The role of CTCF in coordinating the expression of single gene loci.

The CCCTC-binding factor (CTCF), which binds insulator elements in vertebrates, also facilitates coordinated gene expression at several gene clusters, including the β-globin, Igf2/H19 (insulin like growth factor 2/H19 noncoding RNA), and major histocompatibility complex (MHC) class II loci. CTCF controls expression of these genes both by enabling insulator function and facilitating higher order chromatin interactions. While the role of CTCF in gene regulation is best studied at these multi-gene loci, there is also evidence that CTCF contributes to the regulated expression of single genes.

Molecular mechanisms controlling CFTR gene expression in the airway.

The low levels of CFTR gene expression and paucity of CFTR protein in human airway epithelial cells are not easily reconciled with the pivotal role of the lung in cystic fibrosis pathology. Previous data suggested that the regulatory mechanisms controlling CFTR gene expression might be different in airway epithelium in comparison to intestinal epithelium where CFTR mRNA and protein is much more abundant. Here we examine chromatin structure and modification across the CFTR locus in primary human tracheal (HTE) and bronchial (NHBE) epithelial cells and airway cell lines including 16HBE14o- and Calu3.

A population-based twin study on sleep duration and body composition.

The aim of this study is to investigate the relationship between sleep duration and body composition and to estimate the genetic contribution of sleep duration and body composition in a Chinese twin population. This cross-sectional analysis included 738 men and 511 women aged 21-72 year. Anthropometric and dual-energy X-ray absorptiometry (DXA) measures of body composition were used.

A model of early human embryonic stem cell differentiation reveals inter- and intracellular changes on transition to squamous epithelium.

The molecular events leading to human embryonic stem cell (hESC) differentiation are the subject of considerable scrutiny. Here, we characterize an in vitro model that permits analysis of the earliest steps in the transition of hESC colonies to squamous epithelium on basic fibroblast growth factor withdrawal. A set of markers (GSC, CK18, Gata4, Eomes, and Sox17) point to a mesendodermal nature of the epithelial cells with subsequent commitment to definitive endoderm (Sox17, Cdx2, nestin, and Islet1).

MUC6 mucin expression inhibits tumor cell invasion.

The MUC6 mucin has a critical protective function in the normal stomach, pancreas and duodenum and is aberrantly expressed during the progression of some gastrointestinal cancers. Our aim was to determine whether MUC6 contributes to the etiology or progression of pancreatic cancer and elucidate the molecular basis of its involvement. Expression of MUC6 glycoprotein was examined in pancreatic cancer tissues by immunofluorescence and loss of MUC6 was observed.

Associations between gene polymorphisms in fatty acid metabolism pathway and preterm delivery in a US urban black population.

There is increasing evidence suggesting that higher intakes of fish or n-3 polyunsaturated fatty acids supplements may decrease the risk of preterm delivery (PTD). We hypothesized that genetic variants of the enzymes critical to fatty acids biosynthesis and metabolism may be associated with PTD. We genotyped 231 potentially functional single nucleotide polymorphisms (SNPs) and tagSNPs in 9 genes (FADS1, FADS2, PTGS1, PTGS2, ALOX5, ALOX5AP, PTGES, PTGES2, and PTGES3) among 1,110 black mothers, including 542 mothers who delivered preterm (<37 weeks gestation) and 568 mothers who delivered full-term babies (≥37 weeks gestation) at Boston Medical Center.

C-reactive protein in adolescent twins: patterns and relationship to adiposity.

Context: Elevated C-reactive protein (CRP) is a marker of cardiovascular risk in adults. Patterns and determinants of CRP in adolescents have not been well described.

Objective: This study aimed to determine how CRP varies by age, gender, Tanner stage, and body fat composition in rural Chinese adolescents and to what degree adiposity-CRP associations are attributable to shared genetic and environmental factors.

Gene-vitamin D interactions on food sensitization: a prospective birth cohort study.

Background: It has been hypothesized that vitamin D deficiency (VDD) contributes to the development of food sensitization (FS) and then food allergy. However, the epidemiological evidence is conflicting. We aim to examine whether cord blood VDD is associated with FS and whether such association can be modified by genetic variants in a prospective birth cohort.

Gender-specific association of sleep duration with blood pressure in rural Chinese adults.

Background: There are limited data about the role of gender on the relationship between sleep duration and blood pressure (BP) from rural populations.

Methods: We conducted a cross-sectional rural population-based study. This report includes 1033 men and 783 women aged 18-65 years from a cohort of twins enrolled in Anhui, China, between 2005 and 2008.

Identification of an evolutionarily conserved regulatory element of the zebrafish col2a1a gene.

Zebrafish (Danio rerio) is an excellent model organism for the study of vertebrate development including skeletogenesis. Studies of mammalian cartilage formation were greatly advanced through the use of a cartilage specific regulatory element of the Collagen type II alpha 1 (Col2a1) gene. In an effort to isolate such an element in zebrafish, we compared the expression of two col2a1 homologues and found that expression of col2a1b, a previously uncharacterized zebrafish homologue, only partially overlaps with col2a1a.

Association of adipokines with blood pressure in rural Chinese adolescents.

Previous research has demonstrated that adipokines influence blood pressure (BP). Limited data exist in healthy adolescents, who are in a critical period for preventing the development of high BP. This study investigated the association of leptin, adiponectin and the leptin-to-adiponectin ratio (LAR) with BP in rural Chinese adolescents.

Gene polymorphisms, breast-feeding, and development of food sensitization in early childhood.

Background: The effect of breast-feeding on the development of allergic disease is uncertain. There are no data that show whether this relationship varies by individual genotypes.

Objective: We sought to evaluate the effect of breast-feeding and gene-breast-feeding interactions on food sensitization (FS) in a prospective US birth cohort.

MicroRNA regulation of expression of the cystic fibrosis transmembrane conductance regulator gene.

The CFTR (cystic fibrosis transmembrane conductance regulator) gene shows a complex temporal and spatial pattern of expression that is controlled by multiple cis-acting elements interacting with the basal promoter. Although significant progress has been made towards understanding these genomic elements, there have been no reports of post-transcriptional regulation of CFTR by miRNAs (microRNAs). In the present study, we identify two miRNAs, hsa-miR-145 and hsa-miR-494, which regulate CFTR expression by directly targeting discrete sites in the CFTR 3' UTR (untranslated region).

The role of nerve growth factor in caspase-dependent apoptosis in human BE(2)C neuroblastoma.

Purpose: The purpose of the study was to determine if nerve growth factor (NGF) stimulation induces apoptosis in the BE(2)C neuroblastoma cell line in vitro.

Methods: The LPCX retroviral vector was used to achieve stable transduction of NGF complementary DNA into BE(2)C neuroblastoma cells. Wild-type and NGF-transduced cells were then incubated with varying concentrations of NGF for varying periods.

Dynamics of zonula occludens-2 expression during preimplantation embryonic development in the hamster.

The objective was to study the expression of zonula occludens-2, a tight junction protein, during preimplantation hamster embryonic development, to predict its possible localization, source, and roles in trophectoderm differentiation and blastocyst formation in this species. Comparison of zonula occludens-2 expression pattern between the hamster and mouse preimplantation embryos from the zygote up to the blastocyst stage was also an objective of this study. Zonula occludens-2 localization was noted in nuclei of blastomeres in all stages of hamster and mouse embryonic development.

Genomic approaches to studying CFTR transcriptional regulation.

The CFTR gene was identified over 20 years ago, and yet how the gene is transcriptionally regulated is not fully understood. Completion of the human genome sequence has encouraged a new generation of genomic techniques that can be used to identify and characterize the regulatory elements of the genome, which are often hidden in non-coding regions. In this chapter we describe two techniques that we have used to identify regulatory regions of the CFTR locus: DNase-chip, which utilizes DNase I-digested chromatin hybridized to tiled microarrays in order to locate regions of the CFTR locus that are "open" and thus likely regions of transcription factor binding; and quantitative chromosome conformation capture (q3C), which uses quantitative PCR analysis of digested and ligated, crosslinked chromosomes to measure physical interactions between distal genomic regions.

Introduction to Section II: RNA methods to approach CFTR expression.

In this section, we review methods for the analysis of the CFTR gene and its transcript. First, we discuss techniques to accurately measure levels of CFTR mRNA in primary human cells; next, protocols for measuring CFTR transcripts that contain premature termination codons and for evaluating the role of nonsense-mediated decay which targets these transcripts; a further chapter considers methodology to investigate pre-mRNA splicing. The penultimate chapter concentrates on methods for evaluating microRNA regulation of gene expression in the context of airway disease.

Gestational alloimmune liver disease in cases of fetal death.

Objective: To determine whether alloimmune liver disease can be identified as a cause of fetal death.

Study Design: This is a retrospective examination of the autopsy tissue of 6 stillborn fetuses and 2 extreme preterm infants (gestational age, 20 to 34 weeks) drawn from families referred for suspected neonatal hemochromatosis. Thirteen appropriate nondisease controls and 8 cases of neonatal acute liver failure with known etiology were also examined.

The impact of epigenomics on future drug design and new therapies.

The future of drug design and the development of new therapeutics will rely on our ability to unravel the complexities of the epigenome in normal and disease states. Proper epigenetic regulation is essential for normal differentiation in embryogenesis and development. Conversely, abnormal epigenetic regulation is a feature of complex diseases, including cancer, diabetes, heart disease and other pathologies.

An α-synuclein AAV gene silencing vector ameliorates a behavioral deficit in a rat model of Parkinson's disease, but displays toxicity in dopamine neurons.

Effects of silencing ectopically expressed hSNCA in rat substantia nigra (SN) were examined as a novel therapeutic approach to Parkinson's disease (PD). AAV-hSNCA with or without an AAV harboring a short-hairpin (sh)RNA targeting hSNCA or luciferase was injected into one SN. At 9weeks, hSNCA-expressing rats had reduced SN dopamine (DA) neurons and exhibited a forelimb deficit.

High resolution three-dimensional imaging: Evidence for cell cycle reentry in regenerating skeletal muscle.

Newts and other urodele amphibians can replace lost structures including limbs, providing a vertebrate model for the study of regeneration of complex tissues. The composite of different cell and tissue types in the limb, however, presents a challenge for their imaging in three-dimensions (3D) at cellular level resolution. To observe myofibers in vivo without distortion, we developed a streamlined protocol whereby 80 μm thick cryosections are mounted on slides, processed for immunohistochemistry, imaged using confocal microscopy and z-stacks rendered in 3D.

Embryonic signaling in melanoma: potential for diagnosis and therapy.

As the frequency of melanoma diagnosis increases, current treatment strategies are still struggling to significantly impact patient survival. Some promise has been shown in treating certain melanomas by targeting activated signaling pathways resulting from specific mutations in proteins, such as BRAF and NRAS. Recently, the identification of embryonic signaling pathways in melanoma has helped us better understand certain biological characteristics, such as cellular heterogeneity and phenotypic plasticity, and has provided novel insight pertinent to diagnosis and therapy.

Cord blood immune biomarkers in small for gestational age births.

Fetal growth restriction is a risk factor for development of adulthood diseases, but the biological mechanism of this association remains unknown. Limited biomarkers have been studied in settings of preterm birth and maternal inflammation, but the relationship between a wide range of immune biomarkers and fetal growth has not been studied. The hypothesis of this study was that fetal growth restriction is associated with altered immune biomarker levels.

A microRNA embedded AAV α-synuclein gene silencing vector for dopaminergic neurons.

Alpha-synuclein (SNCA), an abundantly expressed presynaptic protein, is implicated in Parkinson's disease (PD). Since over-expression of human SNCA (hSNCA) leads to death of dopaminergic (DA) neurons in human, rodent and fly brain, hSNCA gene silencing may reduce levels of toxic forms of SNCA and ameliorate degeneration of DA neurons in PD. To begin to develop a gene therapy for PD based on hSNCA gene silencing, two AAV gene silencing vectors were designed, and tested for efficiency and specificity of silencing, as well as toxicity in vitro.

Genomic approaches for the discovery of CFTR regulatory elements.

Non-coding regions of the human genome contain vast regulatory potential that contributes to the coordination of gene expression. Indeed, regulatory elements can reside large genomic distances from the promoters of genes they control. Here we describe approaches recently used to identify functional elements within the complex CFTR locus.