Choosing a career in oncology: results of a nationwide cross-sectional study.

BACKGROUND: Little information is currently available concerning young medical students desire to pursue a career in oncology, or their career expectations. METHODS: This project is a cross-sectional epidemiological study. A voluntary and anonymous questionnaire was distributed to all young oncologists studying in France between the 2nd of October 2013 and the 23rd of February 2014. RESULTS: The overall response rate was 75.6%. A total of 505 young oncologists completed the questionnaire. The main determining factors in the decision to practice oncology were the cross-sectional nature of the field (70.8%), the depth and variety of human relations (56.3%) and the multi-disciplinary field of work (50.2%). Most residents would like to complete a rotation outside of their assigned region (59.2%) or abroad (70.2%) in order to acquire additional expertise (67.7%). In addition, most interns would like to undertake a fellowship involving care, teaching and research in order to hone their skills (85.7%) and forge a career in public hospitals (46.4%). Career prospects mainly involve salaried positions in public hospitals. Many young oncologists are concerned about their professional future, due to the shortage of openings (40.8%), the workload (52.8%) and the lack of work-life balance (33.4%). CONCLUSIONS: This investigation provides a comprehensive profile of the reasons young oncologists chose to pursue a career in oncology, and their career prospects.

Complex regulation of LCoR signaling in breast cancer cells.

Ligand-dependent corepressor (LCoR) is a transcriptional repressor of ligand-activated estrogen receptors (ERs) and other transcription factors that acts both by recruiting histone deacetylases and C-terminal binding proteins. Here, we first studied LCOR gene expression in breast cancer cell lines and tissues. We detected two mRNAs variants, LCoR and LCoR2 (which encodes a truncated LCoR protein). Their expression was highly correlated and localized in discrete nuclear foci. LCoR and LCoR2 strongly repressed transcription, inhibited estrogen-induced target gene expression and decreased breast cancer cell proliferation. By mutagenesis analysis, we showed that the helix-turn-helix domain of LCoR is required for these effects. Using in vitro interaction, coimmunoprecipitation, proximity ligation assay and confocal microscopy experiments, we found that receptor-interacting protein of 140 kDa (RIP140) is a LCoR and LCoR2 partner and that this interaction requires the HTH domain of LCoR and RIP140 N- and C-terminal regions. By increasing or silencing LCoR and RIP140 expression in human breast cancer cells, we then showed that RIP140 is necessary for LCoR inhibition of gene expression and cell proliferation. Moreover, LCoR and RIP140 mRNA levels were strongly correlated in breast cancer cell lines and biopsies. In addition, RIP140 positively regulated LCoR expression in human breast cancer cells and in transgenic mouse models. Finally, their expression correlated with overall survival of patients with breast cancer. Taken together, our results provide new insights into the mechanism of action of LCoR and RIP140 and highlight their strong interplay for the control of gene expression and cell proliferation in breast cancer cells.

A novel "correlated ion and neutral time of flight" method: event-by-event detection of neutral and charged fragments in collision induced dissociation of mass selected ions.

A new tandem mass spectrometry (MS/MS) method based on time of flight measurements performed on an event-by-event detection technique is presented. This "correlated ion and neutral time of flight" method allows to explore Collision Induced Dissociation (CID) fragmentation processes by directly identifying not only all ions and neutral fragments produced but also their arrival time correlations within each single fragmentation event from a dissociating molecular ion. This constitutes a new step in the characterization of molecular ions. The method will be illustrated here for a prototypical case involving CID of protonated water clusters H(+)(H2O)n = 1-5 upon collisions with argon atoms.

A new experimental setup designed for the investigation of irradiation of nanosystems in the gas phase: a high intensity mass-and-energy selected cluster beam.

DIAM (Dispositif d'Irradiation d'Agrégats Moléculaires) is a new experimental setup devoted to investigate processes induced by irradiation at the nanoscale. The DIAM apparatus is based on a combination of techniques including a particle beam from high-energy physics, a cluster source from molecular and cluster physics, and mass spectrometry form analytical sciences. In this paper, we will describe the first part of the DIAM apparatus that consists of an ExB double spectrometer connected to a cluster ion source based on a continuous supersonic expansion in the presence of ionizing electrons. This setup produces high intensities of energy-and-mass selected molecular cluster ion beams (1000 s of counts s(-1)). The performance of the instrument will be shown through measurements of 6-8 keV beams of protonated water clusters, (H(2)O)(n)H(+) (n = 0-21) and mixed protonated (or deprotonated) water-pyridine cluster ions: PyrH(+)(H(2)O)(n) (n = 0-15), Pyr(2)H(+) (H(2)O)(n) (n = 0-9), and (Pyr-H)(+) (H(2)O).

Metronidazole resistance in Prevotella spp. and description of a new nim gene in Prevotella baroniae.

Nonduplicate clinical isolates of Prevotella spp. recovered from patients hospitalized between 2003 and 2006 in two French tertiary-care teaching hospitals were investigated for their susceptibility to metronidazole and the presence of nim genes. Of the 188 strains tested, 3 isolates displayed reduced susceptibility to metronidazole after 48 h of incubation, while 27 additional isolates exhibited heterogeneous resistance after prolonged incubation; all 30 of the isolates were nim negative. Among the remaining 158 isolates, 7 nim-positive isolates were detected. All of these strains were identified as Prevotella baroniae by 16S rRNA gene sequence analysis and contained a new nim gene, named nimI, as determined by DNA sequence analysis. Chromosomal localization of this single-copy gene was demonstrated in all clinical isolates as well as in type strain P. baroniae DSM 16972 by using Southern hybridization. No known associated insertion sequence elements were detected upstream of the nimI gene in any of the nim-positive strains by PCR mapping. After prolonged exposure to metronidazole, stable resistant subpopulations could be selected in nimI-positive Prevotella isolates (n = 6) as well as in nim-negative Prevotella isolates (n = 6), irrespective of their initial susceptibility to this antibiotic. This study is the first description of a new nitroimidazole resistance gene in P. baroniae which seems to be silent and which might be intrinsic in this species. Moreover, our findings highlight the fact that high-level resistance to metronidazole may be easily induced in both nim-positive and nim-negative Prevotella sp. strains.

Disposition and metabolism of dipropyl disulphide in vivo in rat.

The metabolism of dipropyl disulphide (DPDS), a sulphur compound from onion, was investigated in vivo in the rat. A single dose (200 mg kg(-1)) was administered by gastric intubation and the time courses of DPDS and its metabolites were followed over 48 h by gas chromatography coupled with mass spectrometry in the stomach, intestine, liver, and blood. DPDS was detected in the stomach where it was transformed into propyl mercaptan, whereas the liver contained only traces of DPDS and none at all in the other examined organs. The metabolites methylpropyl sulphide, methylpropyl sulphoxide (MPSO), and methylpropyl sulphone (MPSO2) were sequentially formed in the liver. The route of elimination from the liver seemed to be mainly via the blood. The bile also participated in the excretory process, but only for MPSO2. The pharmacokinetic parameters were determined for all of the above compounds. Whereas the bioavailability of DPDS was very low (0.008 h mM), the areas under the curve were higher for the S-oxidized metabolites MPSO and MPSO2, i.e. 9.64 and 24.15 h mM, respectively. The half-lives for DPDS and its metabolites varied between 2.0 and 8.25 h, except for MPSO2, which had a half-life of 29.6 h. MPSO2 was the most abundant and persistent of these metabolites.

[Species identification and molecular epidemiology of bacteria belonging to Ochrobactrum genus]. / Identification d'espèce et épidémiologie moléculaire des bactéries du genre Ochrobactrum.

Two species of medical interest belong to the genus Ochrobactrum, Ochrobactrum anthropi and Ochrobactrum intermedium. They are members of the microbiota of soil and an increasing number of works report the isolation of O. anthropi from clinical specimen, especially from immunocompromised patients and nosocomial infection. Involving of each species in human infection is poorly estimated due to unclear differential phenotypic characters. We performed 16S rDNA sequencing for identification of 20 clinical isolates of Ochrobactrum sp. to the species level. Then, we studied the phenotype of each isolate especially, morphology, culture onto different media and at different temperatures, biochemical characters and antibiotics resistance pattern. Colony morphology after growth onto Trypticase-Soy and McConkey agar, culture at 45 degrees C onto Trypticase-Soja agar, presence of urease, and netilmycin, tobramycin and colistin resistance allowed identification of species. Ribotyping using HindIII and EcoRI gave a supplementary criterion for species determination but did not allow typing at the infra-species level. In contrast, Pulsed-Field Gel Electrophoresis showed high degree of polymorphism between strains and proved the clonality of certain isolates. Thus, this method could be a useful tool for molecular epidemiology of Ochrobactrum infections.

Hepatic metabolism of diallyl disulphide in rat and man.

1. The metabolism of diallyl disulphide was investigated in vitro with rat and human liver cell subfractions and ex vivo with an isolated perfused rat liver. 2. Diallyl disulphide was oxidized to diallylthiosulphinate by rat liver microsomes with an apparent K(m) = 0.86 +/- 0.1 mM and an apparent V(max) = 0.47 +/- 0.12 nmol min(-1) mg(-1) protein (mean +/- SE). Both cytochrome P450 (CYP) and flavin-containing monooxygenases were involved, with CYP2B1/2 and CYP2E1 being the most active CYP enzymes. 3. In rat and man, microsomal oxidation of allylmethyl sulphide to allylmethyl sulphoxide and allylmethyl sulphone also occurred, although at a low rate. Diallyl disulphide was also metabolized to allylglutathione sulphide and allylmercaptan. In addition, diallylthiosulphinate reacted non-enzymatically with glutathione to form allylglutathione sulphide. 4. When an isolated rat liver was perfused with diallyl disulphide, the metabolites allyl mercaptan, allylmethyl sulphide, allylmethyl sulphoxide, allylmethyl sulphone and allylglutathione sulphide were detected primarily within the liver tissue, with only small amounts of metabolites found in the bile and perfusion medium. The pharmacokinetic parameters for diallyl disulphide were t(1/2) = 6.09 min; AUC(0- infinity ) = 4.77 min mmol l(-1); clearance = 34.22 ml min(-1). 5. A scheme for the metabolism of diallyl disulphide in rat and man is proposed.

In vivo metabolism of diallyl disulphide in the rat: identification of two new metabolites.

1. Diallyl disulphide (DADS), a compound formed from the organosulphur compounds present in garlic, is known for its anticarcinogenic effects in animal models. 2. The aim was to identify and analyse the metabolites produced in vivo after a single oral administration of 200 mg kg(-1) DADS to rats. The organic sulphur metabolites present in the stomach, liver, plasma and urine were measured by gas chromatography coupled with mass spectrometry over 15 days. 3. Data indicate that DADS is absorbed and transformed into allyl mercaptan, allyl methyl sulphide, allyl methyl sulphoxide (AMSO) and allyl methyl sulphone (AMSO(2)), which are detected throughout the excretion period. Overall, the highest amounts of metabolites were measured 48-72h after the DADS administration. AMSO(2) is the most abundant and persistent of these compounds. The levels of all the sulphur compounds rapidly decline within the first week after administration and disappear during the second week. Only AMSO and AMSO(2) are significantly excreted in urine. 4. These potential metabolites are thought to be active in the target tissues. Our data warrant further studies to check this hypothesis.

Effect of onion consumption by rats on hepatic drug-metabolizing enzymes.

Fruits and vegetables or their natural constituents which increase detoxication enzymes and/or reduce activating enzymes are considered as good candidates to prevent chemically-induced carcinogenesis. In this study, rats were fed a diet supplemented with 20% onion powder for 9 days. Several cytochrome P450 (CYP)s enzymes (CYP 1A, 2B, 2E1, 3A), which are involved in carcinogen activation, were determined by measuring their enzyme activities using specific substrates. In addition, phase II enzymes activities such as UDP-glucuronosyltransferase (UGT) and glutathione S-transferase (GST), involved in detoxication of carcinogens, were measured. Protein levels of CYPs and GST A1/A2, A3/A5, Ml, M2 and P1 were measured using antibodies in Western blots. Consumption of onion induced CYP 1A and CYP 2B activities while it decreased CYP 2E1 activity. This later modification was accompanied by a decrease of CYP 2E1 levels. The same dietary treatment caused a slight increase of the total GST activity. The relative proportions of GST subunits were modified. GST Al/A2 subunits were increased while GST A3/A5 and GST M2 subunits were decreased and GST M1 and P1 were not modified. Onion consumption also increased p-nitrophenol UGT activity. Taken together, these results suggest that the decrease of CYP 2E1 and the increase of phase II enzymes by onion can afford protection against some carcinogens, while the decrease of some GST subunits could increase the genotoxic effects of other chemicals. The modulating effect of onion could be ascribed to alk(en)yl polysulphides and/or glycosides of flavonols, which were identified in the onion powder.

Characterization of the physical interaction between estrogen receptor alpha and JUN proteins.

Activated estrogen receptor alpha (ERalpha) modulates transcription triggered by the transcription factor activator protein-1 (AP-1), which consists of Jun-Jun homodimers and Jun-Fos heterodimers. Previous studies have demonstrated that the interference occurs without binding of ERalpha to DNA but probably results from protein.protein interactions. However, involvement of a direct interaction between ERalpha and AP-1 is still debated. Using glutathione S-transferase pull-down assays, we demonstrated that ERalpha bound directly to c-Jun and JunB but not to FOS family members, in a ligand-independent manner. The interaction could occur when c-Jun was bound onto DNA, as shown in a protein-protein-DNA assay. It implicated the C-terminal part of c-Jun and amino acids 259-302 present in the ERalpha hinge domain. ERalpha but not an ERalpha mutant deleted of amino acids 250-303 (ER241G), also associated with c-Jun in intact cells, in the presence of estradiol, as shown by two-hybrid and coimmunoprecipitation assays. We also show that ERalpha, c-Jun, and the p160 coactivator GRIP1 can form a multiprotein complex in vitro and in intact cells and that the ERalpha.c-Jun interaction could be crucial for the stability of this complex. VP16-ERalpha and c-Jun, which both interact with GRIP1, had synergistic effect on GAL4-GRIP1-induced transcription in the presence of estradiol, and this synergistic effect was not observed with the ERalpha mutant VP16-ER241G or when c-Fos, which bound GRIP1 but not ERalpha, was used instead of c-Jun. Finally, ER241G was inefficient for regulation of AP-1 activity, and an ERalpha truncation mutant encompassing the hinge domain had a dominant negative effect on ERalpha action. These results altogether demonstrate that ERalpha can bind to c-Jun in vitro and in intact cells and that this interaction, by stabilizing a multiprotein complex containing p160 coactivator, is likely to be involved in estradiol regulation of AP-1 responses.

Metabolism of dipropyl disulfide by rat liver phase I and phase II enzymes and by isolated perfused rat liver.

The metabolism of dipropyl disulfide (DPDS), an Allium sulfur compound, was investigated in rat liver cell subfractions and in an isolated perfused rat liver. DPDS is oxidized to dipropyl thiosulfinate (DPDSO) by rat microsomes. The contribution of cytochrome P450 enzymes (CYPs) or flavin-containing monooxygenases (FMO) to the formation of DPDSO from its precursor was investigated. In rat microsomes, the reaction followed Michaelis-Menten kinetics with a K(m) = 0.52 +/- 0.1 mM and a V(max) = 5.91 +/- 0.5 nmol/min/mg of protein, respectively (mean +/- S.E., n = 4). Both FMOs and CYPs were involved in DPDS oxidation, although the contribution of CYPs was predominant. Liver microsomes from phenobarbital-treated rats showed a 3.2-fold increase in the rate of formation of DPDSO. Among many CYP isoform-specific inhibitors, only CYP2B1/2 inhibitors decreased the formation of DPDSO and the best correlation between the rate of DPDS oxidation with specific monooxygenase activities was found with a marker of CYP2B1/2 activity. The action of phase II enzymes on DPDS metabolism was studied by incubating DPDS or DPDSO with liver cytosols or microsomes. Two metabolites were formed from DPDS: propylglutathione sulfide conjugate and propylmercaptan, whereas with DPDSO, only the glutathione conjugate was observed. No conjugate compound was detected in the presence of UDP-glucuronyl transferases. When isolated rat liver was perfused with DPDS, different metabolites were obtained in the output and in the liver tissues: propylmercaptan appeared in the output, whereas methylpropyl sulfide, methylpropyl sulfone, and propylglutathione sulfide were detected in the liver tissue.

Stability and physicochemical properties of the bovine brain phosphatidylethanolamine-binding protein.

The equilibrium behaviour of the bovine phosphatidylethanolamine-binding protein (PEBP) has been studied under various conditions of pH, temperature and urea concentration. Far-UV and near-UV CD, fluorescence and Fourier transform infrared spectroscopies indicate that, in its native state, PEBP is mainly composed of beta-sheets, with Trp residues mostly localized in a hydrophobic environment; these results suggest that the conformation of PEBP in solution is similar to the three-dimensional structure determined by X-ray crystallography. The pH-induced conformational changes show a transition midpoint at pH 3.0, implying nine protons in the transition. At neutral pH, the thermal denaturation is irreversible due to protein precipitation, whereas at acidic pH values the protein exhibits a reversible denaturation. The thermal denaturation curves, as monitored by CD, fluorescence and differential scanning calorimetry, support a two-state model for the equilibrium and display coincident values with a melting temperature Tm = 54 degrees C, an enthalpy change DeltaH = 119 kcal.mol-1 and a free energy change DeltaG(H2O, 25 degrees C) = 5 kcal.mol-1. The urea-induced unfolding profiles of PEBP show a midpoint of the two-state unfolding transition at 4.8 M denaturant, and the stability of PEBP is 4.5 kcal.mol-1 at 25 degrees C. Moreover, the surface active properties indicate that PEBP is essentially a hydrophilic protein which progressively unfolds at the air/water interface over the course of time. Together, these results suggest that PEBP is well-structured in solution but that its conformation is weakly stable and sensitive to hydrophobic conditions: the PEBP structure seems to be flexible and adaptable to its environment.

Metabolism of diallyl disulfide by human liver microsomal cytochromes P-450 and flavin-containing monooxygenases.

The metabolism of diallyl disulfide (DADS), a garlic sulfur compound, was investigated in human liver microsomes. Diallyl thiosulfinate (allicin) was the only metabolite observed and its formation followed Michaelis-Menten kinetics with a Km = 0.61 +/- 0.2 mM and a Vmax = 18.5 +/- 4.2 nmol/min/mg protein, respectively (mean +/- S.E. M., n = 4). Both flavin-containing monooxygenase and the cytochrome P-450 monooxygenases (CYP) were involved in DADS oxidation, but the contribution of CYP was predominant. The cytochrome P-450 isoforms involved in this metabolism were investigated using selective chemical inhibitors, microsomes from cells expressing recombinant CYP isoenzymes, and studying the correlation of the rate of DADS oxidation with specific monooxygenase activities of human liver microsomes. Diethyldithiocarbamate and tranylcypromine inhibited allicin formation, whereas other specific inhibitors had low or no effect. Most of the different human microsomes from cells expressing CYP were able to catalyze this reaction, but CYP2E1 showed the highest affinity with a substantial activity. Furthermore, allicin formation by human liver microsomes was correlated with p-nitrophenol hydroxylase activity, a marker of CYP2E1, and tolbutamide hydroxylase activity, a marker of CYP2C9. Among these approaches only CYP2E1 was identified in each case, which suggested that DADS is preferentially metabolized to allicin by CYP2E1 in human liver. However the minor participation of other CYP forms and flavin-containing monooxygenases is likely.

FRA-1 expression level modulates regulation of activator protein-1 activity by estradiol in breast cancer cells.

We compared the effect of estradiol on activator protein-1 (AP-1) activity in estrogen receptor positive (ER alpha+) and estrogen receptor negative (ER alpha-) human breast cancer cell lines transiently transfected with the AP-1-responsive reporter plasmid AP-1-TK-CAT and an ER alpha expression vector. While estradiol increased AP-1 activity in the ER alpha+ cell lines MCF7, ZR75.1, and T47D, it decreased (MDA-MB231 and BT20 cells) or had no significant effect (MDA-MB435 cells) on AP-1-mediated transcription in ER alpha- cells. Estradiol also inhibited AP-1 activity in ER alpha-MDA-MB231 cells stably transfected with ER alpha and in which ER alpha levels are close to those found in MCF7. Use of ER alpha mutant expression vectors demonstrated that the DNA-binding domain of ER alpha was needed for stimulation or inhibition of AP-1 activity by estradiol but suggested that ER alpha binding to estrogen-responsive elements was not required for these effects. Changes in regulation paralleled quantitative and qualitative changes in protein binding to AP-1 sites, as demonstrated by gel shift assay: protein binding was greater and DNA/protein complexes migrated faster for ER alpha--than for ER alpha+ cells. In fact, by Northern blot, a high level of Fra-1 mRNA was found in BT20 and MDA-MB231 cells as compared with ER alpha+ cells, and MDA-MB435 cells showed an intermediary level of expression. The differential expression of Fra-1 in MCF7 and MDA-MB231 cells was confirmed at the protein level by supershift experiments. In addition, overexpression of Fra-1 in MCF7 cells decreased the positive effect of estradiol while inhibition of Fra-1 expression in MDA-MB231 cells, by transient transfection of the Fra-1 antisense expression vector, abolished the negative effect of the hormone. In conclusion, we demonstrated that ER alpha- breast cancer cell lines differ from ER+ cells by a high level of AP-1 DNA-binding activity due, at least in part, to high Fra-1 constitutive expression. High Fra-1 concentration is crucial for the negative regulation of AP-1 activity by estradiol and thus may take part in estradiol-induced inhibition of cell proliferation in ER alpha- breast cancer cells transfected with ER alpha expression construct.

Preferential binding of the archaebacterial histone-like MC1 protein to negatively supercoiled DNA minicircles.

The interaction of the archaebacterial MC1 protein with 207 bp negatively supercoiled DNA minicircles has been examined by gel retardation assays and compared to that observed with the relaxed DNA minicircle. MC1 binding induces a drastic DNA conformational change of each minicircle, leading to an increase of the electrophoretic mobility of the DNA. A slight increase in salt concentration enhances the amount of bound MC1, and high NaCl concentrations are required to dissociate the complexes. Furthermore, the salt effect on binding depends on the supercoiling state of the DNA. The dissociation rates decrease with increasing linking difference of the minicircles relative to their relaxed configuration to reach a maximum at -2 turns. In addition, differences between the topoisomers are also observed in terms of stoichiometry of the strongest complexes. So with the -2 topoisomer the complex with two MC1 molecules is the most stable, while with the -1 and -3 topoisomers, the strongest ones are those with one MC1 molecule per DNA ring.

Conformational changes of DNA minicircles upon the binding of the archaebacterial histone-like protein MC1.

Binding of the archaebacterial histone-like protein MC1 to DNA minicircles has been examined by gel retardation and electron microscopy. MC1 preferentially binds to a 207-base pair relaxed DNA minicircle as compared with the linear fragment. Random binding is observed at very low ionic strength, and a slight increase in salt concentration highly favors the formation of a complex that corresponds to the binding of two MC1 molecules per DNA ring. Measurements of dissociation rates show that this complex is remarkably stable, and electron microscopy reveals that it is characterized by two diametrically opposed kinks. These results are discussed in regard to the mechanisms by which MC1 affects DNA structure.

Archaebacterial histone-like protein MC1 can exhibit a sequence-specific binding to DNA.

The binding of MC1 protein, the major chromosomal protein of the archaebacterium Methanosarcina sp. CHTI 55, to the region preceding the strongly expressed genes encoding methyl coenzyme reductase in a closely related micro-organism has been investigated. By gel retardation and DNAase I footprinting assays, we identified a preferential binding sequence in an open reading frame of unknown function. The large area of DNA protected against DNAase I is interrupted by a strong cleavage enhancement site on each strand. By circular permutation assays, we showed that the DNA bends upon MC1 binding. Furthermore we observed that the presence of a sequence outside the binding site can induce an unusual electrophoretic behaviour in some complexes.