Treating the placenta to prevent adverse effects of gestational hypoxia on fetal brain development.

Some neuropsychiatric disease, including schizophrenia, may originate during prenatal development, following periods of gestational hypoxia and placental oxidative stress. Here we investigated if gestational hypoxia promotes damaging secretions from the placenta that affect fetal development and whether a mitochondria-targeted antioxidant MitoQ might prevent this. Gestational hypoxia caused low birth-weight and changes in young adult offspring brain, mimicking those in human neuropsychiatric disease. Exposure of cultured neurons to fetal plasma or to secretions from the placenta or from model trophoblast barriers that had been exposed to altered oxygenation caused similar morphological changes. The secretions and plasma contained altered microRNAs whose targets were linked with changes in gene expression in the fetal brain and with human schizophrenia loci. Molecular and morphological changes in vivo and in vitro were prevented by a single dose of MitoQ bound to nanoparticles, which were shown to localise and prevent oxidative stress in the placenta but not in the fetus. We suggest the possibility of developing preventative treatments that target the placenta and not the fetus to reduce risk of psychiatric disease in later life

RhoE Is Regulated by Cyclic AMP and Promotes Fusion of Human BeWo Choriocarcinoma Cells

Fusion of placental villous cytotrophoblasts with the overlying syncytiotrophoblast is essential for the maintenance of successful pregnancy, and disturbances in this process have been implicated in pathological conditions such as pre-eclampsia and intra-uterine growth retardation. In this study we examined the role of the Rho GTPase family member RhoE in trophoblast differentiation and fusion using the BeWo choriocarcinoma cell line, a model of villous cytotrophoblast fusion. Treatment of BeWo cells with the cell permeable cyclic AMP analogue dibutyryl cyclic AMP (dbcAMP) resulted in a strong upregulation of RhoE at 24h, coinciding with the onset of fusion. Using the protein kinase A (PKA)-specific cAMP analogue N6-phenyl-cAMP, and a specific inhibitor of PKA (14–22 amide, PKI), we found that upregulation of RhoE by cAMP was mediated through activation of PKA signalling. Silencing of RhoE expression by RNA interference resulted in a significant decrease in dbcAMP-induced fusion. However, expression of differentiation markers human chorionic gonadotrophin and placental alkaline phosphatase was unaffected by RhoE silencing. Finally, we found that RhoE upregulation by dbcAMP was significantly reduced under hypoxic conditions in which cell fusion is impaired. These results show that induction of RhoE by cAMP is mediated through PKA and promotes BeWo cell fusion but has no effect on functional differentiation, supporting evidence that these two processes may be controlled by separate or diverging pathways

Downregulation of caveolin-1 enhances fusion of human BeWo choriocarcinoma cells

Background: Fusion of placental villous cytotrophoblasts with the overlying syncytiotrophoblast is essential for the maintenance of successful pregnancy, and disturbances in this process have been implicated in pathological conditions such as pre-eclampsia and intra-uterine growth retardation. Caveolin-1 has been shown to be expressed in human villous cytotrophoblast and to be downregulated during fusion into syncytiotrophoblast but it is unclear whether it plays a role in this process.Methodology/Principal Findings: We used RNA interference to determine whether caveolin-1 plays a role in differentiation and fusion in the BeWo choriocarcinoma cell line, a model of villous cytotrophoblast fusion. Assessment of cell fusion by desmosomal protein immunostaining revealed that cells transfected with caveolin-1 siRNA showed significantly enhanced fusion in response to treatment with dibutyryl cyclic AMP compared with cells transfected with a non-silencing control. Furtermore, caveolin-1 knockdown alone was sufficient to promote spontaneous fusion. In addition, biochemical differentiation, assessed by expression of placental alkaline phosphatase, was upregulated in caveolin-1 siRNA-transfected cells, with or without dbcAMP treatment. Assessment of Akt phosphorylation showed that caveolin-1 knockdown resulted in a significant reduction in phosphorylation at Thr308.Conclusions/Significance: Taken together, these results suggest that caveolin-1 regulates BeWo cell differentiation and fusion, possibly through a mechanism involving modulation of Akt activity.</p

siRNA-mediated downregulation of caveolin-1 expression in BeWo cells.

<p><i>A</i>, BeWo cells were transfected with the indicated concentrations of caveolin-1(Cav-1) siRNA or a non-silencing control siRNA. After 48 h cells were lysed and levels of caveolin-1 and β-actin were assessed by immunoblotting. <i>B</i>, densitometric analysis of immunoblots assessed for caveolin-1 expression and normalised to β-actin expression, in cells transfected for 48 h with 50 nM Cav-1 siRNA or non-silencing control siRNA. Results are presented as mean ± SEM for three separate experiments, *p<0.001 compared with control transfected cells (determined by ANOVA).</p

Effect of cyclic AMP on RhoE expression and fusion in BeWo cells.

<p>BeWo cells were treated with or without 1mM dbcAMP and studied at the indicated times. Cell lysates were made and expression of RhoE and β-actin was assessed by immunoblotting (A) and densitometric analysis of blots (B). Cells were fixed, immunostained for desmosomal protein (green) and counterstained with Hoechst 33258 (blue) (C) and cell fusion was quantified (D) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030453#s2" target="_blank">Materials & Methods</a>. Results are presented as mean ± SEM for three separate experiments. *p<0.05, **p<0.01 compared with control (determined by ANOVA).</p

RhoE is expressed in primary human villous cytotrophoblasts.

<p>Lysates were prepared from BeWo cells or freshly isolated primary human villous cytotrophoblasts (three separate preparations) and assessed for RhoE and β-actin expression by immunoblotting.</p

Effect of hypoxia on cAMP-induced RhoE expression.

<p>BeWo cells were cultured at 20% O₂ (normoxia) or 1% O₂ (hypoxia) for 24h, then treated with 1mM dbcAMP at 20% or 1% O₂ for a further 24h. Cell lysates were made and RhoE and β-actin expression was assessed by immunoblotting (A) with densitometric analysis of RhoE expression normalised to β-actin expression (B). A representative blot from three separate experiments is shown.</p

Effect of RhoE knockdown on BeWo cell fusion and differentiation.

<p>BeWo cells were transfected with 50nM RhoE siRNA or a non-silencing control then treated with 1mM dbcAMP and studied at the time points indicated. Cell lysates were made and expression of RhoE and β-actin was assessed by immunoblotting (A). Cells were also fixed and immunostained for desmosomal protein and cell fusion quantified (B) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030453#s2" target="_blank">Materials & Methods</a>. Cell lysates were also studied for expression of PLAP, β-hCG and β-actin by immunoblotting (C) with densitometric analysis normalised to β-actin expression (D). Results are presented as mean ± SEM for four separate experiments. *p<0.05 compared with non-silencing control (determined by ANOVA).</p

Attenuation of RhoE expression in response to cyclic AMP in JEG-3 cells.

<p>BeWo or JEG-3 cells were treated with 1mM dbcAMP. At the indicated times cell lysates were made and expression of RhoE and β-actin was assessed by immunoblotting (A) with densitometric analysis of RhoE expression normalised to β-actin expression (B). Cells were fixed, immunostained for desmosomal protein (C) and cell fusion was quantified (D) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030453#s2" target="_blank">Materials & Methods</a>. Results are presented as mean ± SEM for three separate experiments. *p<0.05, **p<0.01 compared with BeWo cells (determined by ANOVA).</p

Emissions of trace gases and aerosols during the open combustion of biomass in the laboratory

We characterized the gas- and speciated aerosol-phase emissions from the open combustion of 33 different plant species during a series of 255 controlled laboratory burns during the Fire Laboratory at Missoula Experiments (FLAME). The plant species we tested were chosen to improve the existing database for U.S. domestic fuels: laboratory-based emission factors have not previously been reported for many commonly-burned species that are frequently consumed by fires near populated regions and protected scenic areas. The plants we tested included the chaparral species chamise, manzanita, and ceanothus, and species common to the southeastern US (common reed, hickory, kudzu, needlegrass rush, rhododendron, cord grass, sawgrass, titi, and wax myrtle). Fire-integrated emission factors for gas-phase CO{sub 2}, CO, CH{sub 4}, C{sub 2-4} hydrocarbons, NH{sub 3}, SO{sub 2}, NO, NO{sub 2}, HNO{sub 3} and particle-phase organic carbon (OC), elemental carbon (EC), SO{sub 4}{sup 2-}, NO{sub 3}{sup -}, Cl{sup -}, Na{sup +}, K{sup +}, and NH{sub 4}{sup +} generally varied with both fuel type and with the fire-integrated modified combustion efficiency (MCE), a measure of the relative importance of flaming- and smoldering-phase combustion to the total emissions during the burn. Chaparral fuels tended to emit less particulate OC per unit mass of dry fuel than did other fuel types, whereas southeastern species had some of the largest observed EF for total fine particulate matter. Our measurements often spanned a larger range of MCE than prior studies, and thus help to improve estimates for individual fuels of the variation of emissions with combustion conditions