. Nitric oxide (NO) production was examined in primary endothelial cells from mouse aortae. PPARδ agonist GW1516 restored EDRs in mouse aortae under high-glucose conditions or in db/db mouse aortae ex vivo. After oral treatment with GW1516, EDRs in aortae and FMDs in mesenteric resistance arteries were improved in obese mice in a PPARδ-specific manner. The effects of GW1516 on endothelial function were mediated through phosphatidylinositol 3-kinase (PI3K) and Akt with a subsequent increase of endothelial nitric oxide synthase (eNOS) activity and NO production. The current study demonstrates an endothelial-protective effect of PPARδ agonists in diabetic mice through PI3K/Akt/eNOS signaling, suggesting the therapeutic potential of PPARδ agonists for diabetic vasculopathy.
doi:10.2337/db12-0117
PMCID: PMC3501853  PMID: 22933110
7.  Single nucleotide polymorphisms at the TRAF1/C5 locus are associated with rheumatoid arthritis in a Han Chinese population 
BMC Medical Genetics  2011;12:53.
Background
Genetic variants in TRAF1C5 and PTPN22 genes have been shown to be significantly associated with arthritis rheumatoid in Caucasian populations. This study investigated the association between single nucleotide polymorphisms (SNPs) in TRAF1/C5 and PTPN22 genes and rheumatoid arthritis (RA) in a Han Chinese population. We genotyped SNPs rs3761847 and rs7021206 at the TRAF1/C5 locus and rs2476601 SNP in the PTPN22 gene in a Han Chinese cohort composed of 576 patients with RA and 689 controls. The concentrations of anti-cyclic citrullinated peptide antibodies (CCP) and rheumatoid factor (RF) were determined for all affected patients. The difference between the cases and the controls was compared using χ2 analysis.
Results
Significant differences in SNPs rs3761847 and rs7021206 at TRAF1/C5 were observed between the case and control groups in this cohort; the allelic p-value was 0.0018 with an odds ratio of 1.28 for rs3761847 and 0.005 with an odds ratio of 1.27 for rs7021206. This significant association between rs3761847 and RA was independent of the concentrations of anti-CCP and RF. No polymorphism of rs2476601 was observed in this cohort.
Conclusions
We first demonstrated that genetic variants at the TRAF1/C5 locus are significantly associated with RA in Han Chinese, suggesting that TRAF1/C5 may play a role in the development of RA in this population, which expands the pathogenesis role of TRAF1/C5 in a different ethnicity.
doi:10.1186/1471-2350-12-53
PMCID: PMC3094270  PMID: 21492465
rheumatoid arthritis; genetics; TRAF1/C5; association study; Chinese
8.  Impairment of Myocardial Mitochondria in Viral Myocardial Disease and Its Reflective Window in Peripheral Cells 
PLoS ONE  2014;9(12):e116239.
Background
Viral myocardial disease (VMD) is a common disease inducing heart failure. It has not been clear the roles of mitochondrial damage in the pathological changes of cardiomyocytes in VMD.
Methods
Myocardial tissues and lymphocytes were collected from 83 VMD patients. Control groups included 12 cases of healthy accidental death with myocardial autopsy and 23 healthy blood donors. The mouse model of viral myocarditis (VMC) was established by Coxsackie virus B3 infection and myocardial tissues and skeletal muscle were collected. Mitochondrial DNA (mtDNA) deletion rate was quantitatively determined using polymerase chain reaction.
Results
There was significantly difference of myocardial mitochondrial DNA deletion rate between VMD or VMC group and control group (P<0.05). Moreover, the loss of mitochondrial membrane phospholipids was significantly different between VMD or VMC group and control group. In VMC mice, there were negative correlations between myocardial mtDNA3867 deletion rate and left ventricular peak systolic pressure (LVPSP) (r = −0.66, P<0.05), and between myocardial mtDNA3867 deletion rate and +dp/dtmax (r = −0.79, P<0.05), while there was positive correlation between myocardial mtDNA3867 deletion rate and −dp/dtmax (r = 0.80, P<0.05).
Conclusion
Mitochondrial damage is an important pathophysiological mechanism leading to myocardial injury and cardiac dysfunction. The mitochondrial damage in the skeletal muscle and lymphocytes reflect a “window” of myocardial mitochondrial damage.
doi:10.1371/journal.pone.0116239
PMCID: PMC4281208  PMID: 25551390
9.  Recent trends in SELEX technique and its application to food safety monitoring 
Mikrochimica acta  2014;181(5-6):479-491.
The method referred to as “systemic evolution of ligands by exponential enrichment” (SELEX) was introduced in 1990 and ever since has become an important tool for the identification and screening of aptamers. Such nucleic acids can recognize and bind to their corresponding targets (analytes) with high selectivity and affinity, and aptamers therefore have become attractive alternatives to traditional antibodies not the least because they are much more stable. Meanwhile, they have found numerous applications in different fields including food quality and safety monitoring. This review first gives an introduction into the selection process and to the evolution of SELEX, then covers applications of aptamers in the surveillance of food safety (with subsections on absorptiometric, electrochemical, fluorescent and other methods), and then gives conclusions and perspectives. The SELEX method excels by its features of in vitro, high throughput and ease of operation. This review contains 86 references.
doi:10.1007/s00604-013-1156-7
PMCID: PMC4239545  PMID: 25419005
SELEX; Aptamer; Food safety; Rapid detection; Biosensor
10.  Effects of atrazine on the oxidative damage of kidney in Wister rats 
The environmental persistence and bioaccumulation of herbicide atrazine may pose a significant threat to human health. In this experiment, 4 weeks old female Wister rats were treated by 0, 5, 25 and 125 mg/kg atrazine respectively for 28 days, and the oxidative stress responses as well as the activations of Nrf2 signaling pathway in kidney tissues induced by atrazine were observed. The results showed that after be treated by atrazine, the Blood urea nitrogen (BUN) and creatinine (CREA) levels in serum were increased, the contents of nitric oxide (NO) and malondialdehyde (MDA) in the kidney tissue homogenates were increased, the over-expressed Nrf2 transferred into the nuclei and played an antioxidant role by up-regulated the expression of II phase detoxifying enzymes such as heme oxygenase-1 (HO1) and NAD(P)H quinone oxidoreductase (NQO1) and the expression of antioxidant enzymes such as catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px).
PMCID: PMC4238554  PMID: 25419354
Atrazine; oxidative injury; Nrf2; rat; kidney
11.  The REGγ Proteasome Regulates Hepatic Lipid Metabolism through Inhibition of Autophagy 
Cell metabolism  2013;18(3):10.1016/j.cmet.2013.08.012.
SUMMARY
The ubiquitin-proteasome and autophagy-lysosome systems are major proteolytic pathways, whereas function of the Ub-independent proteasome pathway is yet to be clarified. Here, we investigated roles of the Ub-independent REGγ-proteasome proteolytic system in regulating metabolism. We demonstrate that mice deficient for the proteasome activator REGγ exhibit dramatic autophagy induction and are protected against high-fat diet (HFD)-induced liver steatosis through autophagy. Molecularly, prevention of steatosis in the absence of REGγ entails elevated SirT1, a deacetylase regulating autophagy and metabolism. REGγ physically binds to SirT1, promotes its Ub-independent degradation and inhibits its activity to deacetylate autophagy-related proteins, thereby inhibiting autophagy under normal conditions. Moreover, REGγ and SirT1 dissociate from each other through a phosphorylation-dependent mechanism under energy-deprived conditions, unleashing SirT1 to stimulate autophagy. These observations provide a function of the REGγ proteasome in autophagy and hepatosteatosis, underscoring mechanistically a cross-talk between the proteasome and autophagy degradation system in the regulation of lipid homeostasis.
doi:10.1016/j.cmet.2013.08.012
PMCID: PMC3813599  PMID: 24011073
12.  A Class-Information-Based Penalized Matrix Decomposition for Identifying Plants Core Genes Responding to Abiotic Stresses 
PLoS ONE  2014;9(9):e106097.
In terms of making genes expression data more interpretable and comprehensible, there exists a significant superiority on sparse methods. Many sparse methods, such as penalized matrix decomposition (PMD) and sparse principal component analysis (SPCA), have been applied to extract plants core genes. Supervised algorithms, especially the support vector machine-recursive feature elimination (SVM-RFE) method, always have good performance in gene selection. In this paper, we draw into class information via the total scatter matrix and put forward a class-information-based penalized matrix decomposition (CIPMD) method to improve the gene identification performance of PMD-based method. Firstly, the total scatter matrix is obtained based on different samples of the gene expression data. Secondly, a new data matrix is constructed by decomposing the total scatter matrix. Thirdly, the new data matrix is decomposed by PMD to obtain the sparse eigensamples. Finally, the core genes are identified according to the nonzero entries in eigensamples. The results on simulation data show that CIPMD method can reach higher identification accuracies than the conventional gene identification methods. Moreover, the results on real gene expression data demonstrate that CIPMD method can identify more core genes closely related to the abiotic stresses than the other methods.
doi:10.1371/journal.pone.0106097
PMCID: PMC4152128  PMID: 25180509
13.  Potent DGAT1 Inhibitors in the Benzimidazole Class with a Pyridyl-oxy-cyclohexanecarboxylic Acid Moiety 
ACS Medicinal Chemistry Letters  2013;4(8):773-778.
We report the design and synthesis of a series of novel DGAT1 inhibitors in the benzimidazole class with a pyridyl-oxy-cyclohexanecarboxylic acid moiety. In particular, compound 11A is a potent DGAT1 inhibitor with excellent selectivity against ACAT1. Compound 11A significantly reduces triglyceride excursion in lipid tolerance tests (LTT) in both mice and dogs at low plasma exposure. An in vivo study in mice with des-fluoro analogue 10A indicates that this series of compounds appears to distribute in intestine preferentially over plasma. The propensity to target intestine over plasma could be advantageous in reducing potential side effects since lower circulating levels of drug are required for efficacy. However, in the preclinical species, compound 11A undergoes cis/trans epimerization in vivo, which could complicate further development due to the presence of an active metabolite.
doi:10.1021/ml400168h
PMCID: PMC4027454  PMID: 24900745
DGAT1; inhibitor; benzimidazole; ACAT1; cyclohexanecarboxylic acid; lipid tolerance test; epimerization; metabolite
14.  The subcellular distribution and function of MTA1 in cancer differentiation 
Oncotarget  2014;5(13):5153-5164.
The functions and mechanisms of metastasis-associated protein 1 (MTA1) in cancer progression are still unclear due to a lagged recognition of the subcellular localization. In the present study, using multiple molecular technologies we confirmed for the first time that MTA1 localizes to the nucleus, cytoplasm and nuclear envelope. MTA1 is primarily localized in the nucleus of normal adult tissues but in the cytoplasm of embryonic tissues. While in colon cancer, both distributions have been described. Further investigation revealed that MTA1 localizes on the nuclear envelope in a translocated promoter region (TPR)-dependent manner, while in the cytoplasm, MTA1 shows an obvious localization on microtubules. Both nuclear and cytoplasmic MTA1 are associated with cancer progression. However, these functions may be associated with different mechanisms because only nuclear MTA1 has been associated with cancer differentiation. Overexpression of MTA1 in HCT116 cells inhibited differentiation and promoted proliferation, whereas MTA1 knockdown resulted in cell differentiation and death. Theses results not only suggest that nuclear MTA1 is a good marker for cancer differentiation diagnosis and a potential target for the treatment of cancers but also reveal the necessity to differentially examine the functions of nuclear and cytoplasmic MTA1.
PMCID: PMC4148129  PMID: 24970816
MTA1; Subcellular distribution; Cancer; Differentiation
15.  Dose-Dependent Effect of Estrogen Suppresses the Osteo-Adipogenic Transdifferentiation of Osteoblasts via Canonical Wnt Signaling Pathway 
PLoS ONE  2014;9(6):e99137.
Fat infiltration within marrow cavity is one of multitudinous features of estrogen deficiency, which leads to a decline in bone formation functionality. The origin of this fat is unclear, but one possibility is that it is derived from osteoblasts, which transdifferentiate into adipocytes that produce bone marrow fat. We examined the dose-dependent effect of 17β-estradiol on the ability of MC3T3-E1 cells and murine bone marrow-derived mesenchymal stem cell (BMMSC)-derived osteoblasts to undergo osteo-adipogenic transdifferentiation. We found that 17β-estradiol significantly increased alkaline phosphatase activity (P<0.05); calcium deposition; and Alp, Col1a1, Runx2, and Ocn expression levels dose-dependently. By contrast, 17β-estradiol significantly decreased the number and size of lipid droplets, and Fabp4 and PPARγ expression levels during osteo-adipogenic transdifferentiation (P<0.05). Moreover, the expression levels of brown adipocyte markers (Myf5, Elovl3, and Cidea) and undifferentiated adipocyte markers (Dlk1, Gata2, and Wnt10b) were also affected by 17β-estradiol during osteo-adipogenic transdifferentiation. Western blotting and immunostaining further showed that canonical Wnt signaling can be activated by estrogen to exert its inhibitory effect of osteo-adipogenesis. This is the first study to demonstrate the dose-dependent effect of 17β-estradiol on the osteo-adipogenic transdifferentiation of MC3T3-E1 cells and BMMSCs likely via canonical Wnt signaling. In summary, our results indicate that osteo-adipogenic transdifferentiation modulated by canonical Wnt signaling pathway in bone metabolism may be a new explanation for the gradually increased bone marrow fat in estrogen-inefficient condition.
doi:10.1371/journal.pone.0099137
PMCID: PMC4053448  PMID: 24918446
16.  Impaired Frontal-Basal Ganglia Connectivity in Adolescents with Internet Addiction 
Scientific Reports  2014;4:5027.
Understanding the neural basis of poor impulse control in Internet addiction (IA) is important for understanding the neurobiological mechanisms of this syndrome. The current study investigated how neuronal pathways implicated in response inhibition were affected in IA using a Go-Stop paradigm and functional magnetic resonance imaging (fMRI). Twenty-three control subjects aged 15.2 ± 0.5 years (mean ± S.D.) and eighteen IA subjects aged 15.1 ± 1.4 years were studied. Effective connectivity within the response inhibition network was quantified using (stochastic) dynamic causal modeling (DCM). The results showed that the indirect frontal-basal ganglia pathway was engaged by response inhibition in healthy subjects. However, we did not detect any equivalent effective connectivity in the IA group. This suggests the IA subjects fail to recruit this pathway and inhibit unwanted actions. This study provides a clear link between Internet addiction as a behavioral disorder and aberrant connectivity in the response inhibition network.
doi:10.1038/srep05027
PMCID: PMC4030253  PMID: 24848380
17.  Maternal glucose level and body mass index measured at gestational diabetes mellitus screening and the risk of macrosomia: results from a perinatal cohort study 
BMJ Open  2014;4(5):e004538.
Objective
To examine the impact of maternal blood glucose (BG) level and body mass index (BMI) measured at gestational diabetes mellitus (GDM) screening on the risk of macrosomia.
Design
A perinatal cohort of women were followed up from receiving perinatal healthcare to giving birth.
Setting
Beichen District, Tianjin, China between June 2011 and October 2012.
Participants
1951 women aged 19–42 years with valid values of BMI and BG level at GDM screening (24–28 weeks gestation), singleton birth and birth weight (BW)>2500 g.
Main outcomes and measures
Primary outcome was macrosomia (BW>4000 g). BG level and BMI were measured at GDM screening.
Results
191 (9.7%) newborns were macrosomia. The ORs (95% CIs) of macrosomia from multiple logistic regression were 1.14 (1.10 to 1.19, p<0.0001) for BMI and 1.11 (1.01 to 1.23, p=0.03) for BG. When BMI and BG levels (continuous) were modelled simultaneously, the OR for BMI was similar, but significantly attenuated for BG. Areas of receiver operating characteristics (ROC) were 0.6530 (0.6258 to 0.6803) for BMI and 0.5548 (0.5248 to 0.5848) for BG (χ2=26.17, p<0.0001). BG (mmol/L, <6.7, 6.7–7.8 or ≥7.8) and BMI in quintiles (Q1–Q5) were evaluated with BG <6.7 and Q2 BMI as the reference group. The ORs of macrosomia were not statistically different for mothers in Q1 or Q2 of BMI regardless of the BG levels; the ORs for ≥Q3 of BMI were elevated significantly with the highest OR observed in Q5 of BMI and BG levels ≥7.8 (6.93 (2.61 to 18.43), p<0.0001).
Conclusions
High BMI measured at GDM screening was the most important determinant for risk of macrosomia. These findings suggest that GDM screening may be a critical gestational time point to initiate maternal weight control oriented intervention strategy to lower the risk.
doi:10.1136/bmjopen-2013-004538
PMCID: PMC4039786  PMID: 24844269
Epidemiology
18.  Calcium Dependent Ligand Binding and G-protein Signaling of Family B GPCR Parathyroid Hormone 1 Receptor Purified in Nanodiscs 
ACS chemical biology  2013;8(3):617-625.
GPCRs mediate intracellular signaling upon external stimuli, making them ideal drug targets. However, little is known about their activation mechanisms due to the difficulty in purification. Here, we introduce a method to purify GPCRs in nanodiscs, which incorporates GPCRs into lipid bilayers immediately after membrane solubilization, followed by single-step purification. Using this approach, we purified a family B GPCR, parathyroid hormone 1 receptor (PTH1R), which regulates calcium and phosphate homeostasis and is a drug target for osteoporosis. We demonstrated that the purified PTH1R in nanodiscs can bind to PTH(1-34) and activate G protein. We also observed that Ca2+ is a weak agonist of PTH1R and Ca2+ in millimolar concentration can switch PTH(1-34) from an inverse agonist to an agonist. Hence, our results show that nanodiscs are a viable vehicle for GPCR purification, enabling studies of GPCRs under precise experimental conditions without interference from other cellular or membrane components.
doi:10.1021/cb300466n
PMCID: PMC4015634  PMID: 23237450
19.  Validation of Potential Reference Genes for qPCR in Maize across Abiotic Stresses, Hormone Treatments, and Tissue Types 
PLoS ONE  2014;9(5):e95445.
The reverse transcription quantitative polymerase chain reaction (RT-qPCR) is a powerful and widely used technique for the measurement of gene expression. Reference genes, which serve as endogenous controls ensure that the results are accurate and reproducible, are vital for data normalization. To bolster the literature on reference gene selection in maize, ten candidate reference genes, including eight traditionally used internal control genes and two potential candidate genes from our microarray datasets, were evaluated for expression level in maize across abiotic stresses (cold, heat, salinity, and PEG), phytohormone treatments (abscisic acid, salicylic acid, jasmonic acid, ethylene, and gibberellins), and different tissue types. Three analytical software packages, geNorm, NormFinder, and Bestkeeper, were used to assess the stability of reference gene expression. The results revealed that elongation factor 1 alpha (EF1α), tubulin beta (β-TUB), cyclophilin (CYP), and eukaryotic initiation factor 4A (EIF4A) were the most reliable reference genes for overall gene expression normalization in maize, while GRP (Glycine-rich RNA-binding protein), GLU1(beta-glucosidase), and UBQ9 (ubiquitin 9) were the least stable and most unsuitable genes. In addition, the suitability of EF1α, β-TUB, and their combination as reference genes was confirmed by validating the expression of WRKY50 in various samples. The current study indicates the appropriate reference genes for the urgent requirement of gene expression normalization in maize across certain abiotic stresses, hormones, and tissue types.
doi:10.1371/journal.pone.0095445
PMCID: PMC4014480  PMID: 24810581
20.  Directed differentiation of skin-derived precursors into fibroblast-like cells 
Skin-derived precursors (SKPs), which are located at skin’s dermis, display multi-lineage potential and can produce both neural and mesodermal progeny in vitro. SKPs are considered to take part in dermal reconstruction and may be an important source of fibroblast during wound repairing. To explore the possibility of differentiation of SKPs into fibroblasts, the 3rd passage SKPs were treated with 0, 20, 40, 100, or 500 ng/ml human recombinant connective tissue growth factor (CTGF) for 48 h or treated with 100 ng/ml CTGF for 0, 24, 48, 72, or 96 h. Subsequently, a series of methods were to be used to observe cells immunocytochemistry changes under fluorescence microscope, to validate the mRNA expression change of collagen I, collagen III, fibroblast-specific protein 1 (FSP-1) and alpha smooth muscle actin (α-SMA) by quantitative real-time reverse transcriptase polymerase chain reaction (QRT-PCR), to analyze the expression of collagen I and collagen III protein by Enzyme-linked immunosorbent assay (ELISA), to semiquantitatively measure the expression of FSP-1 and α-SMA by western-blot. After differentiation, cells showed that positively staining for collagen I, collagen III, α-SMA, and FSP-1, which are markers for fibroblasts, but negative expression for neural precursors. The effects of CTGF on collagen I, collagen III, FSP-1 and α-SMA in SKPs were detected both on the transcriptional and posttranscriptional levels. These findings indicate that SKPs can be induced to differentiate into fibroblast-like cells with CTGF treatment that may be a key source of fibroblast in wound healing.
PMCID: PMC4014227  PMID: 24817943
Skin-derived precursor (SKP); differentiation; fibroblast-like cell; connective tissue growth factor (CTGF)
21.  Positive effects of porcine IL-2 and IL-4 on virus-specific immune responses induced by the porcine reproductive and respiratory syndrome virus (PRRSV) ORF5 DNA vaccine in swine 
Journal of Veterinary Science  2014;15(1):99-109.
The purpose of this study was to investigate the effects of porcine interleukin (IL)-2 and IL-4 genes on enhancing the immunogenicity of a porcine reproductive and respiratory syndrome virus ORF5 DNA vaccine in piglets. Eukaryotic expression plasmids pcDNA-ORF5, pcDNA-IL-2, and pcDNA-IL-4 were constructed and then expressed in Marc-145 cells. The effects of these genes were detected using an indirect immunofluorescent assay and reverse transcription polymerase chain reaction (RT-PCR). Characteristic fluorescence was observed at different times after pcDNA-ORF5 was expressed in the Marc-145 cells, and PCR products corresponding to ORF5, IL-2, and IL-4 genes were detected at 48 h. Based on these data, healthy piglets were injected intramuscularly with different combinations of the purified plasmids: pcDNA-ORF5 alone, pcDNA-ORF5 + pcDNA-IL-2, pcDNA-ORF5 + pcDNA-IL-4, and pcDNA-ORF5 + pcDNAIL-4 + pcDNA-IL-2. The ensuing humoral immune responses, percentages of CD4+ and CD8+ T lymphocytes, proliferation indices, and interferon-γ expression were analyzed. Results revealed that the piglets co-immunized with pcDNA-ORF5 + pcDNA-IL-4 + pcDNA-IL-2 plasmids developed significantly higher antibody titers and neutralizing antibody levels, had significantly increased levels of specific T lymphocyte proliferation, elevated percentages of CD4+ and CD8+ T lymphocytes, and significantly higher IFN-γ production than the other inoculated pigs (p < 0.05).
doi:10.4142/jvs.2014.15.1.99
PMCID: PMC3973771  PMID: 24136204
IL-2; IL-4; immunogenicity; ORF5 DNA vaccine; porcine reproductive and respiratory syndrome virus
22.  Mass spectrometric analysis of cerebrospinal fluid protein for glioma and its clinical application 
Contemporary Oncology  2014;18(2):100-105.
Aim of the study
To establish and evaluate the fingerprint diagnostic models of cerebrospinal protein profile in glioma with surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) and bioinformatics analysis, in order to seek new tumor markers.
Material and methods
SELDI-TOF-MS was used to detect the cerebrospinal protein bond to ProteinChip H4. The cerebrospinal protein profiles were obtained and analyzed using the artificial neural network (ANN) method. Fingerprint diagnostic models of cerebrospinal protein profiles for distinguishing glioma from non-brain-tumor, and distinguishing glioma from benign brain tumor, were established. The support vector machine (SVM) algorithm was used for verification of established diagnostic models. The tumor markers were screened.
Results
In a fingerprint diagnostic model of cerebrospinal protein profiles for distinguishing glioma from non-brain tumor, the sensitivity and specificity of glioma diagnosis were 100% and 91.7%, respectively. Seven candidate tumor markers were obtained. In a fingerprint diagnostic model for distinguishing glioma from benign brain tumor, the sensitivity and specificity of glioma diagnosis were 88.9% and 100%, respectively, and 8 candidate tumor markers were gained.
Conclusions
The combination of SELDI-TOF-MS and bioinformatics tools is a very effective method for screening and identifying new markers of glioma. The established diagnostic models have provided a new way for clinical diagnosis of glioma, especially for qualitative diagnosis.
doi:10.5114/wo.2014.40455
PMCID: PMC4068817  PMID: 24966792
glioma; cerebrospinal fluid; SELDI-TOF-MS; artificial neural network; support vector machine; diagnostic model; tumor markers
23.  Clinical and Epidemiologic Characteristics of Norovirus GII.4 Sydney during Winter 2012–13 in Beijing, China following Its Global Emergence 
PLoS ONE  2013;8(8):e71483.
Background
Limited information is available on the molecular epidemiology of GII.4 Sydney-associated diarrhea in China in the winter of 2012–13 during the global epidemic associated with the emergence of GII.4 Sydney.
Methods
Fecal specimens collected from 171 diarrhea outpatients (one from each) between late October 2012 and the middle of March 2013 were examined for NoV by reverse transcription-polymerase chain reaction and sequences corresponding to both the NoV partial polymerase and partial capsid regions were analyzed phylogenetically. Clinical characteristics of GII.4 Sydney cases versus other NoV-positive cases detected in a previous study were compared statistically.
Results
Twenty-six (15.2%, 26/171) outpatients with diarrhea were infected with NoV. Twenty-two of the 26 (84.6%) identified NoV strains clustered into GII.4 Sydney. There was a significant difference in symptoms of fever (χ2, P<0.05 ), abdominal pain (χ2, P<0.05 ) and diarrhea frequency (Mann-Whitney U test, P<0.05) between the GII.4 Sydney case group and other NoV-positive case group.
Conclusions
The new NoV variant, GII.4 Sydney, has been circulating in Beijing, China and became the predominant strain in the winter of 2012–13. GII.4 Sydney causes severe fever, abdominal pain and higher diarrhea frequency clinically compared to other NoV infections.
doi:10.1371/journal.pone.0071483
PMCID: PMC3745450  PMID: 23977050
24.  Ferroelectric BaTiO3/SrTiO3 multilayered thin films for room-temperature tunable microwave elements 
Nanoscale Research Letters  2013;8(1):338.
Ferroelectric BaTiO3/SrTiO3 with optimized c-axis-oriented multilayered thin films were epitaxially fabricated on (001) MgO substrates. The microstructural studies indicate that the in-plane interface relationships between the films as well as the substrate are determined to be (001)SrTiO3//(001)BaTiO3//(001)MgO and [100]SrTiO3//[100]BaTiO3//[100]MgO. The microwave (5 to 18 GHz) dielectric measurements reveal that the multilayered thin films have excellent dielectric properties with large dielectric constant, low dielectric loss, and high dielectric tunability, which suggests that the as-grown ferroelectric multilayered thin films can be developed for room-temperature tunable microwave elements and related device applications.
doi:10.1186/1556-276X-8-338
PMCID: PMC3735410  PMID: 23895295
BaTiO3//SrTiO3; Multilayer; Ferroelectric thin films; Epitaxial behavior; Microwave dielectric properties
25.  Genome Sequence of the Biocontrol Agent Microbacterium barkeri Strain 2011-R4 
Journal of Bacteriology  2012;194(23):6666-6667.
Microbacterium barkeri strain 2011-R4 is a Gram-positive epiphyte which has been confirmed as a biocontrol agent against several plant pathogens in our previous studies. Here, we present the draft genome sequence of this strain, which was isolated from the rice rhizosphere in Tonglu city, Zhejiang province, China.
doi:10.1128/JB.01468-12
PMCID: PMC3497549  PMID: 23144410

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