The Unique Non-Catalytic C-Terminus of P37delta-PI3K Adds Proliferative Properties In Vitro and In Vivo

The PI3K/Akt pathway is central for numerous cellular functions and is frequently deregulated in human cancers. The catalytic subunits of PI3K, p110, are thought to have a potential oncogenic function, and the regulatory subunit p85 exerts tumor suppressor properties. The fruit fly, Drosophila melanogaster, is a highly suitable system to investigate PI3K signaling, expressing one catalytic, Dp110, and one regulatory subunit, Dp60, and both show strong homology with the human PI3K proteins p110 and p85. We recently showed that p37δ, an alternatively spliced product of human PI3K p110δ, displayed strong proliferation-promoting properties despite lacking the catalytic domain completely. Here we functionally evaluate the different domains of human p37δ in Drosophila. The N-terminal region of Dp110 alone promotes cell proliferation, and we show that the unique C-terminal region of human p37δ further enhances these proliferative properties, both when expressed in Drosophila, and in human HEK-293 cells. Surprisingly, although the N-terminal region of Dp110 and the C-terminal region of p37δ both display proliferative effects, over-expression of full length Dp110 or the N-terminal part of Dp110 decreases survival in Drosophila, whereas the unique C-terminal region of p37δ prevents this effect. Furthermore, we found that the N-terminal region of the catalytic subunit of PI3K p110, including only the Dp60 (p85)-binding domain and a minor part of the Ras binding domain, rescues phenotypes with severely impaired development caused by Dp60 over-expression in Drosophila, possibly by regulating the levels of Dp60, and also by increasing the levels of phosphorylated Akt. Our results indicate a novel kinase-independent function of the PI3K catalytic subunit.     

Electronic Cigarette and Traditional Cigarette Use among Middle and High School Students in Florida, 2011–2014

Recent youth trends in the prevalence of e-cigarette and traditional cigarette use in Florida were examined in a cross-sectional, representative state sample from 2011 to 2014. Traditional cigarette use among youth declined during the study period. Experimentation with and past 30-day use of e-cigarettes among Florida youth tripled over 4 years. Past 30-day e-cigarette use exceeded traditional cigarette use in 2014; 10.8% of high school and 4.0% of middle school students reported recent e-cigarette use, compared with 8.7% of high school and 2.9% of middle school students for traditional cigarettes (P<0.001). By 2014, 20.5% of high school and 8.5% of middle school students reported ever use of e-cigarettes. Among ever e-cigarette users in 2014, 30.3% of high school and 42.2% of middle school students had never smoked traditional cigarettes. Given the concern that significant rates of e-cigarette use by U.S. adolescents may have a negative effect on public health, further review of e-cigarette advertising, marketing, sales, and use among U.S. youth is warranted.     

Endothelial-Mesenchymal Transition of Brain Endothelial Cells: Possible Role during Metastatic Extravasation

Cancer progression towards metastasis follows a defined sequence of events described as the metastatic cascade. For extravasation and transendothelial migration metastatic cells interact first with endothelial cells. Yet the role of endothelial cells during the process of metastasis formation and extravasation is still unclear, and the interaction between metastatic and endothelial cells during transendothelial migration is poorly understood. Since tumor cells are well known to express TGF-β, and the compact endothelial layer undergoes a series of changes during metastatic extravasation (cell contact disruption, cytoskeletal reorganization, enhanced contractility), we hypothesized that an EndMT may be necessary for metastatic extravasation. We demonstrate that primary cultured rat brain endothelial cells (BEC) undergo EndMT upon TGF-β1 treatment, characterized by the loss of tight and adherens junction proteins, expression of fibronectin, β1-integrin, calponin and α-smooth muscle actin (SMA). B16/F10 cell line conditioned and activated medium (ACM) had similar effects: claudin-5 down-regulation, fibronectin and SMA expression. Inhibition of TGF-β signaling during B16/F10 ACM stimulation using SB-431542 maintained claudin-5 levels and mitigated fibronectin and SMA expression. B16/F10 ACM stimulation of BECs led to phosphorylation of Smad2 and Smad3. SB-431542 prevented SMA up-regulation upon stimulation of BECs with A2058, MCF-7 and MDA-MB231 ACM as well. Moreover, B16/F10 ACM caused a reduction in transendothelial electrical resistance, enhanced the number of melanoma cells adhering to and transmigrating through the endothelial layer, in a TGF-β-dependent manner. These effects were not confined to BECs: HUVECs showed TGF-β-dependent SMA expression when stimulated with breast cancer cell line ACM. Our results indicate that an EndMT may be necessary for metastatic transendothelial migration, and this transition may be one of the potential mechanisms occurring during the complex phenomenon known as metastatic extravasation.     

Effect of anticoagulants in vitro on the viability of lymphocytes and content of free fatty acids in plasma

Summary These authors attempted to test the effect of anticoagulants on lymphocytes viability by reproducing the procedure used for lymphocyte isolation for various immunologic tests in which blood specimens are allowed to stay at room temperature for 2 h before lymphocytes are isolated. Blood was obtained with three different anticoagulants i.e. heparin, citrate, and CPDA (citrate, phosphate, dextrose, and adenine). Plasma was lyophilized and extracted with ethanol. Dried ethanol extracts were suspended in medium (RPMI 1640+10% fetal bovine serum) and incubated with a lymphocyte cell line (MOLT-4). After 24 h of incubation the viability of cells was examined. The following death rates of the cells were observed: heparin −63±4.6% (mean±SEM), citrate −27±6.7%, and CPDA 6.2±0.6% (P<0.0005). A significant correlation was found between these results and changes in the concentrations of free fatty acids in the extracts. These results emphasize the importance of choosing the right anticoagulant when the viability of lymphocytes is obligatory.