Coleus forskholii plant that is widely used in cell physiology to raise intracellular cAMP levels. In the field of trophoblast biology, forskolin is one of the primary treatments used to induce trophoblastic cellular fusion. The syncytiotrophoblast (ST) is a continuous multinucleated cell in the human placenta that separates maternal from fetal circulations and can only expand by fusion with its stem cell, the cytotrophoblast (CT). Functional investigation of any aspect of ST physiology requires in vitro differentiation of CT and de novo ST formation, thus selecting the most appropriate differentiation agent for the hypothesis being investigated is necessary as well as addressing potential off-target effects. Previous studies, using forskolin to induce fusion in trophoblastic cell lines, identified phosphatidylserine (PS) externalization to be essential for trophoblast fusion and showed that widespread PS externalization is present even after fusion has been achieved. PS is a membrane phospholipid that is primarily localized to the inner-membrane leaflet. Externalization of PS is a hallmark of early apoptosis and is involved in cellular fusion of myocytes and macrophages. We were interested to examine whether PS externalization was also involved in primary trophoblast fusion. We show widespread PS externalization occurs after 72 hours when fusion was stimulated with forskolin, but not when stimulated with the cell permeant cAMP analog Br-cAMP. Using a forskolin analog, 1,9-dideoxyforskolin, which stimulates membrane transporters but not adenylate cyclase, we found that widespread PS externalization required both increased intracellular cAMP levels and stimulation of membrane transporters. Treatment of primary trophoblasts with Br-cAMP alone did not result in widespread PS externalization despite high levels of cellular fusion. Thus, we concluded that widespread PS externalization is independent of trophoblast fusion and, importantly, provide evidence that the common differentiation agent forskolin has previously unappreciated pleiotropic effects on trophoblastic cells.Forskolin is an extract of the  A properly formed and well functioning placenta is essential for optimal growth of the fetus and aberrant formation and function of the placenta is associated with the common pregnancy conditions of preeclampsia and intrauterine growth restriction in vitro has not been achieved, thus investigations in to all functional aspects of ST cell physiology requires de novo formation of ST in culture through fusion of CT-like cells. The most popular models used to produce syncytium are the choriocarcinoma cell line Bewo and isolated primary CT. Bewo proliferate normally in culture and fuse to form syncytia only upon exogenous stimulation with differentiation agents. Extensive fusion is most commonly stimulated with cell permeant cAMP analogs or the adenylate cyclase activator forskolin for 48 to 72 hours Isolation of ST fragments capable of functioning Much is known regarding the molecular pathways controlling trophoblast differentiation, though specific aspects of the differentiation process still require elucidation. The signals that initiate trophoblast differentiation have not been conclusively established but it is thought that when the ST is becoming deficient in intracellular organelles and/or functional nuclei it signals to the CT, which lie directly below the ST Another apparently obligate step that has been identified for trophoblast fusion is the externalization of the membrane phospholipid phosphatidylserine (PS) 2+ or apoptotic pathway mediators (though which mediators are involved remains to be identified) PS efflux is an important event in cellular apoptosis, externalization of PS in platelets is involved in the initiation of the clotting cascade, and non-apoptotic PS externalization has been observed in cardiomyocytes As noted previously, PS externalization is considered to be an obligatory event for trophoblast fusion, however this conclusion is based on data produced in trophoblastic cell lines such as Bewo and have never been validated with primary cells. Therefore, we sought to examine whether PS externalization was required for fusion in primary trophoblasts.The Bewo choriocarcinoma cell line was obtained from the American Type Culture Collection . Primary trophoblasts were isolated from normal term placentas at 37-39 weeks gestation that were delivered by caesarean section without labour after written informed consent was obtained and full ethics review by the University of Alberta Ethics Committee. Purified villous CT were isolated by trypsin-DNAse digestion of minced chorionic tissue and immunoabsorbtion onto immunoglobulin (Ig)-coated glass bead columns as previously described 5 cells per well in 96 well plates  in Iscoves Modified Dulbecco's Medium  supplemented with 10% FCS (Gibco) (IMDM-FCS) and antibiotics  and incubated for 4 hours under a fully humidified standard atmosphere. The cells were then washed to remove non-adherent cells and medium was changed to either IMDM-FCS or IMDM-FCS plus 10 µM of the cell permeable cAMP analog Br-cAMP , 10 µM forskolin (Sigma), 10 µM 1,9-dideoxyforskolin (Sigma), the combination of cAMP plus 1,9-dideoxyforskolin, or solvent control DMSO in triplicate wells per treatment. Cells were then returned to the incubator for 24, 48, or 72 hours and the medium was changed after 48 hours.CT were seeded at a density of 1×10Bewo cells were maintained in Nutrient Mixture F12 Hams (Gibco) supplemented with 15% FCS (Gibco) (Hams-FCS) and antibiotics  in 6 well dishes . When 75-85% confluent the cells were passaged using 0.05% trypsin-EDTA (Gibco) for 5 minutes and then seeded into 96 well plates at a density of 10,000 cells per well in Hams-FCS. Cells were incubated for 4 hours in a fully humidified standard atmosphere and medium was changed to Ham-FCS with or without 10 µM forskolin, 10 µM 1,9-dideoxyforskolin, 250 µM Br-cAMP, both Br-cAMP and 1,9-dideoxyforskolin, or solvent control DMSO in triplicate wells per treatment. Medium was changed after 48 hours and cells were incubated for a total of 72 hours. Lower concentrations of Br-cAMP were tested (10–100 µM) but were not found to stimulate statistically significant rates of fusion (30% fusion or less).Primary trophoblasts were fixed after 24, 48, or 72 hours in culture with methanol and stained for the cellular junction protein desmoplakin as previously described Bewo cellular fusion was assessed with E-cadherin antibody. After 72 hours in culture cells were fixed with methanol for 10 minutes followed by a one hour incubation with 20% normal goat serum (Gibco) and then incubated overnight at 4°C with anti-E-cadherin  or the appropriate non-immune control. The cells were then washed and incubated with Alexafluor goat anti-mouse 488  for one hour and the nuclei were visualized with DAPI  for 10 min at room temperature. Triplicate images were randomly captured per well on an Olympus IX2-UCB microscope equipped with a Roper Scientific camera and a Sutter Instruments Lambda DG-4 fluorescent lamp . Slidebook 3.0  was utilized as capture software and Image-J for analysis. The percentage of nuclei lacking expression of E-cadherin were assessed, since E-cadherin expression has been shown to decrease with differentiation into syncytium 2; pH 7.4) then annexin-V-FITC  was added in annexin-V binding buffer (1∶50 for primary cells and 1:25 for Bewo) and the cells were incubated for 1 hour in an incubator under standard conditions. Cells were then washed twice with annexin-V binding buffer and fixed with 4% paraformaldehyde for 10 min. Nuclei were visualized with DAPI and triplicate images of each well were randomly obtained as detailed above. The number of nuclei associated with areas of annexin-V binding were counted. As a positive control primary trophoblasts and Bewo were incubated with 0.3 µM staurosporine for 4 hours and then annexin-V binding was visualized to establish the amount of annexin-V required to visualize PS externalization  for the same time period leads to fusion of ∼75% the cells + channels, to inhibit the glucose transporter GLUT1 and interact with ATP-binding cassette sub-family member B1 (ABCB1) in addition to modulating adenylate cyclase activity In contrast to treatment with cAMP, treatment of primary CT with forskolin for 72 hours led to fusion of ∼60% of nuclei concomitant with the same proportion of cells having externalized PS . Since fTreatment of Bewo choriocarcinoma cells with cAMP stimulated cellular fusion (∼70%), but did not stimulate high levels of PS externalization (∼4%) . ForskolIn this study we examined whether PS externalization was involved in primary villous CT differentiation into syncytium. We found that extensive PS externalization only occurs when differentiation is stimulated with the adenylate cyclase activator forskolin or the combination of cAMP and the forskolin analog 1,9-dideoxyforkolin, and that cAMP treatment alone and spontaneous trophoblast fusion do not result in significant extensive PS externalization in primary trophoblasts. Therefore extensive PS externalization is likely due to modulation of membrane transporters by forskolin and its analog, which does not stimulate adenylate cyclase activity, and is also a process that requires increased intracellular cAMP concentrations. Our data also demonstrated that extensive PS externalization is independent of trophoblast fusion in primary trophoblasts.We also assessed Bewo cells and found that the proportion of cells that were observed to have externalized PS was much lower than the proportion of fused cells observed and that extensive PS externalization was not consistently observed. Previously, using data produced in trophoblastic cell lines stimulated to fuse with forskolin, it was established that PS externalization on the entire surface of syncytium occurred concomitant with cellular fusion and that a monoclonal anti-PS antibody was capable of inhibiting cellular fusion + channels, the glucose transporter GLUT1, and ABCB1 (MDR1) Forskolin and 1,9-dideoxyforskolin are known to interact with and modulate activity of voltage gated KForskolin is used extensively in the trophoblast literature as a differentiation agent of trophoblastic cell lines though, to our knowledge, the pleiotropic effects of forskolin beyond activation of adenylate cyclase have not been widely, if ever, acknowledged. Use of forskolin as a differentiating agent for Bewo was initially presented by Wice et al. (1990) where only the adenylate cyclase activating properties of forskolin are discussed. The data presented here demonstrates that forskolin can have pleiotropic effects on trophoblastic cells independent of adenylate cyclase activity.Though it appears that extensive PS externalization is not involved in trophoblastic cell fusion, our experiments have not directly examined whether transient PS externalization at the site of membrane fusion is occurring in trophoblasts. This pattern of PS externalization has been observed in myoblast and macrophage fusion Ultimately the data presented in this communication suggest that PS externalization is unlikely to be involved in trophoblastic cell fusion and brings to the attention of the reader the pleiotropic effects of forskolin treatment on trophoblastic cells.Figure S1Representative images of annexin-V-FITC binding to trophoblastic cells after induction of apoptosis with staurosporine. A) Primary trophoblasts 4 hours after staurosporine treatment; B) Bewo cells 4 hours after staurosporine treatment.(Tif)Click here for additional data file.


DNA methylation was assessed across the genome with the HumanMethylation450 BeadChip. For each individual sample and CpG site, the proportion of DNA methylation was estimated. The associations between methylation and PTB or GA were evaluated by fitting a separate linear model for each CpG site, adjusting for relevant covariates. Overall, 29 CpG sites associated with PTB  independent of GA. Also, 9637 sites associated with GA , with 61.8% decreasing in methylation with shorter GA. GA-associated CpG sites were depleted in the CpG islands of their respective genes (p<2.2×10−16). Gene set enrichment analysis (GSEA) supported enrichment of GA-associated CpG sites in genes that play a role in embryonic development as well as the extracellular matrix. Additionally, this study replicated the association of several CpG sites associated with gestational age in other studies . Dramatic differences in fetal DNA methylation are evident in fetuses born preterm versus at term, and the patterns established at birth may provide insight into the long-term consequences associated with PTB.Spontaneous preterm birth  is a major public health concern, and children born preterm have a higher risk of morbidity and mortality throughout their lives. Recent studies suggest that fetal DNA methylation of several genes varies across a range of gestational ages (GA), but it is not yet clear if fetal epigenetic changes associate with PTB. The objective of this study is to interrogate methylation patterns across the genome in fetal leukocyte DNA from African Americans with early PTB (24 Despite advances in health care, the rate of preterm birth  has been increasing for the last 25 years Several epidemiologic, behavioral and biological factors  have been associated with PTB, but the mechanistic pathways that underlie the association of the risk factors to PTB are still unclear DNA methylation is an epigenetic modification required for proper gene regulation and cellular differentiation during fetal development This study was approved by the Institutional Review Boards of Centennial Women’s Hospital, Western Institutional Review Board and the University of Texas Medical Branch.The Nashville Birth Cohort (NBC) was established to examine genetic risk factors and changes in the biochemical pathways that distinguish spontaneous preterm from term labor. All subjects were recruited at Centennial Women’s Hospital and the Perinatal Research Center in Nashville, TN beginning in 2003. Pregnant women were enrolled during their first clinical visit after obtaining informed consent. Maternal demographic and clinical data were recorded from medical records or by interviews during the consenting process. Demographic and clinical data specific to the fetus was collected from clinical records. Gestational age of the neonate was determined by maternal reporting of the last menstrual period and corroboration by ultrasound dating. Race was identified by self-reporting that traced back to three generations from maternal and paternal side of the fetus. Only African Americans of non-Hispanic ethnicity were included in this study.1/7 weeks and 340/7 weeks. Controls were delivered (>390/7 weeks) with spontaneous term labor and delivery and no current or history of pregnancy-related complications including PTB and preterm or prelabor rupture of the membranes (pPROM). Subjects who had multiple gestations, preeclampsia, placenta-previa, fetal anomalies, and/or medical or surgical complications during pregnancy were excluded from the study. Subjects with any surgical procedures during pregnancy were treated for preterm labor or for suspected intra-amniotic infection and delivered at term were excluded from the control group. Maternal demographic and clinical data were collected from medical records or thorough self-report at the time of consent.Subjects were included in this study if they had contractions (rate of 2 contractions/10 minutes) leading to delivery either at preterm or term. Cases were delivered preterm with intact membranes between 24Race, socioeconomic , behavioral (cigarette smoking) factors were documented by maternal self-report. Intraamniotic infection was determined by amniotic fluid culture or by PCR for 16 s ribosomal RNA. In cases where culture or PCR data were not available, infection was assessed with four of the following clinical or histologic symptoms: high fever (>102°C), high CRP (>0.8 U/ml), abdominal tenderness, fetal tachycardia, mucopurulent vaginal discharge or histologic chorioamnionitis, funisitis.Umbilical cord blood samples were collected in EDTA tubes soon after placental delivery. Blood samples were centrifuged at 3,000 RPM to separate plasma, and buffy coats were aliquoted and stored at −80°C. DNA was extracted using the Autopure automated system .For each subject, >485,000 CpG sites across the genome were interrogated using the HumanMethylation450 BeadChip  We used MethLAB st exon, 3′UTR, or intragenic regions) to the number of non-GA-associated sites that did or did not occur in that gene region, using Fisher’s exact test. We then performed similar tests of enrichment for CpG-rich regions defined as islands or CpG poor regions defined as shores The location of each CpG site was determined using the Illumina array annotation for the HumanMethylation450 BeadChip based on build 37 of the human genome. We tested for enrichment among GA-associated sites by comparing the number of GA-associated CpG sites that did or did not occur in a particular gene region . Based on annotation with data from the 1000 Genomes Project, 5 of these 29 CpG probes (17.2%) do contain a SNP , suggesting that we could be observing a genetic rather than an epigenetic association for these 5 CpG sites; the methylated and unmethylated signals for these five sites are shown in GSK3B  and decreased methylation of a CpG site (cg25376491) in MAML1  in fetuses with PTB. In addition, 3 other CpG sites in GSK3B and 4 in MAML1 were nominally associated with PTB (p<.05).After accounting for multiple comparisons (FDR<.05) and confounding factors , 29 CpG sites associate with PTB independently of GA  and enriched in CpG shores . Examining the directionality of GA-associated CpG sites, 61.8% (5958 CpG sites) had lower methylation in subjects with lower GA; these CpG sites were twice as likely to be located in CpG islands  and 3′UTR (p = 1.5×10−9). While the sample size was not sufficient to look for sex-specific differences (i.e. interactions between age and sex), we did note a depletion of GA-associated CpG sites on the X chromosome ; both the depletion of GA-associated variants on CpG islands and the X chromosome are consistent with a previous report of age-associated methylation in children Our above analyses of PTB all included GA as a covariate because PTB and GA are by definition correlated (r = .93), and there is overwhelming agreement in the association of DNA methylation with PTB unadjusted for GA, or GA itself . In factHDAC4, 1.3.x10−11<p<9.8×10−4; −.0023<Δβ per week<−.01) have higher methylation levels in fetuses with lower GA. HDAC4 is involved in numerous identified pathways including system development and multicellular organismal development, anatomical structure development, organ development, and nervous system development. Several other CpG sites involved in epigenetic regulation during development were also identified. Specifically, CpG sites in the gene body of DNMT1 , the gene body of DNMT3A , the 5′UTR of DNMT3B  and the 5′UTR of TET1  also associate with GA  was used to gain further insight into the functional context of GA-associated CpG sites FDR<.05; . Promine with GA .MMP9  had higher methylation with increasing gestational age. MMP9 is involved in the breakdown of the extracellular matrix in the process of cervical ripening, and increased expression has been seen in pPROM compared to preterm birth with intact membranes TIMP2 also associates with GA .Among the enriched cellular components are several groups that relate to extracellular regions. Remodeling of the extracellular matrix is required to support pregnancy and parturition −11<p<.05; −8.1<t<8.9; −.01<Δβ per week<.01). Notably, methylation of a CpG site in corticotrophin-releasing hormone binding protein (CRHBP) increased with decreasing GA . CRHBP regulates corticotrophin-releasing hormone (CRH), a principal regulator of the hypothalamic-pituitary-adrenal (HPA) axis. In addition, methylation increased in a CpG site in the promoter of PIK3CD  with decreasing GA . The therapeutic value of PIK3CD inhibitors is currently being explored as anti-inflammatory drugs To complement our discovery approach, we evaluated the association between CpG sites in genes that had been associated with GA in a previous study that used a less dense array with 27,578 CpG sites IGFBP1 has been considered as a marker for preterm in vaginal infection and leaking amniotic fluid  located in the gene body though the direction of the association changed based on proximity of the CpG site to the CpG island.One limitation of this strategy is that GA and PTB represent correlated but etiologically distinct phenotypes. Thus, replicating associations observed with GA may not capture the same breadth of candidate genes that could be explored in a study focused on PTB. For example, GSK3B (glycogen synthase kinase 3 beta), which is involved in neuronal migration, development, and polarization, particularly during early embryonic development GSK3B is a negative regulator of MAML1 (mastermind-like 1) MAML1 also associated with PTB. GSK3B decreases transcription in the notch pathway through inhibition of MAML1GSK3B in regulating MAML1, there was an inverse relationship in the associations for the CpG sites in these genes. During development, the Notch pathway is integral to several developmental processes including neurogenesis, cardiovascular function, angiogenesis as well as intestinal and bone development .By examining DNA methylation across the genome, we identified 29 CpG sites that associated with PTB independently of GA in leukocyte DNA from high-risk African American fetuses. Among these are CpG sites in HDAC4, DNMT1, DNMT3A, DNMT3B, and TET1. For example, CpG sites in TET1 and DNMT3B have lower DNA methylation in subjects with shorter GA. TET1 functions to hydroxylate 5′methylcyctosine(mC) into 5′hydroxymethyl cytosine (hmC) TET1 has been implicated is normal embryogenesis, and the depletion of TET1 leads to low birth weight (LBW) in mouse pups TET1 promotes active demethylation while DNMT3B promotes de novo methylation; these two processes are highly involved in the establishment of tissue-specific DNA methylation patterns during development Additionally, 9637 CpG sites associated with GA when it was modeled separately from PTB. Our analyses suggest enrichment of GA-associated CpG sites in biological processes involved not only in embryonic and organ development but also in neurogenesis, nervous system development and neuron development. These processes involve extensive epigenetic regulation so it is not surprising that we observed associations with CpG sites in genes related to shaping epigenetic patterns during development: MMP9 and TIMP2 are integral to the process of parturition The cellular components most enriched for genes with GA-associated CpG sites were primarily related to the extracellular region. Genes such as (IGF2BP1), a developmentally regulated gene that binds IGF2 and has been a focus of the fetal programming literature Many studies of fetal programming or prenatal exposures focus on fetuses with intrauterine growth restrictions or that were small for gestational age. Recent studies in the field support associations between GA and both DNA methylation and gene expression differences, but note lesser or no associations with birth weight CRHBP associated with GA. CHRBP binds CRH limiting its activity, and changes in the relative ratios of CRH to CRHBP associate with timing of birth Our results were consistent with previous studies of DNA methylation in gestational age. Despite differences between cohorts and study design, we replicated >80% of CpG sites associated with GA in a previous study The goal of this study was to identify associations between DNA methylation and PTB. However, PTB is defined by GA at birth; thus, the differences observed may correspond to differences in the developmental stage versus the causes or consequences of PTB. In this study, the correlation between association tests for PTB and GA is strong r = .93; , and delFigure S1Scatter plots of the unmethylated vs. methylated signals (A versus B) for the five PTB-associated CpG sites that have 1000 Genomes SNPs within the probe.(TIF)Click here for additional data file.Figure S2Correlation between the t-statics depicting association analysis of CpG sites with PTB (x-axis) compared to GA (y-axis). All CpG sites are depicted whether or not they were associated with the outcome. In order to compare more directly compare the results from analyses of PTB and GA, we reversed the sign of the t-statistics for PTB in this plot.(TIF)Click here for additional data file.Table S1CpG sites that associate with GA (FDR<.05).(CSV)Click here for additional data file.


Euphorbia peplus. Crude euphorbia extract causes local toxicity and transient inflammation when applied topically and has been used in the treatment of warts, skin keratoses and skin cancer. PEP005 is a broad range activator of the classical  and novel  protein kinase C isoenzymes. Direct pro-apoptotic effects of this drug have been demonstrated in several malignant cells, including melanoma cell lines and primary human acute myelogenous leukemia cells. At micromolar concentrations required to kill melanoma cells this agent causes PKC-independent secondary necrosis. In contrast, the killing of leukemic cells occurs in the nanomolar range, requires activation of protein kinase C δ (PKCδ) and is specifically associated with translocation of PKCδ from the cytoplasm to the nuclear membrane. However, in addition to this pro-apoptotic effect the agent seems to have immunostimulatory effects, including: (i) increased chemokine release by malignant cells; (ii) a general increase in proliferation and cytokine release by activated T cells, including T cells derived from patients with chemotherapy-induced lymphopenia; (iii) local infiltration of neutrophils after topical application with increased antibody-dependent cytotoxicity; and (iv) development of specific anti-cancer immune responses by CD8+ T cells in animal models. Published studies mainly describe effects from in vitro investigations or after topical application of the agent, and careful evaluation of the toxicity after systemic administration is required before the possible use of this agent in the treatment of malignancies other than skin cancers. The diterpene ester ingenol-3-angelate (referred to as PEP005) is derived from the plant  A wide range of carcinogenesis-associated molecules are now investigated as possible therapeutic targets in human malignancies. These possible targets are usually mediators that show an altered expression in cancer cells or they affect essential cancer cell functions, e.g., regulation of proliferation or viability ,2. The pEuphorbia peplus and crude euphorbia extracts have been used for centuries in traditional Thai and Australian medicine for treating various skin conditions, including warts, keratoses and cancers [PEP005 ingenol 3-angelate) is derived from the plant -angelate cancers . Fractio cancers .N-terminal domain that interacts with phosphatidylserine, Ca2+, diacylglycerol, phorbol ester and/or other lipids [The PKC family was first distinguished by their status as cyclic nucleotide-independent kinases ,6 and isr lipids . The PKCr lipids ). PKCs hr lipids . Membersr lipids . It can r lipids . 2+ and diacylglycerol levels in the cell. These mediators can activate PKC, leading to a wide range of cellular events, depending on the isoenzyme activated [Although PKC mutations are very uncommon in human cancers, the expression of various PKCs, including PKCδ, is often altered in human cancers, as illustrated by the data summarized in ctivated ,38. In cctivated , and likctivated . PEP005 PEP005 is an activator of novel and classical PKC isoenzymes but its pro-apoptotic effects in leukemic cells rely upon the activation of PKCδ and its translocation from the cytoplasm to the plasma membrane, nuclear membrane and mitochondrial membrane in CHO-K1 cells and AML cell lines . PKCδ ac-/- mice develop normally and are fertile, suggesting that PKCδ plays minor roles during development, or that its actions can be taken over by another PKC isoenzyme [in vitro studies have suggested that PKCδ together with PKCα can be essential for monocyte differentiation [PKCδsoenzyme ,48. In csoenzyme . Recentlntiation ,43. The ntiation ,43,44,51ntiation , and oventiation . The molecular structure of the PKCδ isoenzyme is shown in Several PKC isoenzymes show altered expression in human cancers as summarized in -/- mice develop normally and are fertile [-/-) have given important insight into the role of PKCδ in vivo. Studies by Leitges and colleagues showed that vein segments from PKCδ-/- mice, subsequently grafted onto the carotid arteries of recipient mice (either PKCδ-/- or PKCδ+/+), lead to more severe atherosclerosis than was seen with PKCδ+/+ vein grafts [-/- mice contained significantly more smooth muscle cells (SMCs) than were found in the wild-type animals, and that this increased number of cells correlated with decreased SMC death in the lesions of PKCδ-/- mice [-/- mice were resistant to cell death after treatment with a number of apoptosis-inducing stimuli, including UV light, H2O2, and TNF-α [-/- mice when compared to wild-type mice [-/- mice were defective in mitochondrial dependent apoptosis, as shown by a suppression of etoposide-induced cytochrome-c release. Moreover, apoptotic responsiveness was restored by re-introduction of PKCδ [in vivo. Other work with PKCδ-/- mice has demonstrated a role for PKCδ in the negative regulation of B-cell proliferation [-/- mice [As stated above PKCδ fertile ,43,44. Hype mice . It was  of PKCδ . Both offeration . In this-/- mice ,50. SimiHuman neutrophils express the conventional PKCs α, βI, βII, the novel PKC δ and the atypical PKCζ, and PKCs are important in neutrophil function . PKC is The recruitment of neutrophils to sites of inflammation usually occurs across the endothelial cells in postcapillary venules ,53,54. PAn additional mechanism for PEP005-induced recruitment of neutrophils could be direct effects on the endothelial cells with increased expression of adhesion molecules and/or the induction of neutrophil-chemotactic cytokines. A recent study described increased transcriptional upregulation of the expression of E-selectin, ICAM-1 and CXCL8 in umbilical vein endothelial cells after exposure to PEP005 . When usTaken together these results suggest that PEP005 causes local recruitment of neutrophils through: (i) direct effects on endothelial cells with increased adhesion; (ii) indirect effects on the endothelial cells through local release of activating cytokines from neighboring cells; and (iii) the release of neutrophil chemotactic CXCL8 by endothelial cells and perivascular cells. Although one cannot exclude that umbilical cord and microvascular endothelial cells show functional differences, it seems likely that all three mechanisms contribute to the inflammatory response to topical application of PEP005.Topical treatment of skin tumors with PEP005 induces cancer cell necrosis followed by local inflammation characterized by neutrophil infiltration and release of reactive oxygen species . The trenu mice. This effect is associated with local macroscopic inflammation due to leukocyte infiltration dominated by neutrophils. After antibody-depletion of neutrophils topical PEP005 treatment caused a similar initial ablation, but tumors later re-emerged.- Topical application of PEP005 can cure implanted skin cancers without later relapse in the T cell deficient Foxn1- The neutrophil extravasation into the inflamed sites is severely impaired in CD18-deficient mice; topical treatment of implanted tumors in these animals was associated with initial cure followed by a weak local inflammation and later tumor relapse.nu mice, and macrophages are seen in PEP005 induced infiltrates. The local inflammation and relapse rate were not altered by depletion of NK cells. Neither inflammation nor relapse risk was altered for tumors implanted in Csfmop/Csfmop mice that lack functional M-CSF and therefore are severely monocytopenic. - NK cells and macrophages are present in Foxn1nu mice, but a high relapse rate was observed for the B cell-depleted mice.- The effect of topical PEP005 was investigated for LK2 tumors implanted in SCID mice that lack a humoral immune system . Tumors Taken together these results suggest that topical application of PEP005 to skin tumors mediates anticancer effects through three distinct phases . FirstlyTwo randomized studies have investigated the short-course use of topical PEP005 in the treatment of actinic keratosis, a premalignant lesion that can progress to invasive squamous cell carcinoma ,58. BothAcute myelogenous leukemia (AML) is the human malignancy where the effects of low-dose PEP005 have been most extensively studied ,60. In mChemokine release. Primary human AML cells show constitutive release of a wide range of chemokines [- emokines ,62. PEP0Chemokine receptor expression. PEP005 has only minor effects on the expression of most CCR and CXCR receptors , the only exception being CXCR4 that shows decreased expression. CXCR4 is one of the two receptors for the CXCL12 chemokine that is usually not released or only released at low levels by primary human AML cells [- ML cells . HoweverML cells . The CXCML cells . For thiCytokine release. Other cytokines are also released at higher levels, including Hepatocyte growth factor (HGF) and Granulocyte-macrophage colony-stimulating factor (GM-CSF) [- (GM-CSF) ,60.Differentiation. PEP005 decreases the expression of stem cell markers (including CXCR4) and increases the expression of lineage-associated markers, an observation consistent with differentiation induction.- Apoptosis regulation. PEP005 increases the expression of Bax and the activation of caspase 3. These pro-apoptotic effects are seen over a wide concentration range, whereas no induction of apoptosis was evident for normal CD34+ hematopoietic cells when testing concentrations up to 200 nM.- Intracellular signaling. The effect in AML cells is mediated through a PKCδ agonistic effect. The ERK1/2 pathway then seems to be important for the increased chemokine release together with increased expression of the NFκB subunits p50, p52 and p65.- Thus, the effect of PEP005 at these relatively low concentrations is mediated through induction of apoptosis and differentiation. The effect of PEP005 has been investigated only in AML, but various PKC isoenzymes also seem to be important in other leukemias as reviewed by Redig and Platanias . In chroThe studies described above demonstrate that PEP005 has an anticancer effect in different malignancies, but it should be emphasized that human cancer cells can also be generally resistant or the pro-apoptotic effect may be context-dependent . In a rein vitro exposure to PEP005; this additional pharmacological effect was observed for 20% of the cell lines [1 phase for up to 24 hours after in vitro exposure. Optimal combination of PEP005 with conventional cytotoxic drugs therefore seems to require a lag-time between exposure to the different drugs [Another study described induction of senescence in melanoma cells after ll lines . This grnt drugs .via the ETRA receptor. Other studies in colon cancer cells suggest that PEP005 can affect signaling through several pathways with increased phosphorylation of PKCδ, Raf1, ERK1/2, c-jun, p38, mitogen-activated protein kinase (MAPK) and PTEN [Resistance mechanisms to PEP005 have also been investigated in colon cancer cell lines that were cross-resistant to several chemotherapeutics . PEP005 and PTEN . These aTaken together these observations suggest that PEP005 can affect several intracellular signaling pathways and that resistance may occur dependent upon the differential activity of pro- and anti-apoptotic pathways in individual patients and between different malignancies. nu mice [in vitro studies demonstrated that the drug could kill human breast cancer cells and T-leukemia cells, and for all these cell types the LD90s seemed comparable. The mechanisms behind the effects seem to be destabilization of endocytosed vesicles followed by endosome disruption with release of calcium into the cytoplasm and thereby mitochondrial swelling, disturbed energy metabolism, loss of mitochondrial membrane potential, rapid plasma membrane perturbation and cell death due to necrosis [The effect of topical PEP005 has also been tested for other malignancies after skin implantation in Foxn1nu mice . These enu mice . Additionecrosis .in vitro studies suggest that normal urothelial cells may be less sensitive than bladder cancer cells. However, initial animal studies will be required because frequent inspection of the local inflammation is not possible in bladder cancer, and if severe hematuria occurred this complication may require specific therapeutic intervention. Another possibility for topical treatment is bladder cancer . The expin vitro seems to have both anti-leukemic and immunostimulatory effects in cells from AML patients and if this is extended to the in vivo situation, the immunostimulation could also include indirect effects through increased T cell recruitment [The increased release of chemotactic chemokines that enhance recruitment of various leukocytes by PEP005 treated AML cells must be regarded as an immunostimulatory effect . The effruitment  and direruitment ,72.+ response by PEP005 was observed, and this response contributed to regression of distant metastases. PEP005 was also found to have adjuvant properties and upregulated the expression of T cell costimulatory molecules CD80 and CD86 on dendritic cells. These observations further demonstrate that PEP005 has a broad immunostimulatory effect , a recent report reported that specific T cell responses can also be induced following local treatment . Inductiy effect .in vitro studies. In contrast, the immunostimulatory effect is documented both in experimental models, in vitro studies of human T cells and after topical application in humans. In systemic therapy the immunostimulatory effects represent a beneficial effect with regard to anticancer activity but also a potential risk of toxicity if pro-inflammatory effects predominate. Testing of PEP005 in animal models of leukemia is now required to determine if the compound applied systemically can achieve its anti-leukemic effects without significant toxicity. The overall literature described above suggests that PEP005 can mediate anticancer effects in different malignancies , but it PEP005 has been used as a single agent therapy in the topical treatment of skin diseases. Preclinical studies suggest that a combination of PKCδ agonists with conventional chemotherapy should be considered in human cancer therapy. PKCδ activation is induced after exposure of leukemia cells to etoposide, this is also observed after exposure of myeloid leukemia cells to Interferon-α, and in addition PKCδ seems important for anthracyclin-induced pro-apoptotic signaling ,75. Both4 antibody [The proinflammatory effect of PEP005 is clearly seen after topical therapy and involves neutrophils, B cells and T cells. Generally, great care should be taken if a drug with known proinflammatory effects is tried in systemic therapy. A dramatic example was the monoclonal antibody TGN1412, an anti-CD28 specific IgGantibody ,77,78,79antibody . Thus, sThe clinical studies of topical PEP005 therapy showed no evidence for systemic effects. However, when using topical application to other body surfaces the risk of increased absorption must be considered. As an example, PEP005 is now considered for the treatment of bladder cancer . The absThe increased chemokine release by AML cells after PEP005 exposure will also affect NK cells that express receptors for several CCL and CXCL chemokines . It is nOne possible approach to avoid severe adverse events during systemic administration of proinflammatory drugs could be to direct the drug release towards the disease compartment. Several strategies may then be possible. In hematological malignancies the disease is usually detected throughout a large part of the body. These disorders often infiltrate diffusely throughout the bone marrow or affect several lymph node regions. Coupling of drugs to a disease-reactive monoclonal antibody has been used to direct anticancer therapy, e.g., coupling to antibodies against the myeloid marker CD33 or the CD20 lymphoid marker ,83,84. AAnother possibility to avoid adverse events due to proinflammatory effects would be to administer the drug to severely immunocompromised cancer patients. Patients receiving treatment for acute leukemia develop a period of 2-3 weeks with severe leukopenia, and the risk of developing severe side effects may be less in such patients . FurtherSeveral chemokines that show altered release after PEP005 exposure have angioregulatory effects , but wheThe increased chemokine release may have proinflammatory effects that may represent a risk of toxicity. However, in certain clinical contexts the combination of anticancer and proinflammatory effects may be an advantage. Antileukemic immune reactivity is important for the reduced relapse risk after allogeneic stem cell transplantation, and increased T cell reactivity may then strengthen this antileukemic effect ,93,95. WPEP005 has both anticancer and proinflammatory effects . These d


Tympanuchus cupido), a grouse species that shows moderate sex-dimorphism. Greater Prairie-Chickens are native to tallgrass prairies of North America, although populations persist in less natural mosaics of cropland and native habitats. We used stable isotope analysis of carbon and nitrogen in blood, claws and feathers to assess seasonal differences in trophic niche breadth and individual specialization between male and female Greater Prairie-Chickens, and between birds living in continuous and fragmented landscapes. We found that females showed broader niches and higher individual specialization than males, especially in winter and autumn. However, differences between females and males were smaller in spring when birds converge at leks, suggesting that females and males may exhibit similar feeding behaviors during the lekking period. In addition, we found that birds living in native prairies showed greater annual trophic variability than conspecifics in agricultural mosaic landscapes. Native habitats may provide greater dietary diversity, resulting in greater diversity of feeding strategies.Partitioning of ecological niche is expected in lekking species that show marked sexual size dimorphism as a consequence of sex-specific ecological constraints. However, niche partitioning is uncertain in species with moderate sexual dimorphism. In addition, the ecological niche of a species may also be affected by landscape composition; particularly, agricultural fragmentation may greatly influence the trophic behavior of herbivores. We studied trophic niche variation in Greater Prairie-Chickens ( In grouse (Tetraonidae), sexual dimorphism in body size ranges from large (100%) in Capercaillie  might be expected due to differences in habitat structure and resource quality and availability.Prairie-chickens are native to prairies of central North America and were widespread prior to European settlement Trophic ecology is a central aspect of a species' ecological niche. The ability of animals to forage is a large part of their ability to cope with the environment, thus influencing behavior, habitat selection, and demography Stable isotope analysis of consumer tissues is a powerful tool to study intrapopulation niche partitioning native prairie; 533 km2; 39°00′N 96°26′W) was covered by 81% grassland and 10% cropland, with an average patch size of grassland of 51 ha, and a road density of 0.57 km per km2. Grasslands at the Smoky Hills site  were more fragmented, with 53% cover of grassland and 38% cropland, smaller average size of grassland patches (15 ha), and higher road density (1.4 km per km2). Crops within the agricultural mosaic included sorghum, wheat, corn and soybean. The density of prairie-chickens was similar in both sites: 0.19 and 0.17 birds km−2 in the native prairie and the agricultural mosaic, respectively Our study was conducted at two sites that differed in land cover, land use and degree of fragmentation of native tallgrass prairie habitat. The two sites were located 110 km apart in the northern Flint Hills and the Smoky Hills of north-central Kansas, U.S.A. . The FliPrairie-chickens were captured at 20 leks between March and May 2009, by means of walk-in traps and drop-nets We captured 156 prairie-chickens at 8 leks located in native prairie and 12 leks located in agricultural mosaic between 6 March and 6 May, 2009. All samples from birds captured in the native prairie (n = 48) were used in isotopic analysis, whereas from birds captured in the agricultural mosaic (n = 114) we used a 50% random selection of the males and all the females (n = 57), to balance sample sizes. The selection totaled 96 blood samples, 55 claw samples, and 100 feather samples distributed across sex-classes and study sites .In addition we collected vegetation samples to establish the isotopic baseline for prairie-chicken . Stable isotope ratios are expressed in δ notation, as parts per thousand deviations from a standard (‰). We used Pee Dee belemnite limestone as a standard for δ13C and atmospheric nitrogen as a standard for δ15N according to the equation: δX  =  [(Rsample/Rstandard) – 1]×1000 where X is 15N or 13C and R is the ratio of stable isotopes (15N:14N or 13C:12C).Blood samples were dried to constant mass in an oven at 60°C, and then refrigerated until isotopic analysis. Feathers and claws were treated with a 2∶1 chloroform-methanol solution for 24 hours to remove oils and debris, and then dried in an oven at 60°C. Feathers were frozen in liquid nitrogen and immediately ground to fine powder using a MM200 ball mill. Approximately 1 mg (1±0.2 mg) of each of the tissues was subsampled and packed into a 4×6 mm tin capsules for δ15N and 0.18‰ (±0.24) for δ13 C.A random quarter of the samples were analyzed in duplicate, and the analytical error was minimal at 0.13‰ (±0.14) for δ13C-δ15N space δ15N and δ13C were standardized (z-scores) to avoid differential weighting of the axes in the δ13C-δ15N biplot TA) that included the isotopic values of all individuals.To calculate trophic niche breadth and trophic variability we used quantitative metrics based on the position of individuals and Euclidean distances among them in the δ13C-δ15N plane. First, we calculated the distance of each individual to the isotopic centroid of its subset (CD), providing an index of the trophic diversity within each segment of the population. Second, we calculated the coefficient of variation of the distances from each individual to its neighbors in the isotopic space (CVND), which yields a measure of the clustering of values and trophic redundancy within the subset CVND (increased trophic redundancy) than a population in which individuals are, on average, more divergent in terms of their trophic niche CD and CVND were calculated with Quantum Gis 1.6.0.To estimate the trophic variability within each sex and population we calculated Euclidean distances among individuals in the CD and CVND) between males and females, and also between populations in different landscapes  itself is a summary variable, which yields a single value for each season (tissue) and landscape. To test for differences in Sampling methods were approved by Kansas State University's Institutional Animal Care and Use Committee (Protocol numbers 2474 and 2781). Research occurred exclusively on private lands with landowner permission. Field study did not involve endangered or protected species.δ13C values in blood (spring) for males than for females, no differences in isotopic values of feathers (autumn) and a significant interaction for both isotopic values in claws (winter) and for δ15N in blood  was wider in females, both in autumn  and in winter . Total niche space was similar for both sexes at each site during the spring (CD) than males. In winter the interaction between study site and sex for CD was significant. CD did not show differences between males and females in spring. CVND did not differ between males and females in any comparison  was 38% broader in the native prairie than in the agricultural mosaic in autumn (feather samples) and 22% broader in winter (claw samples), whereas no differences were found for during the spring  was significantly higher for prairie-chickens in the native prairie than in the agricultural mosaic both in autumn (feathers) and spring (blood). CD in winter (claws) and the coefficient of variation of the distances from each individual to its neighbors in the isotopic space (CVND) did not differ between landscapes and showed a significant interaction between study site and sex in all comparisons of the three tissues .Generally, wider niches were attained via an increase in individual diversity, supporting the niche variation hypothesis which states that more generalized populations are expect to be more heterogeneous Our study provides the first evidence of sexual niche partitioning in North American prairie-grouse which resembles patterns found in old-world forest grouse In autumn, wider niches and higher trophic diversity  of femalIn winter, we found that differences between sexes were site-specific: females showed wider niches and higher trophic diversity in the agricultural mosaic but not in the native prairie landscape. Males had wider niches and higher trophic diversity in native prairie. We speculate that the reduced availability of diet resources in winter favors opportunistic behaviors. In the agricultural mosaic landscape, different kinds of cultivated grains become available at different times of the season Convergence of females and males on leks in the spring reduces sexual spatial segregation, likely resulting in similar diet resource use during the spring breeding season.Among grouse, sexual differences in trophic strategies have been found for the most sexually dimorphic species of Capercaillie We observed that prairie-chickens showed distinct foraging strategies in two landscapes that differed in the degree of native habitat loss and fragmentation. Generally, the population in the native prairie landscape showed more generalist foraging behavior and a higher individual trophic diversity than prairie-chickens occurring in an agricultural mosaic.Landscape composition and the reduction of native habitat influence availability of food resources for herbivores. Hence, the higher trophic variability found in the native prairie landscape might indicate greater specific and microhabitat structural diversity of less fragmented native habitats Populations adapt to local conditions and distinct trophic behaviors which depart from prevalent view of the species' niche, might be among such local adaptations δ15N and δ13C are appropriately scaled to have equal weighting when combined in metrics, (2) the variation in isotopic ratios of basal sources should be considered 13C and δ15N signatures can be quite variable within and between landscapes, due to complex and poorly understood relations with soil and topographic variables Stable isotope analyses do have several advantages over traditional techniques to study the population trophic structure of threatened or declining species  for each of the 3 main diet categories and for male and female Greater Prairie-Chicken feathers in native prairie and agricultural mosaic.(TIF)Click here for additional data file.Table S1Stable isotopes of carbon and nitrogen  in the main groups of vegetation sampled in the native prairie study site. We also provide isotopic values of Greater Prairie- Chicken feathers  in the same study site for comparison.(DOCX)Click here for additional data file.Table S2Stable isotopes of carbon and nitrogen  for sorghum in the native prairie and agricultural mosaic study sites.(DOCX)Click here for additional data file.Text S1Isotopic baseline analysis.(DOCX)Click here for additional data file.


Structural genetic changes, especially copy number variants (CNVs), represent a major source of genetic variation contributing to human disease. Tetralogy of Fallot (TOF) is the most common form of cyanotic congenital heart disease, but to date little is known about the role of CNVs in the etiology of TOF. Using high-resolution genome-wide microarrays and stringent calling methods, we investigated rare CNVs in a prospectively recruited cohort of 433 unrelated adults with TOF and/or pulmonary atresia at a single centre. We excluded those with recognized syndromes, including 22q11.2 deletion syndrome. We identified candidate genes for TOF based on converging evidence between rare CNVs that overlapped the same gene in unrelated individuals and from pathway analyses comparing rare CNVs in TOF cases to those in epidemiologic controls. Even after excluding the 53 (10.7%) subjects with 22q11.2 deletions, we found that adults with TOF had a greater burden of large rare genic CNVs compared to controls . Six loci showed evidence for recurrence in TOF or related congenital heart disease, including typical 1q21.1 duplications in four (1.18%) of 340 Caucasian probands. The rare CNVs implicated novel candidate genes of interest for TOF, including PLXNA2, a gene involved in semaphorin signaling. Independent pathway analyses highlighted developmental processes as potential contributors to the pathogenesis of TOF. These results indicate that individually rare CNVs are collectively significant contributors to the genetic burden of TOF. Further, the data provide new evidence for dosage sensitive genes in PLXNA2-semaphorin signaling and related developmental processes in human cardiovascular development, consistent with previous animal models. Congenital heart disease affects nearly 1% of all live births. Tetralogy of Fallot (TOF) is the most common form of cyanotic congenital heart disease. This condition is associated with hemizygous deletions of chromosome 22q11.2 and chromosomal trisomies, but little else is known about the genetic heterogeneity of this complex disease. We used high-resolution microarrays and stringent methods to study structural (copy number) variants in a systematically phenotyped cohort of unrelated adults with TOF. We found that individually rare genic copy number variants (CNVs) were collectively significant contributors to the genetic burden in TOF. Among CNVs that implicated candidate genes of interest were loss CNVs overlapping the PLXNA2 gene that codes for plexin A2. This is the first study to show a role for this semaphorin receptor in human congenital heart disease, consistent with a Plxna2 mouse knockout phenotype. Pathway analyses comparing rare exonic loss CNVs in the TOF sample to controls implicated other novel gene sets suggest new pathogenetic mechanisms. Tetralogy of Fallot (TOF) is the most common form of cyanotic congenital cardiac disease in humans. With surgical advances and increased longevity, attention has shifted from immediate outcomes to understanding causation. However, for most patients with TOF, the genetic basis for the disease remains unknown. Recently, there has been a focus on unbalanced structural genomic changes, or copy number variants (CNVs), and disease Of the 495 unrelated adults with TOF recruited, 53  had 22q11.2 deletions associated with 22q11.2 deletion syndrome in silico detection, we tested 68 CNVs across different size ranges using quantitative PCR (qPCR). We observed a high true positive validation rate of 65/68 (95.6%), consistent with our previous studies Using a strict CNV analysis strategy We first compared the CNV burden of large (>500 kb) rare CNVs in the TOF cases and the OPGP controls. Consistent with our hypothesis, a significantly greater proportion of cases harbored large rare CNVs compared to controls  . This waFor those subjects with TOF, large rare CNVs were enriched in the syndromic subgroup however this difference reached statistical significance only for subjects with large rare exonic losses  . These rThere were two other subjects in the non-syndromic subgroup with genomic disorders at loci previously associated with congenital cardiac disease: one proband with a previously undetected 22q11.21 duplication (OMIM #608363) Amongst other large rare CNVs of note, one proband with syndromic features had a novel tandem duplication-deletion in the 18q22.3-q23 region that was transmitted to her daughter. Both had TOF, learning difficulties, short stature, obesity, and thyroid disease. This complex CNV overlapped the region involved in the 18q22 deletion syndrome, e.g., the 16 Mb deletion of a subject excluded from our TOF cohort , BBS9 (n = 1) and SNX8 (n = 1). Primary ciliary dyskinesis has several genetic causes, including mutations in DNAH11, a gene coding for a dynein heavy chain component of the axoneme, the inner cytoskeletal core of cilia (OMIM #6033). Similarly, BBS9 is one of 14 genes known to be responsible for Bardet-Biedl syndrome, a multisystem disorder FGF10 was another plausible candidate gene for human congenital cardiac disease In pathway analyses testing of case and control subjects with rare CNVs that overlapped 6 or fewer genes, only exonic losses led to significant results for the gene-set test   Consortium, it is now suggested that chromosomal microarrays be used as the first-tier diagnostic test for patients with multiple congenital anomalies and/or unexplained developmental delay After 22q11.2 deletions, the most common large rare CNV in our TOF cohort was the recurrent 1q21.1 duplication. The 1.18% prevalence of the 1q21.1 duplication is consistent with results using a targeted assay in two previous studies of TOF This is the first study to report 1q32.2 deletions at the PLXNA2 locus in patients with congenital heart disease. The PLXNA2 gene codes for a transmembrane protein, plexin A2 There are multiple semaphorin-plexin pathways. We identified two subjects with loss CNVs involving semaphorin genes: a 7q21.11 deletion overlapping three exons of semaphorin 3D (SEMA3D) and a 7q21.11 deletion intronic to semaphorin 3E (SEMA3E) . SemaphoThese CNV-related results direct attention to novel genes potentially involved in cardiac development in humans, and extend data from previous animal and human studies. For example, the migration of cardiac neural crest cells into the outflow tract is a process orchestrated, in part, by PLXNA2 signaling There is a known association between ciliopathies and congenital heart disease. In the current study, we found four cases with rare CNVs overlapping genes responsible for ciliary motility disorders: primary ciliary dyskinesis, Bardet-Biedl syndrome and 7p22 deletion syndrome. Two subjects had CNVs overlapping the DNAH11 gene, one of the genes responsible for primary ciliary dyskinesis. The cardiac lesion in this condition is believed to be due to an abnormality of nodal ciliary motility during development. In addition to abnormalities of cardiac situs (left and right sided heterotaxy), other congenital lesions including TOF have been reported in humans Most of these CNV findings remain in the research realm with functional studies essential to determine the true role of such variants and candidate genes in cardiac maldevelopment. Even the possible role for 1q21.1 duplications in the genetic burden of TOF, detectable by clinical genome-wide and targeted microarrays, requires more data to delineate the associated breadth and penetrance of cardiac and extracardiac expression a priori decision to minimize false positives, at the expense of such false negatives, would be expected to affect both cases and controls equally. Although our results overlapped certain previously described CNVs, further replication studies will be important to help define the significance and relative prevalence of the novel rare CNVs identified in this study. Large, multicentre studies may be useful, provided that comparable phenotyping and stringent quality control methods, as highlighted in this study, are maintained de novo status and segregation patterns of CNVs. These data will be essential to determine the true penetrance and variable expression of individual CNVs. Examining CNVs in patients with other forms of conotruncal defects or other forms of congenital heart disease may also be informative, and could reveal a genetically-related spectrum of clinically-distinct cardiac maldevelopment as is increasingly appreciated for, e.g., neuropsychiatric disorders This is the first study of genome-wide CNVs in TOF to use a well-characterized cohort of adult patients, stringent molecular methods, and multiple converging analyses. We have identified novel candidate genes for TOF, in addition to providing replication of previous findings, including some from a smaller genome-wide study of CNVs in TOF In addition to well known 22q11.2 deletions, other structural genomic changes appear to be important contributors to the genetic heterogeneity of TOF. In particular, these include 1q21.1 duplications and other rare copy number changes that disrupt genes involved in cell migration and vasculature development pathways including PLXNA2-semaphorin signaling and perhaps ciliary motility. Further studies will help to improve our understanding of the complex etiology and pathogenesis of TOF and of congenital heart disease in general.The study was approved by institutional research ethics boards at the University Health Network and the Centre for Addiction and Mental Health.We prospectively recruited 495 unrelated adults (≥18 years) with TOF including a subset with TOF-pulmonary atresia or pulmonary atresia and ventricular septal defect (collectively termed “TOF” in this study), without autosomal trisomies, from a single clinic . Patients with pulmonary atresia in the setting of more complex cardiac lesions, such as single ventricle lesions or transposition complexes, were not included. We excluded 62 subjects with documented syndromes, including 53 with 22q11.2 deletion syndrome associated with 1.5 to 3.0 Mb 22q11.2 deletions and genome-wide CNV data reported elsewhere TOF diagnosis was confirmed using echocardiogram and/or cardiac catheterization together with other imaging and surgical data reviewed using lifetime medical records To optimize our analyses, we used an independent Canadian control sample from the Ontario Population Genomics Platform (OPGP) genetic epidemiological project that comprised adults of European ancestry . To maximize quality control and minimize artefactual/laboratory-related findings High quality genomic DNA was genotyped using the high resolution Affymetrix Genome-Wide Human SNP Array 6.0. CNV analysis and adjudication for all TOF case and OPGP control samples were performed at The Centre for Applied Genomics . Arrays meeting Affymetrix-recommended quality control guidelines of contrast QC>0.4 were used for further analysis as outlined below and in Figure 1 in To accurately estimate ancestry, in addition to self-reported ethnicities genotypes of the TOF cases from 1,120 genome-wide unlinked SNPs were clustered by the program STRUCTURE Genome-wide CNVs were determined using a multiple-algorithm approach to maximize sensitivity and specificity of CNV calling, as described previously http://projects.tcag.ca/variation/) CNVs, may be found elsewhere Each stringently defined CNV identified in the TOF case and OPGP control samples was then adjudicated for rarity by comparison to those CNVs identified in two large population-based control cohorts comprising 2,357 individuals of European ancestry from Ontario and Germany, which had already been assessed using an identical microarray platform and CNV analysis strategy  CNVs>6.5 Mb in size, likely to be detectable by karyotype and/or potentially indicating artefactual results, were excluded. To ensure consistency of data, for major analyses we used only autosomal CNVs>10 kb in size in individuals of European ancestry  meeting the following criteria for more detailed examination: a) very rare  Confirmatory studies of possible TOF-associated CNVs used Stratagene SYBR Green based quantitative-PCR (qPCR). Each qPCR assay was performed in triplicate, for both the target region and for a control region at the FOXP2 locus on chromosome 7. Where available, molecular cytogenetic or microarray results from clinical laboratories also confirmed CNVs.http://frodo.wi.mit.edu/primer3/) was used to design primers. The amplified products were sequenced with the Big Dye Terminator kit using the ABI 3730XL capillary sequencer (Applied Biosystems) and analyzed for sequence variants using Sequencher . Putative sequence variants of interest were confirmed by sequencing in the reverse direction. SIFT For candidate gene discovery in TOF we prioritized further sequencing characterization to a single gene selected based on our CNV results and previous animal model studies to be the most likely to be involved in cardiac development. We performed mutation screening of PLXNA2 coding sequence  using standard PCR-based Sanger sequencing. The PLXNA2 gene contains 31 coding exons  that were fully sequenced with 32 amplicons. The program Primer 3 . The main analyses compared rare CNVs in the 340 TOF cases of European ancestry with those in the 416 OPGP controls and within-group comparisons of syndromic versus non-syndromic TOF subjects. Chi-square or Fisher's exact tests were used to compare categorical variables and Student's t tests for continuous variables, as appropriate. All tests were two-side, with statistical significance defined as p<0.05.For pathway analyses, we first assessed if pre-defined gene-sets  displayed a higher rare CNV load in TOF cases than in OPGP controls. Gene-sets were derived from Gene Ontology annotations , pathway databases  and protein domains . Only gene-sets with a number of member genes between 25 and 750 were tested: 2,456 total, with 1,939 from GO, 414 from pathways and 103 from PFAM domains. Gene-sets with fewer than 25 genes decrease the statistical power of the analysis, whereas those with more than 750 genes tend to have a very broad biological scope (e.g. GO “regulation of biopolymer catabolism”) and hinder the visualization of results. Subjects with rare CNVs overlapping more than 6 genes were not considered for the gene-set analysis, as these may have a more promiscuous set of gene functions perturbed by the rare variant. For exonic losses this led to the exclusion of 14 TOF cases and 11 OPGP controls.For each gene-set, we built a contingency matrix with subjects of European ancestry as sampling units. Subjects were categorized as (a) TOF cases or OPGP controls and (b) having at least one gene-set gene harboring a rare CNV or not. On the basis of this contingency table, a one-tailed Fisher's Exact Test was used to test higher prevalence of rare CNVs in TOF probands versus OPGP controls. This test can be regarded as an extension of a single-gene or single-variant association test; however, testing association for groups of genes, unlike single genes or single variants, provides sufficient power to detect significant association even when considering only rare variants The Fisher's Exact Test nominal p-value was corrected for multiple tests using a case/control class permutation procedure to estimate an empirical false discovery rate. We favored a permutation strategy over classical Benjamini-Hochberg false discovery rate owing to the highly complex dependency structure among gene-sets and overly conservative nature of this test Previously known TOF disease genes . Table 4: Rare large CNVs (>500 kb) in 43 of 433 unrelated adults with tetralogy of Fallot. Table 5: Very rare CNVs overlapping 26 candidate genes for tetralogy of Fallot. Table 6: (DOC)Click here for additional data file.


We estimate that hundreds of exonic transcriptional regulatory elements exist, an unexpected finding that highlights a surprising multi-functionality of sequences in the human genome.We sought exonic transcriptional regulatory elements by shotgun cloning human cDNA fragments into luciferase reporter vectors and measuring the resulting expression levels in liver cells. We uncovered seven regulatory elements within coding regions and three within 3' untranslated regions (UTRs). Two of the putative regulatory elements were enhancers and eight were silencers. The regulatory elements were generally but not consistently evolutionarily conserved and also showed a trend toward decreased population diversity. Furthermore, the exonic regulatory elements were enriched in known transcription factor binding sites (TFBSs) and were associated with several histone modifications and transcriptionally relevant chromatin. Evidence was obtained for bidirectional  An important key to deciphering the human genome is to identify the regulatory elements that control gene expression. Indeed, disruption of these elements has been linked to a number of human diseases including cancers Because regulatory elements can be found anywhere in the genome, large-scale, high-throughput screens are needed to identify them efficiently. Genome-wide searches have met with some success by exploiting several features of regulatory elements, for example their enrichment in transcription factor binding sites Comparative genomics were among the first approaches used to search for functional elements and identified sequences more conserved across species than would be expected by chance The current view of transcriptional regulatory elements is that they are clusters of transcription factor binding sites (TFBSs), which when bound by complexes of transcription factors (TFs) can recruit or block various critical components of the transcriptional machinery such as RNA polymerase II Histone modification occurs through recruitment of other regulatory factors and is a means by which gene expression can be controlled with greater finesse than simply relying upon sequence alone. Regulatory elements are often associated with particular chromatin states marked by a number of histone variants, particularly those that are methylated and/or acetylated Since many searches for transcriptional regulatory elements employ surrogate markers, such as chromatin modifications or evolutionary conservation, we have employed direct measures of transcriptional activity using reporter constructs. Here we report an unbiased search for exonic regulatory elements active in liver cells. We expand on previous work in which we evaluated genomic DNA from the ApoE gene cluster on chromosome 19 for regulatory elements To maximize transcript coverage we pooled mRNA from three human cell lines, C3A (liver), HEK-293 (kidney) and SVGp12 (astrocytes). We then synthesized cDNA from the RNA. Pooling RNA from a diverse variety of cell types was an attempt to normalize the cDNA library. Because we were only interested in exonic sequences, we restricted our assays to cDNA rather than whole genomic DNA. Pooled cDNA was digested either with Sau3AI or AluI and subcloned into the multiple cloning site upstream of the basal SV40 early promoter of the pGL3-promoter vector. A total of 1,932 clones were created, 1,008 from Sau3AI and 924 from AluI, with an average fragment size of ∼167 bp based on sequencing.Renilla luciferase vectors into C3A cells in 96-well plates. Expression of the two luciferase channels was assayed independently, and the regulatory activity of the putative element estimated from the log10 ratio of firefly to Renilla reporter gene activity. This measure evaluated expression of the tested element relative to transfection efficiency. Transfection efficiency measured using a CMV-GFP construct  was uniform at approximately seven percent All clone-containing firefly luciferase vectors were co-transfected with Quantile normalization was used to compare luciferase activities across plates, and the activities of clones produced by Sau3AI and AluI digestion were normalized separately . Control−300).The overall screening procedure was designed to identify coding fragments that reliably show strong regulatory signals, with more stringent thresholds for inclusion at each step . From thTwo clones that showed significantly higher activity than the promoter control across the eight replicates were deemed putative enhancers, while eight clones with lower activity were deemed putative silencers . Genomichttp://genome.ucsc.edu/) As judged by phastCons To evaluate whether the exonic regulatory elements tended to be more highly conserved than the rest of the host gene, we compared the phastCons conservation score of each regulatory element to the scores of the other exon sequences within the same gene. We used a sampling approach to obtain the null distribution combined with the Wilcoxon-rank sum test . Seven ohttp://www.1000genomes.org/) −1) were less than for non-regulatory exon sequences (median rate = 0.0031506 bp−1) .The inconsistent pattern of increased evolutionary conservation in the exonic regulatory elements may reflect multiple selection pressures on exons, even in the face of additional selection for transcriptional control. We therefore used the statistical power of single nucleotide polymorphism (SNP) data collected from the 1000 Genomes Project (06 bp−1) , with a −1 and 0.0038306 bp−1 in regulatory elements and host gene coding sequences respectively, Wilcoxon signed rank test, W = 10, Nr = 4, P = 0.1003). In contrast, the synonymous substitution rate showed diminished differences between the regulatory elements and host gene coding sequences . Overall, the trend towards decreased nucleotide substitution rates may imply a selective pressure for coding region regulatory elements in addition to translation alone. A larger database of exonic regulatory elements might further substantiate the notion that these elements possess decreased population diversity.Interestingly, most of the decreased nucleotide divergence in the coding region regulatory elements was due to non-synonymous substitutions (median rates = 0 bp−5) for TFBS along the entire human genome as determined by ChIP-seq. Because most DNA-protein interactions were tested in HepG2 liver cells, we confined our search to that cell line. HepG2 cells and the C3A cells used in our study both originate from liver. Transcription factor binding site peaks found within the putative element sequences are listed in We searched for TFBSs using the UCSC Genome Browser ENCODE/HAIB Transcription Factor Binding Sites “peaks” track, which annotates sites with the best evidence  across human, mouse, and rat. TFBSs are computationally determined, but not experimentally verified, using TFBS sequence data in the Transfac Matrix Database No ChIP-seq data were available for RORA and PPARA from the ENCODE/HAIB Transcription Factor Binding sites “peaks” track. We therefore sought corroborating evidence from related nuclear hormone receptor proteins. ChIP-seq data from ENCODE indicated that the retinoid X receptor, α (RXRA), showed binding to the S5 element  [24]. InAn interplay between retinoic acid receptors and the S5 element was further supported by a significant UniPROBE enrichment score of 0.457 To investigate whether our putative regulatory elements displayed evidence of open chromatin, we used the UCSC Genome Browser ENCODE/DNase I hypersensitivity (HS) “peaks” track. This track displays DNase I HS across the human genome (p<0.05) in various cell lines as determined by sequencing of DNase I digested genomic DNA. Data from three cell lines, GM12878 (lymphoblastoid), K562 (leukemia) and HepG2 , were used. DNase I hypersensitivity sites within putative element sequences and their flanking sequences are shown in GC-rich CpG islands (CGIs) are most often found in the core promoter region immediately upstream of the transcription start site. However, the discovery of a CGI in the intron of the PAX6 gene that may act as an alternative transcription start site has introduced the notion that CGIs not associated with the core promoter may also play a role in transcriptional regulation It is possible that regulatory elements are actually less likely to be in CGIs than other expressed sequences of the host gene. We therefore compared the GC-content of each element to the GC-content of all exons within the same gene using a sampling strategy combined with the Wilcoxon-rank-sum test . One of Well-studied histone modifications associated with enhancers include H3K4me1 Consistent with their diverse contexts, fragments varied in the number and types of histone modifications with which they were associated . The E1 To investigate the enrichment of chromatin states (epigenetic marks) in our putative regulatory elements, we used the UCSC Genome Browser ENCODE Broad ChromHMM track cis-regulation by the PPARA and RORA transcription factors (above). The S5 element was found to be enriched in strong enhancer chromatin state in some cell lines using the ChromHMM track. Possibly the S5 silencer has a bidirectional effect based on how it interacts with cell-type specific transcription factors. In general, the majority of the putative regulatory elements displayed chromatin states that were appropriately related to their functional roles.We found that the regulatory elements varied in their chromatin states . The E1 enhancer element was found to be enriched with a strong enhancer signal that correlated well with our analysis of enhancer histone signatures (described above). The E2 element had no data in the ChromHMM track. The silencer elements S1, S2 and S3 were enriched in chromatin states suggestive of repressive regions that also correlated well with our analysis of repressive histone signatures (described above). Elements S4, S6, S7, and S8 were broadly enriched in the transcriptional transition state in most of the cell lines. The S5 silencer element resides within the coding region of an exon of the TUBA1B gene and exhibits antagonistic cis-regulate expression of their host genes, then manipulation of transcription factors that bind to the regulatory element should alter expression of the host gene. To test this hypothesis, we searched the Gene Expression Omnibus  for studies in which the relevant transcription factor was perturbed and the expression of the target gene was measured. We focused on the target gene tubulin α1b (TUBA1B) and the putative silencer S5, located inside one of the coding exons of the gene. Two transcription factors, peroxisome proliferator-activated receptor α (PPARA) and retinoic acid receptor-related orphan receptor α (RORA) have overlapping binding sites within the boundaries of the S5 silencer. Both PPARA and RORA are involved in regulation of lipid metabolism and so are both highly expressed in liver If exonic enhancers and silencers We first analyzed data from a study in which global gene expression was measured using Affymetrix microarrays in wild-type and PPARA-null mice after administration of either the PPARA agonist WY1463 or after fasting (GEO ID: GSE5475) In short, the PPARA agonist decreased expression of TUBA1B in wild-type mice, but this decrease was abolished in PPARA-null mice. These results suggest that activation of PPARA decreases expression of the TUBA1B gene and that this decrease is mediated through the PPARA gene. In contrast, ANOVA revealed no significant effects of PPARA-genotype , PPARA-agonist , or their interaction  on expression of the tubulin β4 (TUBB4) gene . The absResults from the fasting study were similar . ANOVA sIn contrast, testing using ANOVA showed that the expression of TUBB4  did not To test for association between RORA activity and TUBA1B expression, we used data from a study using Illumina BeadChip arrays in which global gene expression was compared in skeletal muscle taken from wild-type mice and mice with a RORA dominant negative mutation (GEO ID: GSE20646) From the above observations, we propose that PPARA and RORA compete to bind the TUBA1B regulatory element, wherein PPARA represses TUBA1B when bound, while RORA activates . ConsistFrom a pool of 1,932 random fragments we discovered 10 exonic regulatory elements active in liver within coding regions and 3′ UTRs . A previous screen of 1,798 random fragments from a BAC containing both genic and intergenic DNA from the ApoE gene cluster on chromosome 19 also yielded 10 regulatory elements active in liver Many of the exonic regulatory elements resided in housekeeping genes, which tend to be expressed at high levels in most cell types. By pooling RNA from a diverse variety of cell types, we attempted to partially normalize the cDNA library and increase transcript coverage. However, the cDNA library was not subtracted, which will cause a bias toward abundant transcripts. The luciferase assay used to screen for the regulatory elements exhibited considerable noise in the initial screening phase of the workflow. However, this variance was mitigated by the use stringent criteria to identify regulatory elements in the follow-up stages, which incorporated replicates of 3× followed by 8×. In addition, selection of each putative element was restricted to a corrected P value with FDR of <5%.In both our present study of exons and our previous study of the chromosome 19 genome region Nine out of ten of the exonic regulatory fragments were conserved across vertebrates, with seven strongly conserved (phastCons score >0.5). The evolutionary conservation of the regulatory fragment was correlated with conservation of the host gene as a whole, and fragments within coding regions were more conserved than those in 3′ UTRs. Others have shown that computationally predicted exonic regulatory elements have lower nucleotide substitution rates than other coding exons within the same host gene, presumably because of dual selective pressure to preserve both protein-coding sequence and TFBSs Complementing our findings of increased conservation at the evolutionary level, we also showed that the coding region regulatory elements displayed a trend toward decreased polymorphism diversity using data from the 1000 Genomes Project. The decreased diversity of the elements in the human population was largely due to decreased non-synonymous substitution rates. These observations suggest that the coding region regulatory elements are under additional selective pressure than translation alone. The trend towards decreased diversity is also consistent with our findings of increased evolutionary conservation for some of the regulatory elements. Perhaps the exonic elements exert a subtle selective pressure that can best be detected in the context of intraspecies evolution. A larger database of exonic regulatory elements will allow this possibility to be addressed with more certainty.Traditionally, high GC-content has been associated with core promoter sequences, while thus far evidence of association between distal regulatory elements and higher GC-content is scarce Six fragments had predicted TFBSs determined by ChIP-seq and comparative genomics. Three fragments had binding sites for HNF4A, hepatocyte nuclear factor 4α. HNF4A is known to be a master regulator of the expression of a wide variety of genes in liver Using comparative genomics, ChIP-seq and genome-wide binding assays, overlapping conserved TFBSs for PPARA, RORA and other nuclear receptors were predicted within S5, a putative silencer in the coding region of TUBA1B. The S5 silencer also mapped to a processed pseudogene in a different region of the genome  with the same score as TUBA1B. However, unlike the TUBA1B silencer element, the second location did not display any enhancer-like signatures in the UCSC Genome Browser histone modification or ChromHMM ENCODE tracks (not shown), making it less likely that this region acts as a regulatory element.cis-regulatory potential of the TUBA1B silencer by positively and negatively correlating expression of TUBA1B with RORA and PPARA activation, respectively. Nuclear receptors, like RORA and PPARs, have been shown to have opposing effects on downstream gene expression We verified the The exonic regulatory elements exhibited a degree of open chromatin conformation as judged using DNase I HS, but the extent was less than found at traditional transcriptional initiation sites. The versatility and difficulty in identifying regulatory elements is reflected in the diversity of histone modifications associated with them. Most of the signatures previously used to identify enhancers, such as H3K4me1, were associated with several exonic silencers in our work. Because not all histone modifications were mapped in HepG2 liver cells, we also looked at the chromatin state at the position each of our putative exonic regulatory elements in all other cell types tested for the UCSC Genome Browser ENCODE/Broad Histone Modification track. Cross-cell type inferences should be made cautiously, as histone modifications at enhancers are known to vary considerably between cell types To overcome the limitations of examining any one histone modification in isolation, we turned to the UCSC Genome Browser ENCODE Broad ChromHMM track. This track improves the reliability of chromatin assignments by integrating multiple data types, including histone ChIP-seq datasets. In general, we found reasonable agreement between the chromatin states and functions of the exonic elements. However, the function of the exonic regulatory elements can potentially change in various cellular contexts. One strategy to investigate this possibility would be to test the putative elements in different cell lines.(ADAMTS5) Exonic regulatory elements appear to be sequences that are concurrently conserved, enriched in TFBSs and associated with several histone modifications. This likely reflects the biology: a given TFBS motif appears many times in the genome, and only a fraction are likely true binding sites, most likely those which are clustered together, in which access to the TFBSs is permitted by histone modifications and those where the TFBSs are conserved across species. No feature correlates perfectly with regulatory activity, so single-feature based approaches are likely to fail. Integrated approaches have already been successfully used to predict the locations of coding regulatory elements. For example, a search for clusters of TFBS conserved spatially and evolutionary across human, mouse, and rat was used to predict ∼700,000 regulatory elements, including an experimentally-verified coding enhancer for the gene ADAM metallopeptidase with thrombospondin type 1 motif, 5 In sum, the variability of coding regulatory elements requires that genome-wide searches will have to define an appropriately multifaceted signature. The complexity of interactions between DNA, transcription factors and chromatin state should be integrated into tools used to search for the sequences where these interactions occur. Nevertheless, the bioinformatic identification of regulatory elements, both exonic and non-exonic, will clearly benefit from the development of rapid experimental tools that can permit direct genome-wide evaluation of transcriptional control sequences rather than depending upon related, but ultimately, surrogate markers of regulation.To normalize transcript levels used to generate cDNA, RNA was pooled in equal amounts from three human cell lines; HEK-293 (kidney), C3A (liver) and SvGp12 (astrocyte) . Cell lines were grown in Eagle’s Minimum Essential medium (ATCC 30-2003) and 10% fetal bovine serum (Invitrogen) until 75% confluency was reached. For each cell line, the Oligotex mRNA mini kit (Qiagen) was used to isolate and purify mRNA. Pooled mRNA from all cell lines then used to synthesize cDNA using the Just cDNA Double Stranded cDNA Synthesis Kit (Agilent). Random hexamers were chosen as primers to avoid the 3′ bias of oligo-dT primers. Quality of RNA and cDNA were assessed using spectrophotometry and gel electrophoresis, respectively.Samples of pooled cDNA were digested by either Sau3AI or AluI (New England Biolabs) and sub-cloned into the pGL3-promoter vector (Promega), digested with SmaI or BglII, respectively. Vectors were then transformed into MAX efficiency DH5α chemically competent bacteria (Invitrogen), clones isolated, and plasmid DNA purified using 96 Plasmid Miniprep Kit (Qiagen).The pGL3-promoter and pGL3-basic vectors, both from Promega, served as neutral  and negative controls , respectively. The reporter gene for both vectors was firefly luciferase. For a positive control, we used the previously identified human APOE liver-specific enhancer HCR1 inserted into the pGL3- promoter vector Renilla luciferase plasmid  were co-transfected into C3A human liver cells (ATCC) using the Effectene transfection reagent (Qiagen) in 96-well plates. The Renilla plasmid serves as a control for transfection efficiency. Transfection was performed when cells had reached 80% confluency. Cells were then grown in Eagle’s Minimum Essential medium (ATCC 30-2003) and lysed after 24 hours. Luciferase reporter gene activity was assayed using the Dual-Luciferase Assay Kit (Promega).For each clone, 100 ng of firefly experimental luciferase plasmid and 10 ng of control 10 ratio of firefly to Renilla luciferase signal, was used as a measure of expression relative to transfection efficiency. Raw activity ratios were quantile normalized across 96-well plates. Clones were chosen for further screening upon demonstrating activity two standard deviations away from the mean after normalization. Sequencing of putative clones was performed at GenoSeq, the UCLA genotyping and sequencing core.Relative luciferase activity, the logFor each host gene, a sliding window of length equal to the regulatory element length was employed to obtain phastCons conservation and GC content scores from the UCSC genome browser. The window was shifted in single base pair increments along the entire length of the expressed region of the host gene to obtain a null distribution of score values for the gene. The Wilcoxon-rank sum test was then used to compare values of the regulatory elements with the null distributions obtained from the host genes.Figure S1Conservation of fragments. PhastCons scores, which represent the probability that a base is conserved across vertebrates, for all bases in each fragment sequence.(TIF)Click here for additional data file.Figure S2Nucleotide substitution rates. Comparison of nucleotide substitution rate for exonic regulatory elements and remaining exon sequences of host genes.(TIF)Click here for additional data file.Figure S3Conserved transcription factor binding sites. Positions of transcription factor binding sites conserved across human, mouse and rat relative to amino acid sequence of fragment.(TIF)Click here for additional data file.Figure S4A) RXRA binding site on S5 element determined by ChIP-seq. (B) RXRA and RARA binding sites on S5 element determined by protein binding microarray data from UniPROBE.RXRA and RARA transcription factor binding sites on S5 element. ((TIF)Click here for additional data file.Figure S5DNase I hypersensitive sites. Positions of DNase I hypersensitive sites for the exonic regulatory elements in cell lines GM12878, K562 and HepG2.(TIF)Click here for additional data file.Figure S6Chromatin state of the exonic regulatory elements. Signatures of epigenetic marks (chromatin states) obtained using the ChromHMM ENCODE track for the exonic regulatory elements E1, S1, S2, S3, and S4 in nine cell lines.(TIF)Click here for additional data file.Figure S7Chromatin state of the exonic regulatory elements. Signatures of epigenetic marks (chromatin states) obtained using the ChromHMM ENCODE track for the exonic regulatory elements S5, S6, S7 and S8 in nine cell lines.(TIF)Click here for additional data file.Table S1Putative regulatory elements.(DOC)Click here for additional data file.Table S2Fragment sequences.(DOC)Click here for additional data file.Table S3Conservation of regulatory elements and host exon sequences.(DOC)Click here for additional data file.Table S4Nucleotide substitution rate of regulatory elements.(DOC)Click here for additional data file.Table S5Transcription factor binding sites determined by ChIP-seq.(DOC)Click here for additional data file.Table S6GC content of regulatory elements and host exon sequences.(DOC)Click here for additional data file.


In vitro studies of influenza infection have revealed novel effector functions of iNKT cells including IL-22 production and modulation of myeloid-derived suppressor cells, but ex vivo characterization of human iNKT cells during influenza infection are lacking. Similarly, as recent evidence suggests iNKT involvement in dengue virus pathogenesis, iNKT cells may modulate responses to a number of emerging pathogens. This Review will summarize current knowledge of iNKT involvement in responses to viral infections in both human and mouse models and will identify critical gaps in knowledge and opportunities for future study. We will also highlight recent efforts to harness iNKT ligands as vaccine adjuvants capable of improving vaccination-induced cellular immune responses.Natural killer T cells (NKT cells) represent a subset of T lymphocytes that express natural killer (NK) cell surface markers. A subset of NKT cells, termed invariant NKT cells (iNKT), express a highly restricted T cell receptor (TCR) and respond to CD1d-restricted lipid ligands. iNKT cells are now appreciated to play an important role in linking innate and adaptive immune responses and have been implicated in infectious disease, allergy, asthma, autoimmunity, and tumor surveillance. Advances in iNKT identification and purification have allowed for the detailed study of iNKT activity in both humans and mice during a variety of chronic and acute infections. Comparison of iNKT function between non-pathogenic simian immunodeficiency virus (SIV) infection models and chronic HIV-infected patients implies a role for iNKT activity in controlling immune activation.  The immune response to invading pathogens requires the successful activation of innate immunity, which informs the development of the subsequent adaptive immune response. A small subset of T lymphocytes expressing surface markers characteristic of both T cells and natural killer (NK) cells are now appreciated to form an important link between the innate and adaptive immune responses. These NKT cells can be activated in both antigen-dependent and independent manners and respond with robust Th1 and Th2 cytokine production, allowing them to exhibit remarkable functional plasticity with both pro-inflammatory and immunoregulatory characteristics. NKT cells can be grouped into several subsets , but theCurrent knowledge regarding iNKT thymic selection has recently been thoroughly reviewed by Hu et al. ex vivo on the surface of iNKT cells iNKT TCR–mediated responses are restricted by CD1d, a member of the non-polymorphic CD1 antigen presenting protein family Human iNKTs express CD4 and CD8α A hallmark of iNKT activation is the rapid production of a vast array of cytokines and chemokines The functional plasticity of iNKT cells, combined with their ability to modulate activation of other immune cell subsets, suggests that they may play both a protective role in controlling viral infection and a detrimental role in enhancing viral pathogenesis. Here, we review the current understanding of the roles of iNKT cells in human viral infections, with particular focus on HIV and influenza infection  Transgenic mouse models of HBV infection have suggested iNKT control of HBV replication through hepatic IFNα/β/γ induction and NK activation Description of peripheral and hepatic iNKT cells during human HCV infection has been highly inconsistent. One study reported significantly lower peripheral blood iNKT (Vα24+Vβ11+) frequency among viremic compared to aviremic HCV seropositive individuals and healthy controls Functional data suggests that iNKT cells may traffic to the liver during HCV infection and acquire a fibrogenic cytokine producing profile. CXCR3 is upregulated on iNKT cells among HCV+ patients HSV. In the context of herpes virus infections, evidence is emerging to support viral interference of iNKT function. Kaposi's sarcoma-associated herpesvirus (KSHV) was the first to be shown to possess the ability to downregulate CD1d expression, an effect mediated by the viral modulation of immune recognition proteins MIR1 and MIR2 −/− mice exhibit significantly higher HSV-1 viral load within dorsal root ganglia, larger skin lesions, and greater neuronal death than wild-type mice, indicating that efficient early viral control requires intact iNKT cells In murine models, CD1din vitro. Influenza infection of CD1d-deficient mice also suggests that iNKT-mediated IFNγ production is required for full NK and CD8+ T cell activation and antiviral activity −/− mice in vitroA novel role for iNKT cells in modulating immune activity was discovered when De Santo et al. reported the identification of myeloid-derived suppressor cells (MDSCs) that could inhibit influenza-specific immune responses and result in increased viral titres and mortality + T cell response after co-administration of vaccine with the adjuvant. They demonstrated that the adjuvant led to activation of iNKT cells, which in turn resulted in antibody responses even in the absence of CD4+ T cells (MHC class II knockout mice), an effect not reproduced by T cell adjuvants such as alum. The authors concluded that iNKT cells can compensate for the absence of CD4+ T cell help The vast majority of literature on iNKT cells in influenza infection focuses on the role of αGalCer and iNKT activation as a vaccine adjuvant in mouse models. Initially, it was shown that nasal administration of αGalCer with the antigen PR8 HA ) induced high levels of systemic IgG and mucosal S-IgA Abs, high levels of IFN-γ and IL-4 both locally and systemically, and Ag-specific CTLs. These responses were associated with complete protection against an influenza viral challenge 366). Interestingly, increased NP366-specific memory CD8+ responses were demonstrated after 6 weeks in the group that received the adjuvant. Taken together, this study indicates a blunted antigen-specific effector CTL response that is followed by an enhanced CD8+ recall Several lines of evidence also suggest that the stimulation of iNKT cells influences the subsequent cell mediated response to influenza. Administration of αGalCer with a high dose of an inactivated, non-replicating virus had a strong iNKT activating effect; however, this was accompanied by diminished peak CD8+ response to the immunodominant nucleoprotein epitope  infection