Detection of Pleiotropy through a Phenome-Wide Association Study (PheWAS) of Epidemiologic Data as Part of the Environmental Architecture for Genes Linked to Environment (EAGLE) Study

We performed a Phenome-wide association study (PheWAS) utilizing diverse genotypic and phenotypic data existing across multiple populations in the National Health and Nutrition Examination Surveys (NHANES), conducted by the Centers for Disease Control and Prevention (CDC), and accessed by the Epidemiological Architecture for Genes Linked to Environment (EAGLE) study. We calculated comprehensive tests of association in Genetic NHANES using 80 SNPs and 1,008 phenotypes (grouped into 184 phenotype classes), stratified by race-ethnicity. Genetic NHANES includes three surveys (NHANES III, 1999–2000, and 2001–2002) and three race-ethnicities: non-Hispanic whites (n = 6,634), non-Hispanic blacks (n = 3,458), and Mexican Americans (n = 3,950). We identified 69 PheWAS associations replicating across surveys for the same SNP, phenotype-class, direction of effect, and race-ethnicity at p<0.01, allele frequency >0.01, and sample size >200. Of these 69 PheWAS associations, 39 replicated previously reported SNP-phenotype associations, 9 were related to previously reported associations, and 21 were novel associations. Fourteen results had the same direction of effect across more than one race-ethnicity: one result was novel, 11 replicated previously reported associations, and two were related to previously reported results. Thirteen SNPs showed evidence of pleiotropy. We further explored results with gene-based biological networks, contrasting the direction of effect for pleiotropic associations across phenotypes. One PheWAS result was ABCG2 missense SNP rs2231142, associated with uric acid levels in both non-Hispanic whites and Mexican Americans, protoporphyrin levels in non-Hispanic whites and Mexican Americans, and blood pressure levels in Mexican Americans. Another example was SNP rs1800588 near LIPC, significantly associated with the novel phenotypes of folate levels (Mexican Americans), vitamin E levels (non-Hispanic whites) and triglyceride levels (non-Hispanic whites), and replication for cholesterol levels. The results of this PheWAS show the utility of this approach for exposing more of the complex genetic architecture underlying multiple traits, through generating novel hypotheses for future research.     

Nitric oxide causes macrophage migration via the HIF-1-stimulated small GTPases Cdc42 and Rac1

Hypoxia-inducible factor 1 (HIF-1) is a key regulator of tumor development. Recently, the tumor microenvironment, with the presence of tumor-associated macrophages (TAMs), has gained considerable interest. The mechanisms of macrophage/TAM migration as well as the role of HIF-1 in macrophages for tumor progression are still under debate. We present evidence that under normoxic conditions, nitric oxide (NO) promotes macrophage migration. The response was impaired in macrophages from leukocyte conditional HIF-1α^−/− mice. NO production and cell migration in response to cytokines were attenuated in macrophages from iNOS^−/− mice, suggesting that iNOS-derived NO transmits cytokine signaling toward cell migration. We further identified the small GTPases Cdc42 and Rac1 as effectors of the NO–HIF axis to drive macrophage migration by modulating the actin cytoskeleton. Our observations strengthen the role of HIF-1 in macrophages as a target of NO in facilitating functional responses such as migration.     

Biogeochemical mass-balances (C, N, P, Si) in three large reservoirs of the Seine Basin (France)

Three major reservoirs (Marne, Seine and Aube), situated in the upstream basin of the river Seine represent a storage capacity of 800 10⁶ m³. In order to quantify the possible role of these reservoirs as a sink or source of nutrients and organic matter for the river system, an input/output mass-balance of suspended matter, organic carbon, inorganic nitrogen forms, phosphorus and reactive silica was established, providing reliable estimates of their retention/elimination and export. The study was carried out over 3 years (1993, 1994 and 1995) in differing hydrological conditions. The retention times varied from 0.3 to 0.8 year, depending on the reservoir and the year, but was longer in 1993 that was a drier year than 1994 and 1995, hydrologically quite similar.Regarding retention (or elimination) and export, the behaviour of the three studied reservoirs was similar. A clear loss or retention of nitrogen, phosphorus and silica was observed in the reservoirs and represented about 40% of the incoming flux of nitrate, 50% of silica, and 60% of phosphate. The retention was lower for total phosphorus than for phosphate. The reservoirs are also sites of suspended matter deposition except during the decennial drawdown, when suspended matter is exported. For inorganic nitrogen, the average amount of nitrate retained in the Seine basin reservoirs upstream from Paris is 5000 tonnes y^–1 that is almost equal to the estimated retention by deposition or denitrification in river channel sediments for the whole drainage network. The retention in the reservoirs represents about 12% of the total flux of nitrate at the outlet of the basin upstream from Paris, and 5% at the mouth of the Seine River.We also calculated inlake C, N, P, Si budgets on the basis of direct process measurements. Measurements of planktonic primary and bacterial activity production led to annual net production of 4200 and 580 tonnes of carbon, respectively. A reasonable value (450 tonnes of carbon) of grazing was calculated. Corresponding N, P, Si fluxes were drawn from appropriate C:N:P:Si ratios. Benthic fluxes were measured with bell jars. The retention of P and Si represents a small fraction of important internal fluxes of phytoplanktonic uptake and recycling, while inorganic nitrogen retention depends mostly on benthic denitrification. The behaviour of P and Si differs in that P is mainly recycled in the water column, while Si dissolution occurs at the sediment interface. Nitrogen is recycled in both the planktonic and the benthic phase.     

Applicability of green fluorescence protein in the study of endothelin converting enzyme‐1c trafficking

Endothelin‐1 (ET‐1) is one of the most potent peptide vasoconstrictors known. It is produced upon the cleavage of its precursor big endothelin‐1 by endothelin converting enzyme‐1 (ECE‐1). Production of ET‐1 is thought to be dependent upon the expression of ECE‐1 at the cell surface. Therefore, mechanisms inducing the trafficking of ECE‐1 to the cell surface have been the focus of recent research. This research has identified phosphorylation of the cytoplasmic region of ECE‐1 as a main cellular signal inducing its trafficking to the cell surface. Previous studies have used green fluorescent protein (GFP) tagged ECE‐1 to monitor phosphorylation induced trafficking of ECE‐1 to the cell surface. However, it has been speculated that the addition of the GFP tag can itself alter enzyme activity and phosphorylation of ECE‐1, and hence the suitability of GFP or any other protein tag in studying ECE‐1 distribution and trafficking. ECE‐1c is the most widely expressed isoform in endothelial cells. We therefore expressed ECE‐1c with a GFP tag either at the N or C‐terminus of ECE‐1c. Catalytic activity and effect on protein kinase C (PKC) induced phosphorylation was compared between the two chimeras and wild‐type ECE‐1c. Our results indicate that positioning of the GFP tag on the C‐terminus abrogates activity without effecting PKC‐induced phosphorylation. However, GFP tag on the N‐terminus has the opposite effect. Results of this study shed light on the applicability of GFP or perhaps other protein tags in studying ECE‐1c distribution and trafficking.     

Detection of ginkgolide A in Ginkgo biloba cell cultures

Ginkgo biloba cells were cultured in two 500 mL shake flasks and in 2 L and 6 L immobilization bioreactors using MS medium supplemented with 1 mg.L^–1 NAA, 0.1 mg.L^–1 K and 30 g.L^–1 sucrose. Specific growth rates were 0.06 d^–1, 0.11 d^–1 and 0.07 d^–1 for the 2 L and 6 L bioreactors and shake flask cultures, respectively. Extracellular phosphate, nitrate, ammonium and carbohydrate uptake rates of the bio reactor cultures were approximately 17 to 39% slower than those of shake flask cultures. The specific oxygen uptake and carbon dioxide transfer rates of immobilized Ginkgo biloba cells ranged from 0.027 to 0.041 mmol O₂.g^–1.d.w.hr^–1 (maximum uptake at 14 days) and 0.020 to 0.057 mmol CO₂g. ^–1.d.w.hr^–1 (maximum production at 14 days). Extracts from the biomass of the two immobilized and shake flask suspension cultures were analysed for ginkgolide A by GC-MS. Yields of 7, 17, 19 and 7 ng.g. ^–1d.w. of ginkgolide A were determined for shake flask 1, shake flask 2 and the 2 L and 6 L immobilized cultures, respectively. Traces of ginkgolide B were detected with the signal to noise ratio, however, being too low for positive confirmation of this last product.Abbreviations CTR Carbon dioxide transfer rate - DO Dissolved oxygen - g.d.w. Gram dry weight - GA Ginkgolide A - GB Ginkgolide B - GC Gas chromatography - GC-MS Gas chromatography-mass spectrometry - HPLC High performance liquid chromatography - K Kinetin - MS Murashige and Skoog salt medium - N₁K₁MS Complete Murashige and Skoog medium supplemented with 1 mg.L^–1 NAA, 0.1 mg.L^–1 K and 30g.L^–1 sucrose - NAA Naphthaleneacetic acid - OTR Oxygen transfer rate - PAF Platelet Aggregating Factor - q_CO2 Specific carbon dioxide production rate - q_O2 Specific oxygen uptake rate - u Specific growth rate     

Quercetin abrogates IL-6/STAT3 signaling and inhibits glioblastoma cell line growth and migration

Evidence has suggested that STAT3 functions as an oncogene in gliomagenesis. As a consequence, changes in the inflammatory microenvironment are thought to promote tumor development. Regardless of its origin, cancer-related inflammation has many tumor-promoting effects, such as the promotion of cell cycle progression, cell proliferation, cell migration and cell survival. Given that IL-6, a major cancer-related inflammatory cytokine, regulates STAT3 activation and is upregulated in glioblastoma, we sought to investigate the inhibitory effects of the chemopreventive flavonoid quercetin on glioblastoma cell proliferation and migration triggered by IL-6, and to determine the underlying mechanisms of action. In this study, we show that quercetin is a potent inhibitor of the IL-6-induced STAT3 signaling pathway in T98G and U87 glioblastoma cells. Exposure to quercetin resulted in the reduction of GP130, JAK1 and STAT3 activation by IL-6, as well as a marked decrease of the proliferative and migratory properties of glioblastoma cells induced by IL-6. Interestingly, quercetin also modulated the expression of two target genes regulated by STAT3, i.e. cyclin D1 and matrix metalloproteinase-2 (MMP-2). Moreover, quercetin reduced the recruitment of STAT3 at the cyclin D1 promoter and inhibited Rb phosphorylation in the presence of IL-6. Overall, these results provide new insight into the role of quercetin as a blocker of the STAT3 activation pathway stimulated by IL-6, with a potential role in the prevention and treatment of glioblastoma.     

Vascular endothelial growth factor-C stimulates the lymphatic pump by a VEGF receptor-3-dependent mechanism

Vascular endothelial growth factor (VEGF)-C plays an important role in lymphangiogenesis; however, functional responses of lymphatic vessels to VEGF-C have not been characterized. We tested the hypothesis that VEGF-C-induced activation of VEGF receptor (VEGFR)-3 increases lymphatic pump output. We examined the in vivo pump activity of rat mesenteric collecting lymphatics using intravital microscopy during basal conditions and during treatment with 1 nM recombinant VEGF-C, the selective VEGFR-3 agonist VEGF-Cys(156)Ser mutation (C156S; 1 nM), or 0.1 nM VEGF-A. Their specific responses were also analyzed during selective inhibition of VEGFR-3 with MAZ-51. Contraction frequency, end-diastolic diameter, end-systolic diameter, stroke volume index, pump flow index, and ejection fraction were evaluated. We also assessed arteriolar diameter and microvascular extravasation of FITC-albumin. The results show that both VEGF-C and VEGF-C156S significantly increased contraction frequency, end-diastolic diameter, stroke volume index, and pump flow index in a time-dependent manner. VEGF-A caused a different response characterized by a significantly increased stroke volume after 30 min of treatment. MAZ-51 (5 muM) caused tonic constriction and decreased contraction frequency. In addition, 0.5 and 5 muM MAZ-51 attenuated VEGF-C- and VEGF-C156S-induced lymphatic pump activation. VEGF-A caused vasodilation of arterioles, whereas VEGF-C and VEGF-C156S did not significantly alter arteriolar diameter. Also, VEGF-A and VEGF-C caused increased microvascular permeability, whereas VEGF-C156S did not. Our results demonstrate that VEGF-C increases lymphatic pumping through VEGFR-3. Furthermore, changes in microvascular hemodynamics are not required for VEGFR-3-mediated changes in lymphatic pump activity.