ApoFnr binds as a monomer to promoters regulating expression of enterotoxin genes of Bacillus cereus.

International audienceBacillus cereus Fnr is a member of the Crp/Fnr (cAMP-binding protein/fumarate nitrate reduction regulatory protein) family of helix-turn-helix transcriptional regulators. It is essential for the expression of Hbl and Nhe enterotoxin genes independently of the oxygen tension in the environment. We studied aerobic Fnr binding to target sites in promoters regulating the expression of enterotoxin genes. B. cereus Fnr was overexpressed and purified as either a C-terminal His-tagged (FnrHis) fusion protein or an N-terminal fusion protein tagged with the Strep-tag (IBA BioTAGnology) (StrepFnr). Both recombinant Fnr proteins were produced as apoforms (clusterless) and occured as mixtures of monomers and oligomers in solution. However, apoFnrHis was mainly monomeric, while apoStrepFnr was mainly oligomeric, suggesting that the His-tagged C-terminal extremity may interfere with oligomerization. The oligomeric state of apoStrepFnr was dithiothreitol sensitive, underlining the importance of a disulphide bridge for apoFnr oligomerization. Electrophoretic mobility shift assays showed that monomeric apoFnr, but not oligomeric apoFnr, bound to specific sequences located in the promoter regions of the enterotoxin regulators fnr, resDE and plcR and the structural genes hbl and nhe. The question of whether apoFnr binding is regulated in vivo by redox-dependent oligomerization is discussed

ResDE-dependent regulation of enterotoxin gene expression in <em>Bacillus cereus</em>: evidences for multiple modes of binding for ResD and interaction with Fnr

International audienceIn the food-borne pathogen Bacillus cereus F4430/73, the production of major virulence factors hemolysin BL (Hbl) and non-hemolytic enterotoxin (Nhe) is regulated through complex mechanisms. The two-component regulatory system ResDE is involved in the activation of hbl and nhe transcription. Here, the response regulator ResD and the sensor kinase ResE were overexpressed and purified, and autophosphorylation of ResE and transphosphorylation of ResD by ResE were demonstrated in vitro. ResD is mainly monomeric in solution regardless of its phosphorylation state. ResD was shown to interact directly with promoter regions (p) of the enterotoxin regulator genes resDE, fnr and plcR and the enterotoxin structural genes nhe and hbl, but with different affinities. Binding of ResD to pplcR, pnhe and phbl was not dependent on ResD phosphorylation status. In contrast, ResD phosphorylation significantly increased interactions between ResD and presDE and pfnr. Taken together, these results showed that phosphorylation of ResD results in a different target expression pattern. Furthermore, ResD and the redox activator Fnr were found to physically interact and simultaneously bind their target DNAs. We propose that unphosphorylated ResD acts as an anti-activator of Fnr while phosphorylated ResD acts as a co-activator of Fnr. Finally, our findings represent the first molecular evidence for the role of ResDE as a sentinel system capable of sensing redox changes and coordinating a response that modulates B. cereus virulenc

Mise au point d'un protocole d'extraction sélective du 36^{36}Cl contenu dans le combustible nucléaire irradié

National audienceDans le contexte général du recyclage des combustibles nucléaires usés, plusieurs isotopes présentent unintérêt particulier dans le cadre de la veille sur les émetteurs $\beta$-$\gamma$, parmi eux, le chlore 36. Le $^{36}$Cl est unémetteur $\beta$- issu de l’activation de l’impureté chlore 35 contenue dans l’oxyde. La teneur exacte en impuretéest inconnue mais répond aux spécifications de fabrication du combustible

Chlorine 36 analysis in nuclear spent fuel

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Americium, curium and neodymium analysis in ECRIX-H irradiated pellet: sample preparation for TIMS measurements

International audienceThis paper concerns quantitative isotopic analysis of Am, Cm and Nd contained in an irradiated AmO 1.62 /MgO pellet. The complete analysis protocol is described, from dissolution of the pellets in a shielded line to the laboratory glove separation processes box for TIMS analysis. Emphasis is placed on the separation processes: by ion exchange resin in a hot cell and by HPLC in the laboratory. Intermediate measurements by X-ray fluorescence, alpha spectrometry, and ICP-AES are described

Mise au point d’un protocole d’extraction sélective du chlore contenu dans le combustible nucléaire irradié

National audienceDans le contexte général du recyclage des combustibles nucléaires usés, plusieurs isotopes présentent un intérêt particulier dans le cadre de la veille sur les émetteurs $\beta$-$\gamma$, parmi eux, le chlore 36. Le $^{36}$Cl est un émetteur $\beta$- issu de l’activation de l’impureté chlore 35 contenue dans l’oxyde. La teneur exacte en impureté est inconnue mais répond aux spécifications de fabrication du combustible. Lors du retraitement du combustible irradié, sa teneur dans les rejets est soumise à des réglementations (ANDRA, ASN, décrets). Afin de réaliser une cartographie de la répartition de cet isotope dans les différents flux du retraitement du combustible irradié, il est nécessaire de connaître précisément la valeur d’entrée du $^{36}$Cl dans le procédé. Une mesure précise de la teneur du chlore 36 dans le combustible nucléaire irradié est nécessaire

Influence of the decontamination of a high radioactive solution from Cesiumon analytical performances

International audienceThe high rate of irradiation of certain samples is essentially due to the presence of the ^[137}Cs. Removing selectively Cs from these samples provides several advantages 1- a weaker dilution to make possible the samples manipulation and to carry out their analyses by techniques usually implanted in glove boxes (ICP-AES, ICP-QMS, radiometry, etc.), 2- a lower detection limit of analyzed elements, 3- a minimization of the Compton effect on gamma spectra.The method of cations / Cs$^+$ separation is very important. It must lead to the highest possible cesium decontamination factor, the quantitative recovery of the other elements and the lowest possible dilution factor of the sample. The implemented method is extraction chromatography using the AMP-PAN resin (ammonium molybdophosphate embedded in polyacrylnitrile). It consists in adding the sample, firstly adjusted at a nitric acidity of 2M on a AMP-PAN column, then in adding a washing solution ([HNO$_3$] = 2M). During these two stages, the cesium is expected to be retained by the chromatographic support whereas the other cations cross the column.The efficiency of this method was tested by comparison of analyses by gamma and alpha spectrometry, by X-ray fluorescence and by ICP-AES realized with a sample, taken in a storage tank of fissions products, before and after its passage on an AMP-PAN column.The results of analyses were very satisfactory. A strong decontamination of the initial sample from $^{137}$Cs (FD > 5.104) and a quasi-quantitative recovery of the other analyzed elements were observed. The gamma spectra showed in particular several radionuclides which were not quantified on the initial sample and led to detection limits twenty times lower.The description of the chromatographic cycle on AM-PAN resin and all the analyses will be presented during this talk

Effectiveness of nitrous oxide in external cephalic version on success rate: a randomized controlled trial

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Decontamination of high level activity solutions from cesium and strontium by chromatography

International audienceThe high rate of irradiation of certain samples resulting from various programs carried out in ATALANTE facility is essentially due to the presence of $^{137}$+$^{134}$Cs and $^{90}$Sr. The elimination of these radionuclides would make it possible to handle these samples in glove boxes without having to carry out important dilution beforehand. It would allow to analyze these samples by techniques usually implemented in glove boxes (ICP, radiometry, ...) and to reach lower detection limits.Preliminary tests were carried out with real samples by using extraction chromatography as separative technique.The principle of the cations/Cs separation consists in adding on a 2 mL column filled with resin AM-PAN (ammonium molybdophosphate embedded in polyacrylnitrile), the sample adjusted before at a nitric acidity of 2M, then a washing solution ([HNO$_3$] = 2M). During these two steps, Cs$^+$ is expected to be retained by the resin while the other cations cross the column.This separation, tested on an aliquote of a solution of irradiated fuel dissolution, showed that contrary to the cesium, elements measured by ICP-AES (U and Pu) and by gamma spectrometry ($^{154}$+$^{155}$Eu, $^{244}$Ce, $^{241}$Am and $^{106}$Ru) are not retained during the feeding and washing steps. The decontamination factor of cesium is superior to 500.The principle of the cations / Sr separation consists in adding on a 2 mL column filled with a Sr resin ((1.0M 4,4 ' (5 ')-di-t-butylcyclohexano 18-crown-6 (crown ether) in 1-octanol (weighing 40%) / inert chromatographic support (weighing 60%)) the sample, adjusted before at the acidity [HNO$_3$] = 3M / [H$_2$C$_2$O$_4$] = 0.01M, then a washing solution ([HNO$_3$] = 3M / [H$_2$C$_2$O$_4$] = 0.01M). During these two steps, the strontium is expected to be retained by the resin whereas the other cations cross the column.This separation, tested on an aliquote of a solution of irradiated dissolution of fuel, showed that elements measured by ICP-AES (U, Fe, Mo, Zr, Pd, Zr, Pd, Ru, Tc) and by spectrometry gamma ($^{134}$+$^{137}$Cs, $^{154}$+$^{155}$Eu, $^{241}$Am, $^{106}$Ru) are not retained during the feeding and washing stages. The passage on the column of an elution solution ([HNO$_3$] = 0.01M) led to the total recovery of the initial strontium. The results of the cations / Cs and cations / Sr separations are very encouraging. Additional experiments on other high level radioactive samples are in progress to consolidate both tested methods. They consist in using a wider range of samples, studying the influence of fluorides and determining the behavior of a largest number of elements. These studies will be presented on the poster