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Large inconsistencies still exist in nuclear data libraries regarding the kinetic parameters of delayed neutron (DN) precursors. As an example, there is a 17% gap between ENDF-B/VIII.0 and JEFF-3.3 on the average lifetime T1/2 of DN precursors from thermal fission of 235U. This parameter is of major importance for reactivity predictions of nuclear reactors in nominal or accidental configurations. In this context, CEA is actively participating to the ALDEN project (Average number and Lifetime of DElayed Neutrons) which aims at providing the nuclear data community with new data sets of DN from thermal and fast neutron induced fission of various actinides. A dedicated experimental setup was designed and optimized for that purpose and is presented in this paper. It consists of a “long counter” detector containing 16 proportional counters filled with 3He, embedded in a high density polyethylene matrix. The detector surrounds a fissile target prepared in the form of a miniature fission chamber, containing a few hundreds of micro-grams of fissile material. This set-up is connected to fast and efficient neutron shutters that can produce step-irradiations of variable durations. The equations driving the DN counting following step-irradiations of the fissile target are established and discussed in the perspective of DN yield or group parameter measurement. A comprehensive analysis of the different steps of data reduction is detailed: dead time characterization, Region of Interest (ROI) determination, absolute and relative efficiency calibration, fission rate estimation, irradiation time and background determination, DN decay curve production and physical parameter fitting. Following a prototype experiment performed in 2018 at the PF1b cold neutron beam line of Institute Laue Langevin (ILL, Grenoble, France), we discuss here the analysis of two campaigns occurring in 2019 and 2021 in which significant improvements were achieved in terms of background minimization, counting statistics and fission rate determination. The achievements of this work are the measurement of the delayed neutron emission per fission for the thermal neutron induced fission of 235U, estimated at (1.625 ± 0.010) % and the group parameters leading to an estimated lifetime of T 1 / 2 = (8.87 ± 0.10) s. Those results are consistent with the values recommended by the IAEA/CRP work and they come with reduced uncertainties compared with previously published results. [ABSTRACT FROM AUTHOR]

Post-transcriptional regulatory mechanisms play a role in many biological contexts through the control of mRNA degradation, translation and localization. Here, we show that the RING finger protein RNF219 co-purifies with the CCR4-NOT complex, the major mRNA deadenylase in eukaryotes, which mediates translational repression in both a deadenylase activity-dependent and -independent manner. Strikingly, RNF219 both inhibits the deadenylase activity of CCR4-NOT and enhances its capacity to repress translation of a target mRNA. We propose that the interaction of RNF219 with the CCR4-NOT complex directs the translational repressive activity of CCR4-NOT to a deadenylation-independent mechanism. [ABSTRACT FROM AUTHOR]

Ontologies have been increasingly used over the past few decades in a wide range of application domains spanning both academic and industrial communities. As ontologies are the cornerstone of the Semantic Web, the technologies developed in this context, including ontology languages, specialized databases and query languages, have become widely used. However, the expressiveness of the proposed ontology languages does not always cover the needs of specific domains. For instance, engineering is a domain for which the LIAS laboratory has proposed dedicated solutions with a worldwide recognition. The underlying assumptions made in the context of the Semantic Web, an open and distributed environment, do not apply to the controlled environments of our projects where the correctness and completeness of modeling can be guaranteed to a certain degree. As a consequence, we have developed over the last decades a specialized standard ontology language named PLIB associated with the OntoDB/OntoQL platform to manage ontological engineering data within a database. The goal of this paper is threefold: (1) to share our experience in manipulating ontologies in the engineering domain by describing their specificities and constraints; (2) to define a comprehensive classification of ontologies with respect to three main research communities: Artificial Intelligence, Databases and Natural Language Processing and (3) to present a persistent solution, called OntoDB, for managing extremely large semantic data sets associated with an ontological query language, called OntoQL. These objectives are illustrated by several examples that show the effectiveness and interest of our propositions in several industrial projects in different domains including vehicle manufacturing and CO $$_2$$ storage. [ABSTRACT FROM AUTHOR]

Myogenic differentiation requires the coordination between permanent cell cycle withdrawal, mediated by members of the cyclin-dependent kinase inhibitor (CKI) family, and activation of a cascade of myogenic transcription factors, particularly MYOGENIN (MYOG). Recently, it has been reported that the Protein aRginine Methyl Transferase PRMT5 modulates the early phase of induction of MYOG expression. Here, we show that the histone- and PRMT5-associated protein COPR5 (cooperator of PRMT5) is required for myogenic differentiation. C2C12 cells, in which COPR5 had been silenced, could not irreversibly exit the cell cycle and differentiate into muscle cells. This phenotype might be explained by the finding that, in cells in which COPR5 was downregulated, p21 and MYOG induction was strongly reduced and PRMT5 recruitment to the promoters of these genes was also altered. Moreover, we suggest that COPR5 interaction with the Runt-related transcription factor 1 (RUNX1)-core binding factor-β (CBFβ) complex contributes to targeting the COPR5-PRMT5 complex to these promoters. Finally, we present evidence that COPR5 depletion delayed the in vivo regeneration of cardiotoxin-injured mouse skeletal muscles. Altogether, these data extend the role of COPR5 from an adaptor protein required for nuclear functions of PRMT5 to an essential coordinator of myogenic differentiation. [ABSTRACT FROM AUTHOR]

The initiation of DNA synthesis is governed by the licensing of replication origins, which consists of assembling a pre-replication complex (pre-RC) on origins during late M- and G1-phases. In metazoans, functional replication origins do not show defined DNA consensus sequences, thus evoking the involvement of chromatin determinants in the selection of these origins. Here, we show that the onset of licensing in mammalian cells coincides with an increase in histone H4 Lys 20 monomethylation (H4K20me1) at replication origins by the methyltransferase PR-Set7 (also known as Set8 or KMT5A). Indeed, tethering PR-Set7 methylase activity to a specific genomic locus promotes the loading of pre-RC proteins on chromatin. In addition, we demonstrate that PR-Set7 undergoes a PCNA- and Cul4-Ddb1-driven degradation during S phase that contributes to the disappearance of H4K20me1 at origins and the inhibition of replication licensing. Strikingly, expression of a PR-Set7 mutant insensitive to this degradation causes the maintenance of H4K20me1 and repeated DNA replication at origins. These results elucidate a critical role for PR-Set7 and H4K20me1 in the chromatin events that regulate replication origins. [ABSTRACT FROM AUTHOR]

CDK4–pRB–E2F1 cell-cycle regulators are robustly expressed in non-proliferating β cells, suggesting that besides the control of β-cell number the CDK4–pRB–E2F1 pathway has a role in β-cell function. We show here that E2F1 directly regulates expression of Kir6.2, which is a key component of the KATP channel involved in the regulation of glucose-induced insulin secretion. We demonstrate, through chromatin immunoprecipitation analysis from tissues, that Kir6.2 expression is regulated at the promoter level by the CDK4–pRB–E2F1 pathway. Consistently, inhibition of CDK4, or genetic inactivation of E2F1, results in decreased expression of Kir6.2, impaired insulin secretion and glucose intolerance in mice. Furthermore we show that rescue of Kir6.2 expression restores insulin secretion in E2f1−/− β cells. Finally, we demonstrate that CDK4 is activated by glucose through the insulin pathway, ultimately resulting in E2F1 activation and, consequently, increased expression of Kir6.2. In summary we provide evidence that the CDK4–pRB–E2F1 regulatory pathway is involved in glucose homeostasis, defining a new link between cell proliferation and metabolism. [ABSTRACT FROM AUTHOR]

Abstract  The enigmatic arrow worms (Chaetognatha) are marine carnivores and among the most abundant planktonic organisms. Their phylogenetic position has been heavily debated for a long time. Most recent molecular studies still provide a diverging picture and suggest arrow worms to be some kind of basal protostomes. In an effort to understand the organization of the nervous system in this clade for a broad comparison with other Metazoa we analysed the ultrastructure of the ventral nerve centre in Spadella cephaloptera by transmission electron microscopy. We were able to identify six different types of neurons in the bilateral somata clusters by means of the cytoplasmic composition (regarding the structure of the neurite and soma including the shape and eu-/heterochromatin ratio within the nucleus) as well as the size and position of these neurons. Furthermore, our study provides new insights into the neuropil composition of the ventral nerve centre and several other fine structural features. Our second goal was to examine if individually identifiable neurons are present in the ventral nerve centres of four chaetognath species, Sagitta setosa, Sagitta enflata, Pterosagitta draco, and Spadella cephaloptera. For that purpose, we processed whole mount specimens of these species for immunolocalization of RFamide-related neuropeptides and analysed them with confocal laser-scanning microscopy. Our experiments provide evidence for the interspecific homology of individual neurons in the ventral nerve centres of these four chaetognath species suggesting that the potential to generate serially arranged neurons with individual identities is part of their ground pattern. [ABSTRACT FROM AUTHOR]

The E1A-targeted transcription factor E4F1 is a key player in the control of mammalian embryonic and somatic cell proliferation and survival. Mouse embryos lacking E4F die at an early developmental stage, whereas enforced expression of E4F1 in various cell lines inhibits cell cycle progression. E4F1-antiproliferative effects have been shown to depend on its capacity to repress transcription and to interact with pRb and p53. Here we show that full-length E4F1 protein (p120E4F1) but not its E1A-activated and truncated form (p50E4F1), interacts directly in vitro and in vivo with the LIM-only protein FHL2, the product of the p53-responsive gene FHL2/DRAL (downregulated in rhabdomyosarcoma Lim protein). This E4F1-FHL2 association occurs in the nuclear compartment and inhibits the capacity of E4F1 to block cell proliferation. Consistent with this effect, ectopic expression of FHL2 inhibits E4F1 repressive effects on transcription and correlates with a reduction of nuclear E4F1-p53 complexes. Overall, these results suggest that FHL2/DRAL is an inhibitor of E4F1 activity. Finally, we show that endogenous E4F1-FHL2 complexes form in U2OS cells upon UV-light-induced nuclear accumulation of FHL2.Oncogene (2006) 25, 5475–5484. doi:10.1038/sj.onc.1209567; published online 1 May 2006 [ABSTRACT FROM AUTHOR]

The expression of cyclin E and that of a few other bona fide cell cycle regulatory genes periodically oscillates every cycle in proliferating cells. Although numerous experiments have documented the role of E2F sites and E2F activities in the control of these genes as cells exit from G0 to move through the initial G1/S phase transition, almost nothing is known on the role of E2Fs during the subsequent cell cycles. Here we show that a variant E2F-site that is part of the Cyclin E Repressor Module (CERM) (Le Cam et al., 1999b) accounts for the periodic down regulation of the cyclin E promoter observed between the exit from mitosis until the mid/late G1 phase in exponentially cycling cells. This cell cycle-dependent repression correlates with the periodic binding of an atypical G1-specific high molecular weight p107-E2F complex (Cyclin E Repressor Complex: CERC2) that differs in both size and DNA binding behaviors from known p107-E2F complexes. Notably, affinity purified CERC2 displays a TSA-sensitive histone deacetylase activity and, consistent with this, derepression of the cyclin E promoter by trichostatin A depends on the CERM element. Altogether, this shows that the cell cycle-dependent control of cyclin E promoter in cycling cells is embroiled in acetylation pathways via the CERM-like E2F element. Oncogene (2001) 20, 4115–4127. [ABSTRACT FROM AUTHOR]

Rho GTPases control actin reorganization and many other cellular functions. Guanine nucleotide-exchange factors (GEFs) activate Rho GTPases by promoting their exchange of GDP for GTP. Trio is a unique Rho GEF, because it has separate GEF domains, GEFD1 and GEFD2, that control the GTPases RhoG/Rac1 and RhoA, respectively. Dbl-homology (DH) domains that are common to GEFs catalyse nucleotide exchange, and pleckstrin-homology (PH) domains localize Rho GEFs near their downstream targets. Here we show that Trio GEFD1 interacts through its PH domain with the actin-filament-crosslinking protein filamin, and localizes with endogenous filamin in HeLa cells. Trio GEFD1 induces actin-based ruffling in filamin-expressing, but not filamin-deficient, cells and in cells transfected with a filamin construct that lacks the Trio-binding domain. In addition, Trio GEFD1 exchange activity is not affected by filamin binding. Our results indicate that filamin, as a molecular target of Trio, may be a scaffold for the spatial organization of Rho-GTPase-mediated signalling pathways. [ABSTRACT FROM AUTHOR]

The p16-cyclin D-pRB-E2F pathway is frequently deregulated in human tumors. This critical regulatory pathway controls the G1/S transition of the mammalian cell cycle by positive and negative regulation of E2F-responsive genes required for DNA replication. To assess the value of the transcription factors E2Fs as targets for antiproliferative strategies, we have initiated a program aiming to develop inhibitors targeting specifically these proteins in vitro and in vivo. The cellular activity of E2F is the result of the heterodimeric association of two families of proteins, E2Fs and DPs, which then bind DNA. Here, we use a two hybrid approach to isolate from combinatorial libraries peptide aptamers that specifically interact with E2Fs DNA binding and dimerization domains. One of these is a potent inhibitor of E2F binding activity in vitro and in mammalian fibroblasts, blocks cells in G1, and the free variable region from this aptamer has the same effect. Our experiments argue that the variable region of this aptamer is structured, and that it functions by binding E2F with a motif that resembles a DP heterodimerization region, and blocking E2F's association with DP. These results show that cell proliferation can be inhibited using genetically-selected synthetic peptides that specifically target protein-protein interaction motifs within cell cycle regulators. These results also emphasize the critical role of the E2F pathway for cell proliferation and might allow the design of novel antiproliferative agents targeting the cyclin/CDK-pRB-E2F pathway. [ABSTRACT FROM AUTHOR]

Freeze-substitution was used to study the distribution of calcium in sea urchin eggs, and the validity of the technique was assessed. We followed the fate of both total and exchangeable calcium of sea urchin eggs in two species ( Paracentrotus lividus and Arbacia lixula) after the various treatments needed for freeze-substitution and embedding. We compared the calcium content either by X-ray microanalysis of Epon-embedded sections of freeze-substituted eggs (6.2±0.71 mmoles/kg of Epon-embedded tissue) or by flame spectrometry analysis of living eggs (32.3±1.30 nmoles/mg protein). After standardization of units, both values lead to similar total calcium content. We also measured the movements of Ca from prelabelled eggs. Exchangeable Ca as well as total calcium appeared unaffected by the preparative treatment for X-ray microanalysis. In conclusion, our preparative technique for X-ray microanalysis can be considered appropriate for our material and allows us to undertake a subcellular quantification of calcium in various organelles. [ABSTRACT FROM AUTHOR]

E2F-1 is the prototype of a family of transcription factors playing a central role in the control of cell proliferation and apoptosis. E2F DNA binding activity is down-regulated during cellular differentiation, which is correlated with cell division arrest. We report here that the expression of E2F-1 itself is down-regulated in the developing quail neural retina between embryonic days E8-E10. This event occurs just after the massive arrest of the quail neuroretina cell division (E7-E8). To gain further insight into the regulatory mechanisms monitoring E2F-1 expression in differentiating neurons, we have cloned the quail E2F-1 promoter. In vivo DNA footprintings of this promoter have shown that a number of potential SP-1 and C/EBP response elements are constitutively occupied in the entire quail neuroretina of E5 and E14, whereas the two consensus palindromic E2F binding sites are only protected at E5. This suggests that these E2F elements participate in down-regulation of E2F-1 gene expression during avian neuroretina development. CAT reporter assays have shown that E2F-1 in association with its partner DP-1 transactivates its own promoter, whereas p105Rb inhibits the E2F-1 promoter. Both E2F-1/DP-1 and p105Rb require the presence of the E2F binding sites to mediate their effects. However, experiments performed with deletion mutants of the promoter strongly suggest that other regions located upstream of the E2F binding sites also mediate part of the E2F-1 transactivating effect on its own promoter. Altogether, these results suggest that the down-regulation of E2F-1 gene expression in differentiating neurons could be due, in part, to the E2F/Rb complexes binding to the E2F-1 promoter. [ABSTRACT FROM AUTHOR]

Using the yeast two-hybrid system we have identified novel potential Cdk4 interacting proteins. Here we described the interaction of Cdk4 with a human homologue of the yeast Drosophila CDC37 gene products. Cdc37 protein specifically interacts with Cdk4 and Cdk6, but not with Cdc2, Cdk2, Cdk3, Cdk5 and any of a number of cyclins tested. Cdc37 is not an inhibitor nor an activator of the Cdk4/cyclin D1 kinase, while it appears to facilitate complex assembly between Cdk4 and cyclin D1 in vitro. Cdc37 competes with p16 for binding to Cdk4, suggesting that p16 might exert part of its inhibitory function by affecting the formation of Cdk4/cyclin D1 complexes via Cdc37. [ABSTRACT FROM AUTHOR]