Amino-nanopolystyrene exposures of oyster (Crassostrea gigas) embryos induced no apparent intergenerational effects.

Early life stages (ELS) of numerous marine invertebrates mustcope with man-made contaminants, including plastic debris, during their pelagic phase. Among the diversity of plastic particles, nano-sized debris, known as nanoplastics, can induce effects with severe outcomes in ELS of various biological models, including the Pacific oyster Crassostrea gigas. Here, we investigated the effects of a sub-lethal dose (0.1 µg mL-1) of 50 nm polystyrene nanobeads (nano-PS) with amine functions on oyster embryos (24 h exposure) and we assessed consequences on larval and adult performances over two generations of oysters. Only a few effects were observed. Lipid analyses revealed that first-generation (G1) embryos exposed to nano-PS displayed a relative increase in cardiolipin content (+9.7%), suggesting a potential modification of mitochondrial functioning. G1-larvae issued from exposed embryos showed decreases in larval growth (-9%) and lipid storage (-20%). No effect was observed at the G1 adult stage in terms of growth, ecophysiological parameters (clearance and respiration rates, absorption efficiency), or reproductive outputs (gonadic development, gamete quality). Second generation (G2) larvae issued from control G1 displayed a significant growth reduction after G2 embryonic exposure to nano-PS (-24%) compared to control (as observed at the first generation), while no intergenerational effect was detected on G2 larvae issued from G1 exposed embryos. Overall, the present experimental study suggests a low incidence of a short embryonic exposure to nano-PS on oyster phenotypes along the entire life cycle until the next larval generation.

Nanopolystyrene beads affect motility and reproductive success of oyster spermatozoa (Crassostrea gigas).

Oysters are keystone species that use external fertilization as a sexual mode. The gametes are planktonic and face a wide range of stressors, including plastic litter. Nanoplastics are of increasing concern because their size allows pronounced interactions with biological membranes, making them a potential hazard to marine life. In the present study, oyster spermatozoa were exposed for 1 h to various doses (from 0.1 to 25 µg mL-1) of 50-nm polystyrene beads with amine (50-NH2 beads) or carboxyl (50-COOH beads) functions. Microscopy revealed adhesion of particles to the spermatozoa membranes, but no translocation of either particle type into cells. Nevertheless, the 50-NH2 beads at 10 µg mL-1 induced a high spermiotoxicity, characterized by a decrease in the percentage of motile spermatozoa (-79%) and in the velocity (-62%) compared to control spermatozoa, with an overall drop in embryogenesis success (-59%). This major reproduction failure could be linked to a homeostasis disruption in exposed spermatozoa. The 50-COOH beads hampered spermatozoa motility only when administered at 25 µg mL-1 and caused a decrease in the percentage of motile spermatozoa (-66%) and in the velocity (-38%), but did not affect embryogenesis success. Microscopy analyses indicated these effects were probably due to physical blockages by microscale aggregates formed by the 50-COOH beads in seawater. This toxicological study emphasizes that oyster spermatozoa are a useful and sensitive model for (i) deciphering the fine interactions underpinning nanoplastic toxicity and (ii) evaluating adverse effects of plastic nanoparticles on marine biota while waiting for their concentration to be known in the environment.

Thrombin generation and procoagulant phospholipids in patients with essential thrombocythemia and reactive thrombocytosis.

Thrombocytosis is a commonly encountered clinical scenario and can be either a secondary process (reactive thrombocytosis), or due to clonal disorder (i.e., essential thrombocythemia). This distinction is important as it carries implications for evaluation, prognosis and treatment. In this study we compared procoagulant potential in essential thrombocythemia and reactive thrombocytosis by measuring the thrombin generation and the level of circulating procoagulant phospholipids with functional tests. Twenty nine patients with essential thrombocythemia and 24 with reactive thrombocytosis were studied. Thrombin generation was determined by calibrated automated thrombography. Procoagulant phospholipids were detected by a chronometric standardised method (STA-Procoag-PPL). Patients with reactive thrombocytosis had a longer lag time, higher endogenous thrombin potential, peak of thrombin generation and velocity index than patients with essential thrombocythemia. The level of circulating procoagulant phospholipids was increased in patients with essential thrombocythemia as observed with the procoagulant phospholipids assay. Each parameter was analysed using ROC curves. Highest areas under the curve (AUC) were found for lag time and procoagulant phospholipids ratio (0.817 and 0.853, respectively), associated with high negative predictive value for ET (92.3% and 80%, respectively). In conclusion, patients with essential thrombocythemia and reactive thrombocytosis displayed significant differences in terms of thrombin generation and levels of procoagulant phospholipids. Among these parameters, lag time and procoagulant phospholipids ratio could help to differentiate between reactive thrombocytosis and essential thrombocythemia patients.

Investigation of fatty acids accumulation in Nannochloropsis oculata for biodiesel application.

Lipids production of the marine microalga species Nannochloropsis oculata was deeply investigated by studying under continuous light the effects of different nitrogen starvation strategies in photobioreactors of various thicknesses. Operating parameters like incident photons flux density (PFD), initial nitrogen (progressive starvation strategy) or biomass concentrations (sudden starvation strategy) were examined, with a detailed analysis of their effects on the quality and production kinetics of total (TL) and triglycerides (TG). In addition to the already known effect of nitrogen starvation to trigger reserve lipids accumulation (mainly TG), it was demonstrated the relevance of the light received per cell affecting TG content and productivities, as well as fatty acids (FA) profiles. With appropriate optimization, N. oculata was confirmed as an interesting candidate for biodiesel application, with high FA accumulation (up to around 50%DW with 43%DW in TG-FA), high productivity (maximum 3.6×10(-3)kg(TG-FA)m(-2)d(-1)) and a TG-FA profile close to palm oil.

Comparison of three methods of sampling for endometrial cytology in the mare. Preliminary study.

OBJECTIVE: This prospective study aims to compare three different sampling techniques for the collection of endometrial cytological specimens in the mare: the guarded culture swab, the uterine cytobrush and the low volume uterine flush. MATERIAL AND METHODS: The study population consisted of six healthy Standardbred mares in dioestrus. In each mare an acute endometritis was induced by performing a low- volume uterine flush 6days after ovulation using a sterile isotonic solution (lactated Ringer's solution or ViGro™ Complete Flush Solution). Two days after initiating inflammation, samples were collected from each mare using the three compared techniques: the double guarded cotton swab, the uterine cytobrush and the low volume uterine flush. The cytological evaluation of the samples was based on following criteria: the quality and cellularity of the samples and the number of neutrophils recovered. RESULTS: The uterine cytobrush yielded slides of significantly (p=0.02) better quality than the low volume uterine flush. There was no significant difference between the cytobrush and the double guarded swab technique for the quality. There was no difference between techniques in the number of endometrial cells (p=0.55) and neutrophils recovered (p=0.28). CONCLUSION AND CLINICAL RELEVANCE: Endometrial cytology is a practical method for the diagnosis of acute endometrial inflammation in the mare. Since no difference in the number of neutrophils was found between the three techniques, the choice of the sampling method should be based on other factors such as practicability, costs and disadvantages of each technique.

Analysis of genomic breakpoints in p190 and p210 BCR-ABL indicate distinct mechanisms of formation.

We sought to understand the genesis of the t(9;22) by characterizing genomic breakpoints in chronic myeloid leukemia (CML) and BCR-ABL-positive acute lymphoblastic leukemia (ALL). BCR-ABL breakpoints were identified in p190 ALL (n=25), p210 ALL (n=25) and p210 CML (n=32); reciprocal breakpoints were identified in 54 cases. No evidence for significant clustering and no association with sequence motifs was found except for a breakpoint deficit in repeat regions within BCR for p210 cases. Comparison of reciprocal breakpoints, however, showed differences in the patterns of deletion/insertions between p190 and p210. To explore the possibility that recombinase-activating gene (RAG) activity might be involved in ALL, we performed extra-chromosomal recombination assays for cases with breakpoints close to potential cryptic recombination signal sequence (cRSS) sites. Of 13 ALL cases tested, 1/10 with p190 and 1/3 with p210 precisely recapitulated the forward BCR-ABL breakpoint and 1/10 with p190 precisely recapitulated the reciprocal breakpoint. In contrast, neither of the p210 CMLs tested showed functional cRSSs. Thus, although the t(9;22) does not arise from aberrant variable (V), joining (J) and diversity (D) (V(D)J) recombination, our data suggest that in a subset of ALL cases RAG might create one of the initiating double-strand breaks.

Bivalve population health: multistress to identify hot spots.

This study investigated some stress (metals, parasites) and response (immunity, metallothionein) factors in two cockle and two Manila clam populations. Data from eight seasons were averaged to obtain global baseline values. Stress/response characteristics of each population were compared to population health status that was determined through population dynamics parameters. Four different scenarios were discussed: (1) a lightly stressed cockle population with correct population health but with a risk of deterioration (hot spot); (2) a lightly stressed introduced cockle population threatened of extinction. In this case ecological factors were suspected; (3) a moderately stressed clam population with moderate adaptative response. The population was sustainable but the level of stress should not increase (hotspot); and (4) a stressed clam population and unfavourable ecological conditions preventing clam settlement. This monitoring highlighted that the discrepancy between population health and stress levels could be due to insufficient response by bivalves and/or by unfavourable ecological factors.

[DIC and peripheral gangrene in a severe Plasmodium falciparum malaria: the coagulation-inflammation cycle with Plasmodium falciparum as a model]. / CIVD et gangrène périphérique dans un cas de paludisme sévère : le cycle coagulation-inflammation appliqué au Plasmodium falciparum.

Peripheral gangrene with disseminated intravascular coagulation (DIC) during severe Plasmodium falciparum malaria has already been described but is unfrequent. We report here the case of a 62-year-old man admitted in the intensive care unit of our hospital for severe Plasmodium falciparum malaria with disseminated intravascular coagulation (DIC) and peripheral gangrene of his toes that needed amputation. Pathophysiological mechanisms leading to DIC in malaria can be used as a model to explain the relation between coagulation and inflammation. Therapeutic targeting of coagulation, by acting on inflammation, could be useful to limit the coagulation-inflammation cycle.

Detection and molecular monitoring of FIP1L1-PDGFRA-positive disease by analysis of patient-specific genomic DNA fusion junctions.

To evaluate current detection methods for FIP1L1-PDGFRA in hypereosinophilic syndrome (HES), we developed a means to rapidly amplify genomic break points. We screened 202 cases and detected genomic junctions in all samples previously identified as RT-PCR positive (n=43). Genomic fusions were amplified by single step PCR in all cases whereas only 22 (51%) were single step RT-PCR positive. Importantly, FIP1L1-PDGFRA was detected in two cases that initially tested negative by RT-PCR or fluorescence in situ hybridization. Absolute quantitation of the fusion by real-time PCR from genomic DNA (gDNA) using patient-specific primer/probe combinations at presentation (n=13) revealed a 40-fold variation between patients (range, 0.027-1.1 FIP1L1-PDGFRA copies/haploid genome). In follow up samples, quantitative analysis of gDNA gave 1-2 log greater sensitivity than RQ-PCR of cDNA. Minimal residual disease assessment using gDNA showed that 11 of 13 patients achieved complete molecular response to imatinib within a median of 9 months (range, 3-17) of starting treatment, with a sensitivity of detection of up to 1 in 10(5). One case relapsed with an acquired D842V mutation. We conclude that detection of FIP1L1-PDGFRA from gDNA is a useful adjunct to standard diagnostic procedures and enables more sensitive follow up of positive cases after treatment.

Imatinib sensitivity as a consequence of a CSF1R-Y571D mutation and CSF1/CSF1R signaling abnormalities in the cell line GDM1.

Imatinib is usually a highly effective treatment for myeloproliferative neoplasms (MPNs) associated with ABL, PDGFRA or PDGFRB gene fusions; however, occasional imatinib-responsive patients have been reported without abnormalities of these genes. To identify novel imatinib-sensitive lesions, we screened 11 BCR-ABL-negative cell lines and identified GDM1, derived from a patient with an atypical MPN (aMPN), as being responsive to imatinib. Screening of genes encoding known imatinib targets revealed an exon 12 mutation in the colony-stimulating factor 1 receptor (CSF1R; c-FMS) with a predicted Y571D amino-acid substitution. CSF1R in GDM1 was constitutively phosphorylated, but rapidly dephosphorylated on exposure to imatinib. Y571D did not transform FDCP1 cells to growth factor independence, but resulted in a significantly increased colony growth compared with controls, constitutive CSF1R phosphorylation and elevated CSF1R signaling. We found that GDM1 expresses CSF1, and CSF1 neutralization partially inhibited proliferation, suggesting the importance of both autocrine and intrinsic mechanisms of CSF1R activation. An extensive screen of CSF1R in aMPNs and acute myeloid leukemia identified three additional novel missense variants. None of these variants were active in transformation assays and are therefore likely to be previously unreported rare polymorphisms or non-pathogenic passenger mutations.

The molecular regulation of muscle stem cell function.

Muscle satellite cells are responsible for the postnatal growth and robust regeneration capacity of adult skeletal muscle. A subset of satellite cells purified from adult skeletal muscle is capable of repopulating the satellite cell pool, suggesting that it has direct therapeutic potential for treating degenerative muscle disease. Satellite cells uniformly express the transcription factor Pax7, and Pax7 is required for satellite cell viability and to give rise to myogenic precursors that express the basic helix-loop-helic (bHLH) transcription factors Myf5 and MyoD. Pax7 activates expression of target genes such as Myf5 and MyoD through recruitment of the Wdr5/Ash2L/MLL2 histone methyltransferase complex. Extensive genetic analysis has revealed that Myf5 and MyoD are required for myogenic determination, whereas myogenin and MRF4 have roles in terminal differentiation. Using a Myf5-Cre knockin allele and an R26R-YFP Cre reporter, we observed that in vivo about 10% of satellite cells only express Pax7 and have never expressed Myf5. Moreover, we found that Pax7(+)/Myf5(-) satellite cells give rise to Pax7(+)/Myf5(+) satellite cells through basal-apical asymmetric cell divisions. Therefore, satellite cells in skeletal muscle are a heterogeneous population composed of satellite stem cells (Pax7(+)/Myf5(-)) and satellite myogenic cells (Pax7(+)/Myf5(+)). Evidence is accumulating that indicates that satellite stem cells represent a true stem cell reservoir, and targeting mechanisms that regulate their function represents an important therapeutic strategy for the treatment of neuromuscular disease.

NMR signal analysis to characterize solid, aqueous, and lipid phases in baked cakes.

Proton mobility was studied in molecular fractions of some model systems and of cake using a 1H nuclear magnetic resonance (NMR) relaxation technique. For cake, five spin-spin relaxation times (T2) were obtained from transverse relaxation curves: T2 (1) approximately 20 micros, T2 (2) approximately 0.2 ms, T2 (3) approximately 3 ms, T2 (4) approximately 50 ms, and T2 (2) approximately 165 ms. The faster component was attributed to the solid phase, components 2 and 3 were associated with the aqueous phase, and the two slowest components were linked to the lipid phase. After cooking, the crust contained more fat but less water than the center part of the cake. The amount of gelatinized starch was lower in the crust, and water was more mobile due to less interaction with macromolecules. This preliminary study revealed different effects of storage on the center and crust.

Identification of a novel imatinib responsive KIF5B-PDGFRA fusion gene following screening for PDGFRA overexpression in patients with hypereosinophilia.

Idiopathic hypereosinophilic syndrome (IHES) is a disease that is difficult to classify, and diagnosis is one of exclusion. The identification of a cytogenetically invisible interstitial deletion resulting in the fusion of FIP1-Like-1 (FIP1L1) to platelet-derived growth factor receptor alpha (PDGFRA) has enabled many IHES cases to be reclassified as chronic eosinophilic leukemia. As it is likely that PDGFRA may fuse to other partner genes, we established a reverse transcriptase-PCR test to detect specific overexpression of the PDGFRA kinase domain as an indicator of the presence of a fusion gene. Overexpression was detected in 12/12 FIP1L1-PDGFRA-positive patients, plus 9/217 (4%) patients with hypereosinophilia who had tested negative for FIP1L1-PDGFRA. One of the positive cases was investigated in detail and found to have a complex karyotype involving chromosomes 3, 4 and 10. Amplification of the genomic breakpoint by bubble PCR revealed a novel fusion between KIF5B at 10p11 and PDGFRA at 4q12. Imatinib, a known inhibitor of PDGFRalpha, produced a complete cytogenetic response and disappearance of the KIF5B-PDGFRA fusion by PCR, from both genomic DNA and mRNA. This study demonstrates the utility of screening for PDGFRA kinase domain overexpression in patients with IHES and has identified a third PDGFRA fusion partner in chronic myeloproliferative disorders.

Disruption and aberrant expression of HMGA2 as a consequence of diverse chromosomal translocations in myeloid malignancies.

Chromosomal translocations that target HMGA2 at chromosome band 12q14 are seen in a variety of malignancies, notably lipoma, pleomorphic salivary adenoma and uterine leiomyoma. Although some HMGA2 fusion genes have been reported, several lines of evidence suggest that the critical pathogenic event is the expression of truncated HMGA2 isoforms. We report here the involvement of HMGA2 in six patients with myeloid neoplasia, dysplastic features and translocations or an inversion involving chromosome bands 12q13-15 and either 7p12, 8q22, 11q23, 12p11, 14q31 or 20q11. Breaks within or very close to HMGA2 were found in all six cases by molecular cytogenetic analysis, leading to overexpression of this gene as assessed by RT-PCR. Truncated transcripts consisting of HMGA2 exons 1-2 or exons 1-3 spliced to intron-derived sequences were identified in two patients, but were not seen in controls. These findings suggest that abnormalities of HMGA2 play an important and previously unsuspected role in myelodysplasia.

Histoplasmosis as the cause of a pathological fracture.

We report the case of an 82-year-old man with a pathological fracture of the hip caused by infection with Histoplasma capsulatum var capsulatum. He was treated by a hemiarthroplasty and with oral itraconazole.

Continuous flow isotope ratio mass spectrometry for the measurement of nanomole amounts of (13)CO(2) by a reverse isotope dilution method.

A simple method for the determination of nanomole amounts of (13)CO(2) generated from an in vitro reaction is reported. The incubation medium contains a known amount of unlabeled sodium bicarbonate and the gaseous (13)CO(2) enriches the atmosphere upon which a measurement of the isotopic enrichment ((13)CO(2)/(12)CO(2)) is made corresponding to a reverse isotope dilution. The quantification of the (13)CO(2) was performed by gas chromatography/isotope ratio mass spectrometry. This assay was validated in terms of linearity, accuracy and precision using three different substrates which produce (13)CO(2) either by enzymatic reaction [(13)C]urea, sodium [(13)C]formate) or by chemical reaction (sodium [(13)C]bicarbonate). Four calibration curves were tested for each (13)C-labeled substrate, allowing the quantification of (13)CO(2) from 25 pmol to 150 nmol. The dynamics of the assay were obtained as a function of the quantity of unlabeled sodium bicarbonate added to each sample.

[Optimisation of the measurement of nitrite and nitrate in serum by the Griess reaction]. / Optimisation des paramètres du dosage des nitrites et nitrates sériques par la technique de Griess.

Nitrite (NO2-) and nitrate (NO3-) concentrations are usually measured as a marker of NO metabolism. Indeed, this radical plays an important role in a lot of pathological processes. The aim of this study was to validate and standardize a method for the quantification of these two metabolites in serum. The most commonly method involved to determine nitrite and nitrate is the Griess reaction. From the different methods which are reported in literature, we tested several parameters to define operating conditions: the samples were deproteinized with zinc sulfate and reduced with cadmium granules. Analytical recovery of NO3- added to serum samples after reduction was 99.6% +/- 3.2% (n = 4). Within-run precision was 6.1% (n = 17) and between-day precision was 6.6% (n = 12). Usual values were determined from healthy fasted subjects: the mean concentration of NO2- and NO3- was 47.8 muM +/- 15.7 muM (n = 25). The detection limit of the assay was 1.3 muM and the quantitation limit was 2.5 muM. We tested also an HPLC method. However, it was not possible to use it from a biological matrice.

Contact-mediated inhibition of human haematopoietic progenitor cell proliferation may be conferred by stem cell antigen, CD34.

INTRODUCTION: The function of CD34, a transmembrane sialomucin expressed by human haematopoietic progenitor cells, is poorly understood. Its structure suggests it may act as a cell adhesion and signalling molecule. MATERIALS AND METHODS: KGIa cells and primary CD34-positive marrow cells were tested for their ability to aggregate in the presence of the anti-CD34 antibody QBEND10; CFU-GM colonies were grown using standard methods and tested for their content of colony-forming cells by replating; 'haematons' were isolated from marrow by filtration; the phosphorylation of CD34 was investigated by immunoprecipitation and Western blotting DISCUSSION: CD34-positive cells in human bone marrow, like KG1a cells, aggregate when incubated with QBEND10. Staining aggregates with anti-CD34-FITC revealed that aggregation involved co-localisation of CD34 at intercellular binding sites. We examined myeloid colonies (CFU-GM) grown from normal human bone marrow cells, and multicellular aggregates ('haematons') separated from freshly aspirated marrow by filtration, and found CD34-positive cells bound together with co-localisation of the CD34 at the binding sites. This finding shows that CD34-positive cell-cell adhesion occurs physiologically in vitro and in vivo. QBEND10-induced aggregation of KG1a and CD34-positive cells was enhanced by staurosporine (a protein kinase C inhibitor) and inhibited by genistein (a protein tyrosine kinase inhibitor). Moreover, aggregated cells had increased phosphorylation of tyrosine on CD34 and translocation of protein kinase C (PKC) to the cytoplasm, compared with non-aggregated cells. We used the ability of primary colonies to produce secondary colonies on replating as a functional parameter and found that the replating ability of the colonies was increased by treatment with genistein (P=0.003). In addition, the ability of individual samples of primary CD34-positive cells to undergo QBEND10-induced aggregation and the ability of CD34-positive cell-derived colonies to produce secondary clones on replating were inversely related (r=0.86). CONCLUSION: Our results suggest that homotypic aggregation of haematopoietic progenitor cells may be an important mechanism for preventing inappropriate proliferation in vivo. Thus, regulation of expression of the CD34 molecule may play an important role in maintaining the normal level of haematopoietic activity by contact-mediated inhibition of progenitor cell proliferation.