Regulation of cell survival by sphingosine-1-phosphate receptor S1P1 via reciprocal ERK-dependent suppression of bim and PI-3-kinase/protein kinase C-mediated upregulation of Mcl-1

Although the ability of bioactive lipid sphingosine-1-phosphate (S1P) to positively regulate anti-apoptotic/pro-survival responses by binding to S1P1 is well known, the molecular mechanisms remain unclear. Here we demonstrate that expression of S1P1 renders CCL39 lung fibroblasts resistant to apoptosis following growth factor withdrawal. Resistance to apoptosis was associated with attenuated accumulation of pro-apoptotic BH3-only protein Bim. However, although blockade of extracellular signal-regulated kinase (ERK) activation could reverse S1P1-mediated suppression of Bim accumulation, inhibition of caspase-3 cleavage was unaffected. Instead S1P1-mediated inhibition of caspase-3 cleavage was reversed by inhibition of phosphatidylinositol-3-kinase (PI3K) and protein kinase C (PKC), which had no effect on S1P1 regulation of Bim. However, S1P1 suppression of caspase-3 was associated with increased expression of anti-apoptotic protein Mcl-1, the expression of which was also reduced by inhibition of PI3K and PKC. A role for the induction of Mcl-1 in regulating endogenous S1P receptor-dependent pro-survival responses in human umbilical vein endothelial cells was confirmed using S1P receptor agonist FTY720-phosphate (FTY720P). FTY720P induced a transient accumulation of Mcl-1 that was associated with a delayed onset of caspase-3 cleavage following growth factor withdrawal, whereas Mcl-1 knockdown was sufficient to enhance caspase-3 cleavage even in the presence of FTY720P. Consistent with a pro-survival role of S1P1 in disease, analysis of tissue microarrays from ER+ breast cancer patients revealed a significant correlation between S1P1 expression and tumour cell survival. In these tumours, S1P1 expression and cancer cell survival were correlated with increased activation of ERK, but not the PI3K/PKB pathway. In summary, pro-survival/anti-apoptotic signalling from S1P1 is intimately linked to its ability to promote the accumulation of pro-survival protein Mcl-1 and downregulation of pro-apoptotic BH3-only protein Bim via distinct signalling pathways. However, the functional importance of each pathway is dependent on the specific cellular context

Steel-based applications in earthquake-prone areas

Steel-Earth project aims at distributing among technicians, engineers, design companies and standardization bodies the results of three past RFCS projects (Steel-Retro [3], Opus [2] and PrecaSteel [1]), providing useful tools for the design and for the retrofit of existing buildings. Technical documents and practical applications to case studies, regarding design of steel and composite steel/concrete buildings and innovative steel-based techniques for the retrofit of existing r.c. and masonry constructions, have been elaborated and collected into a volume distributed during the final workshop of the dissemination project. Pre-normative and background documents concerning the design of steel and composite structures and the rehabilitation of existing constructions have been prepared. A lot of attention has been paid to the analysis of the influence of overstrength factors on the seismic design of steel and composite structures. The prepared documents have been distributed to the attending people and to the members of WG 2 (CEN/TC 250/SC 8/WG 2 “Steel and Composite Structures”) during the final workshop of the project. Technical sheets, working examples and background documents have been translated into several languages (German, French, Italian, Romanian and Greek) and are free available on the website of the project (https://www.steelconstruct.com/site/), where information regarding Steel-Earth are also presented.11 Workshops in Italy, Greece, Germany, Belgium, Portugal, Spain and Romania and 5 conferences in Emilia-Romagna have been organized, as well as 2 practical courses for engineers and academic people in Pavia (Italy). Flash-drives with the technical documents and applications elaborated in Steel-Earth have been distributed to the attending people

ERK2 phosphorylation of serine 77 regulates Bmf pro-apoptotic activity

B-cell lymphoma 2 (Bcl-2) homology 3 (BH3)-only proteins represent a class of pro-apoptotic factors that neutralize pro-survival Bcl-2 proteins, and, in some cases, directly activate Bax. The mechanisms of control and the role of BH3-only proteins, such as Bcl-2 like protein 11 extra large and Bad are well studied. By contrast, relatively little is known about the regulation and role of Bcl-2 modifying factor (Bmf). The B-RAF oncogene is mutated in ∼8% of human tumors. We have previously shown that Bmf is upregulated at the transcript level and is required for apoptosis induced by targeting B-RAF signaling in tumor cells harboring mutant B-RAF. In this study, we show that Bmf is regulated at the post-translational level by mutant B-RAF-MEK-ERK2 signaling. Extracellular signal-regulated kinase (ERK2) directly phosphorylates Bmf on serine 74 and serine 77 residues with serine 77 being the predominant site. In addition, serine 77 phosphorylation reduces Bmf pro-apoptotic activity likely through a mechanism independent of altering Bmf localization to the mitochondria and/or interactions with dynein light chain 2 and the pro-survival proteins, B-cell lymphoma extra large, Bcl-2 and Mcl-1. These data identify a novel mode of regulation in Bmf that modulates its pro-apoptotic activity in mutant B-RAF tumor cells

Correlation of SHOX2 Gene Amplification and DNA Methylation in Lung Cancer Tumors

<p>Abstract</p> <p>Background</p> <p>DNA methylation in the <it>SHOX2 </it>locus was previously used to reliably detect lung cancer in a group of critical controls, including 'cytologically negative' samples with no visible tumor cell content, at a high specificity based on the analysis of bronchial lavage samples. This study aimed to investigate, if the methylation correlates with <it>SHOX2 </it>gene expression and/or copy number alterations. An amplification of the <it>SHOX2 </it>gene locus together with the observed tumor-specific hypermethylation might explain the good performance of this marker in bronchial lavage samples.</p> <p>Methods</p> <p><it>SHOX2 </it>expression, gene copy number and DNA methylation were determined in lung tumor tissues and matched morphologically normal adjacent tissues (NAT) from 55 lung cancer patients. Quantitative HeavyMethyl (HM) real-time PCR was used to detect <it>SHOX2 </it>DNA methylation levels. <it>SHOX2 </it>expression was assayed with quantitative real-time PCR, and copy numbers alterations were measured with conventional real-time PCR and array CGH.</p> <p>Results</p> <p>A hypermethylation of the <it>SHOX2 </it>locus in tumor tissue as compared to the matched NAT from the same patient was detected in 96% of tumors from a group of 55 lung cancer patients. This correlated highly significantly with the frequent occurrence of copy number amplification (p < 0.0001), while the expression of the <it>SHOX2 </it>gene showed no difference.</p> <p>Conclusions</p> <p>Frequent gene amplification correlated with hypermethylation of the <it>SHOX2 </it>gene locus. This concerted effect qualifies <it>SHOX2 </it>DNA methylation as a biomarker for lung cancer diagnosis, especially when sensitive detection is needed, i.e. in bronchial lavage or blood samples.</p

Multiple recombinant events in human T-Cell leukemia virus type 1: complete sequences of recombinant African strains.

Africa is the largest known endemic area for HTLV-1, with a high diversity of molecular genotypes. We previously demonstrated that some HTLV-1 strains from North Africa (the a-NA clade) are the result of a recombinant event between Senegalese and West African strains. To better characterize these recombinants and their distribution, we sequenced the LTR region and/or a env gene fragment of a series of 52 HTLV-1 strains from 13 different countries in North and West Africa. Four samples from descendants of African slaves and native to French Guiana were also added. Furthermore, we characterized the complete sequence for 7 viral strains, from the different genotypes. Until now, no complete sequence was available for a-NA clade. Phylogenetic analysis demonstrates that most of the new African strains belong to the Cosmopolitan a-genotype. Ten new strains from the recombinant North African (a-NA) clade could be found in Morocco, Western Sahara, Mali, Guinea, Côte d'Ivoire, and Ghana. We also identified a new clade (named a-G-Rec) comprising 4 strains from Guinea and Ghana that arose from a distinct recombination event between strains from Senegal and West Africa. The analyses of complete sequences suggest that recombination does not only occur in the LTR but also in other regions (env/pol) of the genome. Our data strongly suggest that a-NA and a-G-Rec strains have a mosaic profile with genetic segments deriving from either a-WA or a-Sen strains. In conclusion, our work demonstrates that recombination in HTLV-1 may not be such a rare event as previously proposed

Development and initial validation of the EDIN scale, a new tool for assessing prolonged pain in preterm infants

OBJECTIVE—To develop and validate a scale suitable for use in clinical practice as a tool for assessing prolonged pain in premature infants.
METHODS—Pain indicators identified by observation of preterm infants and selected by a panel of experts were used to develop the EDIN scale (Échelle Douleur Inconfort Nouveau-Né, neonatal pain and discomfort scale). A cohort of preterm infants was studied prospectively to determine construct validity, inter-rater reliability, and internal consistency of the scale.
RESULTS—The EDIN scale uses five behavioural indicators of prolonged pain: facial activity, body movements, quality of sleep, quality of contact with nurses, and consolability. The validation study included 76 preterm infants with a mean gestational age of 31.5weeks. Inter-rater reliability was acceptable, with a κ coefficient range of 0.59-0.74. Internal consistency was high: Cronbach's α coefficients calculated after deleting each item ranged from 0.86 to 0.94. To establish construct validity, EDIN scores in two extreme situations (pain and no pain) were compared, and a significant difference was observed.
CONCLUSIONS—The validation data suggest that the EDIN is appropriate for assessing prolonged pain in preterm infants. Further studies are warranted to obtain further evidence of construct validity by comparing scores in less extreme situations.