A new HPF specimen carrier adapter for the use of high-pressure freezing with cryoscanning electron microscope: two applications: stearic acid organization in a hydroxypropyl methylcellulose matrix and mice myocardium.

Cryogenic transmission electron microscopy of high-pressure freezing (HPF) samples is a well-established technique for the analysis of liquid containing specimens. This technique enables observation without removing water or other volatile components. The HPF technique is less used in scanning electron microscopy (SEM) due to the lack of a suitable HPF specimen carrier adapter. The traditional SEM cryotransfer system (PP3000T Quorum Laughton, East Sussex, UK; Alto Gatan, Pleasanton, CA, USA) usually uses nitrogen slush. Unfortunately, and unlike HPF, nitrogen slush produces water crystal artefacts. So, we propose a new HPF specimen carrier adapter for sample transfer from HPF system to cryogenic-scanning electronic microscope (Cryo-SEM). The new transfer system is validated using technical two applications, a stearic acid in hydroxypropyl methylcellulose solution and mice myocardium. Preservation of samples is suitable in both cases. Cryo-SEM examination of HPF samples enables a good correlation between acid stearic liquid concentration and acid stearic occupation surface (only for homogeneous solution). For biological samples as myocardium, cytoplasmic structures of cardiomyocyte are easily recognized with adequate preservation of organelle contacts and inner cell organization. We expect this new HPF specimen carrier adapter would enable more SEM-studies using HPF.

Charon's size and an upper limit on its atmosphere from a stellar occultation.

Pluto and its satellite, Charon (discovered in 1978; ref. 1), appear to form a double planet, rather than a hierarchical planet/satellite couple. Charon is about half Pluto's size and about one-eighth its mass. The precise radii of Pluto and Charon have remained uncertain, leading to large uncertainties on their densities. Although stellar occultations by Charon are in principle a powerful way of measuring its size, they are rare, as the satellite subtends less than 0.3 microradians (0.06 arcsec) on the sky. One occultation (in 1980) yielded a lower limit of 600 km for the satellite's radius, which was later refined to 601.5 km (ref. 4). Here we report observations from a multi-station stellar occultation by Charon, which we use to derive a radius, R(C) = 603.6 +/- 1.4 km (1sigma), and a density of rho = 1.71 +/- 0.08 g cm(-3). This occultation also provides upper limits of 110 and 15 (3sigma) nanobar for an atmosphere around Charon, assuming respectively a pure nitrogen or pure methane atmosphere.

NUP98 is rearranged in 3.8% of pediatric AML forming a clinical and molecular homogenous group with a poor prognosis.

Pediatric acute myeloid leukemia (AML) is a rare disease whose prognosis is highly variable according to factors such as chromosomal abnormalities. Recurrent genomic rearrangements are detected in half of pediatric AML by karyotype. NUcleoPorin 98 (NUP98) gene is rearranged with 31 different fusion partner genes. These rearrangements are frequently undetected by conventional cytogenetics, as the NUP98 gene is located at the end of the chromosome 11 short arm (11p15). By screening a series of 574 pediatric AML, we detected a NUP98 rearrangement in 22 cases (3.8%), a frequency similar to CBFB-MYH11 fusion gene (4.0%). The most frequent NUP98 fusion gene partner is NSD1. These cases are homogeneous regarding their biological and clinical characteristics, and associated with bad prognosis only improved by bone marrow transplantation. We detailed the biological characteristics of these AML by exome sequencing which demonstrated few recurrent mutations (FLT3 ITD, WT1, CEBPA, NBPF14, BCR and ODF1). The analysis of the clonal structure in these cases suggests that the mutation order in the NUP98-rearranged pediatric AML begins with the NUP98 rearrangement leading to epigenetic dysregulations then followed by mutations of critical hematopoietic transcription factors and finally, activation of the FLT3 signaling pathway.

Production of insulin-like growth factors and their binding proteins by rabbit articular chondrocytes: relationships with cell multiplication.

Articular chondrocytes from 2- to 3-month-old rabbits were cultured in serum-free medium supplemented with fibroblast growth factor. The effects were studied of GH, insulin-like growth factors (IGFs), and insulin on the production of IGF-I, IGF-II, and their binding proteins (BPs) and on cell multiplication. In the control culture medium, IGF-I levels were about one fifth those of IGF-II. Western blot analysis of the BPs revealed a predominant 30K form and 24K and 20K forms which appeared inconsistently and in small quantities. Ten to 100 ng/ml human GH had no mitogenic effect, and even had a slightly inhibitory effect. IGF-I at 10 ng/ml stimulated cell multiplication above the control level by 41% and at 50 ng/ml by 74%, whereas the mean increase obtained with IGF-II (10 and 50 ng/ml) was only 19%. At the same doses, insulin had no effect, but at 5 micrograms/ml it stimulated cell multiplication by a mean of 67%. There was a positive correlation between cell number and release into the medium of both IGF-I (r = 0.86) and IGF-II (r = 0.77). Neither IGF-I nor IGF-II production was affected by GH. Insulin (5 micrograms/ml) increased IGF-I production by a factor of 2.6, but increased IGF-II production by a factor of only 1.4. Under the various conditions of culture with different doses of GH and insulin, cell multiplication, relative to the control value was positively correlated to the IGF-I/IGF-II production ratio (r = 0.77). It would, therefore, seem that IGF-I secreted by the chondrocytes may stimulate their own proliferation. When IGFs or insulin were added to the culture medium, changes in the electrophoretic profiles of the BPs included an increase in the 30K form and an increase in or the appearance of the 24K and 20K forms. Ten and 50 ng/ml IGF-I or IGF-II had effects equal to or greater than those induced by 5 micrograms/ml insulin. These results indicate that the syntheses of BPs and IGFs are coordinated and that IGFs may be implicated in the control of the synthesis of their BPs.

Morphological and metabolic characterization of a new species of strictly anaerobic rumen fungus: Neocallimastix joyonii.

A new strain of strictly anaerobic fungi was isolated from the rumen of sheep. This strain is characterized by a polycentric thallus, an extensive and polynuclear rhizomycelium, polyflagellated zoospores with gamma particle-like bodies. We propose to assign this strain in a new species: Neocallimastix joyonii.

Presence of soluble lignin-carbohydrate complexes in the bovine rumen.

The cell-free rumen liquor of a steer on a diet of spear grass has been shown to contain macromolecular substances in which carbohydrates and lignin-derived compounds are covalently bound to each other. The lignin-carbohydrate complexes are soluble at pH 7 or higher, but precipitate at pH 3. At the latter pH, small amounts of a polymer, assumed to be glycoprotein, remain in solution. Some of the lignin-carbohydrate linkages are broken by treatment with alkali. Treatment with 50mM sulphuric acid for a few minutes at room temperature converts part of the complex into an acetone-soluble product, which still contains both carbohydrate and lignin-derived compounds. The formation of soluble lignin-carbohydrate complexes by the action of rumen micro-organisms on the grass may account for the dissolution (and hence the apparent digestion) of about half of the total lignin-intake.

[Transplacental passage of epirubicin]. / Passage transplacentaire de l'épirubicine.

We studied the transplacental transfer of epirubicin, an anthracycline used for the treatment of different neoplastic disorders, in particular breast cancers, by in vitro perfusion of term human placenta. Placenta from women with uncomplicated pregnancy were collected immediately after vaginal delivery and put into 37 degrees C thermostated hood. Perfusion of foetal surface of the placenta by modified Earle's solution was started immediately after catheterisation at a flow rate of 6 ml/min and then so was the perfusion of the intervillous space at the rate of 12 ml/min. Samples were collected at different times after the initiation of the perfusion from arterial inflow and venous outflow respective of the maternal and foetal compartment. The transplacental transfer of epirubicin was investigated for two doses: 5 and 9 micrograms/ml. The mean transfer value of epirubicin is low (3.66 +/- 1.07%) for the two tested doses and is only slightly higher than doxorubicin transfer, which drug has provided rare accidents in the clinical reports. These results are in favour of a low placental toxicity of epirubicin. Clinical data have to be collected from pregnant women to confirm the low foetal toxicity of epirubicin.

Lipid metabolism of liquid-associated and solid-adherent bacteria in rumen contents of dairy cows offered lipid-supplemented diets.

The lipid distribution and fatty acid (FA) composition of total lipids, polar lipids and free fatty acids (FFA) were determined in liquid-associated bacteria (LAB) and solid-adherent bacteria (SAB) isolated from the rumen contents of seven dairy cows fitted with rumen fistulas. Two experiments, arranged according to a 4 x 4 and 3 x 3 Latin Square design, were performed using two basal diets consisting of one part hay and one part barley-based concentrate, and five lipid-supplemented diets consisting of the basal diet plus (g/kg dry matter):53 or 94 rapeseed oil, 98 tallow, 87 soya-bean oil or 94 palmitostearin. For all diets used, total lipids were 1.7-2.2 times higher in SAB than in LAB (P less than 0.05); this probably resulted from a preferential incorporation of dietary FA absorbed onto food particles. Addition of oil or fat to the diets did not modify the polar lipid content but increased the FFA content of SAB and LAB by 150%. Lipid droplets were observed in the cytoplasm in SAB and LAB using transmission electron microscopy, which suggested that part of the additional FFA was really incorporated into the intracellular FFA rather than associated with the cell envelope by physical adsorption. Linoleic acid was specifically incorporated into the FFA of SAB, which emphasized the specific role of this bacterial compartment in the protection of this acid against rumen biohydrogenation.