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Articles by L Chen
Total Records ( 30 ) for L Chen
  Q Wang , M Liu , X Li , L Chen and H. Tang
 

Kazrin has recently been identified as a functional protein that is involved in cell–cell junctions and in signal transduction. Here, we identified a new isoform, Kazrin F, which is 518 aa in length and has 97 aa unique at the N-terminus. Knockdown of Kazrin F using siRNA caused cell apoptosis and a marked decrease in cell viability measured by MTT and TUNEL assays. Co-immunoprecipitation analysis revealed that Kazrin F interacts with ARC (apoptosis repressor with caspase recruitment domain) and Bax (Bcl-2-associated X protein). Co-localization of Kazrin F with ARC and Bax in the cytoplasm was determined by immunofluorescence analysis. These results suggested that Kazrin F might play an important role in regulating cellular apoptosis by interacting with ARC and Bax.

  J Du , L Chen and J. Shen
 

This study sought to isolate and identify proteins that interact with centromere-associated protein E (CENP-E), provide new clues for exploring the function of CENP-E in cell cycle control and the pathogenesis of tumor. Yeast two-hybrid screen and regular molecular biologic techniques were undertaken to screen human HeLa cDNA library with the kinetochore binding domain of CENP-E. The bait from the C-terminus of CENP-E was created by subcloning methods to find out optimal candidate proteins that interact with the kinetochore binding domain of CENP-E. Eight novel CENP-E interacting proteins including Homo sapiens Fanconi anemia complementation group A (FANCA) were obtained. In yeast two-hybrid assay, the N-terminal 260 amino acids of FANCA were found to be necessary and sufficient for the interaction with the C-terminus of CENP-E. The interaction was confirmed by in vitro glutathione S-transferase pull-down assay and in vivo co-immunoprecipitation assay. Our finding of the interaction of CENP-E with FANCA demonstrates that CENP-E and FANCA may play important roles in the functional regulation of the mitotic checkpoint signal pathway.

  L Chen , S Xu , X Zeng , J Li , W Yin , Y Chen , Z Shao and W. Jin
 

Chemokine C-X-C motif ligand 12 (CXCL12) is a potent chemotactic and angiogenic factor that has been proposed to play a role in organ-specific metastasis and angiogenic activity in several malignancies. In this study, we found that the overexpression of c-myb could elevate CXCL12 mRNA level and CXCL12 promoter activity in human T47D and MCF-7 breast cancer cells. Chromatin immunoprecipitation assay demonstrated that c-myb could bind to the CXCL12 promoter in the cells transfected with c-myb expression vector. c-myb siRNA attenuated CXCL12 promoter activity and the binding of c-myb to the CXCL12 promoter in T47D and MCF-7 cells. These results indicated that c-myb could activate CXCL12 promoter transcription.

  Y Zhao , J Liu , Q Hong , C Yang , L Chen , Y Chen , Q Wang , K Zhao and W. Jin
 

Overexpression of multidrug resistance 1 (MDR1) in cancer remains one of the major causes for the failure of chemotherapy. In the present study, we found that MyoD and PEA3 could activate P-glycoprotein (P-gp) expression in SGC7901 cells. Knockdown of MyoD and PEA3 attenuated MDR1 expression and increased the sensitivity of multidrug resistant cancer cells to cytotoxic drugs that were transported by P-gp in SGC7901/VCR cells. MyoD or PEA3 could bind to the E-box and PEA3 sites on the MDR1 promoter and activate its transcription. The regulation of MDR1 expression by MyoD and PEA3 may provide potential ways to overcome MDR in cancer treatment.

  M Kubo , K Egashira , T Inoue , J. i Koga , S Oda , L Chen , K Nakano , T Matoba , Y Kawashima , K Hara , H Tsujimoto , K Sueishi , R Tominaga and K. Sunagawa
 

Objective— Recent clinical studies of therapeutic neovascularization using angiogenic growth factors demonstrated smaller therapeutic effects than those reported in animal experiments. We hypothesized that nanoparticle (NP)-mediated cell-selective delivery of statins to vascular endothelium would more effectively and integratively induce therapeutic neovascularization.

Methods and Results— In a murine hindlimb ischemia model, intramuscular injection of biodegradable polymeric NP resulted in cell-selective delivery of NP into the capillary and arteriolar endothelium of ischemic muscles for up to 2 weeks postinjection. NP-mediated statin delivery significantly enhanced recovery of blood perfusion to the ischemic limb, increased angiogenesis and arteriogenesis, and promoted expression of the protein kinase Akt, endothelial nitric oxide synthase (eNOS), and angiogenic growth factors. These effects were blocked in mice administered a nitric oxide synthase inhibitor, or in eNOS-deficient mice.

Conclusions— NP-mediated cell-selective statin delivery may be a more effective and integrative strategy for therapeutic neovascularization in patients with severe organ ischemia.

  L Chen , D. Y Lin and D. Zeng
 

Attributable fractions are commonly used to measure the impact of risk factors on disease incidence in the population. These static measures can be extended to functions of time when the time to disease occurrence or event time is of interest. The present paper deals with nonparametric and semiparametric estimation of attributable fraction functions for cohort studies with potentially censored event time data. The semiparametric models include the familiar proportional hazards model and a broad class of transformation models. The proposed estimators are shown to be consistent, asymptotically normal and asymptotically efficient. Extensive simulation studies demonstrate that the proposed methods perform well in practical situations. A cardiovascular health study is provided. Connections to causal inference are discussed.

  H Wang , A Zhao , L Chen , X Zhong , J Liao , M Gao , M Cai , D. H Lee , J Li , D Chowdhury , Y. g Yang , G. P Pfeifer , Y Yen and X. Xu
 

Human Rap1-interacting protein 1 (RIF1) contributes to the ataxia telangiectasia, mutated-mediated DNA damage response against the dexterous effect of DNA lesions and plays a critical role in the S-phase checkpoint. However, the molecular mechanisms by which human RIF1 conquers DNA aberrations remain largely unknown. We here showed that inhibition of RIF1 expression by small interfering RNA led to defective homologous recombination-mediated DNA double-strand break repair and sensitized cancer cells to camptothecin or staurosporine treatment. RIF1 underwent caspase-dependent cleavage upon apoptosis. We further found that RIF1 was highly expressed in human breast tumors, and its expression status was positively correlated with differentiation degrees of invasive ductal carcinoma of the breast. Our results suggest that RIF1 encodes an anti-apoptotic factor required for DNA repair and is a potential target for cancer treatment.

  W. H Jia , Q. H Pan , H. D Qin , Y. F Xu , G. P Shen , L Chen , L. Z Chen , Q. S Feng , M. H Hong , Y. X Zeng and Y. Y. Shugart
 

Nasopharyngeal carcinoma (NPC) is rare in most parts of the world but is more prevalent in Southern China, especially in Guangdong. The cytochrome P450 2E1 (CYP2E1) has been recognized as one of the critically important enzymes involved in oxidizing carcinogens and is probably to be associated with NPC carcinogenesis. To systematically investigate the association between genetic variants in CYP2E1 and NPC risk in Cantonese, two independent studies, a family-based association study and a case–control study, were conducted using the haplotype-tagging single-nucleotide polymorphism approach. A total of 2499 individuals from 546 nuclear families were initially genotyped for the family-based association study. Single-nucleotide polymorphisms (SNPs) rs9418990, rs915908, rs8192780, rs1536826, rs3827688 and one haplotype h2 (CGTGTTAA) were revealed to be significantly associated with the NPC phenotype (P = 0.045–0.003 and P = 0.003, respectively). To follow up the initial study, a case–control study including 755 cases and 755 controls was conducted. Similar results were observed in the case–control study in individuals <46 years of age and had a history of cigarette smoking, with odds ratios (ORs) of specific genotypes ranging from 1.88 to 2.99 corresponding to SNP rs9418990, rs3813865, rs915906, rs2249695, rs8192780, rs1536826, rs3827688 and of haplotypes h2 with OR = 1.65 (P = 0.026), h5 (CCCGTTAA) with OR = 2.58 (P = 0.007). The values of false-positive report probability were <0.015 for six SNPs, suggesting that the reported associations are less probably to be false. This study provides robust evidence for associations between genetic variants of CYP2E1 and NPC risk.

  Y Ding , J. D Paonessa , K. L Randall , D Argoti , L Chen , P Vouros and Y. Zhang
 

Sulforaphane (SF) is a well-known chemopreventive phytochemical and occurs in broccoli and to a lesser extent in other cruciferous vegetables, whereas 4-aminobiphenyl (ABP) is a major human bladder carcinogen and is present at significant levels in tobacco smoke. Here, we show that SF inhibits ABP-induced DNA damage in both human bladder cells in vitro and mouse bladder tissue in vivo, using dG-C8-ABP as a biomarker, which is the predominant ABP-DNA adduct formed in human bladder cells and tissues. SF activates NF-E2 related factor-2 (Nrf2), which is a well-recognized chemopreventive target and activates the Nrf2-regulated cytoprotective signaling pathway. Comparison between wild-type mice and mice without Nrf2 shows that Nrf2 activation is required by SF for inhibition of ABP-induced DNA damage. Moreover, Nrf2 activation by SF in the bladder occurs primarily in the epithelium, which is the principal site of bladder cancer development. These data, together with our recent observation that SF-enriched broccoli sprout extracts strongly inhibits N-butyl-N-(4-hydroxybutyl)nitrosamine-induced bladder cancer development, suggest that SF is a highly promising agent for bladder cancer prevention and provides a mechanistic insight into the repeated epidemiological observation that consumption of broccoli is inversely associated with bladder cancer risk and mortality.

  S. D Russell , J. G Rogers , C. A Milano , D. B Dyke , F. D Pagani , J. M Aranda , C. T Klodell , A. J Boyle , R John , L Chen , H. T Massey , D. J Farrar , J. V Conte and for the HeartMate II Clinical Investigators
 

Background— The effects of continuous blood flow and reduced pulsatility on major organ function have not been studied in detail.

Methods and Results— We evaluated renal (creatinine and blood urea nitrogen) and hepatic (aspartate transaminase, alanine transaminase, and total bilirubin) function in 309 (235 male, 74 female) advanced heart failure patients who had been supported with the HeartMate II continuous-flow left ventricular assist device for bridge to transplantation. To determine whether patients with impaired renal and hepatic function improve over time with continuous-flow left ventricular assist device support or whether there are any detrimental effects in patients with normal organ function, we divided patients into those with above-normal and normal laboratory values before implantation and measured blood chemistry over time during left ventricular assist device support. There were significant improvements over 6 months in all parameters in the above-normal groups, with values in the normal groups remaining in the normal range over time. Mean blood urea nitrogen and serum creatinine in the above-normal groups decreased significantly from 37±14 to 23±10 mg/dL (P<0.0001) and from 1.8±0.4 to 1.4±0.8 mg/dL (P<0.01), respectively. There were decreases in aspartate transaminase and alanine transaminase in the above-normal groups from 121±206 and 171±348 to 36±19 and 31±22 IU (P<0.001), respectively. Total bilirubin for the above-normal group was 2.1±0.9 mg/dL at baseline; after an acute increase at week 1, it decreased to 0.9±0.5 mg/dL by 6 months (P<0.0001). Both renal and liver values from patients in the normal groups remained normal during support with the left ventricular assist device.

Conclusions— The HeartMate II continuous-flow left ventricular assist device improves renal and hepatic function in advanced heart failure patients who are being bridged to transplantation, without evidence of detrimental effects from reduced pulsatility over a 6-month time period.

Clinical Trial Registration Information— URL: http://www.clinicaltrials.gov. Unique identifier: NCT00121472.

  M. C Tsai , L Chen , J Zhou , Z Tang , T. F Hsu , Y Wang , Y. T Shih , H. H Peng , N Wang , Y Guan , S Chien and J. J. Chiu
 

Rationale: Phenotypic modulation of smooth muscle cells (SMCs), which are located in close proximity to endothelial cells (ECs), is critical in regulating vascular function. The role of flow-induced shear stress in the modulation of SMC phenotype has not been well defined.

Objective: The objective was to elucidate the role of shear stress on ECs in modulating SMC phenotype and its underlying mechanism.

Methods and Results: Application of shear stress (12 dyn/cm2) to ECs cocultured with SMCs modulated SMC phenotype from synthetic to contractile state, with upregulation of contractile markers, downregulation of proinflammatory genes, and decreased percentage of cells in the synthetic phase. Treating SMCs with media from sheared ECs induced peroxisome proliferator-activated receptor (PPAR)-, -, and - ligand binding activities; transfecting SMCs with specific small interfering (si)RNAs of PPAR- and -, but not -, inhibited shear induction of contractile markers. ECs exposed to shear stress released prostacyclin (PGI2). Transfecting ECs with PGI2 synthase-specific siRNA inhibited shear-induced activation of PPAR-/, upregulation of contractile markers, downregulation of proinflammatory genes, and decrease in percentage of SMCs in synthetic phase. Mice with PPAR- deficiency (compared with control littermates) showed altered SMC phenotype toward a synthetic state, with increased arterial contractility in response to angiotensin II.

Conclusions: These results indicate that laminar shear stress induces synthetic-to-contractile phenotypic modulation in SMCs through the activation of PPAR-/ by the EC-released PGI2. Our findings provide insights into the mechanisms underlying the EC-SMC interplays and the protective homeostatic function of laminar shear stress in modulating SMC phenotype.

  L Chen , F. G Fulcoli , S Tang and A. Baldini
 

Rationale: TBX1 encodes a T-box transcription factor implicated in DiGeorge syndrome, which affects the development of many organs, including the heart. Loss of Tbx1 results into hypoplasia of heart regions derived from the second heart field, a population of cardiac progenitors cells (CPCs). Thus, we hypothesized that Tbx1 is an important player in the biology of CPCs.

Objective: We asked whether Tbx1 is expressed in multipotent CPCs and, if so, what role it may play in them.

Methods and Results: We used clonal analysis of Tbx1-expressing cells and loss and gain of function models, in vivo and in vitro, to define the role of Tbx1 in CPCs. We found that Tbx1 is expressed in multipotent heart progenitors that, in clonal assays, can give rise to 3 heart lineages expressing endothelial, smooth muscle and cardiomyocyte markers. In multipotent cells, Tbx1 stimulates proliferation, explaining why Tbx1–/– embryos have reduced proliferation in the second heart field. In this population, Tbx1 is expressed while cells are undifferentiated and it disappears with the onset of muscle markers. Loss of Tbx1 results in premature differentiation, whereas gain results in reduced differentiation in vivo. We found that Tbx1 binds serum response factor, a master regulator of muscle differentiation, and negatively regulates its level.

Conclusions: The Tbx1 protein marks CPCs, supports their proliferation, and inhibits their differentiation. We propose that Tbx1 is a key regulator of CPC homeostasis as it modulates positively their proliferation and negatively their differentiation.

  S. C Kim , J. P Stice , L Chen , J. S Jung , S Gupta , Y Wang , G Baumgarten , J Trial and A. A. Knowlton
 

Rationale: Previously, we have found that changes in the location of intracellular heat shock protein (HSP)60 are associated with apoptosis. HSP60 has been reported to be a ligand of Toll-like receptor (TLR)-4.

Objective: We hypothesized that extracellular HSP60 (exHSP60) would mediate apoptosis via TLR4.

Methods and Results: Adult rat cardiac myocytes were treated with HSP60, either recombinant human or with HSP60 purified from the media of injured rat cardiac myocytes. ExHSP60 induced apoptosis in cardiac myocytes, as detected by increased caspase 3 activity and increased DNA fragmentation. Apoptosis could be reduced by blocking antibodies to TLR4 and by nuclear factor B binding decoys, but not completely inhibited, even though similar treatment blocked lipopolysaccharide-induced apoptosis. Three distinct controls showed no evidence for involvement of a ligand other than exHSP60 in the mediation of apoptosis.

Conclusions: This is the first report of HSP60-induced apoptosis via the TLRs. HSP60-mediated activation of TLR4 may be a mechanism of myocyte loss in heart failure, where HSP60 has been detected in the plasma.

  R. W Davies , S Dandona , A. F. R Stewart , L Chen , S. G Ellis , W. H Wilson Tang , S. L Hazen , R Roberts , R McPherson and G. A. Wells
  Background—

Genome-wide association studies (GWAS) have identified single-nucleotide polymorphisms (SNPs) at multiple loci that are significantly associated with coronary artery disease (CAD) risk. In this study, we sought to determine and compare the predictive capabilities of 9p21.3 alone and a panel of SNPs identified and replicated through GWAS for CAD.

Methods and Results—

We used the Ottawa Heart Genomics Study (OHGS) (3323 cases, 2319 control subjects) and the Wellcome Trust Case Control Consortium (WTCCC) (1926 cases, 2938 control subjects) data sets. We compared the ability of allele counting, logistic regression, and support vector machines. Two sets of SNPs, 9p21.3 alone and a set of 12 SNPs identified by GWAS and through a model-fitting procedure, were considered. Performance was assessed by measuring area under the curve (AUC) for OHGS using 10-fold cross-validation and WTCCC as a replication set. AUC for logistic regression using OHGS increased significantly from 0.555 to 0.608 (P=3.59x10–14) for 9p21.3 versus the 12 SNPs, respectively. This difference remained when traditional risk factors were considered in a subgroup of OHGS (1388 cases, 2038 control subjects), with AUC increasing from 0.804 to 0.809 (P=0.037). The added predictive value over and above the traditional risk factors was not significant for 9p21.3 (AUC 0.801 versus 0.804, P=0.097) but was for the 12 SNPs (AUC 0.801 versus 0.809, P=0.0073). Performance was similar between OHGS and WTCCC. Logistic regression outperformed both support vector machines and allele counting.

Conclusions—

Using the collective of 12 SNPs confers significantly greater predictive capabilities for CAD than 9p21.3, whether traditional risks are or are not considered. More accurate models probably will evolve as additional CAD-associated SNPs are identified.

  M Preuss , I. R Konig , J. R Thompson , J Erdmann , D Absher , T. L Assimes , S Blankenberg , E Boerwinkle , L Chen , L. A Cupples , A. S Hall , E Halperin , C Hengstenberg , H Holm , R Laaksonen , M Li , W Marz , R McPherson , K Musunuru , C. P Nelson , M Susan Burnett , S. E Epstein , C. J O'Donnell , T Quertermous , D. J Rader , R Roberts , A Schillert , K Stefansson , A. F. R Stewart , G Thorleifsson , B. F Voight , G. A Wells , A Ziegler , S Kathiresan , M. P Reilly , N. J Samani , H Schunkert and on behalf of the CARDIoGRAM Consortium
  Background—

Recent genome-wide association studies (GWAS) of myocardial infarction (MI) and other forms of coronary artery disease (CAD) have led to the discovery of at least 13 genetic loci. In addition to the effect size, power to detect associations is largely driven by sample size. Therefore, to maximize the chance of finding novel susceptibility loci for CAD and MI, the Coronary ARtery DIsease Genome-wide Replication And Meta-analysis (CARDIoGRAM) consortium was formed.

Methods and Results—

CARDIoGRAM combines data from all published and several unpublished GWAS in individuals with European ancestry; includes >22 000 cases with CAD, MI, or both and >60 000 controls; and unifies samples from the Atherosclerotic Disease VAscular functioN and genetiC Epidemiology study, CADomics, Cohorts for Heart and Aging Research in Genomic Epidemiology, deCODE, the German Myocardial Infarction Family Studies I, II, and III, Ludwigshafen Risk and Cardiovascular Heath Study/AtheroRemo, MedStar, Myocardial Infarction Genetics Consortium, Ottawa Heart Genomics Study, PennCath, and the Wellcome Trust Case Control Consortium. Genotyping was carried out on Affymetrix or Illumina platforms followed by imputation of genotypes in most studies. On average, 2.2 million single nucleotide polymorphisms were generated per study. The results from each study are combined using meta-analysis. As proof of principle, we meta-analyzed risk variants at 9p21 and found that rs1333049 confers a 29% increase in risk for MI per copy (P=2x10–20).

Conclusion—

CARDIoGRAM is poised to contribute to our understanding of the role of common genetic variation on risk for CAD and MI.

  S Bhashyam , A. V Fields , B Patterson , J. M Testani , L Chen , Y. t Shen and R. P. Shannon
  Background—

We have shown that glucagon-like peptide-1 (GLP-1[7–36] amide) stimulates myocardial glucose uptake in dilated cardiomyopathy (DCM) independent of an insulinotropic effect. The cellular mechanisms of GLP-1–induced myocardial glucose uptake are unknown.

Methods and Results—

Myocardial substrates and glucoregulatory hormones were measured in conscious, chronically instrumented dogs at control (n=6), DCM (n=9) and DCM after treatment with a 48-hour infusion of GLP-1 (7–36) amide (n=9) or vehicle (n=6). GLP-1 receptors and cellular pathways implicated in myocardial glucose uptake were measured in sarcolemmal membranes harvested from the 4 groups. GLP-1 stimulated myocardial glucose uptake (DCM: 20±7 nmol/min/g; DCM+GLP-1: 61±12 nmol/min/g; P=0.001) independent of increased plasma insulin levels. The GLP-1 receptors were upregulated in the sarcolemmal membranes (control: 98±2 density units; DCM: 256±58 density units; P=0.046) and were expressed in their activated (65 kDa) form in DCM. The GLP-1–induced increases in myocardial glucose uptake did not involve adenylyl cyclase or Akt activation but was associated with marked increases in p38 MAP kinase activity (DCM+vehicle: 97±22 pmol ATP/mg/min; DCM+GLP-1: 170±36 pmol ATP/mg/min; P=0.051), induction of nitric oxide synthase 2 (DCM+vehicle: 151±13 density units; DCM+GLP-1: 306±12 density units; P=0.001), and GLUT-1 translocation (DCM+vehicle: 21±3% membrane bound; DCM+GLP-1: 39±3% membrane bound; P=0.005). The effects of GLP-1 on myocardial glucose uptake were blocked by pretreatment with the p38 MAP kinase inhibitor or the nonspecific nitric oxide synthase inhibitor nitro-l-arginine.

Conclusions—

GLP-1 stimulates myocardial glucose uptake through a non–Akt-1–dependent mechanism by activating cellular pathways that have been identified in mediating chronic hibernation and the late phase of ischemic preconditioning.

  K Nosho , K Shima , N Irahara , S Kure , Y Baba , G. J Kirkner , L Chen , S Gokhale , A Hazra , D Spiegelman , E. L Giovannucci , R Jaenisch , C. S Fuchs and S. Ogino
 

Purpose: DNA methyltransferase-3B (DNMT3B) plays an important role in de novo CpG island methylation. Dnmt3b can induce colon tumor in mice with methylation in specific CpG islands. We hypothesized that cellular DNMT3B level might influence the occurrence of widespread CpG island methylation (i.e., the CpG island methylator phenotype, CIMP) in colon cancer.

Experimental Design: Utilizing 765 colorectal cancers in two cohort studies, we detected DNMT3B expression in 116 (15%) tumors by immunohistochemistry. We assessed microsatellite instability, quantified DNA methylation in repetitive long interspersed nucleotide element-1 (LINE-1) by Pyrosequencing, eight CIMP-specific promoters [CACNA1G, CDKN2A (p16), CRABP1, IGF2, MLH1, NEUROG1, RUNX3, and SOCS1], and eight other CpG islands (CHFR, HIC1, IGFBP3, MGMT, MINT1, MINT31, p14, and WRN) by real-time PCR (MethyLight).

Results: Tumoral DNMT3B overexpression was significantly associated with CIMP-high [≥6/8 methylated CIMP-specific promoters; odds ratio (OR), 3.34; 95% confidence interval, 2.11-5.29; P < 0.0001]. The relations between DNMT3B and methylation in 16 individual CpG islands varied substantially (OR, 0.80-2.96), suggesting variable locus-to-locus specificities of DNMT3B activity. DNMT3B expression was not significantly related with LINE-1 hypomethylation. In multivariate logistic regression, the significant relation between DNMT3B and CIMP-high persisted (OR, 2.39; 95% confidence interval, 1.11-5.14; P = 0.026) after adjusting for clinical and other molecular features, including p53, β-catenin, LINE-1, microsatellite instability, KRAS, PIK3CA, and BRAF. DNMT3B expression was unrelated with patient outcome, survival, or prognosis.

Conclusions: Tumoral DNMT3B overexpression is associated with CIMP-high in colorectal cancer. Our data support a possible role of DNMT3B in nonrandom de novo CpG island methylation leading to colorectal cancer.

  S Filipovic Sadic , S Sah , L Chen , J Krosting , E Sekinger , W Zhang , P. J Hagerman , T. T Stenzel , A. G Hadd , G. J Latham and F. Tassone
 

Background: Fragile X syndrome (FXS) is a trinucleotide-repeat disease caused by the expansion of CGG sequences in the 5' untranslated region of the FMR1 (fragile X mental retardation 1) gene. Molecular diagnoses of FXS and other emerging FMR1 disorders typically rely on 2 tests, PCR and Southern blotting; however, performance or throughput limitations of these methods currently constrain routine testing.

Methods: We evaluated a novel FMR1 gene–specific PCR technology with DNA templates from 20 cell lines and 146 blinded clinical samples. The CGG repeat number was determined by fragment sizing of PCR amplicons with capillary electrophoresis, and results were compared with those for FMR1 Southern blotting analyses with the same samples.

Results: The FMR1 PCR accurately detected full-mutation alleles up to at least 1300 CGG repeats and consisting of >99% GC character. All categories of alleles detected by Southern blotting, including 66 samples with full mutations, were also identified by the FMR1 PCR for each of the 146 clinical samples. Because all full mutation alleles in samples from heterozygous females were detected by the PCR, allele zygosity was reconciled in every case. The PCR reagents also detected a 1% mass fraction of a 940-CGG allele in a background of 99% 23-CGG allele—a roughly 5- fold greater sensitivity than obtained with Southern blotting.

Conclusions: The novel PCR technology can accurately categorize the spectrum of FMR1 alleles, including alleles previously considered too large to amplify; reproducibly detect low abundance full mutation alleles; and correctly infer homozygosity in female samples, thus greatly reducing the need for sample reflexing to Southern blotting.

  C Zhang , C Wang , X Chen , C Yang , K Li , J Wang , J Dai , Z Hu , X Zhou , L Chen , Y Zhang , Y Li , H Qiu , J Xing , Z Liang , B Ren , K Zen and C. Y. Zhang
  BACKGROUND:

Sensitive and specific biomarkers for the early detection of esophageal squamous cell carcinoma (ESCC) are urgently needed to reduce the high morbidity and mortality of the disease. The discovery of serum microRNAs (miRNAs) and their unique concentration profiles in patients with various diseases makes them attractive, novel noninvasive biomarkers for tumor diagnosis. In this study, we investigated the serum miRNA profile in ESCC patients to develop a novel diagnostic ESCC biomarker.

METHODS:

Serum samples were taken from 290 ESCC patients and 140 age- and sex-matched controls. Solexa sequencing technology was used for an initial screen of miRNAs in serum samples from 141 patients and 40 controls. A hydrolysis probe–based stem–loop quantitative reverse-transcription PCR (RT-qPCR) assay was conducted in the training and verification phases to confirm the concentrations of selected miRNAs in serum samples from 149 patients and 100 controls.

RESULTS:

The Solexa sequencing results demonstrated marked upregulation of 25 serum miRNAs in ESCC patients compared with controls. RT-qPCR analysis identified a profile of 7 serum miRNAs (miR-10a, miR-22, miR-100, miR-148b, miR-223, miR-133a, and miR-127-3p) as ESCC biomarkers. The area under the ROC curve for the selected miRNAs ranged from 0.817 to 0.949, significantly higher than for carcinoembryonic antigen (0.549; P < 0.0005). More importantly, this panel of 7 miRNAs clearly distinguished stage I/II ESCC patients from controls.

CONCLUSIONS:

This panel of 7 serum miRNAs holds promise as a novel blood-based biomarker for the diagnosis of ESCC.

  G Zhang , G Guo , X Hu , Y Zhang , Q Li , R Li , R Zhuang , Z Lu , Z He , X Fang , L Chen , W Tian , Y Tao , K Kristiansen , X Zhang , S Li , H Yang , J Wang and J. Wang
 

Understanding the dynamics of eukaryotic transcriptome is essential for studying the complexity of transcriptional regulation and its impact on phenotype. However, comprehensive studies of transcriptomes at single base resolution are rare, even for modern organisms, and lacking for rice. Here, we present the first transcriptome atlas for eight organs of cultivated rice. Using high-throughput paired-end RNA-seq, we unambiguously detected transcripts expressing at an extremely low level, as well as a substantial number of novel transcripts, exons, and untranslated regions. An analysis of alternative splicing in the rice transcriptome revealed that alternative cis-splicing occurred in ~33% of all rice genes. This is far more than previously reported. In addition, we also identified 234 putative chimeric transcripts that seem to be produced by trans-splicing, indicating that transcript fusion events are more common than expected. In-depth analysis revealed a multitude of fusion transcripts that might be by-products of alternative splicing. Validation and chimeric transcript structural analysis provided evidence that some of these transcripts are likely to be functional in the cell. Taken together, our data provide extensive evidence that transcriptional regulation in rice is vastly more complex than previously believed.

  Y He , Y Li , Z Peng , H Yu , X Zhang , L Chen , Q Ji , W Chen and R. Wang
 

A prominent feature of the rodent Muc3 SEA module is the precursor cleavage event that segregates the O-glycosylated N-terminal fragment and transmembrane domain into the noncovalently attached heterodimer. There are seven potential N-glycosylation sites that occur in a cluster in the SEA module of Muc3. However, it is unknown if these sites are modified or what the function of these N-glycans may be in the SEA module. Our data show that the proteolytic cleavage of the rodent Muc3 SEA module was partially prevented by treatment with tunicamycin, an inhibitor of N-glycosylation. Each single mutant of the seven N-glycosylation sites (N1A, N2A, N3A, N4A, N5A, N6A, and N7A) and multiple mutants, including double (N34A) and triple (N345A) mutants, and mutants with four (N3457A), five (N34567A), six (N134567A and N234567A), seven (N1234567A) mutations, confirmed that all seven of these potential sites are N-glycosylated simultaneously. The proteolytic cleavage of the SEA module was not affected when it lacked only one, two, or three N-glycans, but was partially inhibited when lacking four, five, and six N-glycans. In all, 2%, 48%, 85%, and 73% of the products from N3457A, N34567A, N134567A, and N234567A transfectants, respectively, remained uncleaved. The proteolytic cleavage was completely prevented in the N1234567A transfectant, which eliminated all seven N-glycans in the SEA module. The interaction of the heterodimer was independent of the N-glycans within the rodent Muc3 SEA module. Thus, the N-glycosylation pattern constituted a control point for the modulation of the proteolytic cleavage of the SEA module.

  L. E Flagel , L Chen , B Chaudhary and J. F. Wendel
 

Within polyploid plant species, it has been demonstrated that homoeologous genes (genes duplicated by polyploidy) often display dynamic expression patterns. To determine if chromosomal location plays a role in establishing these expression patterns, we analyzed the relative levels of homoeolog expression among linked genes from 2 locations in the cotton genome. Genes from the region containing the alcohol dehydrogenase A gene show coordinated expression across several tissues, whereas genes from the region containing cellulose synthase A do not. These results indicate that changes in homoeolog expression may be constrained by linkage in some genomic regions, whereas in other regions, homoeolog expression is largely decoupled from physical proximity. Furthermore, these results suggest that both large- and small-scale regulatory mechanisms may control homoeolog expression patterns.

  T. J Maures , L Chen and C. Carter Su
 

The adapter protein SH2B1 (SH2-B, PSM) is recruited to multiple ligand-activated receptor tyrosine kinases, including the receptors for nerve growth factor (NGF), insulin, and IGF-I as well as the cytokine receptor-associated Janus kinase family kinases. In this study, we examine SH2B1’s function in NGF signaling. We show that depleting endogenous SH2B1 using short hairpin RNA against SH2B1 inhibits NGF-dependent neurite outgrowth, but not NGF-mediated phosphorylation of Akt or ERKs 1/2. SH2B1 has been hypothesized to localize and function at the plasma membrane. We identify a nuclear localization signal within SH2B1 and show that it is required for nuclear translocation of SH2B1β. Mutation of the nuclear localization signal has no effect on NGF-induced activation of TrkA and ERKs 1/2 but prevents SH2B1β from enhancing NGF-induced neurite outgrowth. Disruption of SH2B1β nuclear import also prevents SH2B1β from enhancing NGF-induced transcription of genes important for neuronal differentiation, including those encoding urokinase plasminogen activator receptor, and matrix metalloproteinases 3 and 10. Disruption of SH2B1β nuclear export by mutation of its nuclear export sequence similarly prevents SH2B1β enhancement of NGF-induced transcription of those genes. Nuclear translocation of the highly homologous family member SH2B2(APS) was not observed. Together, these data suggest that rather than simply acting as an adapter protein linking signaling proteins to the activated TrkA receptor at the plasma membrane, SH2B1β must shuttle between the plasma membrane and nucleus to function as a critical component of NGF-induced gene expression and neuronal differentiation.

  X. j Cai , L Chen , L Li , M Feng , X Li , K Zhang , Y. y Rong , X. b Hu , M. x Zhang , Y Zhang and M. Zhang
 

Adiponectin is an important antiatherogenic adipocytokine that inhibits inflammation, insulin resistance, and oxide stress. Inflammation in the vascular adventitia is a crucial factor in the pathogenesis of atherosclerosis. Adventitial fibroblasts (AFs) can proliferate, divide into myofibroblasts, and migrate to the intima to become a new component of atherosclerotic plaque under inflammation and atherosclerosis. We investigated whether adiponectin might prevent AFs from proliferating, migrating, and transforming into myofibroblasts. Cultured AFs were stimulated with lipopolysaccharide (LPS) in the presence or absence of adiponectin. Methyl thiazolyl tetrazolium assay and migration and scratch-wound assays demonstrated that adiponectin reduced the AF proliferation and migration induced by LPS, respectively, whereas treatment with AdipoR1 small interfering (si) RNA (siAdipoR1), AMP-activated protein kinase (AMPK) siRNA (siAMPK), and an AMPK inhibitor reversed the effect. Immunocytochemistry and Western blot revealed that adiponectin reduced the transition of AFs to myofibroblasts, and treatment with siAdipoR1, siAMPK, and the AMPK inhibitor increased the transition. RT-PCR, Western blotting, and nitric oxide (NO) assay showed that adiponectin reduces induced NO synthase (iNOS) and nitrotyrosine expression and NO and ONOO production induced by LPS. Treatment with siAdipoR1, siAMPK, and the AMPK inhibitor significantly attenuated adiponectin-induced phosphorylation of AMPK and its downstream target acetyl-coenzyme A carboxylase and up-regulated iNOS mRNA and protein expression, which resulted in a marked increase of NO and ONOO production. In apolipoprotein E-deficient mice, immunohistochemistry of treated vascular adventitia showed that both iNOS expression and ONOO production could be reversed with an adenovirus-adiponectin vector. Taken together, these results suggest that adiponectin reduces LPS-induced NO production and nitrosative stress and prevents AFs from proliferating, transforming to myoflbroblasts, and migrating to the intima, thus worsening atherosclerosis, by inhibiting the AdipoR1-AMPK-iNOS pathway in AFs.

  L Chen , P Collin Dufresne and R. S. Goldstein
 

Structural models of default calibrated to historical default rates, recovery rates, and Sharpe ratios typically generate Baa–Aaa credit spreads that are significantly below historical values. However, this "credit spread puzzle" can be resolved if one accounts for the fact that default rates and Sharpe ratios strongly covary; both are high during recessions and low during booms. As a specific example, we investigate credit spread implications of the Campbell and Cochrane (1999) pricing kernel calibrated to equity returns and aggregate consumption data. Identifying the historical surplus consumption ratio from aggregate consumption data, we find that the implied level and time variation of spreads match historical levels well.

  L Chen and X. Zhao
 

A crucial issue in asset pricing is to understand the relative importance of discount rate (DR) news and cash flow (CF) news in driving the time-series and cross-sectional variations of stock returns. Many studies directly estimate the DR news but back out the CF news as the residual. We argue that this approach has a serious limitation because the DR news cannot be accurately measured due to the small predictive power, and the CF news, as the residual, inherits the large misspecification error of the DR news. We apply this residual-based decomposition approach to Treasury bonds and equities and find results that are either counterintuitive or unrobust. Potential solutions, including modeling both DR news and CF news directly, the Bayesian model averaging approach, and the principal component analysis, are explored.

  W Xu , L Yi , Y Feng , L Chen and J. Liu
 

Pancreatic phospholipase A2 (phospholipase A2 group 1B, G1B) belongs to the superfamily of secreted phospholipase A2 (PLA2) enzymes. G1B has been proposed to be a potential target for diseases such as hypertension, obesity, and diabetes. Human pancreatic prophospholipase A2 (pro-hG1B) is activated by cleavage of the first seven-residue propeptide (phospholipase A2 propeptide, PROP). However, questions still remain on the mode of action for pro-hG1B. In this work, we expressed pro-hG1B in Pichia pastoris and determined the crystal structure at 1.55-Å resolution. The x-ray structure demonstrates that pro-hG1B forms a trimer. In addition, PROP occupies the catalytic cavity and can be self-cleaved at 37 °C. A new membrane-bound surface and activation mechanism are proposed based on the trimeric model of pro-hG1B. We also propose a new autoproteolytic mechanism for pro-hG1B by the reaction triad Asp49-Arg0-Ser(-2) that is similar to the serine protease catalytic triad.

  P. A Darrah , S. T Hegde , D. T Patel , R. W. B Lindsay , L Chen , M Roederer and R. A. Seder
 

The quality of a Th1 response can be a prospective correlate of vaccine-mediated protection against certain intracellular pathogens. Using two distinct vaccine platforms, we evaluate the influence of interleukin (IL) 10 production on the magnitude, quality, and protective capacity of CD4+ T cell responses in the mouse model of Leishmania major infection. Multiparameter flow cytometry was used to delineate the CD4+ T cell production of interferon (IFN) , IL-2, tumor necrosis factor (TNF), and IL-10 (or combinations thereof) after vaccination. Immunization with a high dose of adenovirus (ADV) expressing leishmanial proteins (MML-ADV) elicited a limited proportion of multifunctional IFN-+IL-2+TNF+ Th1 cells, a high frequency of IL-10–producing CD4+ T cells, and did not protect against subsequent challenge. Surprisingly, in the absence of IL-10, there was no change in the magnitude, quality, or protective capacity of the Th1 response elicited by high-dose MML-ADV. In contrast, after immunization with MML protein and CpG (MML + CpG), IL-10 limited the production of IL-12 by DCs in vivo, thereby decreasing the generation of multifunctional Th1 cells. Consequently, three immunizations with MML + CpG were required for full protection. However, inhibiting IL-10 at the time of immunization enhanced the magnitude and quality of the Th1 response sufficiently to mediate protection after only a single immunization. Overall, we delineate distinct mechanisms by which vaccines elicit protective Th1 responses and underscore the importance of multifunctional CD4+ T cells.

  K. M Hoque , O. M Woodward , D. B van Rossum , N. C Zachos , L Chen , G. P.H Leung , W. B Guggino , S. E Guggino and C. M. Tse
 

Intestinal Cl secretion is stimulated by cyclic AMP (cAMP) and intracellular calcium ([Ca2+]i). Recent studies show that protein kinase A (PKA) and the exchange protein directly activated by cAMP (Epac) are downstream targets of cAMP. Therefore, we tested whether both PKA and Epac are involved in forskolin (FSK)/cAMP-stimulated Cl secretion. Human intestinal T84 cells and mouse small intestine were used for short circuit current (Isc) measurement in response to agonist-stimulated Cl secretion. FSK-stimulated Cl secretion was completely inhibited by the additive effects of the PKA inhibitor, H89 (1 µM), and the [Ca2+]i chelator, 1,2-bis-(o-aminophenoxy)-ethane-N,N,N’,N’-tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM; 25 µM). Both FSK and the Epac activator 8-pCPT-2’-O-Me-cAMP (50 µM) elevated [Ca2+]i, activated Ras-related protein 2, and induced Cl secretion in intact or basolateral membrane–permeabilized T84 cells and mouse ileal sheets. The effects of 8-pCPT-2’-O-Me-cAMP were completely abolished by BAPTA-AM, but not by H89. In contrast, T84 cells with silenced Epac1 had a reduced Isc response to FSK, and this response was completely inhibited by H89, but not by the phospholipase C inhibitor U73122 or BAPTA-AM. The stimulatory effect of 8-pCPT-2’-O-Me-cAMP on Cl secretion was not abolished by cystic fibrosis transmembrane conductance (CFTR) inhibitor 172 or glibenclamide, suggesting that CFTR channels are not involved. This was confirmed by lack of effect of 8-pCPT-2’-O-Me-cAMP on whole cell patch clamp recordings of CFTR currents in Chinese hamster ovary cells transiently expressing the human CFTR channel. Furthermore, biophysical characterization of the Epac1-dependent Cl conductance of T84 cells mounted in Ussing chambers suggested that this conductance was hyperpolarization activated, inwardly rectifying, and displayed a Cl>Br>I permeability sequence. These results led us to conclude that the Epac-Rap-PLC-[Ca2+]i signaling pathway is involved in cAMP-stimulated Cl secretion, which is carried by a novel, previously undescribed Cl channel.

 
 
 
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