dataesr/scientific-paragraphs-categorization huggingface dataset:
S5 D  and E ). At least 10 force curves, with a preset contact force of 1 nN, a preset contact time of 0 s, pressing/pulling speeds of 1 μm/s and an acquisition frequency of 1024 Hz, were acquired per oocyst (Fig. 3 A  and B ). Each force curve was evaluated by eye and processed on a PC using the built-in JPK IP software (JPK Instruments), resulting in three to more than 15 data points extracted for each structure. We first observed that the maximal indentation depth at 1 nN was on average ∼20 nm (Fig. 3  C and D); this was less than the thickness of the bilayered wall structure, so the model was accurate enough to allow us to extract the Young modulus of the structures, E, one per valid force curve. To quantify the mechanics, we used as a first approximation a Hertz model for contact to fit the pressing part of the force curves assuming a pyramidal tip of α = 21°half angle and incompressibility of the material (ν = 0.
ACKNOWLEDGMENTS. We thank  M. L. Dardé  for providing 4B6 hybridoma and  PARIS Biotech  for continuous support. This work was supported by  Aix-Marseille University  ( Préciput 2011 Program ) and the  Agence Nationale de la Recherche Jeunes Chercheuses Jeunes Chercheurs DissecTion Program  (P. -H. P. ). P. H. P. thanks  JPK Instruments  for continuous support.
Impact of ICU Stay on AAM Infection To study the effects of a prolonged ICU stay on exposure to AAMs, we separately computed mean number of microorganisms against which the patients exhibited an antibody response for admission serum samples (n = 29) and for samples collected every 7 days thereafter (weekly serum samples; n = 56). Data indicate that the number of organisms against which patients (with or without pneumonia) exhibit an immune response (essentially with IgM antibodies) is positively correlated with the length of the patient ICU stay (Fig. 1 ). Overall, our patients developed an antibody response to an increasing number of AAMs over time, especially from days 7 to 21 following their admission (Fig. 1 ). These findings highlight the high seroconversion rates exhibited by ICU patients during their stay (Table  4 ) and confirm that immune response to AAMs increases when the ICU stay is prolonged.
Hierarchical Cluster Analysis of Serologic Data Hierarchical cluster analysis was used to group sera exhibiting similar antibody responses. This evaluation helped us to identify clusters of microorganisms specific to each patient group. To perform this analysis, T-Mev software was used. In the software, number ''3'' was attributed to positive sera and represented by red color. Number ''23'' was attributed to negative sera and represented by green color. Number ''2'' was attributed to the low-positive sera and represented by dark red sera color. Results show the presence of a cluster associated to IgM antibody response to seven microorganisms (Afipia genospecies 3, M. amorphae, A. felis genospecies A, N. oligomobilis, Mimivirus, B. alpica and B. massiliensis) (yellow cluster in Fig. 2 ). These microorganisms were mainly found in patient sera collected from the acute phase of VAP and CAP, as well as in these from convalescent patients who had a prolonged ICU stay (Fig.
Multiplexed serology was performed using amoebaassociated microorganism slides (Inodiag, Signe, France). Slides also targeted 5 non-AAMs (Afipia clevelandensis, Balneatrix alpica, Chlamydia pneumoniae, Chlamydia psittaci and Mycoplasma pneumoniae). Among them, A. clevelandensis and B. alpica were tested for both IgG and IgM antibodies, whereas C. pneumoniae, C. psittaci and M. pneumoniae were only tested for IgG or IgM antibodies respectively (Table  S2 ). Antibody titers were automatically determined and expressed as arbitrary fluorescence unit values (AUV). All cut-off values used in the present study were pre-programmed into the ''InoSoft 1. 4. 4. 4'' software that was supplied with the slides. These cut-off values were determined as previously reported  [17]. For antigens that had one cut-off value, the serum was considered positive if the fluorescence value was higher than the cut-off value.
Hierarchical Clustering Analysis and Statistical Analysis The final results for all of the serum samples (both IgG and IgM antibodies) were hierarchically clustered with the T-Mev software (www. tm4. org/mev. html) [39]. Statistical analysis was performed with a Chi-square test. Pvalues less than or equal to 0. 05 were considered to be significant. All statistical tests have been done without correction for multiple statistical comparisons due to the fact that the importance of such statistical correction for multiple tests remains paradoxical  [40] [41] [42].
Results New Set of Proteins Functionally Linked to the SKI Complex. To identify additional factors involved in the recognition and degradation of aberrant mRNAs or associated nascent peptides, we performed genetic screens, using the genetic interaction mapping (GIM) strategy  (16)  with mutants of the SKI complex (ski2Δ, ski3Δ, and ski8Δ) and with ltn1Δ. GIM genome-wide genetic screens enable the identification of functional interactions, whether synthetic lethal, synthetic slow growth, or epistatic, by combining a query mutation with a collection of mutant strains: either complete gene deletions  (17)  or DamP mutants for essential genes  (18). Among the 20 mutants having the strongest growth defect when combined with gene deletions of the SKI complex, we identified four gene deletions that were common in all of the screens: ltn1Δ, vms1Δ, ypl009cΔ (tae2Δ), and ydr333cΔ (rqc1Δ) (Dataset S1).
2D  and Dataset S2). In parallel, we examined the localization of Cdc48 in a polysome gradient. We observed that, even if most of the Cdc48 hexamers were localized in lighter fractions of the gradient, a subset of Cdc48 sedimented in the 60S fractions (Fig. 2A ). This subset is likely to correspond to Cdc48 physically interacting with 60S particles associated with Tae2, Rqc1, and Ltn1. Ltn1 and Rqc1 Are Essential for the Recruitment of Cdc48 to Ribosomal Subunits. Cdc48 (called p97 in mammals) is a highly conserved member of the AAA-ATPase family involved in a large number of cellular functions and forming homohexamers able to interact with diverse partners that confer specificity toward various pro-cesses  (14). One of the main functions of Cdc48 is to recognize polyubiquitinated molecules, extract them from a complex, and make these substrates accessible to the proteasome for degradation  (27).
Therefore, the ubiquitination activity of Ltn1 could be an important step for the recruitment of the Cdc48-Ufd1-Npl4 complex to 60S particles associated with Tae2 and Rqc1. We thus tested whether the absence of Ltn1 affected the binding of Cdc48 to the Tae2-and Rqc1-associated complexes. We observed that the deletion of LTN1 resulted in a decrease in the amount of Cdc48 and its partners Ufd1 and Npl4 from Tae2-TAP (Fig. 3  A and B and Dataset S3)-and Rqc1-TAP (Fig. 4A  and Dataset S4)-associated complexes. In addition, we examined the effect of the absence of Rqc1 in Tae2-TAP purifications and noticed a similar Cdc48 decrease (Fig. 3C  and Dataset S3 ). These data were strengthened by an independent experiment showing that the absence of Ltn1 or, to a somewhat lesser extent, Rqc1 efficiently reduced the amount of Cdc48 found in the 60S fractions of a polysome gradient (Fig.
S2 ). The amount of Cdc48 was also decreased to some extent from the Rqc1-TAP complex in the absence of Tae2 (Fig. 4B  and Dataset S4), an observation also corroborated by the diminution of the amount of Cdc48 sedimenting with the 60S in a polysome gradient when Tae2 is absent (Fig. S2 ). This decrease was paralleled by a similar diminution of Ltn1 (Fig. 4B ), suggesting that the decrease of Cdc48 might be indirect, resulting from the lower stability or recruitment of Ltn1 on the complex. In addition, we observed that the Ltn1-3HA fusion, which localizes with the 60S in a polysome gradient in a wild type, is shifted for a large part toward the lighter fractions in the absence of Tae2 (Fig. 4C ). This confirms the importance of Tae2 for the stable association of Ltn1 with the complex. In summary, the Cdc48-Ufd1-Npl4 complex requires both Ltn1 and Rqc1 to efficiently bind 60S particles, which suggests that ubiquitination by Ltn1 is essential for Cdc48 recruitment.
Materials and Methods Yeast Strains, Plasmids, and GIM Screens. The yeast strains used in this study are listed in Tables  S1  and S2. They were constructed by high-fidelity PCR transformation. Serial dilutions were spotted on rich medium plates with or without hygromycin B (40 μg/mL) and incubated for 2 d at 30 °C. Details of plasmid constructions and GIM screens (  16 ) can be found in SI Materials and Methods. Polysome Gradients and Western Blotting. Polysome gradients analyses were conducted as described in SI Materials and Methods. Collected fractions were precipitated and separated on a 10% (wt/vol) acrylamide gel. The proteins were detected by hybridization with the appropriate antibodies (Table  S3 ). Tandem Affinity Purifications Mass Spectrometry and Data Analysis. Biochemical purifications were performed using strains expressing TAP fusion proteins.
A first-step purification was done with magnetic beads coupled with IgG (SI Materials and Methods). An aliquot of the Tobacco Etch Virus protease (TEV) eluate was separated on acrylamide gels and silver stained. The rest of the eluates were analyzed by mass spectrometry. Briefly, after digestion, peptides were identified on an LTQ-Orbitrap and raw MS data were analyzed using the MaxQuant software as described in SI Materials and Methods.
ACKNOWLEDGMENTS. We thank  A. Van Hoof ,  A. Buchberger , and  G. Dieci  for providing plasmids and antibodies against Cdc48 and Rps8, respectively. We thank the proteomics platform of the  Pasteur Institute  for the availability of the Orbitrap. Q. D. and Y. Y. were supported by a fellowship from the  Ministère de l'Enseignement Supérieur et de la Recherche. This work was supported by grants from the  Agence Nationale de la Recherche  ( ANR ),  ANR-2011-BSV6-011-02  and  ANR-08-JCJC-0019-01.
Cloning, Expression, and Purification of AMADHs from Tomato and Maize-The total RNA from apical meristems and leaves of 7-day-old tomato seedlings (S. lycopersicum cv. Amateur) and 5-day-old maize seedlings (Z. mays cv. Sachara) was extracted using the Midi spin column kit (Macherey-Nagel, Du ¨ren, Germany), and the cDNA was synthesized using Superscript II reverse transcriptase (Invitrogen). SlAMADH1 and -2 and ZmAMADH1a, -1b, and -2 cDNA sequences were originally obtained in this work and submitted to the EMBL/ GenBank TM database. The SlAMADH1 ORF (1515 bp; EMBL/ GenBank TM accession number AY796114) was amplified using a primer pair with a BamHI (5Ј-CAGGGATCCGGCAAATC-GTAATGTACCA-3Ј) and XhoI site (5Ј-CGTCTCGAGCTA-ATTCTTTGAAGGTGACTTAT-3Ј). For the SlAMADH2 ORF (1518 bp; FJ228482), an upstream primer with the EcoRI site (5Ј-CATGAATTCGGCGATTCCTAATATACGGAT-3Ј) and a downstream primer with the KpnI site (5Ј-AGTGGTA-CCTTACAGCTTTGAAGGAGACT-3Ј) were used.
The enzyme reaction was started by the addition of APAL (or another aminoaldehyde) at a final 1 mM concentration. NAD ϩ analogs were assayed at 0. 5 mM concentration with 1 mM APAL as described previously  (4). Tris-HCl buffers (150 mM) in the pH range from 7. 0 to 9. 0 and 150 mM glycine-NaOH buffers in the pH range from 8. 8 to 10. 6 were used to determine pH optima using 1 mM APAL and 0. 5 mM NAD ϩ. Kinetic constants were determined with GraphPad Prism 5. 0 data analysis software (GraphPad Software, Inc. , La Jolla, CA). In those cases where no substrate inhibition was observed, the data were fitted to the Michaelis-Menten equation. The data were analyzed by a nonlinear regression using the Michaelis-Menten equation that includes terms in the numerator and denominator to account for partial substrate inhibition  (25) , V ϭ V max ⅐ [S]/͑K m ⅐ ͑1/͑1 ϩ ␤ ⅐ [S]/K i ͒͒ ϩ [S] ⅐ ͑͑1 ϩ [S]/K i ͒/ ͑1 ϩ ␤ ⅐ [S]/K i ͒͒͒ (Eq.
1) where v represents the determined initial velocity, V max is the maximal velocity, [S] is the concentration of the substrate, K m is the substrate concentration at half-maximal velocity, K i is the substrate inhibition constant, and ␤ is the factor that describes the effect of substrate inhibition on V max. Plant AMADHs share a similar pH optimum The pH optima of ZmAMADH1a, ZmAMADH2, and SlAMADH2 fall within the pH range of 9. 4 -9. 8 (i. e. similarly to that of pea or rice AMADHs)  (4, 6). The pH optima of SlAMADH1 and ZmAMADH1b are slightly more basic (pH ϳ10. 2). Crystallization and Data Collection-Crystallization conditions using commercial kits (Classics, PEGs II, and JCSG I suites from Qiagen) were screened in sitting-drop vapor diffusion experiments using a Cartesian nanodrop robot (Genomic Solutions) on IMAGIF (CNRS, Gif sur Yvette, France).
SlAMADH1 with 5 mM NAD ϩ was crystallized over reservoirs containing 23% PEG 1000, 0. 1 M HEPES, pH 7. 5. ZmAMADH1a with 5 mM NAD ϩ was crystallized over reservoirs containing 16% PEG 4000, 0. 1 M HEPES, pH 7. 5, and 10% isopropyl alcohol. The E260A mutant of SlAMADH1 with 5 mM NAD ϩ was crystallized over reservoirs containing 15% PEG 1500, 0. 1 M imidazole, pH 7. 0, and 10% glycerol. Crystals were transferred to a cryoprotectant solution (mother liquor supplemented with 25% PEG 400 for SlAMADH1 or 20% glycerol for the E260A mutant and ZmAMADH1a) and flash-frozen in liquid nitrogen. Diffraction data were collected at 100 K on the PROXIMA 1 beamline (SOLEIL Synchrotron, Saint-Aubin, France). Diffraction intensities were integrated with the program XDS  (26). Data collection and processing statistics are given in Table  2.
Molecular Replacement, Refinement, and Final Model-The structures of SlAMADH1 and of ZmAMADH1a were solved by molecular replacement with Phaser (27) using the dimer structure of PsAMADH2 (Protein Data Bank code 3IWJ) as a search model. The structure of the SlAMADH1-E260A mutant was also solved with Phaser using a monomer of the WT structure as a search model. Model refinement was performed with BUSTER-TNT  (28). Electron density maps were evaluated using COOT (  29 ) for manual refinement. In the SlAMADH1 structure, the maps revealed clear density for the ADP moiety of the NAD ϩ coenzyme. In contrast, the side chain of the active-site base Glu-260 is disordered. For this reason, we set the occupancy of each atom forming the carboxylate at 0. In the ZmAMADH1a and SlAMADH1-E260A structures, the electron density of the whole NAD ϩ molecule was observed.
8  (30) , and then the maximum likelihood phylogeny with bootstrap analysis was performed with PhyML version 3. 0 (31), using an LG amino acid replacement matrix. AMADH sequences were obtained from the EMBL/GenBank TM database, TGI database (tentative consensus numbers), or Phytozome version 8. 0 database: Amaranthus hypochondriacus 1 and 2 (AF017150/O04895); Avicennia marina 1/2 (mangrove, AF170094, and AB043540); Arabidopsis thaliana 1/2 (AY093071/AF370333); Beta vulgaris (sugar beet, AB221006); Brassica napus 1/2 (rapeseed, AY351634/ TC111063); Glycine max 1/2 (soybean, AB333793/AB333794); Gossypium hirsutum (cotton, AY461804); Hordeum vulgare 1/2 (barley, AB063179/AB063178); Lycium barbarum (wolfberry, FJ514799); Leymus chinensis 1/2 (wild rye, AB183715/ AB183716); Medicago truncatula 1/2 (barrel medic, TC151999/BT053176); Oryza sativa 1/2 (rice, AB001348/ AB096083); Physcomitrella patens (moss, EDQ78577); Picea sitchensis (spruce, BT070941); Pinus sylvestris (pine, TC133801); P.
sativum 1/2 (pea, AJ315852/AJ315853); Populus trichocarpa 1/2 (poplar, XM_002322111/DR448910); Prunus persica 1/2 (peach, PPA004563M/PPA022568M); S. lycopersicum 1/2 (tomato, this work); Solanum tuberosum 1/2 (potato, PGSC0003DMC400055759/PGSC0003DMC400042549); Sorghum bicolor (sorghum, XM_002444312); Spinacia oleracea (spinach, M31480); Triticum aestivum 1/2 (wheat, AY050316/ TC389888 ϩ AK332255); Z. mays 1a/1b/2 (maize, this work); Vitis vinifera 1/2 (grapevine, XM_002283654/XM_ 002281948); and Zoysia tenuifolia 1/2 (velvetgrass, AB161712/AB161702). Accession Numbers-The atomic coordinates and structure factors have been deposited in the Protein Data Bank with the following accession codes: 4I9B for SlAMADH1 in complex with NAD ϩ and a thiohemiacetal intermediate, 4I8Q for the Glu-260 mutant of SlAMADH1 in complex with NAD ϩ , and 4I8P for ZmAMADH1a in complex with NAD ϩ.
SlAMADH1 SlAMADH2 All K m and k cat values are given in M and s Ϫ1 , respectively, and k cat /K m ratios are expressed in M Ϫ1 s Ϫ1. Activities were measured in 0. 15 M Tris-HCl buffer, pH 9. 0. Specific activity values with 1 mM APAL were 2. 6 units mg Ϫ1 for ZmAMADH1a, 1. 9 units mg Ϫ1 for ZmAMADH1b, and 4. 1 units mg Ϫ1 for ZmAMADH2. Kinetic constants were measured using a saturating NAD ϩ concentration of 500 M. The K m value for NAD ϩ was measured at a fixed 1 mM concentration of APAL. The data were further analyzed by a nonlinear regression using either a simpler equation for substrate inhibition, v ϭ V max ⅐͓S͔/(K m ϩ ͓S͔⅐(1 ϩ ͓S͔/K i )), or an equation for partial substrate inhibition, v ϭ K m k cat k cat /K m K i ␤ K m k cat k cat /K m K i ␤ M s Ϫ1 M Ϫ1 s Ϫ1 M M s Ϫ1 M Ϫ1 s Ϫ1 M NAD ϩ 72 Ϯ 7 Ϫ Ϫ Ϫ Ϫ 89 Ϯ 3 Ϫ Ϫ Ϫ Ϫ TMABAL V max ⅐͓S͔/(K m ⅐(1/(1 ϩ ␤⅐͓S͔/K i )) ϩ ͓S͔⅐((1 ϩ ͓S͔/K i )/(1 ϩ ␤⅐͓S͔/K i ))) , where v is the determined initial velocity, V max is the maximal velocity, ͓S͔ is the concentration of the substrate, K m is the substrate concentration at half-maximal velocity, K i is the substrate inhibition constant, and ␤ is the factor that describes the effect of substrate inhibition on V max.
The presence of an aldehyde in PEG solution was also mentioned in an hALDH2 crystallographic study, where we believe that the soaked crotonaldehyde substrate forms an intermediate (Protein Data Bank code 1O01)  (15). In consequence, instead of a di(hydroxyethyl)ether molecule, we modeled a 2-(hydroxyethoxy)acetaldehyde molecule in two conformations in subunit B of the SlAMADH1 structure because there was no continuous electron density proving the absence of a covalent bond (Fig. 4C ). Indeed, the catalytic cysteine points toward the coenzyme cavity. However, we cannot rule out the presence of a carboxylate product because the NiR moiety of the coenzyme molecule is disordered in both subunits, indicating the presence of an NAD ϩ /NADH mixture in the crystals as previously shown in the hALDH2 study  (15).
Indeed, its side chain is shifted by 1. 6 Å (corresponding to a rotation of 23°a long the C␤-C␥ axis) away from Tyr-163, leading to an enlargement of the substrate channel favorable for binding of the bulky trimethylammonium group (Fig. 6D ). Ile-444 in PsAMADHs (Ile-445 in SlAMADH1) prevents Trp-459 from adopting the observed position in ZmAMADH1a due to a steric hindrance. Thus, the equivalent cysteine (Cys-446) in ZmAMADH1a, located in the second sphere of the substrate interaction, is responsible for the high activity with BAL, as shown in spinach AMADH/BADH (SoBADH; Protein Data Bank code 4A0M)  (48). Notably, the concerned tryptophan and cysteine residues adopt the same position in ZmAMADH1a and SoBADH structures. HvAMADH2, which also contains a cysteine, exhibits a good BAL activity  (18).
Interestingly, human ALDH9 (ALDH9A1, also called E3 isoenzyme) has been shown to prefer TMABAL (K m ϭ 1. 4 M) over ABAL (K m ϭ 8 -14 M) and BAL (K m ϭ 120 M)  (23). The low K m value for ABAL indicates that this enzyme is involved in GABA formation (reviewed in Ref. 53 ). So far, APAL and GBAL have not been tested with this enzyme. The other well studied ALDH9 from rat liver also shows a high preference for TMABAL. Fish ALDH9 from cod liver (Protein Data Bank codes 1A4S and 1BPW) (54) shows a higher k cat /K m ratio for BAL over aliphatic and aromatic aldehydes, and this is why the enzyme is called BADH  (55). To our knowledge, other -aminoaldehydes have not yet been tested, but the active site preserves aromatic residues responsible for AMADH activity, typical for the ALDH10 family, namely Tyr-167 (Tyr-163 in ALDH10), Trp-174 (Trp-170), and Phe-466 (Trp-459).
The enzymes from maize (ZmAMADHs) and tomato (SlAMADHs) analyzed in this work are shown in boldface type, as are the previously characterized reference enzymes from peas (PsAMADHs) (4), rice (OsAMADHs)  (6) , and barley (HvAMADHs)  (18). The internal labels give bootstrap frequencies for each clade. See "Experimental Procedures" for the corresponding GenBank TM accession numbers. Based on results of the entire study, we can now state that a tryptophan in PsAMADHs), which is found in more than three-quarters of AMADH sequences, together with the conserved cluster of Tyr-163, Trp-170, and Trp-459, is responsible for the high affinity to -aminoaldehydes with APAL as the best substrate. AMADHs devoid of this tryptophan prefer TMABAL either directly due to the increase in the substrate channel diameter (and consequent loss of possible -electron stacking interaction with the electrophilic protonated amino group of the substrate) or indirectly by affecting the position of the Tyr-163 side chain involved in substrate interaction.
Acknowledgments-We are grateful to  Beatriz Guimaraes  for help in data collection on PROXIMA 1 at the SOLEIL synchrotron and to  Dr. Jacques Sne  ´garoff ( Institut Jean-Pierre Bourgin ,  INRA Versailles ) and  Prof. Thomas D. Hurley  ( Indiana University School of Medicine ) for helpful discussion during manuscript preparation. This work benefited from IMAGIF platform facilities at the  Centre de Recherche de Gif-sur-Yvette  ( FRC3115 ,  CNRS, France ) for crystallization work at the  Structural Biology and Proteomic Unit in the Laboratoire d'Enzymologie et Biochimie Structurales.
* This work was supported by  Czech Science Foundation  Grants  P501/11/ 1591  and  522/08/0555  and  OP RD&I  Grant  ED0007/01/01  ( Centre of the Region Hanafor Biotechnological and Agricultural Research ) and supported in part by  CNRS  (to S. M. and A. V. ). The atomic coordinates and structure factors (codes  4I9B ,  4I8Q , and  4I8P ) have been deposited in the Protein Data Bank (http://wwpdb. org/). 1 These authors contributed equally to this work.
Source Common name Systematic name GenBank TM accession ALDH10 Z. mays ZmAMADH1a ALDH10A8 GQ184593 ZmAMADH1b ALDH10A9 JN635700 ZmAMADH2 ALDH10A5 GQ184594 S. lycopersicum SlAMADH1 ALDH10A12 AY796114 SlAMADH2 ALDH10A13 FJ228482.
Antibodies used The anti-MAFA antibody (ab26405, Abcam) and a panel of anti-MAFB antibodies were subjected to different tests to assess their specificity toward MAFA and MAFB proteins both in mouse and in human tissues. The anti-MAFA (ab26405, Abcam) and the noncommercial anti-MAFB2 (mouse monoclonal antibodies, clone 1F4  [12] ) antibodies were validated and used for the current study. Anti-glucagon (G2654, Sigma), the anti-insulin (A056401, DAKO), the anti-PDX1 (ab47383, Abcam) and anti-ARX (noncommercial,  [13] ), mMafB1 (anti mouse MafB, IHC-00351, Bethyl), and hMAFB3 (Anti human MAFB, HPA005653, Sigma) were also used in the study.
All nuclear co-localization experiments were performed and controlled to ensure that no non-specific crossreaction occurred. Images were captured and analyzed on a TCS-SP5 confocal microscope (Leica-Microsystems) or a Zeiss 780 confocal microscope. Cell counting was manually performed with ImageJ cell counter module (U. S. National Institutes of Health, Bethesda, Maryland, USA) on multiple channel pictures. Graphs and results of counting are represented as mean 6 S. E. M.
Acknowledgments We are grateful to  Denis Ressnikoff  ( CCQ, Lyon ),  Annabelle Bouchardon  and  Christophe Vanbelle  ( CeCILE, Lyon ) for expert assistance of confocal microscopy,  Jean-Michel Vicat  (Alecs SPF animal house) for the maintenance of the mouse colonies,  Dr Kanako Miybayashi  for anti-ARX antibody.