Ablation of the Sam68 RNA Binding Protein Protects Mice from Age-Related Bone Loss

The Src substrate associated in mitosis of 68 kDa (Sam68) is a KH-type RNA binding protein that has been shown to regulate several aspects of RNA metabolism; however, its physiologic role has remained elusive. Herein we report the generation of Sam68-null mice by homologous recombination. Aged Sam68(−/−) mice preserved their bone mass, in sharp contrast with 12-month-old wild-type littermates in which bone mass was decreased up to approximately 75%. In fact, the bone volume of the 12-month-old Sam68(−/−) mice was virtually indistinguishable from that of 4-month-old wild-type or Sam68(−/−) mice. Sam68(−/−) bone marrow stromal cells had a differentiation advantage for the osteogenic pathway. Moreover, the knockdown of Sam68 using short hairpin RNA in the embryonic mesenchymal multipotential progenitor C3H10T1/2 cells resulted in more pronounced expression of the mature osteoblast marker osteocalcin when differentiation was induced with bone morphogenetic protein-2. Cultures of mouse embryo fibroblasts generated from Sam68(+/+) and Sam68(−/−) littermates were induced to differentiate into adipocytes with culture medium containing pioglitazone and the Sam68(−/−) mouse embryo fibroblasts shown to have impaired adipocyte differentiation. Furthermore, in vivo it was shown that sections of bone from 12-month-old Sam68(−/−) mice had few marrow adipocytes compared with their age-matched wild-type littermate controls, which exhibited fatty bone marrow. Our findings identify endogenous Sam68 as a positive regulator of adipocyte differentiation and a negative regulator of osteoblast differentiation, which is consistent with Sam68 being a modulator of bone marrow mesenchymal cell differentiation, and hence bone metabolism, in aged mice

A chemical survey of exoplanets with ARIEL

Thousands of exoplanets have now been discovered with a huge range of masses, sizes and orbits: from rocky Earth-like planets to large gas giants grazing the surface of their host star. However, the essential nature of these exoplanets remains largely mysterious: there is no known, discernible pattern linking the presence, size, or orbital parameters of a planet to the nature of its parent star. We have little idea whether the chemistry of a planet is linked to its formation environment, or whether the type of host star drives the physics and chemistry of the planet’s birth, and evolution. ARIEL was conceived to observe a large number (~1000) of transiting planets for statistical understanding, including gas giants, Neptunes, super-Earths and Earth-size planets around a range of host star types using transit spectroscopy in the 1.25–7.8 μm spectral range and multiple narrow-band photometry in the optical. ARIEL will focus on warm and hot planets to take advantage of their well-mixed atmospheres which should show minimal condensation and sequestration of high-Z materials compared to their colder Solar System siblings. Said warm and hot atmospheres are expected to be more representative of the planetary bulk composition. Observations of these warm/hot exoplanets, and in particular of their elemental composition (especially C, O, N, S, Si), will allow the understanding of the early stages of planetary and atmospheric formation during the nebular phase and the following few million years. ARIEL will thus provide a representative picture of the chemical nature of the exoplanets and relate this directly to the type and chemical environment of the host star. ARIEL is designed as a dedicated survey mission for combined-light spectroscopy, capable of observing a large and well-defined planet sample within its 4-year mission lifetime. Transit, eclipse and phase-curve spectroscopy methods, whereby the signal from the star and planet are differentiated using knowledge of the planetary ephemerides, allow us to measure atmospheric signals from the planet at levels of 10–100 part per million (ppm) relative to the star and, given the bright nature of targets, also allows more sophisticated techniques, such as eclipse mapping, to give a deeper insight into the nature of the atmosphere. These types of observations require a stable payload and satellite platform with broad, instantaneous wavelength coverage to detect many molecular species, probe the thermal structure, identify clouds and monitor the stellar activity. The wavelength range proposed covers all the expected major atmospheric gases from e.g. H2O, CO2, CH4 NH3, HCN, H2S through to the more exotic metallic compounds, such as TiO, VO, and condensed species. Simulations of ARIEL performance in conducting exoplanet surveys have been performed – using conservative estimates of mission performance and a full model of all significant noise sources in the measurement – using a list of potential ARIEL targets that incorporates the latest available exoplanet statistics. The conclusion at the end of the Phase A study, is that ARIEL – in line with the stated mission objectives – will be able to observe about 1000 exoplanets depending on the details of the adopted survey strategy, thus confirming the feasibility of the main science objectives.Peer reviewedFinal Published versio

Farmed bivalve loss due to seabream predation in the French Mediterranean Prevost Lagoon

Bivalve predation by seabream has been observed worldwide and is a major concern for bivalve farmers. Farmed bivalve-seabream interactions must be better understood to ensure the sustainability of bivalve aquaculture. The objectives of this study were to characterize gilthead seabream Sparus aurata presence in a bivalve farm in Prevost Lagoon (Mediterranean Sea) using acoustic telemetry and to evaluate monthly losses of mussels Mytilus galloprovincialis and oysters Crassostrea gigas due to seabream predation over an 18 mo period inside the farm and at an unprotected experimental platform. Large (281 to 499 mm TL) seabream were more commonly detected in the bivalve farm than were small (200 to 280 mm TL) seabream. In contrast to small seabream, 90% of large seabream returned to and spent extended periods in the study area the following year, suggesting inter-annual site fidelity for large fish that used the bivalve farm as a feeding site. Signs of predation were observed on mussels and oysters throughout the year at the unprotected experimental platform. Farmers noted losses in the farm from April to September. Maximal losses (90 to 100%) were observed post-oyster ‘sticking’ and mussel socking. Despite the deployment of nets as mechanical protection to reduce predation, oyster losses represented 28% of the annual value of oysters sold while mussel losses were estimated at ca. 1%. These results suggest that bivalves must be protected by nets throughout the year to avoid predation, particularly post-handling. A collaboration between shellfish farmers and fishermen could be a sustainable solution for bivalve farming, by regularly fishing for seabream in farms, between tables and inside protective nets

Old Sam68^−/− Mice Are Protected from the Development of Fatty Bone Marrow

<p>Sections of undecalcified bone were stained with von Kossa and toluidine blue and images captured at original magnifications of ×2 (A), ×40 (B and C) to evaluate mineralized tissue (A, black), marrow adipocytes (B, white), and the mineralization fronts (C, yellow and green). The 12-month-old Sam68^+/+ bone demonstrated a significant reduction in bone (A) and increase in marrow adipocytes (B) and a decrease in the distance between two consecutive fluorochrome labels (C). Magnification at source was ×40. Micrographs are representative of four to six screened in each group of animals.</p

Enhanced Osteogenic Differentiation of the C3HT101/2 Embryonic Cell Line Depleted of Endogenous Sam68

<div><p>(A) C3HT101/2 cells transfected with an empty vector (pSuper-retro) or a vector containing an shRNA (Sam68 shRNA) were selected with puromycin, and knockdown populations depleted of Sam68 were identified. The reduction in Sam68 protein was analyzed by immunoblotting with anti-Sam68 (AD1) antibody and anti–β-actin antibodies as loading controls.</p><p>(B) Osteogenic differentiation was carried out with conditioned medium containing BMP-2 for the indicated times. To assess the level of osteogenic differentiation in these cells, expression of late osteoblast marker, osteocalcin (OCN), was analyzed by RT-PCR and compared with β-actin and GAPDH controls. The DNA fragments were visualized by agarose gel stained with ethidium bromide.</p></div

Immunohistochemical Localization of Sam68 in Embryonic Mice

<div><p>(A) Embryonic mice were removed from pregnant dams at E14.5 and E16.5, fixed in 4% paraformaldehyde, and embedded in paraffin. The entire embryo was immunostained with the AD1 anti-Sam68 antibody and counterstained with methyl green, and the image was captured at ×1.2 magnification.</p><p>(B) Embryonic soft tissues from the brain, heart and gut were stained with hematoxylin (left) and immunostained with anti-Sam68 antibody (right), and images were captured at ×20 magnification.</p><p>(C) Intense anti-Sam68 immunoreactivity was seen in chondrocytes in the nasal septum (panels A–D), in developing vertebra (panels E–H), and in the femoral epiphysis (panels I–K), as well as in diaphyseal osteoblasts (panel L). Adjacent sections were stained with hematoxylin and eosin (panels A, E, and I) or with antibody preadsorbed with the immunizing peptide (panels B, F, and J). Sam68 was localized primarily in the nucleus of cells in a variety of tissues but was also found occasionally in the cytoplasm. Magnification at source ×20, except for panels D, H, and L, which were ×40. Staining patterns are representative of three to five embryos.</p></div

Ex Vivo Activity of Sam68^+/+ and Sam68^−/− Osteoblasts and Osteoclasts

<div><p>Marrow stromal cells were isolated from the long bones of juvenile mice and maintained under conditions that promote osteoblast differentiation.</p><p>(A) Cultures were fixed in 4% paraformaldehyde after 6 or 18 days and stained in situ for ALP activity and with silver nitrate (von Kossa) to detect mineralized nodules. Sam68^−/− cultures stained more intensely for ALP at early and late time points and produced significantly more mineralized nodules after 18 days. Asterisks represent <i>p</i> < 0.01.</p><p>(B) Primary osteoclasts were isolated from the crushed long bones of the same mice and plated on glass coverslips or on dentin slices to quantify numbers and activity, respectively. Osteoclasts were identified as cells with three or more nuclei that stained positive for TRAP activity (upper) and excavated pits in dentin slices, as demonstrated by SEM (lower, bar = 20 μm). No statistical differences were observed either in the number of TRAP-positive cells or in their resorptive activity.</p></div

Generation of Sam68-Deficient Mice

<div><p>(A) The genomic organizations of the wild-type and targeted <i>sam68</i> alleles after homologous recombination are depicted. The location of the DNA fragment used as a probe for the Southern blot analysis is shown, as well as the sizes of the two BglII fragments detected for wild-type and targeted <i>sam68</i> alleles. The targeted allele replaces exon 4 and part of exon 5 of <i>sam68</i> with a PGK-neomycin cassette.</p><p>(B) Southern-blot analysis of genomic DNA from wild-type (+/+), heterozygous (+/−), and homozygous (−/−) mice. DNA fragments corresponding to wild-type (4.5 kb) and the targeted (5.5 kb) alleles are illustrated.</p><p>(C) Western blot analysis of Sam68 expression. Protein extracts from wild-type, heterozygous, and homozygous cells subjected to immunoblot analyses using normal rabbit serum, anti-Sam68 AD1 antibody, the peptide antibody AD1 preabsorbed with the immunogenic peptide corresponding to amino acids 330–348 of mouse Sam68, anti-Sam68 Sc333 antibody that recognizes the C-terminal 20 amino acids of Sam68, and anti-actin antibodies as loading control. The migration of the molecular mass markers is known on the left in kDa.</p></div

Ex Vivo Adipogenesis Analysis of Sam68^−/− Mouse Embryonic Fibroblasts

<div><p>MEFs were isolated from mouse embryos at embryonic day 14.5. Equal number of MEFs from Sam68^+/+ and Sam68^−/− was plated on glass cover slips in 24 well-plates. Adipocyte differentiation was carried out at indicated times by the addition of complete media containing the pioglitazone.</p><p>(A) Cultures were fixed in 4% paraformaldehyde and stained with Oil Red O to detect the fat droplets stored in adipocytes and photographed (top). The cell images were magnified ×10 and ×20 as indicated.</p><p>(B) RT-PCR was carried out on total cellular RNA isolated after differentiation of the MEFs for day 0, 2, 4, 6, and 12. The DNA fragments were visualized on agarose gels stained with ethidium bromide. The expression of adipogenic markers C/EBPβ, C/EBPδ, PPARα, and KLF5 was examined as well as the expression of controls including Sam68, β-actin, and GAPDH.</p></div

Histologic Analysis of Undecalcified Bone from Sam68<b>^+/+</b> and Sam68^−/− Mice

<p>Sections of tibia fixed in 4% paraformaldehyde and embedded in plastic were stained for ALP (A–C, E–G) activity to identify osteoblasts or for TRAP (B–D, F–H) activity to identify osteoclasts. Staining patterns were similar in 4-month-old Sam68^+/+ (A and B), 4-month-old Sam68^−/− (C and D), and 12-month-old Sam68^−/− (G and H) mice compared with 12-month-old Sam68^+/+ mice (E and F). Magnification at source, left panels ×10 and right panels ×40. Micrographs are representative of those taken from five to seven sections in each group of animals.</p