Publications by authors named "Weiguo Han"

33 Publications

Cascade Oxidative C-H Annulation of Thiophenes: Heck-Type Pathway Enables Concise Access to Thienoacenes.

Angew Chem Int Ed Engl 2021 Mar 23. Epub 2021 Mar 23.

Key Laboratory of Green Chemistry and Technology of Ministry of Education, College of Chemistry, Sichuan University, 29 Wangjiang Road, Chengdu, 610064, P. R. China.

The pursuit of efficient synthetic route to thienoacenes represents an appealing yet challenging task in the fields of both organic synthetic chemistry and organic functional materials. In this work, we disclose a rhodium-catalyzed cascade C-H annulation of phenacyl phosphoniums with (benzo)thiophenes via a Heck-type pathway to provide a new class of planar thienoacenes, which involves the formation of three C -C bonds and one C -O bond in a single operation. The neutral S,O-heteroacenes exhibit superior stability and adopt a herringbone-like packing mode with efficient π-π stacking in the crystals, suggesting their potential in organic semiconducting materials. This work first exemplifies the superiority of cascade oxidative C-H annulation involving a Heck-type pathway in the development of concise access to heteroacenes.
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http://dx.doi.org/10.1002/anie.202103160DOI Listing
March 2021

Nickel-Catalyzed 3,3-Dialkynylation of 2-Aryl Acrylamides: Direct Access to -Diethynylethenes via Double Vinylic C-H Bond Activation.

Org Lett 2021 Feb 1;23(4):1199-1203. Epub 2021 Feb 1.

Key Laboratory of Green Chemistry and Technology of Ministry of Education, College of Chemistry, Sichuan University, 29 Wangjiang Road, Chengdu 610064, P.R. China.

Direct access to -diethynylethenes is achieved by a Ni-catalyzed 3,3-dialkynylation of 2-aryl acrylamides with 1-bromotriisopropylsilylacetylene. The preliminary mechanism study reveals that the reaction goes through a sequential double vinylic C-H bond activation with the assistance of an 8-aminoquinolinyl directing group.
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http://dx.doi.org/10.1021/acs.orglett.0c04137DOI Listing
February 2021

Epigenetic targeting of Waldenström macroglobulinemia cells with BET inhibitors synergizes with BCL2 or histone deacetylase inhibition.

Epigenomics 2021 Jan 24;13(2):129-144. Epub 2020 Dec 24.

Department of Molecular, Cellular and Biomedical Sciences, University of New Hampshire, Durham, NH, 03824, USA.

Waldenström macroglobulinemia (WM) is a low-grade B-cell lymphoma characterized by overproduction of monoclonal IgM. To date, there are no therapies that provide a cure for WM patients, and therefore, it is important to explore new therapies. Little is known about the efficiency of epigenetic targeting in WM. WM cells were treated with BET inhibitors (JQ1 and I-BET-762) and venetoclax, panobinostat or ibrutinib. BET inhibition reduces growth of WM cells, with little effect on survival. This finding was enhanced by combination therapy, with panobinostat (LBH589) showing the highest synergy. Our studies identify BET inhibitors as effective therapy for WM, and these inhibitors can be enhanced in combination with BCL2 or histone deacetylase inhibition.
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http://dx.doi.org/10.2217/epi-2020-0189DOI Listing
January 2021

GLI2-Mediated Inflammation in the Tumor Microenvironment.

Adv Exp Med Biol 2020 ;1263:55-65

Department of Molecular, Cellular and Biomedical Sciences, University of New Hampshire, Durham, NH, USA.

The tumor microenvironment (TME) plays an important role in the development and progression of cancer and has been shown to contribute to resistance to therapy. Inflammation is one of the hallmarks of cancer implicated in disease phenotype. Therefore, understanding the mechanisms that regulate inflammation in cancer and consequently how inflammatory mediators promote cancer progression is important for our understanding of cancer cell biology. The transcription factor GLI2 was initially identified as a member of the Hedgehog (HH) signaling pathway. During the last decade, studies have shown a novel mechanism of GLI2 regulation independent of HH signaling, where GLI2 consequently modulated several cytokine genes in the TME. These studies highlight a novel role for GLI2 as an inflammatory mediatory independent of HH stimulation. This chapter will discuss canonical and noncanonical pathways of GLI2 regulation and some of the downstream cytokine target genes regulated by GLI2.
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http://dx.doi.org/10.1007/978-3-030-44518-8_5DOI Listing
July 2020

Double -C-H Activation/Annulation of Benzamides with Aryl Alkynes: A Route to Double-Helical Polycyclic Heteroaromatics.

J Org Chem 2019 Dec 8;84(23):15697-15705. Epub 2019 Nov 8.

Key Laboratory of Green Chemistry and Technology of Ministry of Education, College of Chemistry , Sichuan University , 29 Wangjiang Road , Chengdu 610064 , People's Republic of China.

It remains a challenge to achieve ,-double annulations of primary benzamides with aryl alkynes due to competitive ,-double annulations. Herein, we employed sterically hindered 1-methylcyclohexane-1-carboxylic acid to address this challenge, the double -C-H activation of benzamides and subsequent ,-double annulations with aryl alkynes have been accomplished for the first time. The resulting product can be further transformed into a double-helical extended π-conjugated polycyclic heteroarene via Scholl oxidation, which exhibits blue emission with high fluorescence quantum yields.
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http://dx.doi.org/10.1021/acs.joc.9b02339DOI Listing
December 2019

Targeting MYC activity in double-hit lymphoma with MYC and BCL2 and/or BCL6 rearrangements with epigenetic bromodomain inhibitors.

J Hematol Oncol 2019 07 9;12(1):73. Epub 2019 Jul 9.

Department of Hematology, Mayo Clinic, Rochester, MN, USA.

Double/triple-hit lymphomas (DHL/THL) account for 5-10% of diffuse large B cell lymphoma (DLBCL) with rearrangement of MYC and BCL2 and/or BCL6 resulting in MYC overexpression. Despite the poor prognosis of DHL, R-CHOP chemotherapy remains the treatment backbone and new targeted therapy is needed. We performed comprehensive cytogenetic studies/fluorescence in situ hybridization on DLBCL and Burkitt lymphoma cell lines (n = 11) to identify the DHL/THL DLBCL in vitro model. We identified MYC/IG in Raji and Ramos (single hit); MYC/IG-BCL2 (DHL) in DOHH2, OCI-LY1, SUDHL2, and OCI-LY10; MYC/IG-BCL2/BCL6 (THL) in VAL; and no MYC rearrangement in U2932 and HBL1 (WT-MYC). Targeting MYC in the DHL/THL DLBCLs through bromodomain extra-terminal inhibitors (BETi) (JQ1, I-BET, and OTX015) significantly (p < 0.05) reduced proliferation, similar to WT-MYC cells, accompanied by decreased MYC but not BCL2 protein. Moreover, BETi suppressed MYC transcription and decreased BRD4 binding to MYC promoter in DHL cells. CD47 and PD-L1 are immunoregulatory molecules often expressed on tumors and regulated by MYC. High levels of surface CD47 but not surface PD-L1 was observed in DHL/THL, which was reduced by JQ1 treatment. BETi in combination with Pan-HDAC inhibitor had a limited effect on survival of DHL/THL, while combination of BETi and BCL2 inhibitor (ABT-199) had a significant (p < 0.005) inhibitory effect on survival followed by BCL-XL inhibition. Overall, the data suggests that MYC-expressing DLBCLs are probably addicted to the MYC-oncogenic effect regardless of MYC rearrangements. In summary, we identified an in vitro model for DHL/THL DLBCLs and provide evidence for the therapeutic potential of BET inhibitor alone or in combination with BCL2 inhibitor.
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http://dx.doi.org/10.1186/s13045-019-0761-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6617630PMC
July 2019

Targeting IL-6 receptor reduces IgM levels and tumor growth in Waldenström macroglobulinemia.

Oncotarget 2019 May 21;10(36):3400-3407. Epub 2019 May 21.

Department of Biological Sciences, Northern Illinois University, DeKalb, IL, USA.

The tumor microenvironment (TME) plays an important role in cancer cell biology and is implicated in resistance to therapy. In Waldenström macroglobulinemia (WM), a subtype of Non-Hodgkin lymphoma, the TME modulates WM biology by secreting cytokines that promote the malignant phenotype. In previous work, we have shown that TME-IL-6 promotes WM cell growth and IgM secretion in WM. Tocilizumab/Actemra is an anti-IL-6R antibody, which can competitively block IL-6 binding to the IL-6R. We investigated the efficacy of Tocilizumab in a preclinical mouse model of WM that considers the role of the TME in disease biology. Hairless SCID mice were subcutaneously implanted with BCWM.1 or RPCI-WM1 and bone marrow stromal cells. Groups of mice were treated with Tocilizumab or control antibody three times/week for 5 weeks and the effect on tumor burden and disease biology were evaluated. Although Tocilizumab had no effect on mice survival, there was a significant reduction in tumor growth rate in mice injected with RPCI-WM1 cells treated with Tocilizumab. In mice injected with BCWM.1 cells, there was a significant reduction in human IgM secretion in mice sera with Tocilizumab treatment. There was no significant change in mice weight suggesting Tocilizumab induced no toxicities to the mice. Taken together, our data found that administration of Tocilizumab to tumor bearing mice, results in a significant reduction in tumor volume and IgM secretion. Therefore, the evaluation of the role of Tocilizumab in WM patients may provide therapeutic efficacy by reducing IgM production and slowing the rate of tumor growth.
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http://dx.doi.org/10.18632/oncotarget.26946DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6534366PMC
May 2019

Bone marrow stromal cells interaction with titanium; Effects of composition and surface modification.

PLoS One 2019 22;14(5):e0216087. Epub 2019 May 22.

Department of Molecular, Cellular and Biomedical Sciences, University of New Hampshire, Durham, New Hampshire, United States of America.

Inflammation and implant loosening are major concerns when using titanium implants for hard tissue engineering applications. Surface modification is one of the promising tools to enhance tissue-material integration in metallic implants. Here, we used anodization technique to modify the surface of commercially pure titanium (CP-Ti) and titanium alloy (Ti-6Al-4V) samples. Our results show that electrolyte composition, anodization time and voltage dictated the formation of well-organized nanotubes. Although electrolyte containing HF in water resulted in nanotube formation on Ti, the presence of NH4F and ethylene glycol was necessary for successful nanotube formation on Ti-6Al-4V. Upon examination of the interaction of bone marrow stromal cells (BMSCs) with the modified samples, we found that Ti-6Al-4V without nanotubes induced cell proliferation and cluster of differentiation 40 ligand (CD40L) expression which facilitates B-cell activation to promote early bone healing. However, the expression of glioma associated protein 2 (GLI2), which regulates CD40L, was reduced in Ti-6Al-4V and the presence of nanotubes further reduced its expression. The inflammatory cytokine interleukin-6 (IL-6) expression was reduced by nanotube presence on Ti. These results suggest that Ti-6Al-4V with nanotubes may be suitable implants because they have no effect on BMSC growth and inflammation.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0216087PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6530826PMC
January 2020

Co(iii)-catalyzed Z-selective oxidative C-H/C-H cross-coupling of alkenes with triisopropylsilylacetylene.

Chem Commun (Camb) 2019 May 9;55(43):6118-6121. Epub 2019 May 9.

Key Laboratory of Green Chemistry and Technology of Ministry of Education, College of Chemistry, Sichuan University, 29 Wangjiang Road, Chengdu 610064, P. R. China.

A Co(iii)-catalyzed direct oxidative C-H/C-H cross-coupling reaction of acrylamides with triisopropylsilylacetylene is presented. It is applicable to unsubstituted, internal and terminal acrylamides with a broad functionality tolerance. The feasibility of this protocol is successfully demonstrated by the late-stage alkynylation of a derivative of steroid drug Epristeride.
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http://dx.doi.org/10.1039/c9cc02347jDOI Listing
May 2019

Sinus Surgery Is Associated with a Decrease in Aspirin-Induced Reaction Severity in Patients with Aspirin Exacerbated Respiratory Disease.

J Allergy Clin Immunol Pract 2019 May - Jun;7(5):1580-1588. Epub 2018 Dec 21.

National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle, NC.

Background: Nasal polyps influence the burden of aspirin-exacerbated respiratory disease (AERD) by contributing to eicosanoid production. AERD is diagnosed through graded aspirin challenges. It is not known how sinus surgery affects aspirin challenge outcomes.

Objective: To investigate the effects of endoscopic sinus surgery (ESS) on aspirin-induced reaction severity and on the levels of eicosanoids associated with these reactions.

Methods: Twenty-eight patients with AERD were challenged with aspirin before and 3 to 4 weeks after ESS. Respiratory parameters and plasma and urine levels of eicosanoids were compared before and after challenges.

Results: Before ESS, AERD diagnosis was confirmed in all study patients by aspirin challenges that resulted in hypersensitivity reactions. After ESS, reactions to aspirin were less severe in all patients and 12 of 28 patients (43%, P < .001) had no detectable reaction. A lack of clinical reaction to aspirin was associated with lower peripheral blood eosinophilia (0.1 K/μL [interquartile range (IQR) 0.1-0.3] vs 0.4 K/μL [IQR 0.2-0.8]; P = .006), lower urinary leukotriene E levels after aspirin challenge (98 pg/mg creatinine [IQR 61-239] vs 459 pg/mg creatinine [IQR 141-1344]; P = .02), and lower plasma prostaglandin D to prostaglandin E ratio (0 [±0] vs 0.43 [±0.2]; P = .03), compared with those who reacted.

Conclusions: Sinus surgery results in decreased aspirin sensitivity and a decrease in several plasma and urine eicosanoid levels in patients with AERD. Diagnostic aspirin challenges should be offered to patients with suspected AERD before ESS to increase diagnostic accuracy. Patients with established AERD could undergo aspirin desensitizations after ESS as the severity of their aspirin-induced hypersensitivity reactions lessens.
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http://dx.doi.org/10.1016/j.jaip.2018.12.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6511299PMC
August 2020

P450-Humanized and Human Liver Chimeric Mouse Models for Studying Xenobiotic Metabolism and Toxicity.

Drug Metab Dispos 2018 11 9;46(11):1734-1744. Epub 2018 Aug 9.

Baylor College of Medicine, Houston, Texas (K.-D.B., M.B., F.P.P.); and Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson, Arizona (W.H., N.K., L.D., X.F., Q.-Y.Z., X.D.)

Preclinical evaluation of drug candidates in experimental animal models is an essential step in drug development. Humanized mouse models have emerged as a promising alternative to traditional animal models. The purpose of this mini-review is to provide a brief survey of currently available mouse models for studying human xenobiotic metabolism. Here, we describe both genetic humanization and human liver chimeric mouse models, focusing on the advantages and limitations while outlining their key features and applications. Although this field of biomedical science is relatively young, these humanized mouse models have the potential to transform preclinical drug testing and eventually lead to a more cost-effective and rapid development of new therapies.
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http://dx.doi.org/10.1124/dmd.118.083303DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6199624PMC
November 2018

Global, integrated analysis of methylomes and transcriptomes from laser capture microdissected bronchial and alveolar cells in human lung.

Epigenetics 2018 10;13(3):264-274. Epub 2018 May 10.

a Department of Genetics , Albert Einstein College of Medicine , 1301 Morris Park Ave, Bronx , New York 10461 , USA.

Gene regulatory analysis of highly diverse human tissues in vivo is essentially constrained by the challenge of performing genome-wide, integrated epigenetic and transcriptomic analysis in small selected groups of specific cell types. Here we performed genome-wide bisulfite sequencing and RNA-seq from the same small groups of bronchial and alveolar cells isolated by laser capture microdissection from flash-frozen lung tissue of 12 donors and their peripheral blood T cells. Methylation and transcriptome patterns differed between alveolar and bronchial cells, while each of these epithelia showed more differences from mesodermally-derived T cells. Differentially methylated regions (DMRs) between alveolar and bronchial cells tended to locate at regulatory regions affecting promoters of 4,350 genes. A large number of pathways enriched for these DMRs including GTPase signal transduction, cell death, and skeletal muscle. Similar patterns of transcriptome differences were observed: 4,108 differentially expressed genes (DEGs) enriched in GTPase signal transduction, inflammation, cilium assembly, and others. Prioritizing using DMR-DEG regulatory network, we highlighted genes, e.g., ETS1, PPARG, and RXRG, at prominent alveolar vs. bronchial cell discriminant nodes. Our results show that multi-omic analysis of small, highly specific cells is feasible and yields unique physiologic loci distinguishing human lung cell types in situ.
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http://dx.doi.org/10.1080/15592294.2018.1441650DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5997142PMC
February 2019

Elevated GLI3 expression in germinal center diffuse large B cell lymphoma.

Leuk Lymphoma 2018 11 21;59(11):2743-2745. Epub 2018 Feb 21.

a Department of Molecular, Cellular and Biomedical Sciences , University of New Hampshire , Durham , NH , USA.

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http://dx.doi.org/10.1080/10428194.2018.1439169DOI Listing
November 2018

Novel Molecular Mechanism of Regulation of CD40 Ligand by the Transcription Factor GLI2.

J Immunol 2017 06 1;198(11):4481-4489. Epub 2017 May 1.

Department of Biological Sciences, Northern Illinois University, DeKalb, IL 60115

The interaction between tumor cells and their surrounding microenvironment is essential for the growth and persistence of cancer cells. This interaction is mediated, in part, by cytokines. Although the role of cytokines in normal and malignant cell biology is well established, many of the molecular mechanisms regulating their expression remain elusive. In this article, we provide evidence of a novel pathway controlling the transcriptional activation of CD40L in bone marrow-derived stromal cells. Using a PCR-based screening of cytokines known to play a role in the biology of bone marrow malignancies, we identified CD40L as a novel GLI2 target gene in stromal cells. CD40L plays an important role in malignant B cell biology, and we found increased Erk phosphorylation and cell growth in malignant B cells cocultured with CD40L-expressing stromal cells. Further analysis indicated that GLI2 overexpression induced increased CD40L expression, and, conversely, GLI2 knockdown reduced CD40L expression. Using luciferase and chromatin immunoprecipitation assays, we demonstrate that GLI2 directly binds and regulates the activity of the CD40L promoter. We found that the CCR3-PI3K-AKT signaling modulates the GLI2-CD40L axis, and GLI2 is required for CCR3-PI3K-AKT-mediated regulation of the CD40L promoter. Finally, coculture of malignant B cells with cells stably expressing human CD40L results in increased Erk phosphorylation and increased malignant B cell growth, indicating that CD40L in the tumor microenvironment promotes malignant B cell activation. Therefore, our studies identify a novel molecular mechanism of regulation of CD40L by the transcription factor GLI2 in the tumor microenvironment downstream of CCR3 signaling.
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http://dx.doi.org/10.4049/jimmunol.1601490DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5473292PMC
June 2017

Plasma 15-Hydroxyeicosatetraenoic Acid Predicts Treatment Outcomes in Aspirin-Exacerbated Respiratory Disease.

J Allergy Clin Immunol Pract 2017 Jul - Aug;5(4):998-1007.e2. Epub 2017 Jan 31.

Albert Einstein College of Medicine/Montefiore Medical Center, Bronx, NY.

Background: Aspirin desensitization followed by daily aspirin provides therapeutic benefits to patients with aspirin-exacerbated respiratory disease (AERD). It is not well understood how eicosanoid levels change during aspirin treatment.

Objective: To investigate associations between clinical outcomes of aspirin treatment and plasma eicosanoid levels in patients with AERD.

Methods: Thirty-nine patients with AERD were offered aspirin treatment (650 mg twice daily) for 4 weeks. Respiratory parameters and plasma levels of multiple eicosanoids were recorded at baseline and after 4 weeks of aspirin therapy using the Asthma Control Test and Rhinoconjunctivitis Quality of Life Questionnaire. Respiratory function was evaluated using the FEV and nasal inspiratory peak flow.

Results: After aspirin treatment, respiratory symptoms improved in 16 patients, worsened in 12 patients, and did not change in 4 patients. Seven patients were unable to complete the desensitization protocol. Patients with symptom improvement had higher baseline plasma 15-hydroxyeicosatetraenoic acid (15-HETE) levels than did patients with symptom worsening: 7006 pg/mL (interquartile range, 6056-8688 pg/mL) versus 4800 pg/mL (interquartile range, 4238-5575 pg/mL), P = .0005. Baseline 15-HETE plasma levels positively correlated with the change in Asthma Control Test score (r = 0.61; P = .001) and in FEV after 4 weeks of aspirin treatment (r = 0.49; P = .01). It inversely correlated with Rhinoconjunctivitis Quality of Life Questionnaire score (r = -0.58; P = .002). Black and Latino patients were more likely to have symptom worsening on aspirin or fail to complete the initial desensitization than white, non-Latino patients (P = .02).

Conclusions: In patients with AERD, low baseline 15-HETE plasma levels and black or Latino ethnicity are associated with worsening of respiratory symptoms during aspirin treatment.
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http://dx.doi.org/10.1016/j.jaip.2016.11.021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5491381PMC
January 2018

Modeling Heterogeneity in Direct Infectious Disease Transmission in a Compartmental Model.

Int J Environ Res Public Health 2016 Feb 24;13(3). Epub 2016 Feb 24.

State Key Laboratory of Management and Control for Complex Systems, Institute of Automation, Chinese Academy of Science, Beijing 100190, China.

Mathematical models have been used to understand the transmission dynamics of infectious diseases and to assess the impact of intervention strategies. Traditional mathematical models usually assume a homogeneous mixing in the population, which is rarely the case in reality. Here, we construct a new transmission function by using as the probability density function a negative binomial distribution, and we develop a compartmental model using it to model the heterogeneity of contact rates in the population. We explore the transmission dynamics of the developed model using numerical simulations with different parameter settings, which characterize different levels of heterogeneity. The results show that when the reproductive number, R₀, is larger than one, a low level of heterogeneity results in dynamics similar to those predicted by the homogeneous mixing model. As the level of heterogeneity increases, the dynamics become more different. As a test case, we calibrated the model with the case incidence data for severe acute respiratory syndrome (SARS) in Beijing in 2003, and the estimated parameters demonstrated the effectiveness of the control measures taken during that period.
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http://dx.doi.org/10.3390/ijerph13030253DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4808916PMC
February 2016

Genome Wide Methylome Alterations in Lung Cancer.

PLoS One 2015 18;10(12):e0143826. Epub 2015 Dec 18.

Department of Medicine/Pulmonary, Albert Einstein College of Medicine, Bronx, New York, United States of America.

Aberrant cytosine 5-methylation underlies many deregulated elements of cancer. Among paired non-small cell lung cancers (NSCLC), we sought to profile DNA 5-methyl-cytosine features which may underlie genome-wide deregulation. In one of the more dense interrogations of the methylome, we sampled 1.2 million CpG sites from twenty-four NSCLC tumor (T)-non-tumor (NT) pairs using a methylation-sensitive restriction enzyme- based HELP-microarray assay. We found 225,350 differentially methylated (DM) sites in adenocarcinomas versus adjacent non-tumor tissue that vary in frequency across genomic compartment, particularly notable in gene bodies (GB; p<2.2E-16). Further, when DM was coupled to differential transcriptome (DE) in the same samples, 37,056 differential loci in adenocarcinoma emerged. Approximately 90% of the DM-DE relationships were non-canonical; for example, promoter DM associated with DE in the same direction. Of the canonical changes noted, promoter (PR) DM loci with reciprocal changes in expression in adenocarcinomas included HBEGF, AGER, PTPRM, DPT, CST1, MELK; DM GB loci with concordant changes in expression included FOXM1, FERMT1, SLC7A5, and FAP genes. IPA analyses showed adenocarcinoma-specific promoter DMxDE overlay identified familiar lung cancer nodes [tP53, Akt] as well as less familiar nodes [HBEGF, NQO1, GRK5, VWF, HPGD, CDH5, CTNNAL1, PTPN13, DACH1, SMAD6, LAMA3, AR]. The unique findings from this study include the discovery of numerous candidate The unique findings from this study include the discovery of numerous candidate methylation sites in both PR and GB regions not previously identified in NSCLC, and many non-canonical relationships to gene expression. These DNA methylation features could potentially be developed as risk or diagnostic biomarkers, or as candidate targets for newer methylation locus-targeted preventive or therapeutic agents.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0143826PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4684329PMC
June 2016

Modulation of the IL-6 Receptor α Underlies GLI2-Mediated Regulation of Ig Secretion in Waldenström Macroglobulinemia Cells.

J Immunol 2015 Sep 3;195(6):2908-16. Epub 2015 Aug 3.

Department of Biological Sciences, Northern Illinois University, DeKalb, IL 60115; and

Ig secretion by terminally differentiated B cells is an important component of the immune response to foreign pathogens. Its overproduction is a defining characteristic of several B cell malignancies, including Waldenström macroglobulinemia (WM), where elevated IgM is associated with significant morbidity and poor prognosis. Therefore, the identification and characterization of the mechanisms controlling Ig secretion are of great importance for the development of future therapeutic approaches for this disease. In this study, we define a novel pathway involving the oncogenic transcription factor GLI2 modulating IgM secretion by WM malignant cells. Pharmacological and genetic inhibition of GLI2 in WM malignant cells resulted in a reduction in IgM secretion. Screening for a mechanism identified the IL-6Rα (gp80) subunit as a downstream target of GLI2 mediating the regulation of IgM secretion. Using a combination of expression, luciferase, and chromatin immunoprecipitation assays we demonstrate that GLI2 binds to the IL-6Rα promoter and regulates its activity as well as the expression of this receptor. Additionally, we were able to rescue the reduction in IgM secretion in the GLI2 knockdown group by overexpressing IL-6Rα, thus defining the functional significance of this receptor in GLI2-mediated regulation of IgM secretion. Interestingly, this occurred independent of Hedgehog signaling, a known regulator of GLI2, as manipulation of Hedgehog had no effect on IgM secretion. Given the poor prognosis associated with elevated IgM in WM patients, components of this new signaling axis could be important therapeutic targets.
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http://dx.doi.org/10.4049/jimmunol.1402974DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4561187PMC
September 2015

Lung cancer transcriptomes refined with laser capture microdissection.

Am J Pathol 2014 Nov 14;184(11):2868-84. Epub 2014 Aug 14.

Division of Pulmonary Medicine, Department of Medicine, Albert Einstein College of Medicine, Bronx, New York. Electronic address:

We evaluated the importance of tumor cell selection for generating gene signatures in non-small cell lung cancer. Tumor and nontumor tissue from macroscopically dissected (Macro) surgical specimens (31 pairs from 32 subjects) was homogenized, extracted, amplified, and hybridized to microarrays. Adjacent scout sections were histologically mapped; sets of approximately 1000 tumor cells and nontumor cells (alveolar or bronchial) were procured by laser capture microdissection (LCM). Within histological strata, LCM and Macro specimens exhibited approximately 67% to 80% nonoverlap in differentially expressed (DE) genes. In a representative subset, LCM uniquely identified 300 DE genes in tumor versus nontumor specimens, largely attributable to cell selection; 382 DE genes were common to Macro, Macro with preamplification, and LCM platforms. RT-qPCR validation in a 33-gene subset was confirmatory (ρ = 0.789 to 0.964, P = 0.0013 to 0.0028). Pathway analysis of LCM data suggested alterations in known cancer pathways (cell growth, death, movement, cycle, and signaling components), among others (eg, immune, inflammatory). A unique nine-gene LCM signature had higher tumor-nontumor discriminatory accuracy (100%) than the corresponding Macro signature (87%). Comparison with Cancer Genome Atlas data sets (based on homogenized Macro tissue) revealed both substantial overlap and important differences from LCM specimen results. Thus, cell selection via LCM enhances expression profiling precision, and confirms both known and under-appreciated lung cancer genes and pathways.
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http://dx.doi.org/10.1016/j.ajpath.2014.06.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4215031PMC
November 2014

Site-specific methylated reporter constructs for functional analysis of DNA methylation.

Epigenetics 2013 Nov 4;8(11):1176-87. Epub 2013 Sep 4.

Pulmonary Medicine; Albert Einstein College of Medicine; Bronx, NY USA; Genetics; Albert Einstein College of Medicine; Bronx, NY USA.

Methods to experimentally alter and functionally evaluate cytosine methylation in a site-specific manner have proven elusive. We describe a site-specific DNA methylation method, using synthetically methylated primers and high fidelity PCR coupled with ligation of reporter constructs. We applied this method to introduce methylated cytosines into fragments of the respective DAPK and RASSF1A promoters that had been cloned into luciferase reporters. We found that methylation of 3-7 residue CpG clusters that were 5' adjacent to the transcription start site (TSS) of the DAPK gene produced up to a 54% decrease in promoter activity (p<0.01). Similarly, for RASSF1A promoter reporter constructs, the methylation of either of two clusters of four CpGs each, but not an intervening cluster, produced a 63% decrease in promoter activity (p<0.01), suggesting that precise mCpG position is crucial, and factors other than simple proximity to the TSS are at play. Chromatin immunoprecipitation analysis of these reporter constructs demonstrated that transcription factor Oct-1 and Sp1 preferentially bound the unmethylated vs. methylated DAPK or RASSF1A promoter reporter constructs at the functional CpG sites. Histone H1, hnRNP1, and MeCP2 showed preferential binding to methylated sequence at functional sites in these reporter constructs, as well as highly preferential (> 8-80-fold) binding to native methylated vs. unmethylated chromatin. These results suggest that: (1) site-specific, precision DNA methylation of a reporter construct can be used for functional analysis of commonly observed gene promoter methylation patterns; (2) the reporter system contains key elements of the endogenous chromatin machinery.
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http://dx.doi.org/10.4161/epi.26195DOI Listing
November 2013

A quantitative method to identify microRNAs targeting a messenger RNA using a 3'UTR RNA affinity technique.

Anal Biochem 2013 Dec 9;443(1):1-12. Epub 2013 Aug 9.

Division of Pulmonary Medicine, Albert Einstein College of Medicine, Bronx, NY 10461, USA.

The identification of specific microRNAs (miRNAs) that target a given messenger RNA (mRNA) is essential for studies in gene regulation, but the available bioinformatic software programs are often unreliable. We have developed a unique experimental miRNA affinity assay whereby a 3'UTR RNA is end-labeled with biotin, immobilized, and then used as a bait sequence for affinity pull-down of miRNAs. After washes and release, cloning and sequencing identify the miRNAs. Binding affinity is quantitated by quantitative polymerase chain reaction (qPCR), comparing released and original input concentrations. As an initial demonstration, the TCF8/ZEB1 mRNA affinity pull-down yielded miR-200 family member miRs in the majority of clones, and binding affinity was approximately 100%; virtually all copies of miR-200c bound the immobilized mRNA transcript. For validation in cells, miR-200c strongly inhibited expression of a TCF8 luciferase reporter, native TCF8 mRNA, and protein levels, which contrasted with other recovered miRNAs with lower binding affinities. For Smad4 mRNA, miR-150 (and others) displayed a binding affinity of 39% (or less) yet did not inhibit a Smad4 reporter, native Smad4 mRNA, or protein levels. These results were not predicted by available software. This work demonstrates this miRNA binding affinity assay to be a novel yet facile experimental means of identification of miRNAs targeting a given mRNA.
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http://dx.doi.org/10.1016/j.ab.2013.08.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4112567PMC
December 2013

Genetic and epigenetic regulation of AHR gene expression in MCF-7 breast cancer cells: role of the proximal promoter GC-rich region.

Biochem Pharmacol 2012 Sep 21;84(5):722-35. Epub 2012 Jun 21.

Wadsworth Center, New York State Department of Health, Albany, NY 12201, USA.

The aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor, contributes to carcinogenesis through its role in the regulation of cytochrome P450 1 (CYP1)-catalyzed metabolism of carcinogens. Here, we investigated genetic and epigenetic mechanisms that affect AhR expression. Analyses of the human AHR proximal promoter in MCF-7 human breast cancer cells using luciferase assays and electrophoretic mobility shift assays revealed multiple specificity protein (Sp) 1 binding sequences that are transcriptional activators in vitro. The regulation of AhR expression was evaluated in long-term estrogen exposed (LTEE) MCF-7 cells, which showed increased AhR expression, enhanced CYP1 inducibility, and increased capacity to form DNA adducts when exposed to the dietary carcinogen, 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine. The increased AhR expression in LTEE cells was found not to result from increased mRNA stability, differential RNA processing, or decreased DNA methylation. Analysis of the AHR proximal promoter region using chromatin immunoprecipitation confirmed that enhanced expression of AhR in LTEE cells involves changes in histone modifications, notably decreased trimethylation of histone 3, lysine 27. Upon further examination of the GC-rich Sp1-binding region, we confirmed that it contains a polymorphic (GGGGC)(n) repeat. In a population of newborns from New York State, the allele frequency of (GGGGC)(n) was n = 4 > 5 ≫ 6, 2. Circular dichroism spectroscopy revealed the ability of sequences of this GC-rich region to form guanine-quadruplex structures in vitro. These studies revealed multiple levels at which AhR expression may be controlled, and offer additional insights into mechanisms regulating AhR expression that can ultimately impact carcinogenesis.
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http://dx.doi.org/10.1016/j.bcp.2012.06.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3965201PMC
September 2012

High-throughput library screening identifies two novel NQO1 inducers in human lung cells.

Am J Respir Cell Mol Biol 2012 Mar 20;46(3):365-71. Epub 2011 Oct 20.

M.D. Department of Health Science Research, Mayo Clinic, 200 First Street S.W., Rochester, MN 55905, USA.

Many phytochemicals possess antioxidant and cancer-preventive properties, some putatively through antioxidant response element-mediated phase II metabolism, entailing mutagen/oxidant quenching. In our recent studies, however, most candidate phytochemical agents were not potent in inducing phase II genes in normal human lung cells. In this study, we applied a messenger RNA (mRNA)-specific gene expression-based high throughput in vitro screening approach to discover new, potent plant-derived phase II inducing chemopreventive agents. Primary normal human bronchial epithelial (NHBE) cells and immortalized human bronchial epithelial cells (HBECs) were exposed to 800 individual compounds in the MicroSource Natural Products Library. At a level achievable in humans by diet (1.0 μM), 2,3-dihydroxy-4-methoxy-4'-ethoxybenzophenone (DMEBP), triacetylresveratrol (TRES), ivermectin, sanguinarine sulfate, and daunorubicin induced reduced nicotinamide adenine dinucleotide phosphate:quinone oxidoreductase 1 (NQO1) mRNA and protein expression in NHBE cells. DMEBP and TRES were the most attractive agents as coupling potency and low toxicity for induction of NQO1 (mRNA level, ≥3- to 10.8-fold that of control; protein level, ≥ two- to fourfold that of control). Induction of glutathione S-transferase pi mRNA expression was modest, and none was apparent for glutathione S-transferase pi protein expression. Measurements of reactive oxygen species and glutathione/oxidized glutathione ratio showed an antioxidant effect for DMEBP, but no definite effect was found for TRES in NHBE cells. Exposure of NHBE cells to H(2)O(2) induced nuclear translocation of nuclear factor erythroid 2-related factor 2, but this translocation was not significantly inhibited by TRES and DMEBP. These studies show that potency and low toxicity may align for two potential NQO1-inducing agents, DMEBP and TRES.
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http://dx.doi.org/10.1165/rcmb.2011-0301OCDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3326428PMC
March 2012

Candidate dietary phytochemicals modulate expression of phase II enzymes GSTP1 and NQO1 in human lung cells.

J Nutr 2010 Aug 16;140(8):1404-10. Epub 2010 Jun 16.

Division of Pulmonary Medicine, Albert Einstein College of Medicine, Bronx, NY, USA.

Many phytochemicals possess cancer-preventive properties, some putatively through phase II metabolism-mediated mutagen/oxidant quenching. We applied human lung cells in vitro to investigate the effects of several candidate phytopreventive agents, including green tea extracts (GTE), broccoli sprout extracts (BSE), epigallocatechin gallate (EGCG), sulforaphane (SFN), phenethyl isothiocyanate (PEITC), and benzyl isothiocyanate (BITC), on inducing phase II enzymes glutathione S-transferase P1 (GSTP1) and NAD(P)H:quinone oxidoreductase 1 (NQO1) at mRNA and protein levels. Primary normal human bronchial epithelial cells (NHBE), immortalized human bronchial epithelial cells (HBEC), and lung adenocarcinoma cells (A549) were exposed to diet-achievable levels of GTE and BSE (0.5, 1.0, 2.0 mg/L), or individual index components EGCG, SFN, PEITC, BITC (0.5, 1.0, 2.0 micromol/L) for 24 h, 48 h, and 6 d, respectively. mRNA assays employed RNA-specific quantitative RT-PCR and protein assays employed Western blotting. We found that in NHBE cells, while GSTP1 mRNA levels were slightly but significantly increased after exposure to GTE or BSE, NQO1 mRNA increased to 2- to 4-fold that of control when exposed to GTE, BSE, or SFN. Effects on NQO1 mRNA expression in HBEC cells were similar. NQO1 protein expression increased up to 11.8-fold in SFN-treated NHBE cells. Both GSTP1 and NQO1 protein expression in A549 cells were constitutively high but not induced under any condition. Our results suggest that NQO1 is more responsive to the studied chemopreventive agents than GSTP1 in human lung cells and there is discordance between single agent and complex mixture effects. We conclude that modulation of lung cell phase II metabolism by chemopreventive agents requires cell- and agent-specific discovery and testing.
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http://dx.doi.org/10.3945/jn.110.121905DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2903300PMC
August 2010

Smoking-Related Gene Expression in Laser Capture-Microdissected Human Lung.

Clin Cancer Res 2009 Dec;15(24):7562-7570

Authors' Affiliations: Division of Pulmonary Medicine, Department of Medicine, Department of Epidemiology and Population Health, and Department of Genetics, Albert Einstein College of Medicine, Bronx, New York; and Laboratory of Human Toxicology and Molecular Epidemiology, Wadsworth Center, New York State Department of Health, Albany, New York.

PURPOSE: Interindividual differences in quantitative expression could underlie a propensity for lung cancer. To determine precise individual gene expression signatures on a lung compartment-specific basis, we investigated the expression of carcinogen metabolism genes encoding cytochromes P450 (CYP) 1B1, 2A13, GSTP1, and a tumor suppressor gene p16 in laser capture-microdissected samples of human alveolar compartment (AC) and bronchial epithelial compartment (BEC) lung tissue from 62 smokers and nonsmokers. EXPERIMENTAL DESIGN: Tobacco exposure was determined by plasma nicotine, cotinine, and smoking history. Precise mRNA expression was determined using our RNA-specific qRT-PCR strategy, and correlated with detailed demographic and clinical characteristics. RESULTS: Several correlations of mRNA expression included (a) CYP1B1 in AC (positively with plasma nicotine level, P = 0.008; plasma cotinine level, P = 0.001), (b) GSTP1 in AC (positively with plasma cotinine level, P = 0.003), and (c) GSTP1 in BEC (negatively with smoke dose, P = 0.043; occupational risk, P = 0.019). CYP2A13 was rarely expressed in AC and not expressed in BEC. p16 expression was not correlated with any measured factor. For each gene, subjects showed expression that was individually concordant between these compartments. No clear association of mRNA expression with lung cancer risk was observed in this pilot analysis. CONCLUSIONS: The association between lung mRNA expression and tobacco exposure implies that gene-tobacco interaction is a measurable quantitative trait, albeit with wide interindividual variation. Gene expression tends to be concordant for alveolar and bronchial compartments for these genes in an individual, controlling for proximate tobacco exposure. (Clin Cancer Res 2009;15(24):7562-70).
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http://dx.doi.org/10.1158/1078-0432.CCR-09-1694DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4238890PMC
December 2009

Gene promoter methylation assayed in exhaled breath, with differences in smokers and lung cancer patients.

Respir Res 2009 Sep 25;10:86. Epub 2009 Sep 25.

Wadsworth Center, Human Toxicology & Molecular Epidemiology, Albany, NY, USA.

Background: There is a need for new, noninvasive risk assessment tools for use in lung cancer population screening and prevention programs.

Methods: To investigate the technical feasibility of determining DNA methylation in exhaled breath condensate, we applied our previously-developed method for tag-adapted bisulfite genomic DNA sequencing (tBGS) for mapping of DNA methylation, and adapted it to exhaled breath condensate (EBC) from lung cancer cases and non-cancer controls. Promoter methylation patterns were analyzed in DAPK, RASSF1A and PAX5beta promoters in EBC samples from 54 individuals, comprised of 37 controls [current- (n = 19), former- (n = 10), and never-smokers (n = 8)] and 17 lung cancer cases [current- (n = 5), former- (n = 11), and never-smokers (n = 1)].

Results: We found: (1) Wide inter-individual variability in methylation density and spatial distribution for DAPK, PAX5beta and RASSF1A. (2) Methylation patterns from paired exhaled breath condensate and mouth rinse specimens were completely divergent. (3) For smoking status, the methylation density of RASSF1A was statistically different (p = 0.0285); pair-wise comparisons showed that the former smokers had higher methylation density versus never smokers and current smokers (p = 0.019 and p = 0.031). For DAPK and PAX5beta, there was no such significant smoking-related difference. Underlying lung disease did not impact on methylation density for this geneset. (4) In case-control comparisons, CpG at -63 of DAPK promoter and +52 of PAX5beta promoter were significantly associated with lung cancer status (p = 0.0042 and 0.0093, respectively). After adjusting for multiple testing, both loci were of borderline significance (p(adj) = 0.054 and 0.031). (5) The DAPK gene had a regional methylation pattern with two blocks (1) approximately -215--113 and (2) -84-+26; while similar in block 1, there was a significant case-control difference in methylation density in block 2 (p = 0.045); (6)Tumor stage and histology did not impact on the methylation density among the cases. (7) The results of qMSP applied to EBC correlated with the corresponding tBGS sequencing map loci.

Conclusion: Our results show that DNA methylation in exhaled breath condensate is detectable and is likely of lung origin. Suggestive correlations with smoking and lung cancer case-control status depend on individual gene and CpG site examined.
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http://dx.doi.org/10.1186/1465-9921-10-86DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2759916PMC
September 2009

Haplotype-tagging single nucleotide polymorphisms in the GSTP1 gene promoter and susceptibility to lung cancer.

Cancer Detect Prev 2009 17;32(5-6):403-15. Epub 2009 Mar 17.

Laboratory of Human Toxicology and Molecular Epidemiology, Wadsworth Center, New York State Department of Health, Albany, NY, USA.

Background: Glutathione S-transferase (GST) P1 is a major phase II xenobiotic-metabolizing enzyme in the human lung. Our laboratory had previously identified nine single nucleotide polymorphisms (SNPs) in the GSTP1 gene promoter, which were then grouped into three main haplotypes (Hap1, Hap2, and Hap3) based on statistical inference. Hap3 was found to display a high expression phenotype. The main objective of the current study was to test the association between GSTP1 promoter haplotypes with the risk of lung cancer after determining the promoter haplotypes experimentally through cloning and sequencing.

Methods: We conducted a case-control analysis of 150 subjects with lung cancer and 329 controls with no personal history of the disease. The three statistically inferred GSTP1 promoter haplotypes were confirmed experimentally through cloning and sequencing. Haplotype-tagging SNPs were selected and GSTP1 haplotypes were tested for genetic association to lung cancer using unconditional logistic regression after adjusting for confounders. Statistical interaction between GSTP1 promoter haplotypes with either cigarette smoking or dietary fruit and vegetable intake were tested using the likelihood ratio test.

Results: We did not find protective effects of Hap3 against lung cancer, despite an adequately powered design for this main effect. Homozygous variants of tagSNPs -1738 T>A and -354 G>T, which tag Hap2, showed an increased (but statistically non-significant) risk of lung cancer among all subjects as well as among individuals with low fruit and vegetable intake, compared to homozygous wildtypes for these SNPs. We did not find significant interactions between Hap2 and dietary intake of fruits and vegetables.

Conclusions: Our results do not support significant main and modifying effects for GSTP1 promoter haplotypes on susceptibility to lung cancer in this population, but reinforce the protective effects of dietary intake of fruits and vegetables.
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http://dx.doi.org/10.1016/j.cdp.2009.02.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3730463PMC
August 2009

The expression and antigenicity of a truncated spike-nucleocapsid fusion protein of severe acute respiratory syndrome-associated coronavirus.

BMC Microbiol 2008 Nov 28;8:207. Epub 2008 Nov 28.

Beijing Institute of Genomics, Chinese Academy of Sciences, Beijing, PR China.

Background: In the absence of effective drugs, controlling SARS relies on the rapid identification of cases and appropriate management of the close contacts, or effective vaccines for SARS. Therefore, developing specific and sensitive laboratory tests for SARS as well as effective vaccines are necessary for national authorities.

Results: Genes encoding truncated nucleocapsid (N) and spike (S) proteins of SARSCoV were cloned into the expression vector pQE30 and fusionally expressed in Escherichia coli M15. The fusion protein was analyzed for reactivity with SARS patients' sera and with anti-sera against the two human coronaviruses HCoV 229E and HCoV OC43 by ELISA, IFA and immunoblot assays. Furthermore, to evaluate the antigen-specific humoral antibody and T-cell responses in mice, the fusion protein was injected into 6-week-old BALB/c mice and a neutralization test as well as a T-cell analysis was performed. To evaluate the antiviral efficacy of immunization, BALB/c mice were challenged intranasally with SARSCoV at day 33 post injection and viral loads were determined by fluorescent quantitative RT-PCR. Serological results showed that the diagnostic sensitivity and specificity of the truncated S-N fusion protein derived the SARS virus were > 99% (457/460) and 100.00% (650/650), respectively. Furthermore there was no cross-reactivity with other two human coronaviruses. High titers of antibodies to SRASCoV appeared in the immunized mice and the neutralization test showed that antibodies to the fusion protein could inhibit SARSCoV. The T cell proliferation showed that the fusion protein could induce an antigen-specific T-cell response. Fluorescent quantitative RT-PCR showed that BALB/c mice challenged intranasally with SARSCoV at day 33 post injection were completely protected from virus replication.

Conclusion: The truncated S-N fusion protein is a suitable immunodiagnostic antigen and vaccine candidate.
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http://dx.doi.org/10.1186/1471-2180-8-207DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2613400PMC
November 2008

Spatial dynamics of an epidemic of severe acute respiratory syndrome in an urban area.

Bull World Health Organ 2006 Dec;84(12):965-8

Institute of Geographical Sciences and Natural Resources Research, Chinese Academy of Sciences, Anwai, Beijing, China.

Objective: To map risk of exposure to severe acute respiratory syndrome (SARS) in an urban area and assess the ability of traditional interventions to control dispersion of the disease.

Methods: Data on the Beijing SARS epidemic were used to map spatial clusters of identified contacts and to estimate transmission of SARS using a model with a time-dependent transmission rate.

Results: The estimated transmission rate decreased dramatically from 20 to 30 April 2003. The total number of cases in the epidemic in Beijing was estimated to be 2521. Hierarchical clustering revealed that risk-exposures were widespread, but clustered in a pattern that is distinctly related to the Beijing urban ring roads.

Conclusion: Traditional control measures can be very effective at reducing transmission of SARS. Spatial patterns of risk-exposures can inform disease surveillance, prediction and control by identifying spatial target areas on which interventions should be focused.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2627565PMC
http://dx.doi.org/10.2471/blt.06.030247DOI Listing
December 2006

DNA methylation mapping by tag-modified bisulfite genomic sequencing.

Anal Biochem 2006 Aug 2;355(1):50-61. Epub 2006 Jun 2.

Laboratory of Human Toxicology and Molecular Epidemiology, Wadsworth Center, NYS Department of Health, Albany, NY 12201, USA.

A tag-modified bisulfite genomic sequencing (tBGS) method employing direct cycle sequencing of polymerase chain reaction (PCR) products at kilobase scale, without conventional DNA fragment cloning, was developed for simplified evaluation of DNA methylation sites. The method entails subjecting bisulfite-modified genomic DNA to a second-round PCR amplification employing GC-tagged primers. Qualitative results from tBGS closely correlated with those from conventional BGS (R=0.935, p=0.002). In application, the intertissue and interindividual CpG methylation differences in promoter sequence for two genes, CYP1B1 and GSTP1, were then explored across four human tissue types (peripheral blood cells, exfoliated buccal cells, paired nontumor-tumor lung tissues), and two lung cell types in culture (normal NHBE and malignant A549). Predominantly conserved methylation maps for the two gene promoters were apparent across donors and tissues. At any given CpG site, variation in the degree of methylation could be determined by the relative height of C and T peaks in the sequencing trace. Methylation maps for the GSTP1 promoter diverged between NHBE (unmethylated) and A549 (completely methylated) cells in a previously unexplored upstream region, correlating with a 2.7-fold difference in GSTP1 mRNA expression (p<0.01). The tBGS method simplifies detailed methylation scanning of kilobase-scale genomic DNA, facilitating more ambitious genomic methylation mapping studies.
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http://dx.doi.org/10.1016/j.ab.2006.05.010DOI Listing
August 2006