Visualization, quantification and coordination of Ag+ ions released from silver nanoparticles in hepatocytes.

Silver nanoparticles (AgNPs) can enter eukaryotic cells and exert toxic effects, most probably as a consequence of the release of Ag+ ions. Due to the elusive nature of Ag+ ionic species, quantitative information concerning AgNP intracellular dissolution is missing. By using a synchrotron nanoprobe, silver is visualized and quantified in hepatocytes (HepG2) exposed to AgNPs; the synergistic use of electron microscopy allows for the discrimination between nanoparticular and ionic forms of silver within a single cell. AgNPs are located in endocytosis vesicles, while the visualized Ag+ ions diffuse in the cell. The averaged NP dissolution rates, measured by X-ray absorption spectroscopy, highlight the faster dissolution of citrate-coated AgNPs with respect to the less toxic PVP-coated AgNPs; these results are confirmed at the single-cell level. The released Ag+ ions recombine with thiol-bearing biomolecules: the Ag-S distances measured in cellulo, and the quantitative evaluation of gene expression, provide independent evidence of the involvement of glutathione and metallothioneins in Ag+ binding. The combined use of cutting-edge imaging techniques, atomic spectroscopy and molecular biology brings insight into the fate of AgNPs in hepatocytes, and more generally into the physicochemical transformations of metallic nanoparticles in biological environments and the resulting disruption of metal homeostasis.

Exposure-dependent Ag+ release from silver nanoparticles and its complexation in AgS2 sites in primary murine macrophages.

Silver nanoparticle (AgNP) toxicity is related to their dissolution in biological environments and to the binding of the released Ag(+) ions in cellulo; the chemical environment of recombined Ag(+) ions is responsible for their toxicological outcome, moreover it is indicative of the cellular response to AgNP exposure, and can therefore shed light on the mechanisms governing AgNP toxicity. This study probes the chemistry of Ag species in primary murine macrophages exposed to AgNPs by making use of X-ray Absorption Fine Structure spectroscopy under cryogenic conditions: the linear combination analysis of the near-edge region of the spectra provides the fraction of Ag(+) ions released from the AgNPs under a given exposure condition and highlights their complexation with thiolate groups; the ab initio modelling of the extended spectra allows measuring the Ag-S bond length in cellulo. Dissolution rates depend on the exposure scenario, chronicity leading to higher Ag(+) release than acute exposure; Ag-S bond lengths are 2.41 ± 0.03 Å and 2.38 ± 0.01 Å in acute and chronic exposure respectively, compatible with digonal AgS2 coordination. Glutathione is identified as the most likely putative ligand for Ag(+). The proposed method offers a scope for the investigation of metallic nanoparticle dissolution and recombination in cellular models.

Efficacy of modafinil on excessive daytime sleepiness in Prader-Willi syndrome.

Excessive daytime sleepiness is a frequent and a highly disruptive symptom to the daily routine of children with Prader-Willi Syndrome (PWS) and their families. The objective of the study was to evaluate the efficacy of modafinil, a central stimulant, on excessive daytime sleepiness in children and adolescents with PWS. The efficacy of modafinil was evaluated in this open label pilot study comparing the Epworth sleepiness scale before and after treatment. Ten patients with molecularly confirmed PWS and a complaint of excessive daytime sleepiness underwent a night-time sleep recording and multiple sleep latency tests. One patient was excluded because of severe obstructive sleep apnea syndrome. Nine patients (4 males) with median age of 16 years (8-21) received modafinil at a starting dose of 100 mg/day. We found that all patients had excessive daytime sleepiness with an Epworth sleepiness scale at 14 (11-20) and mean sleep latency on multiple sleep latency tests at 5 (3-6) minutes. Moreover, six patients had at least two sleep-onset rapid eye movement periods showing a narcolepsy-like phenotype. Modafinil significantly improved sleepiness in all patients on the Epworth sleepiness scale from 14 (11-20) to 4 (3-12), (P = 0.007). Body mass index of the patients did not change significantly under treatment. No side effects were reported, and the drug was well-tolerated. We posit that this open label case series shows good efficacy of modafinil in nine children and adolescents with PWS.

Monitoring microbial redox transformations of metal and metalloid elements under high pressure using in situ X-ray absorption spectroscopy.

X-ray absorption spectroscopy is a well-established method for probing local structural and electronic atomic environments in a variety of systems. We used X-ray absorption near-edge structure (XANES) spectroscopy for monitoring in real-time conditions selenium reduction in situ in live cultures of Shewanella oneidensis MR-1 under high hydrostatic pressure. High-quality XANES data show that Shewanella oneidensis MR-1 reduces selenite Se(IV) to red elemental selenium Se(0) up to 150 MPa without any intermediate redox state. MR-1 reduces all selenite provided (5-10 mM) between 0.1 and 60 MPa. Above 60 MPa the selenite reduction yield decreases linearly with pressure and the activity is calculated to stop at 155 ± 5 MPa. The analysis of cultures recovered after in situ measurements showed that the decrease in activity is linked to a decrease in viability. This study emphasizes the promising potential of XANES spectroscopy for real-time probing in situ microbial redox transformations of a broad range of metal and metalloid elements in live samples, including under high hydrostatic pressure.

[Vitamin K supplementation in the exclusively breast-fed infant: how much, how long?]. / Vitamine K orale chez les bébés allaités exclusivement: quelle dose, combien de temps?

There are various ways to prevent late vitamin K deficiency bleeding in exclusively breast-fed infants. The French paediatric society recommends weekly doses of 2mg of mixed micellar preparation of vitamin K during the entire period of exclusive breastfeeding, i.e. 24 doses for a period of six months, which matches recommendations for optimal duration of exclusive breastfeeding by the French paediatric society, WHO and AAP. This significantly exceeds recommendations in other European countries. We describe the risks of vitamin K deficiency; we provide a review of recent literature about administrating vitamin K in other countries, and give a recommendation for daily practice that seems to be acceptable. Nevertheless, a comprehensive randomised prospective study is needed in France to answer the question of the best ways of preventing vitamin K deficiency bleeding.

High-resolution angular beam stability monitoring at a nanofocusing beamline.

Two semi-transparent imaging beam-position monitors developed at the ESRF have been installed at the micro-analysis beamline ID22 for monitoring the angular stability of the X-ray beam. This system allows low-frequency (10 Hz) angular beam stability measurements at a submicroradian range. It is demonstrated that the incoming macro-beam angular fluctuations are one of the major sources of focal spot instabilities downstream of the Kirkpatrick-Baez mirrors. It is also shown that scanning the energy by rotating the so-called fixed-exit monochromator induces some unexpected angular beam shifts that are, to a large extent, deterministic.

Differential mitotic degradation of the CDC25B phosphatase variants.

CDC25 phosphatases control cell-cycle progression by dephosphorylating and activating cyclin-dependent kinases. CDC25B, one of the three members of this family in human cells, is thought to regulate initial mitotic events. CDC25B is an unstable protein whose proteasomal degradation is proposed to be controlled by beta-TrCP. Here, we have investigated the regulation of CDC25B during mitosis, using time-lapse video microscopy. We found that CDC25B expression is high during early mitosis, and that its degradation occurs after the metaphase-anaphase transition and cyclin B1 destruction. We also show that CDC25B degradation after metaphase is dependent on the integrity of the KEN-box and RRKSE motifs that are located within the alternatively spliced B domain, and that the CDC25B2 splice variant, that lacks this domain, is stable during mitosis. Furthermore, we show that the N-terminal region of CDC25B, encompassing the B domain, undergoes major conformational changes during mitosis that can be monitored by intramolecular fluorescence resonance energy transfer variation of specific CDC25B biosensors. This study demonstrates that CDC25B splice variants have differential mitotic stabilities, a feature that is likely to have major consequences on the local control of cyclin-dependent kinase-cyclin activities during mitotic progression.

Estrogen dependent expression of the receptor tyrosine kinase axl in normal and malignant human breast.

BACKGROUND: Axl, a member of a family of receptor tyrosine kinases characterized by an extracellular domain resembling cell adhesion molecules and an intracellular conserved tyrosine kinase domain has been reported to induce cell proliferation and transformation. In mice, axl is expressed in the normal mammary gland and over-expressed in aggressive mammary tumors. PATIENTS AND METHODS: We have investigated the expression of axl immunohistochemically in 23 normal human breast samples and in 111 consecutive breast carcinomas. Expression of axl was correlated with tumour characteristics (lymph node involvement, stage, grade) and immunohistochemical expression of ER, PR, Ki-67 and c-erbB-2. RESULTS: In normal tissue, axl localizes to the membrane of breast epithelial cells. Axl protein shows membrane associated staining in high correlation (P = 0.004) with the expression of the estrogen receptor (ER). Axl expression was found in a subset of breast carcinomas and was also correlated with high significance (P < 0.0001) with the presence of ER. CONCLUSION: Our results suggest that axl may serve as a mediator of estrogen stimulation preventing the completion of the breast epithelial life cycle and that estrogen induced axl expression may give a survival signal to cancerous cells, preventing them from dying through apoptosis.

Juvenile myelomonocytic leukemia: analyses of treatment results in the EORTC Children's Leukemia Cooperative Group (CLCG).

Forty-six children with juvenile myelomonocytic leukemia (JMML) diagnosed between 1978 and 1993 in 12 centers were retrospectively studied. There is no evidence that any conventional treatment influences the long-term evolution of JMML. Among 28 patients treated without bone marrow transplantation (BMT), 26 died (median survival: 17 months), two are alive, one in complete remission (CR) after intensive chemotherapy. Allogenic BMT is the best treatment: 18 patients underwent BMT, 11 are in CR (at 9, 15, 22, 25, 41, 45, 49, 53, 66, 90 and 108 months). Conditioning regimens using chemotherapy alone may cure some patients (3/6) occasionally despite autologous reconstitution (1/3); if relapse occurs, a second BMT may be curative (2/3). Among the 12 patients conditioned immediately with TBI, six are in CR, one is in relapse, five died (one of them in durable autologus CR from Schwannoma). It is our opinion that splenectomy is of therapeutic value and seems not to have influenced the incidence of infections complications. We found no argument in favor of intensive chemotherapy before conditioning. Results with HLA-matched unrelated donors are satisfactory. One patient relapsed at 4 months after an unrelated BMT and entered a new CR after discontinuation of cyclosporine.

Successful cord blood stem cell transplantation for congenital erythropoietic porphyria (Gunther's disease).

Congenital erythropoietic porphyria (Gunther's disease, GD) is a rare autosomal recessive disease. It results from the deficiency of uroporphyrinogen III synthase, the fourth enzyme on the metabolic pathway of heme synthesis. GD leads to severe scarring of the face and hands as a result of photosensitivity and fragility of the skin due to uroporphyrin I and coproporphyrin I accumulation. It also causes erythrocyte fragility leading to haemolytic anaemia. The other clinical features include hirsutism, red discolouration of teeth, finger-nails and urine and stunted growth. The outcome is poor, and the disfiguring nature of GD may partly explain the legend of the werewolf. No curative treatment was known until 1991, when the first case of BMT in GD was reported. The clinical and biological outcome after transplantation was encouraging, with an important regression of the symptoms of the disease, but the child died of CMV-infection 11 months after BMT. We report the second case of GD treated successfully by stem cell transplantation using umbilical cord blood from an HLA-identical brother in a 4-year-old girl suffering from severe GD. Our patient is very well 10 months after transplantation. We confirm that stem cell transplantation is curative for GD.

Nicotine analysis in neonates' hair for measuring gestational exposure to tobacco.

Hair samples were collected at time of delivery from 40 neonates whose mothers were known to be smokers during the prenatal period. Hair was decontaminated in dichloromethane, homogenized in NaOH, and nicotine was extracted in diethyl ether. After separation on a BP-5 capillary column, nicotine was identified and quantified by GC/MS using selected ion monitoring. In all cases, nicotine was found in the neonatal hair and in the hair of the corresponding mother. The ranges of nicotine levels were 0.15 to 11.80 ng/mg, and 0.37 to 63.50 ng/mg, for the neonates, and their mothers, respectively. It was possible to establish a significant correlation between both concentrations, and the correlation coefficient was 0.83. These findings suggest the possibility of monitoring the transfer of maternal nicotine through the placenta by measuring nicotine concentration in neonatal hair.