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The PETALE program aims to provide new experimental data to constrain the stainless steel nuclear data. In this frame, a preliminary measurement campaign has been performed to characterize the neutron flux in key positions of the CROCUS reactor and to develop analysis tools. During this preliminary campaign detailed in the present paper, an efficiency ratio technique has been developed and tested to speed up HPGe measurements by a factor of 30. A second objective of the campaign concerns the propagation of nuclear data uncertainty from the core neutron cross-sections to the reaction rates in the dosimeters. Uncertainties in the core cross sections, such as the uranium cross section, are nuisance parameters that add uncertainty to the dosimeter reaction rate calculation. This component must be fully characterized with covariances to constrain the metal reflector component for Bayesian assimilation. The experimental results are compared to the calculations with different nuclear databases for the nuclear data uncertainty propagation. A good agreement is obtained with the ENDF/B-VII.1 database and a systematic underestimation of around 5–10% in the fast range is observed with the ENDF/B-VIII.0 and JEFF-3.3 databases.

Neutron inspection of sea-going cargo containers has been widely studied in the past 20 yr to non-intrusively detect terrorist threats, like explosives or Special Nuclear Materials (SNM), and illicit goods, like narcotics or smuggling materials. Fast 14 MeV neutrons are produced by a portable generator with the t(d, n)α fusion reaction, and tagged in both direction and time thanks to the alpha particle detection. This Associated Particle Technique (APT) allows focusing inspection on specific areas of interest in the containers, previously identified as containing suspicious items with X-ray radiographic scanners or radiation portal monitors. We describe the principle of APT for non-nuclear material identification, and for nuclear material detection, then we provide illustrations of the performances for 10 min inspections with significant quantities (kilograms) of explosives, illicit drugs, or SNM, in different cargo cover loads (e.g. metallic, organic, or ceramic matrices).

In the normal operation of nuclear reactors, the kinetic behavior of the neutron population in the core is driven by the so-called delayed neutrons (DN). The DN yield per fission, their average lifetime and their groups’ abundances are the main physical parameters used to predict the kinetic behavior of the reactor and its reactivity. The overall uncertainty associated to reactivity predictions, which is linked directly to the nuclear reactor safety margins, is thus closely dependent on a few parameters associated with DN. Depending on the nuclear data library, DN kinetic parameters present significant discrepancies, even for major fissile isotopes such as 235U or 239Pu. In this context, CEA has long been working for improving DN nuclear data. In 2018, CEA launched the ALDEN project (Average Lifetime of DElayed Neutrons) in the framework of a collaboration between CEA/DES, CEA/DRF, CNRS/IN2P3 (LPSC, CENBG, LPC), ENSICAEN and Caen University. This program aims at measuring the kinetics of the delayed neutrons to derive the DN yield, their average lifetime and abundances. Two experimental campaigns focusing on the thermal fission of 235U were conducted in 2018 and 2019. They demonstrated the concept feasibility and gave high quality estimations of the DN multiplicity (1.631 ± 0.014 %) and average lifetime (8.82 ± 0.6 s) for 235U. More recently in March 2021, a more ambitious irradiation campaign was conducted on 239Pu and 233U. This paper details the new experimental setup, which was upgraded to meet ILL safety requirements for handling plutonium. The data analysis process is presented, with a focus on the problem of dead time correction. Finally, some preliminary delayed neutron decay curves are showed and discussed.

Thermo-luminescent detectors are currently used to measure gamma doses in the medical and environmental surveillance fields. During the past few years, the CEA Reactor Studies Division tested and validated the use of these detectors for gamma flux characterization and nuclear heating measurements in mixed neutron/gamma fields of low power reactors. Doses were comprised between a few mGy and a few Gy for dose rates up to a few Gy.h-1. However, in MTR or TRIGA reactors, the gamma flux level is much higher (> 1012 n/cm2/s) and the TLD currently in use (CaF2:Mn and 7LiF:Mg,Ti) and their readout protocols were no longer suitable for the resulting doses. In order to extend the applicable dose range up to ∼1 MGy (dose rate of a few kGy.h-1), several options were explored. On one side, some adjustments were made to the readout protocols of CaF2:Mn and 7LiF:Mg,Ti, notably by testing the use of filters to reduce the amount of light received by the reader PMT to avoid saturation. On the other side, a new type of TLD (LiF:Mg,Cu,P) with different Li enrichments (natural or enriched in 7Li) was tested. This paper presents the calibration measurements results performed in pure gamma fields, at the irradiation platform of the CEA Cadarache Radioprotection Division, between 250 mGy and 3 Gy for all detector types. In addition to the calibration, these measurements also studied the Mg,Cu,P-doped detectors response: reproducibility, dose rate dependence, incoming photon energy dependence, high temperature effect when reading TLD, etc. Results show that at low doses Mg,Cu,P-doped TLDs are slightly less stable than CaF2:Mn and 7LiF:Mg,Ti. The sensitivity modification after a high dose exposure seems to indicate that a new protocol readout should be defined for Mg,Cu,P-doped sensors (high temperature peak).

The Nuclear Measurement Laboratory (LMN) at CEA Cadarache in France is developing a high-energy tomograph reaching energies up to 21 MeV with high dose rates. It allows performing tomographies on massive objects (5 tons, 140 cm diameter) with a multimillimetric spatial resolution. For the control of absence of cracks, bubbles or defects in the concrete coating of some CEA waste drums, the laboratory needs a “high-resolution” version of this tomograph. The purpose of this study is to design an imaging detector able to provide a high spatial resolution on large objects with high dose resistance and scanning times of a few hours. To achieve this design, several steps are necessary and will be presented: – A first step consists in characterizing the current detection elements of the tomograph, such as the camera and its lens with experimental measurements. This enables to compare performance of these elements with that available on the market and to consider a replacement. – Then, the scintillator is selected according to experimental measurements on few scintillator types and a state of the art. – Next, the detector configuration is optimized to achieve the higher spatial resolution. Throughout these steps, we present the design of a highresolution detector with a spatial resolution around 300 µm for a 10%contrast.

The neutron field of various irradiation positions of the TRIGA Mark II reactor of the Jožef Stefan Institute has been thoroughly characterized by neutron activation dosimetry and miniature fission chambers techniques. In order to have a fully validated calculation scheme to analyze and plan experiments, the gamma field also has to be experimentally validated. The 10-year long collaboration between CEA and JSI is a perfect framework to carry out such a study, and measurements of the gamma field started in late 2016. Several measurement techniques were investigated in in-core and ex-core positions. On-line measurements were carried out using miniature ionization chambers manufactured by the CEA and PTW Farmer ionization chambers. Positional dependence was studied, showing a decrease in the delayed gamma contribution to the total gamma flux with increasing distance from the reactor core center. To characterize the gamma dose in the core, as well as in the periphery, thermo- and optically stimulated luminescent detectors were tested. These detectors are commonly used at CEA to measure the gamma dose in a given material in order to study the nuclear heating in various core elements (control rod, baffle, structural material). Different filters were used in order to assess an integrated dose ranging from a few Gy up to several kGy. The feasibility of such measurements demonstrates the complementarity between measurements with dosimetry and ionization chambers from low to very high gamma-dose environment, such as in material testing reactors.

Delayed-neutron (DN) data is essential in inherent reactor safety and reactor control since it is needed for the estimation of the reactivity. Nowadays, discrepancies among the data in various international databases (JEFF, ENDF, JENDL) are large and bring excessive conservatism in the safety margins. The ALDEN (Average Lifetime of DElayed Neutrons) experiment, built in a collaboration between CEA and CNRS, aimed at re-measuring the DN data associated with several fissioning systems (average delayed-neutron yield and kinetic parameters). The first experimental campaign consisted in the integral measurement of the DN activity after the irradiation of an 235U target. It took place under the cold neutron flux of ILL (Institut Laue-Langevin) at the beginning of September 2018, in the PF1b experimental zone (doi:10.1016/j.nima.2006.03.020). The data analysis gave an average DN yield of 1.631E-02(2) DN/fiss and a mean precursors’ half-life of 8.93(9) s. The results are consistent with the literature, but they are affected by one third of the uncertainty.

In the frame of C-BORD project (H2020 program of the EU), a Rapidly relocatable Tagged Neutron Inspection System (RRTNIS) is being developed to non-intrusively detect explosives, chemical threats, and other illicit goods in cargo containers. Material identification is performed through gamma spectroscopy, using twenty NaI detectors and four LaBr3 detectors, to determine the different elements composing the inspected item from their specific gamma signatures induced by fast neutrons. This is performed using an unfolding algorithm to decompose the energy spectrum of a suspect item, selected by X-ray radiography and on which the RRTNIS inspection is focused, on a database of pure element gamma signatures. This paper reports on simulated signatures for the NaI and LaBr3 detectors, constructed using the MCNP6 code. First experimental spectra of a few elements of interest are also presented.

Abstract Background Interstitial lung diseases (ILDs) in children represent a heterogeneous group of rare respiratory disorders that affect the lung parenchyma. After the launch of the French Reference Centre for Rare Lung Diseases (RespiRare®), we created a national network and a web-linked database to collect data on pediatric ILD. Methods Since 2008, the database has been set up in all RespiRare® centres. After patient's parents' oral consent is obtained, physicians enter the data of children with ILD: identity, social data and environmental data; specific aetiological diagnosis of the ILD if known, genetics, patient visits to the centre, and all medical examinations and tests done for the diagnosis and/or during follow up. Each participating centre has a free access to his own patients' data only, and cross-centre studies require mutual agreement. Physicians may use the system as a daily aid for patient care through a web-linked medical file, backed on this database. Results Data was collected for 205 cases of ILD. The M/F sex ratio was 0.9. Median age at diagnosis was 1.5 years old [0–16.9]. A specific aetiology was identified in 149 (72.7%) patients while 56 (27.3%) cases remain undiagnosed. Surfactant deficiencies and alveolar proteinosis, haemosiderosis, and sarcoidosis represent almost half of the diagnoses. Median length of follow-up is 2.9 years [0–17.2]. Conclusions We introduce here the French network and the largest national database in pediatric ILDs. The diagnosis spectrum and the estimated incidence are consistent with other European databases. An important challenge will be to reduce the proportion of unclassified ILDs by a standardized diagnosis work-up. This database is a great opportunity to improve patient care and disease pathogenesis knowledge. A European network including physicians and European foundations is now emerging with the initial aim of devising a simplified European database/register as a first step to larger European studies.

The Green Edge project was designed to investigate the onset, life, and fate of a phytoplankton spring bloom (PSB) in the Arctic Ocean. The lengthening of the ice-free period and the warming of seawater, amongst other factors, have induced major changes in Arctic Ocean biology over the last decades. Because the PSB is at the base of the Arctic Ocean food chain, it is crucial to understand how changes in the Arctic environment will affect it. Green Edge was a large multidisciplinary, collaborative project bringing researchers and technicians from 28 different institutions in seven countries together, aiming at understanding these changes and their impacts on the future. The fieldwork for the Green Edge project took place over two years (2015 and 2016) and was carried out from both an ice camp and a research vessel in Baffin Bay, in the Canadian Arctic. This paper describes the sampling strategy and the dataset obtained from the research cruise, which took place aboard the Canadian Coast Guard ship (CCGS) Amundsen in late spring and early summer 2016. The sampling strategy was designed around the repetitive, perpendicular crossing of the marginal ice zone (MIZ), using not only ship-based station discrete sampling but also high-resolution measurements from autonomous platforms (Gliders, BGC-Argo floats …) and under-way monitoring systems. The dataset is available at https://doi.org/10.17882/86417 (Bruyant et al., 2022).

La fabrication additive, métallique en particulier, est en plein essor, mais les pièces ainsi produites peuvent présenter des défauts tels que des anomalies d'impression, de la rétention de poudre ou des fissures. Pour contrôler l'intégrité de ces pièces, la tomographie par transmission haute résolution reste la méthode de référence. Cependant, pour inspecter des pièces massives et fortement absorbantes, la tomographie haute énergie avec un accélérateur linéaire d'électrons est nécessaire. Le Laboratoire de Mesures Nucléaires du CEA IRESNE dispose d'un tomographe haute énergie et a souhaité améliorer la qualité des projections acquises en mettant en place des post-traitements numériques. Afin d’essayer de dépasser les performances des méthodes de restauration classiques, basées sur des algorithmes de déconvolution, une approche de post-traitement novatrice a été étudiée : la déconvolution de flou par réseaux de neurones convolutifs. Pour ce faire, un jeu de données d’images a tout d’abord été généré par simulation. Un réseau de neurones convolutifs, basé sur la structure du réseau SRCNN (Super-Resolution Convolutional Neural Network), a ensuite été adapté, entraîné et évalué. Chaque hyperparamètre du réseau a alors été spécialement optimisé. Enfin, ce réseau a été validé sur des tomographies à 9 MeV d’objets réels afin d’évaluer les performances finales obtenues, mais aussi comprendre les limitations de ce type d’approche. Le réseau de neurones convolutifs ainsi optimisé démontre de bonnes performances de défloutage tout en limitant l’amplification du bruit.

L’identification des matériaux présents dans des objets volumineux comme des colis de déchets nucléaires nécessite la plupart du temps soit une opération destructive (carottage, découpe) pour les colis bétonnés, soit une intervention humaine (tri, inventaire) sur des colis de faible activité dont le déchet n’est pas bloqué. Dans le cadre de ses activités de R&D sur la gestion des colis de déchets, l’ONDRAF/NIRAS (Belgique) a sollicité le CEA (Institut IRESNE) pour évaluer les performances et les limites de détection et d’identification de matériaux dans des colis bétonnés étalons de 400 l/ 900 kg. La technique mise en oeuvre est la tomographie par transmission au moyen d’un accélérateur linéaire dont l’énergie est réglable de 9 à 21 MeV (avec un maximum de dose à 15 MeV). Des essais ont ainsi été réalisés sur cinq fantômes spécialement conçus par l’ONDRAF/NIRAS pour refléter la gamme de matériaux devant être identifiés (plexiglas, cellulose, bois, métal, eau, huile, filtres, déchets compactés, etc). Les premières séries de mesures ont consisté en des tomographies demi-champ Cone-Beam (3D) afin d’avoir une vue globale des colis et de leur contenu. La diffusion Compton étant majoritaire à ces énergies, il n’a pas été possible, à cause du rayonnement diffusé parasite, de réaliser une calibration en densité. Une seconde série de mesures a alors visé à comprendre les perturbations observées afin de les corriger et obtenir la meilleure précision possible sur la densité à l’aide d’une géométrie collimatée Fan-Beam (2D). Les précisions obtenues sur la densité sont de l’ordre de quelques pourcents pour la majorité des matériaux, la sensibilité variant suivant leur nature. Par exemple, l’eau et l’huile contenues dans un même récipient sont distinguées autant par la valeur de leur densité que par leur stratification alors même que la nature des métaux est plus difficile à extraire à cause du durcissement de spectre. La tomographie 3D et la tomodensitométrie 2D apparaissent donc comme des examens efficaces pour identifier des matériaux contenus dans des objets volumineux et lourds comme des colis de déchets nucléaires.

Le Laboratoire de Mesures Nucléaires de l’institut IRESNE du CEA (centre de Cadarache) réalise dans la cellule d’irradiation CINPHONIE (INB CHICADE) des expertises radiographiques et tomographiques sur des objets denses et/ou de grandes dimensions. Pour ce faire, la cellule est équipée d’un accélérateur linéaire de type SATURNE permettant la production de photons ayant une énergie allant de 9 à 21 MeV (débit de dose maximal à 1 m d’environ 62 Gy/mn). Un banc mécanique de haute précision et d’une capacité de 5 tonnes permet les mouvements en translation (horizontale et verticale) et en rotation d’objets ayant un diamètre maximal de 1,50 m. La détection est assurée au moyen d’un détecteur dit « Grand Champ », composé d’un écran scintillateur de Gadox monté sur un renforçateur en tantale (80 x 60 cm²) et filmé, via un renvoi d’angle, par une caméra sCMOS bas bruit. Compte-tenu des contraintes géométriques de la cellule, le champ de vue du tomographe permettait de mesurer au maximum des objets de diamètre 102 cm en demi-champ. En 2018, une nouvelle méthode de traitement des projections a permis d’étendre le champ de vue à des objets de toute taille. Cette extension du champ de vue est effectuée en enregistrant, pour une coupe tomographique donnée, plusieurs séries de rotation sur 2π obtenues en décalant le centre de rotation de l’objet. Les projections résultantes sont ensuite fusionnées puis utilisées comme données d’entrée des algorithmes de reconstruction. Celle-ci est effectuée au moyen du logiciel RTK (algorithme FDK). Après une phase de validation par simulation au moyen du logiciel MODHERATO, développé au sein du laboratoire, la méthode a été appliquée à divers objets de gros volumes et de densité variées tels que des coques de déchets nucléaires bétonnés (Ø110 cm × 130 cm, d = 2,5) ou encore le crâne du Mammouth de Durfort (110 cm × 110 cm, d = 1,5) appartenant au Museum National d’Histoire Naturelle. Les dimensions maximales des objets sont ainsi définies par les capacités physiques (et non algorithmiques) du système tomographique à disposer d’un rapport signal sur bruit suffisant. Ce rapport dépend uniquement des caractéristiques du faisceau ainsi que du diamètre et de la densité des objets tomographiés.

Abstract 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.

BackgroundAbout 15% of Triple-Negative-Breast-Cancer (TNBC) present silencing of the BRCA1 promoter methylation and are assumed to be Homologous Recombination Deficient (HRD). BRCA1-methylated (BRCA1-Me) TNBC could, thus, be eligible to treatment based on PARP-inhibitors or Platinum salts. However, their actual HRD status is discussed, as these tumors are suspected to develop resistance after chemotherapy exposure.MethodsWe interrogated the sensitivity to olaparib vs. carboplatin of 8 TNBC Patient-Derived Xenografts (PDX) models. Four PDX corresponded to BRCA1-Me, of which 3 were previously exposed to NeoAdjuvant-Chemotherapy (NACT). The remaining PDX models corresponded to two BRCA1-mutated (BRCA1-Mut) and two BRCA1-wild type PDX that were respectively included as positive and negative controls. The HRD status of our PDX models was assessed using both genomic signatures and the functional BRCA1 and RAD51 nuclear foci formation assay. To assess HR restoration associated with olaparib resistance, we studied pairs of BRCA1 deficient cell lines and their resistant subclones.ResultsThe 3 BRCA1-Me PDX that had been exposed to NACT responded poorly to olaparib, likewise BRCA1-WT PDX. Contrastingly, 3 treatment-naïve BRCA1-deficient PDX (1 BRCA1-Me and 2 BRCA1-mutated) responded to olaparib. Noticeably, the three olaparib-responsive PDX scored negative for BRCA1- and RAD51-foci, whereas all non-responsive PDX models, including the 3 NACT-exposed BRCA1-Me PDX, scored positive for RAD51-foci. This suggested HRD in olaparib responsive PDX, while non-responsive models were HR proficient. These results were consistent with observations in cell lines showing a significant increase of RAD51-foci in olaparib-resistant subclones compared with sensitive parental cells, suggesting HR restoration in these models.ConclusionOur results thus support the notion that the actual HRD status of BRCA1-Me TNBC, especially if previously exposed to chemotherapy, may be questioned and should be verified using the BRCA1- and RAD51-foci assay.

Recent data support the notion that a group of key transcriptional regulators involved in tumorigenesis, including MYC, p53, E2F1, and BMI1, share an intriguing capacity to simultaneously regulate metabolism and cell cycle. Here, we show that another factor, the multifunctional protein E4F1, directly controls genes involved in mitochondria functions and cell-cycle checkpoints, including Chek1, a major component of the DNA damage response. Coordination of these cellular functions by E4F1 appears essential for the survival of p53-deficient transformed cells. Acute inactivation of E4F1 in these cells results in CHK1-dependent checkpoint deficiency and multiple mitochondrial dysfunctions that lead to increased ROS production, energy stress, and inhibition of de novo pyrimidine synthesis. This deadly cocktail leads to the accumulation of uncompensated oxidative damage to proteins and extensive DNA damage, ending in cell death. This supports the rationale of therapeutic strategies simultaneously targeting mitochondria and CHK1 for selective killing of p53-deficient cancer cells.

Protein arginine methyl transferase 5 (Prmt5) regulates various differentiation processes, including adipogenesis. Here, we investigated adipogenic conversion in cells and mice in which Copr5, a Prmt5- and histone-binding protein, was genetically invalidated. Compared to control littermates, the retroperitoneal white adipose tissue (WAT) of Copr5 KO mice was slightly but significantly reduced between 8 and 16 week/old and contained fewer and larger adipocytes. Moreover, the adipogenic conversion of Copr5 KO embryoid bodies (EB) and of primary embryo fibroblasts (Mefs) was markedly delayed. Differential transcriptomic analysis identified Copr5 as a negative regulator of the Dlk-1 gene, a Wnt target gene involved in the control of adipocyte progenitors cell fate. Dlk-1 expression was upregulated in Copr5 KO Mefs and the Vascular Stromal Fraction (VSF) of Copr5 KO WAT. Chromatin immunoprecipitation (ChIP) show that the ablation of Copr5 has impaired both the recruitment of Prmt5 and β-catenin at the Dlk-1 promoter. Overall, our data suggest that Copr5 is involved in the transcriptional control exerted by the Wnt pathway on early steps of adipogenesis.

This Data in Brief report describes the experimental and bioinformatic procedures that we used to analyze and interpret E4F1 ChIP-seq experiments published in Rodier et al. (2015) [10]. Raw and processed data are available at the GEO DataSet repository under the subseries # GSE57228. E4F1 is a ubiquitously expressed zinc-finger protein of the GLI-Kruppel family that was first identified in the late eighties as a cellular transcription factor targeted by the adenoviral oncoprotein E1A13S (Ad type V) and required for the transcription of adenoviral genes (Raychaudhuri et al., 1987) [8]. It is a multifunctional factor that also acts as an atypical E3 ubiquitin ligase for p53 (Le Cam et al., 2006) [2]. Using KO mouse models we then demonstrated that E4F1 is essential for early embryonic development (Le Cam et al., 2004), for proliferation of mouse embryonic cell (Rodier et al., 2015), for the maintenance of epidermal stem cells (Lacroix et al., 2010) [6], and strikingly, for the survival of cancer cells (Hatchi et al., 2007) [4]; (Rodier et al., 2015) [10]. The latter survival phenotype was p53-independent and suggested that E4F1 was controlling a transcriptional program driving essential functions in cancer cells. To identify this program, we performed E4F1 ChIP-seq analyses in primary Mouse Embryonic Fibroblasts (MEF) and in p53−/−, H-RasV12-transformed MEFs. The program directly controlled by E4F1 was obtained by intersecting the lists of E4F1 genomic targets with the lists of genes differentially expressed in E4F1 KO and E4F1 WT cells (Rodier et al., 2015). We describe hereby how we improved our ChIP-seq analyses workflow by applying prefilters on raw data and by using a combination of two publicly available programs, Cisgenome and QESEQ.

Ecological succession provides a widely accepted description of seasonal changes in phytoplankton and mesozooplankton assemblages in the natural environment, but concurrent changes in smaller (i.e. microbes) and larger (i.e. macroplankton) organisms are not included in the model because plankton ranging from bacteria to jellies are seldom sampled and analyzed simultaneously. Here we studied, for the first time in the aquatic literature, the succession of marine plankton in the whole-plankton assemblage that spanned 5 orders of magnitude in size from microbes to macroplankton predators (not including fish or fish larvae, for which no consistent data were available). Samples were collected in the northwestern Mediterranean Sea (Bay of Villefranche) weekly during 10 months. Simultaneously collected samples were analyzed by flow cytometry, inverse microscopy, FlowCam, and ZooScan. The whole-plankton assemblage underwent sharp reorganizations that corresponded to bottom-up events of vertical mixing in the water-column, and its development was top-down controlled by large gelatinous filter feeders and predators. Based on the results provided by our novel whole-plankton assemblage approach, we propose a new comprehensive conceptual model of the annual plankton succession (i.e. whole plankton model) characterized by both stepwise stacking of four broad trophic communities from early spring through summer, which is a new concept, and progressive replacement of ecological plankton categories within the different trophic communities, as recognised traditionally.

Use of antibodies is a cornerstone of biological studies and it is important to identify the recognized protein with certainty. Generally an antibody is considered specific if it labels a single band of the expected size in the tissue of interest, or has a strong affinity for the antigen produced in a heterologous system. The identity of the antibody target protein is rarely confirmed by purification and sequencing, however in many cases this may be necessary. In this study we sought to characterize the myoplasm, a mitochondria-rich domain present in eggs and segregated into tadpole muscle cells of ascidians (urochordates). The targeted proteins of two antibodies that label the myoplasm were purified using both classic immunoaffinity methods and a novel protein purification scheme based on sequential ion exchange chromatography followed by two-dimensional gel electrophoresis. Surprisingly, mass spectrometry sequencing revealed that in both cases the proteins recognized are unrelated to the original antigens. NN18, a monoclonal antibody which was raised against porcine spinal cord and recognizes the NF-M neurofilament subunit in vertebrates, in fact labels mitochondrial ATP synthase in the ascidian embryo. PMF-C13, an antibody we raised to and purified against PmMRF, which is the MyoD homolog of the ascidian Phallusia mammillata, in fact recognizes mitochondrial HSP60. High resolution immunolabeling on whole embryos and isolated cortices demonstrates localization to the inner mitochondrial membrane for both ATP synthase and HSP60. We discuss the general implications of our results for antibody specificity and the verification methods which can be used to determine unequivocally an antibody's target.

The structure, robustness, and dynamics of ocean plankton ecosystems remain poorly understood due to sampling, analysis, and computational limitations. The Tara Oceans consortium organizes expeditions to help fill this gap at the global level.

When guinea pigs are fed cholesterol, the cholesterol content of their red cells increases progressively, a large number of cells become spurred, and a hemolytic anemia develops. Unesterified cholesterol is readily transferred from plasma, HDL, or LDL of cholesterol-fed, anemic guinea pigs to normal red cells in vitro. This transfer is reversible and is proportional to the concentration of unesterified cholesterol in the incubation medium. Red cells loaded in vitro with cholesterol develop spurs identical with those on red cells in the circulation of cholesterol-fed, anemic guinea pigs. Neither the cholesterol content nor the morphology of normal red cells is altered by incubation in control plasma or in concentrated control lipoproteins. Plasma infranates (d > 1.21 g/ml) of either group do not cause spurring of control red cells. We conclude: (a) that accumulation of cholesterol by guinea pig red cells in vitro requires an increased concentration of unesterified cholesterol in lipoprotein rather than an increased concentration of normal lipoproteins, and (b) that an increased cholesterol content in guinea pig red cell membranes is necessary for their abnormal morphology. The flux of cholesterol between cholesterol-loaded cells and plasma from cholesterol-fed guinea pigs is three times greater than that between control red cells and control plasma, and the fractional exchange rates are altered.

Dietary cholesterol induces a hemolytic anemia in guinea pigs, accompanied by changes in the lipid composition of red cells and of plasma lipoproteins. This report presents a characterization of the lipoprotein species present in each main density class in both control and cholesterol-fed guinea pigs. Traces of a typical high density lipoprotein (HDL) were detected in control plasma. HDL from cholesterol-fed, anemic guinea pigs differed from control HDL in electron microscopic appearance and lipid and peptide composition. Long stacks of discs were observed in the electron microscope in addition to smaller, spherical particles characteristic of control HDL.Low density lipoproteins (LDL) from cholesterol-fed, anemic guinea pigs had two main populations, which were separated by gel chromatography. One population appeared in the electron microscope as large transparent discs and contained mainly unesterified cholesterol and phospholipids in a 2:1 molar ratio. The other population resembled control LDL in size and composition except for its high unesterified cholesterol content. Dietary cholesterol also altered the composition and decreased the electrophoretic mobility of very low density lipoproteins. Gel electrophoretic and immunochemical evidence indicates that a peptide (mol wt 35,000) appears in lipoproteins from cholesterol-fed, anemic guinea pigs that is undetectable in those of controls.Similarities between the cholesterol-induced lipoprotein abnormalities in guinea pigs and those reported in patients with obstructive jaundice, biliary cirrhosis, type III hyperlipoproteinemia, or familial lecithin:cholesterol acyltransferase deficiency are discussed.