Ring chromosomes are unusual abnormalities that are observed in prenatal diagnosis. A 23-year-old patient  referred for amniocentesis due to abnormal maternal serum screening result in the 16th week of second pregnancy. Cytogenetic analysis of cultured amniyotic fluid cells revealed out ring chromosome 4. Both maternal and paternal karyotypes were normal. Terminal deletion was observed in both 4p and 4q arms of ring chromosome 4 by fluorescence in situ hybridization (FISH). However deletion was not observed in the WHS critical region of both normal and ring chromosome 4 by an additional FISH study. These results were confirmed by means of array-CGH showing terminal deletions on 4p16.3 (130 kb) and 4q35.2 (2.449 Mb). In the 21th week of pregnancy, no gross anomalia, except two weeks symmetric growth retardation, was present in the fetal ultrasonographic examination. According to our review of literature, this is the first prenatal case with 4p and 4q subtelomeric deletion of ring chromosome 4 without the involvement of WHS critical region. Our report describes the prenatal case with a ring chromosome 4 abnormality completely characterized by array-CGH which provided complementary data for genetic counseling of prenatal diagnosis. Autosomal ring chromosomes are uncommon cytogenetic aberrations in prenatal diagnosis. Estimated frequency ranges from 1/27000 to 1/62000 in consecutive newborn and prenatal diagnosis studies . Cases wIn the present study, our aim was to determine whether ring chromosome leads to abnormality in the following weeks of pregnancy or in the postnatal period of fetus which has no gross anomalia in the second trimester of pregnancy, except growth retardation, and the second aim was to manage genetic counselling to family members. For this purpose, we performed classical cytogenetic, molecular cytogenetic (FISH), and array-CGH techniques in amniocentesis and chordosynthesis samples to establish whether any deletion exists in ring chromosome 4 with breakpoints of deleted segments and identify genes involving deletions.A 23-years-old patient with a history of a pregnancy resulted with abortus referred for amniocentesis due to abnormal maternal serum screening test in the 16th week of the 2nd pregnancy. Down syndrome risk was 1,97 according to maternal serum screening test in the 12th week of pregnancy. Cytogenetic analysis of the cultured amniocytes using flask culture method revealed ring chromosome 4 ] . To confThe ring chromosome 4 was characterized by FISH using the 4p and 4q specific subtelomeric probe . FISH study showed deletions at the subtelomeric regions of 4p and 4q on the ring chromosome 4 . WHS criArray-CGH was performed to determine breaking points and involved genes on terminal deletions of ring chromosome 4.CytoSure microarray platform (Oxford Gene Technology) was used for aCGH analysis. This platform has approximately 44,000 oligonucleotide probes. The arrays were scanned on an Agilent G2505B scanner and quantified using Agilent's Feature Extraction software. Data was then normalised using CytoSure visualisation software which uses a standard LOWESS method . SubsequIn the 21th week of pregnancy, no gross anomalia, except 2 weeks symmetric growth retardation, existed in the fetal ultrasonographic examination. Parents were informed about the probable prenatal and postnatal complications of subtelomeric deletion of p and q arms of chromosome 4. However, parents denied to terminate pregnancy.In the present report, prenatal case with 4p16.3 and 4q35.2 subtelomeric deletion of ring chromosome 4 without the involvement of WHS critical region was diagnosed.Ring chromosome 4 is an uncommon cytogenetic abnormality that has been rarely diagnosed in the prenatal stage. Up to now, three cases with prenatal diagnosis of ring chromosome 4 were reported in the English literature. In the first case, Sherer et al. reported a severely and symmetrically growth-retarded fetus with microcephaly, hypertelorism, and hypoplastic genitalia with a two-vessel umbilical cord, a characteristic “Greek warrior helmet” facial profile and clubfoot in a 29th week fetus of ring chromosome 4 carrier with 4p15 and 4q35 deletions . In the Among published cases with 4p16 deletions, South et al. reported a case of ring 4 with 1.27–1.46 Mb deletion at 4p16.3 presented with significant postnatal growth retardation, mild developmental retardation, and nutritional disturbances. In addition to abnormalities of the first case, the second reported that a case exhibited terminal 4p microdeletion of approximately 1.78 Mb caused complex seizure disorder. No deletion was observed at WHS region in both cases . KhonsarIn the present case, genes that encode 6 zinc finger protein exist on deleted segment of 130 kb at 4p16.3 as shown in According to our knowledge, this is the first de novo ring chromosome 4 case with 4p and 4q subtelomeric deletion without deletion of WHS critical region and that had no fetal anomalia except intrauterine growth retardation determined by second trimester fetal ultrasonographic examination. In the light of literature, genetic counselling was performed to family members by considering genes on deleted segments. The role of FISH and array-CGH on genetic diagnosis and phenotypic correlation in the prenatal evaluation is confirmed.


In C4 plants CO2 is supplied to Rubisco by an auxiliary CO2-concentrating pathway that helps to maximize the carboxylase activity of the enzyme while suppressing its oxygenase activity. As a consequence, C4 Rubisco exhibits a higher maximum velocity but lower substrate specificity compared with the C3 enzyme. Specific amino-acids in Rubisco are associated with C4 photosynthesis in monocots, but it is not known whether selection has acted on Rubisco in a similar way in eudicots.Rubisco  catalyses the key reaction in the photosynthetic assimilation of COsensu lato (including Chenopodiaceae), the third-largest family of C4 plants, using phylogeny-based maximum likelihood and Bayesian methods to detect Darwinian selection on the chloroplast rbcL gene in a sample of 179 species. Two Rubisco residues, 281 and 309, were found to be under positive selection in C4 Amaranthaceae with multiple parallel replacements of alanine by serine at position 281 and methionine by isoleucine at position 309. Remarkably, both amino-acids have been detected in other C4 plant groups, such as C4 monocots, illustrating a striking parallelism in molecular evolution.We investigated Rubisco evolution in Amaranthaceae Our findings illustrate how simple genetic changes can contribute to the evolution of photosynthesis and strengthen the hypothesis that parallel amino-acid replacements are associated with adaptive changes in Rubisco. Observations of significant variation in Rubisco kinetics between plant species 2 and O2 as substrates, and under present-day atmospheric conditions (385 p.p.m. CO2), the carboxylase activity of Rubisco is undersaturated in C3 plants, and the oxygenase activity gives rise directly to the competing process of photorespiration. Photorespiratory rates in C3 plants increase steeply with increasing temperature and give rise to a distinct temperature optimum for net photosynthesis, above which plant yields decline steeply. Increased carbon loss via photorespiration at higher temperatures is attributable mainly to the declining specificity of Rubisco for CO2 relative to O2 (Sc/o). In fact, it has been proposed that the very slow turnover of Rubisco (kcat ≈3 s−1) is a direct consequence of the enzyme's particular reaction mechanism, in which Sc/o is maximized by tight binding of the transition-state intermediate kcat and of the large size of the holoenzyme (560 kDa) is that Rubisco comprises up to 50% of soluble protein in photosynthetic tissues and is probably the most abundant enzyme on Earth Rubisco discriminates imperfectly between CO4 photosynthesis or crassulacean acid metabolism (CAM), and in many aquatic organisms, photorespiration is partially or completely suppressed by the operation of an auxiliary CO2-concentrating mechanism. C4 plants initially fix atmospheric carbon in the mesophyll cells using phosphoenolpyruvate carboxylase, an enzyme with a high effective affinity for CO2 (HCO3− being the true substrate of the enzyme). Further four-carbon compounds  produced by this fixation are transported to the specialized bundle-sheath cells, where CO2 is released and fixed by Rubisco. Rubisco from C4 plants, which experiences ∼10-fold higher CO2 concentrations in bundle-sheath cells than does the enzyme in C3 plants 2 but a higher kcat (≈4 s−1). Having less specific but faster Rubisco and no photorespiration losses, C4 plants require 60 to 75% less Rubisco to match the photosynthetic capacity of C3 plants 4 plants such as maize, sugarcane and sorghum are among the most productive of all species cultivated agriculturally. Although C4 plants appeared relatively recently in evolutionary terms and constitute only 3% of terrestrial plant species, they are already among the most successful and abundant groups in warm climates and are responsible for about 20% of terrestrial gross primary productivity In terrestrial plants with C4 photosynthesis evolved independently in at least 62 recognizable lineages of angiosperms and represents one of the most striking examples of a convergent biochemical adaptation in plants 4 monocots in the Poaceae, while C4 eudicots have been studied less intensively. The family Amaranthaceae sensu lato (i.e. including Chenopodiaceae) 4 species 4 family among eudicots and the third-largest among angiosperms (after Poaceae and Cyperaceae). C4 photosynthesis evolved at least 15 times within Amaranthaceae 4 photosynthesis and Rubisco. Notably, the Amaranthaceae exceed the Poaceae and Cyperaceae in the diversity of photosynthetic organ anatomy 4 plants that lack Kranz anatomy, with three species having a single-cell rather than the more usual dual-cell C4 system sensu stricto and primarily temperate and subtropical Chenopodiaceae have long been treated as two closely related families (see review in sensu lato (henceforth referred to as Amaranthaceae) constitutes the most diverse lineage of the Caryophyllales. Both C3 and C4 species from this family are adapted to a range of conditions from temperate meadows to the tropics, hot deserts and salt marshes. However, it has been shown that the abundance of C4 Amaranthaceae is correlated with precipitation but not temperature, in contrast to the abundance of C4 Poaceae and Cyperaceae, which is correlated with temperature but not precipitation C4 Amaranthaceae showing different suites of anatomical and biochemical adaptations as well as ecological preferences compared to C4 Poaceae and Cyperaceae, like C4 monocots they possess faster but less CO2-specific Rubiscos than their C3 relatives 4 eudicots and monocots represents a notable example of convergent evolution of enzyme properties in phylogenetically distant groups. However, it is not known whether this functional convergence in Rubisco kinetics evolved via similar or different structural changes in protein dN) and synonymous  mutations along a phylogenetic tree using maximum likelihood and Bayesian frameworks rbcL, which encodes the large subunit of Rubisco that forms the enzyme's active site, and showed that positive Darwinian selection is acting within most lineages of plants 4 lineages of Poaceae and Cyperaceae Flaveria3 and C4 species. However, no specific analysis has yet been made of Rubisco sequence evolution in a large group of C4 eudicots. In this study, we investigate positive selection on the rbcL gene of plants from the Amaranthaceae family and, in particular, focus on coevolution of Rubisco and C4 photosynthesis asking whether positive selection on the rbcL gene occured on branches leading to C4 clades and/or within C4 clades. Finally, we address the following question: which amino-acid replacements were associated with transitions from C3 to C4 photosynthesis in Amaranthaceae, and are these replacements unique to this lineage or shared with C4 monocots and/or Flaveria?Despite CrbcL nucleotide sequences available in GenBank and aligned them. Sequences shorter than 1341 base pairs and sequences with missing data were excluded. The resulting trimmed alignment consisted of 179 rbcL sequences of 1341 base pairs long which represented 94% of the rbcL coding region and corresponded to positions 64 to 1404 of the rbcL sequence of Spinacia oleracea (GenBank AJ400848). The analysed dataset consisted of 95 C3 and 84 C4 species  and synonymous (dS) mutations along a phylogenetic tree dN/dS = 1), while purifying (negative) or adaptive (positive) selection is expected to deflate (dN/dS<1) or inflate (dN/dS>1) this ratio, respectively. One can use likelihood ratio tests to detect positive selection that affects only a subset of codons in a protein-coding gene, with positive selection indicated by accelerated nonsynonymous substitutions. Models assuming positive selection along all phylogeny or prespecified branches only (e.g. C4 lineages in our case) can be employed within Phylogenetic Analysis by Maximum Likelihood (PAML) framework Positive, neutral, or purifying selection at the molecular level can be inferred by comparing rates of non-synonymous . Further, codeml was used to perform likelihood ratio tests (LRTs) for positive selection among amino acid sites. The tree length value obtained from the model M0 was compared with tree length values obtained from other models to control for consistency among models. We performed two LRTs to compare null models which assume the same selective pressure along all branches of a phylogeny and do not allow positive selection (dN/dS >1) with nested models which do allow it dN/dS ≤1 with the M2a model . The second LRT, M8a-M8, compares the M8a model which assumes a discrete beta distribution for dN/dS, which is constrained between 0 and 1 including a class with dN/dS  = 1 with the M8 model which allows the same distribution as M8a but an extra class under positive selection with dN/dS >1.We used the 4 branches only where positive selection was allowed only on branches leading to C4 clades. The second was the A model for all C4 branches where positive selection was allowed on branches leading to C4 clades and branches within C4 clades. The A1-A LRT compares the null model A1 with the nested model A. Both the A1 and A models allow dN/dS ratios to vary among sites and among lineages. The A1 model allows 0< dN/dS <1 and dN/dS  = 1 for all branches, and also two additional classes of codons with fixed dN/dS  = 1 along prespecified foreground branches while restricted as 0< dN/dS <1 and dN/dS  = 1 on background branches. The alternative A model allows 0< dN/dS <1 and dN/dS  = 1 for all branches, and also two additional classes of codons under positive selection with dN/dS >1 along prespecified foreground branches while restricted as 0< dN/dS <1 and dN/dS  = 1 on background branches. C4 lineages were marked as foreground branches.Finally, we performed two branch-site tests of positive selection along prespecified foreground branches dN/dS >1, whereas the M2a, M8 and A models are alternative models which do allow codons with dN/dS >1. The significance of the LRTs was calculated assuming that twice the difference in the log of maximum likelihood between the two models was distributed as a chi-square distribution with the degrees of freedom (df) given by the difference in the numbers of parameters in the two nested models dN/dS values (0.1 and 0.4) to test for suboptimal local peaks. To identify amino acid sites potentially under positive selection, the parameter estimates from M2a, M8 and A models were used to calculate the posterior probabilities that an amino acid belongs to a class with dN/dS >1 using the Bayes Empirical Bayes (BEB) approaches implemented in PAML codeml we used the SLR program which implements “sitewise likelihood-ratio” (SLR) method for detecting non-neutral evolution, a statistical test that can identify sites under positive selection even when the strength of selection is low dN/dS  = 1) against an alternative model (dN/dS ≠1). SLR method is a test of whether a given site has undergone selection or not, and the test statistic summarizes the strength of the evidence for selection rather than the strength of the selection itself codeml and SLR.For all LRTs, the first model is a simplified version of the second, with fewer parameters, and is thus expected to provide a poorer fit to the data (lower maximum likelihood). The M1a, M8a and A1 models are null models which do not allow codons with 4 photosynthesis and (2) the presence/absence of particular amino-acid at sites found to be under positive selection along C4 branches in the A model of codeml. For this purpose, we used the phylogeny obtained using RAxML (see above) and performed Pagel's test of correlated (discrete) character evolution 4 branches and Bonferroni correction was performed for simultaneous statistical testing.Closely related taxa are not independent data points and they consequently violate the assumptions of conventional statistical methods Spinacia oleracea, Amaranthaceae) from data file 1RBO We used the published Rubisco protein structure from spinach  to test for suboptimal local peaks produced identical results. LRTs for positive selection p-value <0.00001; 4 clades we used two branch site models  for variation in 4 clades while under relaxed or purifying selection within C3 clades with a posterior probability >0.99 by BEB in the A model for C4 branches. Both sites had only two alternative amino acids in this dataset  between the presence of C4 photosynthesis and the presence of ‘C4’ amino acids at sites 281 and 309, shown to be under positive selection along C4 branches.Four sites were identified as evolving under positive selection with a posterior probability >0.95 by BEB  dataset . One of  species , but therbcL showed that positive selection is widespread among all main lineages of land plants, but is restricted to a relatively small number of Rubisco amino acid residues within functionally important sites rbcL is under positive selection in particular taxonomic groups As the performance of Rubisco can directly affect plant growth and crop yields, substantial efforts have been made to study its structure and function, with the ultimate aim of trying to improve Rubisco performance 4 photosynthesis has increased the availability of CO2 for Rubisco in numerous independently evolved lineages of C4 plants, including Amaranthaceae, driving selection for less specific but faster enzymes which have both higher KM(CO2) and kcat values 4 branches provided a significantly better fit to the analysed Amaranthaceae dataset than the null model without selection  and hence allows detection only of the most typical substitutions, potentially missing ones that are unique for a particular branch. Other possible explanations are variation in Rubisco kinetic properties not only between C3 and C4 groups of species but also within these groups 3 and C4 plants rbcS genes encoding small subunits have been shown under positive selection in C4FlaveriaInterestingly, both selected residues in C4 Amaranthaceae and C4 Cyperaceae and Poaceae, representing eudicots and monocots with significantly different anatomy and ecological preferences 3 plants are considered as well. Various groups of C3 plants such as some aquatic species and C3 species from cold habitats have faster but less CO2-specific Rubisco compared with their C3 relatives from terrestrial and warm conditions, respectively 3 plants can arrive at the same evolutionary solutions for Rubisco fine-tuning as C4 plants. Indeed, ‘C4’ amino acids shown for C4 Amaranthaceae in the present study and for C4 monocots and Flaveria previously 3 plants by Kapralov and Filatov 4’ amino acids, 281S and 309I, evolved in parallel in various phylogenetically distant lineages of C3 and C4 plants in which faster but less specific Rubisco was needed.Identical amino-acids in Rubisco of CThe residue 309 is located on the interface of large subunits within a large subunit dimer, while the residue 281 is involved into dimer-dimer interactions . MethionFlaveria changed Rubisco kinetics from “C3-like” to “C4-like” making the enzyme faster but less CO2-specific. Importance of M309I replacement for changes in kinetics of Flaveria Rubisco was predicted using in silico approach similar to one used in the present study in planta by the study of Whitney et al. Effects of A281S replacement on kinetics of land plants Rubisco has not been studied, while recent study by Whitney et al. Continuing population growth creating increasing demand for food, coupled with future climate change and its potentially dire consequences such as biome collapse and crop failure, both call for an improved understanding of mechanisms allowing plant species to adapt the photosynthetic process to a wide range of conditions. Hence, there is a necessity for more phylogeny-based studies of genes encoding Rubisco from various lineages of phototrophs established in different conditions to better understand Rubisco evolution at the molecular level. The integration of phylogenetic and biochemical research is required to study how Darwinian selection has created a range of enzymes with different kinetic and physical properties tailored to function in virtually all ecosystems on our planet. Knowledge of the role of specific residues in Rubisco adaptation to the particular conditions may provide clues for engineering better enzymes suited to contemporary agricultural needs as well as helping to understand what modifications in the enzyme may have been (and perhaps will be) driven by adaptation to different environmental conditions.Table S1List of studied species.(XLSX)Click here for additional data file.


Mesenchymal stem cells (MSCs) are able to reduce systemic inflammatory response in an experimental sepsis. One of the limiting factors of MSC therapy is a high degree of apoptosis of transplanted cells. On the basis of recently detected receptors for erythropoietin (EPO) on the surface of MSCs we hypothesized that introduction of EPO together with MSCs may increase survival of grafted MSCs and improve the clinical efficacy of cell transplantation.5 allogeneic MSCs (Group 3), 8.5 μg recombinant EPO-β (Group 4), MSCs and EPO in the same doses (Group 5). Surviving animals were euthanized on the fourth day. The morphological study, white blood cell count (WBC) and serum level of IL-1β, IL-2, IL-6, and TNFα measurement were performed.Fifty Wistar male rats were randomized into five groups with 10 animals in each: Group 1 were healthy controls, Groups 2 to 5 were intraperitoneally introduced bacterial LPS 20 mg/kg. Two hours later LPS injection animals received the following intravenous treatment: 4 × 10The highest WBC was found in the group of combined treatment EPO + MSCs. The serum IL-1β level in Groups 2 and 4 was significantly higher than in healthy and treated with MSCs and EPO + MSCs animals. The main results of the morphologic analysis of different tissues in the groups are presented in Table Combined treatment with EPO and MSCs can reduce acute lung injury and kidney damage, cause hyperplasia of lymphoid tissue and enhance the immune response more than separate treatment in an experimental model of endotoxemia in rats.


In vertebrates, poly(A) binding protein (PABP) is known to exist in five different isoforms. PABPs are primarily cytosolic with the exception of the nuclear PABP (PABPN1), which is located in the nucleus. Within the nucleus, PABPN1 is believed to bind to the poly(A) tail of nascent mRNA and along with cleavage and polyadenylation specificity factor (CPSF) define the length of the newly synthesized poly(A) tail.The cellular role of PABP1 has been extensively studied over the years; however, the function of other PABPs remains poorly defined. In order to understand the role of PABPN1 in cellular mRNA metabolism and it’s interrelation with other PABPs, we depleted PABPN1 using RNAi in HeLa and HEK293 cells. Our results show that PABPN1 depletion did not have any effect on the poly(A) tail length, nuclear export of mRNA, mRNA translation, and transcription. Rather, PABPN1 depletion resulted in a compensatory response as observed by increased level of PABP5 and nuclear accumulation of PABP4. In addition, PABP4 was associated with the poly(A) tract of pre-mRNA and CPSF in PABPN1 depleted cells. Nevertheless, PABPN1 depletion significantly affected cell survival as evidenced by an increase in apoptosis markers: phosphorylated p53 and PUMA and as judged by the expression of ER stress marker GRP78.Our results suggest that although function of PABPN1 may be compensated by nuclear translocation of PABP4 and perhaps by increase in the cytoplasmic abundance of PABP5, these were not sufficient to prevent apoptosis of cells. Thus PABPN1 may have a novel anti apoptotic role in mammalian cells. Mammalian nuclear poly(A) binding protein (PABPN1) is a highly conserved nuclear RNA binding protein with specificity towards the poly(A) tract of eukaryotic mRNAs. It consists of one typical RNA recognition motifs (RRM) domain with consensus RNP1 and RNP2 motifs in the central region of the polypeptide, and an arginine rich C- terminal domain Results of biochemical studies suggest that the main cellular function of PABPN1 is to stimulate the elongation of poly(A) tract of eukaryotic mRNA, and control its length Xenopus laevis suggests that both PABP1 and PABP4 are required for their normal development In contrast to the presence of only one nuclear poly(A) binding protein, vertebrates express several cytoplasmic PABPs with conserved RNA binding motifs Deletion of yeast pabp2 gene, which is the homolog of mammalian PABPN1, leads to extension of poly(A) tract of mRNAs The Stealth RNAi sequence used for silencing PABPN1 expression was the same as described earlier 2. Approximately 30% confluent cells, grown on 35 mm dishes were transfected with double stranded si RNA using lipofectamine2000™  according to manufacturer’s instructions. In short, for each 35 mm plate 1 µl of siRNA  was mixed with 5 µl lipofectamine2000™ and 500 µl of Opti-MEM  medium and incubated for 20 min before being added to the culture dish. After 24 h, the medium containing the RNA-lipofectamine complex was replaced by fresh DMEM containing 10% FBS and 1% glutamine. For co-transfection of the cells, 1 µg of plasmid was mixed with siRNA, and transfection was performed as described above. Cells were harvested between 45–80 h after transfection depending on the experiment.HeLa and HEK293 cells were grown in Dulbecco’s Modified Eagle’s medium (DMEM) containing 1% glutamine  and 10% FBS  at 37°C in presence of 5% CO2HPO4, 2H2O, 1.47 mM KH2PO4, pH 7.4). 200 µl of cytoplasmic lysis buffer  was added to the plate and incubated for 10–15 min on ice. Cells were scrapped off and the cell suspension was passed through a 30 gauge needle until 90% cells were lysed as judged by the presence of cytoplasmic tag free nuclei. Cell lysates were centrifuged at 2500 g for 7 min to obtain the cytoplasmic fraction. The pellet (nuclear fraction) was washed 3 times with the lysis buffer and lysed in (a) 150 µl of Laemmli buffer , 10% (v/v) glycerol, 5% (v/v) β-mercaptoethanol, 0.01% (w/v) bromophenol blue) for protein extraction or (b) in 1 ml of Trizol  for RNA extraction.For isolation of nuclear proteins, cells grown on a 35 mm plate were washed three times with PBS  according to manufacturer’s instructions. The quality and quantity of the RNA were determined by 1.5% agarose gel electrophoresis and spectrophotometric measurements respectively. The levels of specific mRNAs were determined by RT-PCR. An aliquot of total RNA (100–500 ng) was reverse transcribed using High Capacity cDNA transcription kit . After the reaction, 2 µL of the cDNA sample was amplified by PCR in a total master mix  reaction volume of 50 µL, which included 100 ng of primers specific for diffeent mRNAs  2, 1 mM EGTA, 100 mM NaPPi, 100 mM NaF, and 1% Triton X-100) by gentle sonication using XL2020 Sonicator (Mandel Scientific) at setting 1, pulse 3 (4 s each) to break the cytoplasmic membrane. Cells were then centrifuged at 2500 g for 10 min to separate the nuclear fraction (pellet). The pellet was resuspended in PLC buffer with 1% formaldehyde (Electron Microscopy Sciences) for 10 min at room temperature (RT) to crosslink the RNA and proteins of RNP complexes. The nuclear pellet was then incubated in 0.25 M of glycine, centrifuged and washed three times with PLC buffer. The nuclear pellet was finally resuspended in 400 µl of chilled PLC buffer supplemented with protease inhibitor (Roche) and again sonicated at a higher setting . Cell debris was removed by centrifugation at 12000 rpm for 10 min. The nuclear extract was incubated overnight with PABPC4 or PABPN1 antibody and rabbit or goat IgG-Agarose beads (Sigma). Following Immunoprecipitation, beads were washed extensively with PLC buffer and the RNA-protein cross-link was reversed by incubation at 70°C for 45 min in 100 µL of elution buffer . RNA was then extracted with three volumes of a mixture of phenol/chloroform (50/50). The RNA was precipitated using one volume of ethanol, and left overnight at −20°C. The precipitated RNA was collected by centrifugation at 10,000 g for 10 min at 4°C and resuspended in RNAse free water. Contaminating DNA was removed from RNA samples by RQ1 RNase-free DNAse (Promega) treatment prior to being reverse transcribed and PCR amplified using β-actin pre-mRNA primers Immunoprecipitation was done as previously described For immunoprecipitation studies nuclear extracts were prepared as described above and incubated with the designated antibody and appropriate IgG agarose beads for 16 h at 4°C. The eluted samples and nuclear extracts were subjected to SDS-PAGE and Western blotting.12–18 in a reaction containing 0.1 M DTT, 10 mM dNTP, 10 mM ATP, SuperscriptH− buffer, and 10 U/µl T4 DNA ligase (New England Biolabs). The resultant mixture was hybridized with oligo dT anchor (5′GCGAGCTCCGCGGCCGCGT12 3′) followed by reverse transcription with RNAseH− reverse transcriptase (Invitrogen). The cDNA was then PCR amplified with oligo dT primer and β-actin specific primer (CAC ACA GGG GAG GTG ATA GCA).PAT assay used to determine the poly(A) tail length was done as described previously by Salles and Strikeland 1999 35S]-methionine. For this, the cells were pre-incubated in methionine free DMEM medium  with 10% dialyzed FBS  for 2 h at 37°C to reduce the pool of endogenous methionine. Samples were incubated with 50 µCi [35S]-Methionine  in 1 ml of fresh methionine free DMEM medium with 10% dialyzed FBS  at 37°C for 30 min. Following incubation, cells were lysed with 200 µL of Lamelli buffer. The samples were boiled for 5 min and separated by SDS-10% polyacrylamide gel electrophoresis (PAGE). The gel was fixed in 10% trichloroacetic acid (TCA), at 95°C and allowed to cool down for 5 min at RT. The gel was then washed twice with 10% TCA at 20°C, and twice with 10% ethanol and finally with 100% ethanol. The gel was then soaked in 1 M Na-Salicylate for 30 min and dried at 50°C for one hour, and autofluorographed with an X-ray film for the desired time before being developed.Protein synthesis was measured by radioactively labelling cellular proteins with [35S] isotope.10 µL of each sample was spotted on small strips of filter paper. The filter paper was then placed in 100 mL of 10% TCA, and brought to boil and allowed to cool for 10 min; this process was then repeated three times. The filters were washed twice with 10% TCA at RT followed by 100% ethanol. The filter papers were placed in scintillation vials with sufficient amount of liquid scintillation counting fluid and counted using a liquid scintillation counter at a setting for -Methionine into polypeptides. Results of analyses of protein synthesis by SDS-PAGE followed by auto fluorography of equivalent amount proteins from NT, UTR and Si-PABPN1 transfected cells showed that there was no detectable difference in protein synthesis amongst these samples  tract addition (8) we tested whether the nuclear PABP4 in PABPN1 depleted cells also interacts with CPSF. The results of co-immunoprecipitation studies using PABP4 antibody show that CPSF1 was immunoprecipitated by PABP4 antibody from the nuclear extract of PABPN1-Si treated cells but not from the same of NT cells  G. As poExamination of the presence of apoptotic cells by AO/EtBr and DAPI staining revealed that significantly more cells were apoptotic in the cultures following PABPN1-Si treatment than what was observed for the NT and PABPN1-UTR treated cells. After 72 hours almost 40% of PABPN1 depleted cells were undergoing apoptosis while negligible amount of cells in our control cultures were apoptotic . In addiFurthermore, we measured the abundance of additional markers of cell stress, such as, the constitutive HSC70 and the inducible HSP70 by western blotting with an antibody that recognizes both proteins. The result show that HSC/HSP70 abundance did not increase following PABPN1 depletion. Therefore, PABPN1 depletion produced a distinct ER stress response. In our studies the abundance of eIF2-α and GAPDH remained unchanged by PABPN1 depletion suggesting that the increase of phospho p53, Puma and GRP78 was not due to non specific effect of PABPN1 depletion on protein abundance. Taken collectively results of our studies suggest that in spite of the effort of PABP4 and PABP5 to rescue cells from the adverse effect of PABPN1 depletion, it was not sufficient to prevent cell death. Perhaps PABPN1 plays a more critical anti apoptotic function than its previously believed role in poly(A) tail elongation.We tested two different target sequence of PABPN1 mRNA for SiRNA mediated ablation of PABPN1 expression. The first target was within the coding region of mRNA and the second target sequence was in the 5′UTR. Our studies demonstrated that the coding region was an effective target and the 5′UTR was probably not accessible for base pairing with the SiRNA due to presence of extensive secondary structures, therefore, did not produce any effect on PABPN1 expression. Examination of various parameters of cellular mRNA metabolism demonstrated that there were no detectable effects of PABPN1 depletion on mRNA export, mRNA abundance, and mRNA translation. In addition, cellular transcription as judged by the presence of pre-mRNA transcript of β-actin and localization of splicing factor1 in nuclear speckles was unaffected by PABPN1 depletion. Although it is well accepted that the central role of PABPN1 is to stimulate addition of poly(A) tract we did not observe any shortening of the length of the poly(A) tract. We examined the poly(A) tract length for a specific mRNA such as the β-actin mRNA, and found that the average poly(A) tract length was approximately 150–200 nucleotides for both control and PABPN1 depleted cells.We have demonstrated that amongst the cytoplasmic PABPs only PABP5 showed a significant increase in abundance in PABPN1 depleted cells. The cellular level of other cytoplasmic PABPs including PABP3 and PABP4 remained unchanged. The significance of the increase of PABP5 abundance is not clear at this time. Since PABP5 is also capable of interacting with eIF4G albeit with a lower efficiency than PABP1 (results not shown), perhaps the increase in PABP5 abundance stimulates protein synthesis in PABPN1 depleted cells as cellular homeostasis. Interestingly, a small but reproducible increase of global protein synthesis was observed following PABPN1 depletion. Beside its ability to bind poly(A), little is known regarding the cellular function of PABP5. It is possible that PABP5 is involved in regulating the metabolism of a specific sub-family of mRNAs (18). As such, it will be important to examine whether translation and/or stability of mRNAs encoding regulators of apoptosis can be modulated by PABP5.Although the abundance of PABP4 did not change in PABPN1 depleted cells, an increase in its nuclear localization was evident following depletion of cellular PABPN1. It was previously shown that UV damage results in nuclear translocation of both PABP1 and PABP4 In spite of the cellular homeostasis by increasing PABP5 abundance and nuclear translocation of a related poly(A) binding protein almost 40% PABPN1 depleted cells were apoptotic within 72 hours. Recent evidences show that beside poly(A) addition PABPN1 has other vital functions in RNA metabolism , such as, processing of snoRNAs (20) and selection of cleavage and polyadenylation site (21). It was shown recently that depletion of PABPN1 in mammalian cells leads to a switch to the use of the proximal polyadenylation site for a large number of cellular mRNAs resulting in transcripts with shorter 3′ untranslated region (UTR). Shortening of the 3′ UTR results in the loss of micro RNA target sites, therefore, PABPN1 is important for 3′UTR mediated repression of gene expression (21). It is likely that PABP4 only compensates some but not all the cellular functions of PABPN1.PUMA gene We showed here that in response to PABPN1 depletion p53 was phosphorylated in serine 46 which is known to activate p53 for inducing expression of PUMA. We demonstrated PUMA expression is activated in PABPN1 depleted cells. Phosphorylation alters the structural conformation of p53 leading to its stabilization and activation Figure S1Dose response of PCR reaction for different mRNAs. 250 ng of total RNA from non-transfected (NT) Hela cells was reverse transcribed as described in (TIF)Click here for additional data file.


Understanding the dynamics of influenza transmission on international flights is necessary for prioritizing public health response to pandemic incursions. A retrospective cohort study to ascertain in-flight transmission of pandemic (H1N1) 2009 and influenza-like illness (ILI) was undertaken for 2 long-haul flights entering Australia during May 2009. Combined results, including survey responses from 319 (43%) of 738 passengers, showed that 13 (2%) had an ILI in flight and an ILI developed in 32 (5%) passengers during the first week post arrival. Passengers were at 3.6% increased risk of contracting pandemic (H1N1) 2009 if they sat in the same row as or within 2 rows of persons who were symptomatic preflight. A closer exposed zone  increased the risk for postflight disease to 7.7%. Efficiency of contact tracing without compromising the effectiveness of the public health intervention might be improved by limiting the exposed zone. The emergence of pandemic influenza A (H1N1) 2009 in Mexico and the United States, with rapid spread to Europe, Asia, and the Pacific, is testament to the ease of spread of infectious disease across the globe (–Mycobacterium tuberculosis (–Reports documenting spread of disease during airline flight are limited (A retrospective cohort study designed to determine exposure risk to known pandemic (H1N1) 2009 virus was undertaken for 2 long-haul flights that entered Australia the weekend of May 23–24, 2009. Flight 1 was chosen after identification of 6 passengers with confirmed pandemic (H1N1) 2009 infection within 24 hours after flight arrival from the United States. Flight 2 was chosen after identification of a confirmed case of pandemic (H1N1) 2009. This flight came from an area that lacked community transmission. Passenger details were obtained through collection of Health Declaration Cards and comparing the cards to flight manifests obtained from the airlines.>1 symptom  within 7–14 days before the flight or during the flight or <7 days after arrival. The time periods were put in place to help determine when passengers were most likely to have contracted their ILI. Passengers were excluded who indicated bacterial infection  or regular health issues, such as migraines. All but 4 passengers reporting symptoms had confirmation of ILI status from a qualified health professional.The definition of ILI was broad to capture as many persons as possible within the dataset. Passengers were asked to self-report development of any of the following signs or symptoms: fever, cough, sore throat, headache, runny nose, muscle aches, diarrhea, and lethargy. ILI was defined as Self-identification of other health conditions that were considered potential concurrent conditions for purpose of this study included obesity, diabetes mellitus, immunosuppression, asthma, chronic lung disease, and pregnancy. Seat location, concurrent condition status, and contraction of disease were compared. Ethics approval was given by the Australian Government Department of Health and Ageing Ethics Committee and the Australian National University Human Research Ethics Committee.Surveys were distributed to passengers 3 months after flight arrival. The survey asked about influenza-like symptoms, symptom onset time, concurrent conditions, antiviral prophylaxis and treatment, isolation or quarantine dates, other potential exposure to ILI before and after the flight, contact with health professionals after the flight, and details of testing for pandemic (H1N1) 2009 virus. Two reminders were sent to improve the response rate of the study.As a triangulation method, all passenger names, passenger sex, disease onset dates, and postal codes were cross-checked against those of passengers with known pandemic (H1N1) 2009 cases that were notified to national authorities for 1 month after flight arrival to verify information received from the survey responses and identify additional cases. Travel details were verified for pandemic (H1N1) 2009 case-patients identified through national notification. Contact tracing through public health authorities also identified ILI case-patients who had negative laboratory test results for pandemic (H1N1) 2009 virus.The increased risk of passengers contracting either laboratory-confirmed pandemic (H1N1) 2009 or an ILI (including pandemic [H1N1] 2009) was separately estimated by dividing the number of persons with the illness by the number of susceptible persons in the contact zones as described. For pandemic (H1N1) 2009, only passengers sitting in the economy class were considered exposed because of the location of persons displaying symptoms preflight or during the flight and the sectional layout of the aircraft.Of the 738 passengers on the 2 flights, 319 (43%) responded to a questionnaire; 143 (18%) passengers could not be contacted. Cross-checking of mandatory notifications of pandemic (H1N1) 2009 against all passengers on both flights found 2 additional pandemic (H1N1) 2009 infection cases while also confirming symptom data on survey responses. Contact tracing by public health authorities found 5 additional passengers with ILI who had negative test results for pandemic (H1N1) 2009.>1 potential concurrent conditions . Limited analysis demonstrated that the concurrent condition did not make these persons more susceptible than other passengers to contracting an ILI.No passengers who had positive test results for pandemic (H1N1) 2009 had underlying health conditions considered to make them more susceptible to influenza; however, 5 of 32 passengers reporting ILI postflight had Flight 1, an Airbus A380, embarked from Los Angeles and arrived in Sydney on May 24, 2009, carrying 445 passengers. Of the 188 (42%) passengers who responded to a survey, 169 (90%) were Australian residents. Response rate varied with class of travel, with 11 (79%) of 14 first class passengers, 40 (56%) of 71 business class passengers, 19 (59%) of 32 premium economy class passengers, and 117 (36%) of 327 economy class passengers responding.Combined results from the survey and disease notification data sources identified 8 passengers who had an ILI at the beginning of the 14-hour flight. For 4 of these passengers, pandemic (H1N1) 2009 infection was later laboratory confirmed; for 1, pandemic (H1N1) 2009 infection was confirmed as negative; 3 passengers were not tested. ILI symptoms developed in 2 other passengers during the flight, and pandemic (H1N1) 2009 was confirmed in both.<7 days after arrival in Australia. Of these, 2 passengers had laboratory-confirmed pandemic (H1N1) 2009 infection, 15 had illness confirmed as negative for the pandemic virus, and 7 were not tested. Most passengers experienced onset of symptoms <3 days after flight arrival; however, 6 passengers did not state exact date of disease onset (Twenty-four passengers were identified as developing ILI symptoms se onset .Self-reporting of symptoms from passengers did not distinguish between different causes of ILI . Fever wTwenty (83%) of 24 passengers in whom an ILI developed postflight sat in aisle seats . This seSome clustering of cases was seen with the potential for spread of either ILI or pandemic (H1N1) 2009 from passengers who were infectious before and during the flight . The pasIn the cabin section of rows 66–77 was a cluster of passengers with symptoms before boarding who were later found to have positive test results for pandemic (H1N1) 2009. ILI symptoms developed in 7 passengers after the flight. In 2 of these passengers, pandemic (H1N1) 2009 was laboratory confirmed; for 4, ILI was confirmed as negative for pandemic (H1N1) 2009; and 1 person (seat 75G) was not tested.Similarly, in the cabin section of rows 50–64, symptoms developed in 2 passengers during the flight or on the day of arrival (seats 52C and 58B); these passengers were found to be pandemic (H1N1) 2009 positive. Symptoms developed in 7 passengers after flight arrival; 5 of whom had negative test results for pandemic (H1N1) 2009, 2 (seats 54F and 62D) were not tested.>3 days after flight arrival.We examined the risk of contracting pandemic (H1N1) 2009 infection postflight to all susceptible passengers seated in the economy section of the aircraft. Health authorities contacted 145 passengers on flight 1 for quarantine and prophylactic treatment after potential exposure to pandemic (H1N1) 2009  2009 by sitting in those seats was 1.4% . If the contact zone was modified to sitting in the same row as or within 2 rows either side of passengers who had preflight symptoms (rows 67–72) and not those in whom symptoms developed during the flight, the risk of contracting pandemic (H1N1) 2009 postflight increased to 3.6% (95% CI –1.3% to 8.6%). No passengers were detected as acquiring pandemic (H1N1) 2009 from either of the passengers with symptoms that developed during the flight; however, 2 passengers in the same section of the aircraft who responded to the survey indicated having had ILI symptoms but not being tested for pandemic (H1N1) 2009.The current zone for contact tracing is defined by passengers in the same row as and within 2 rows either side of the index case-patient. A closer zone forming a square delimited by 2 seats in front, 2 seats behind, and 2 seats on either side of the index case-patient could be prescribed  passengers responding to a survey regarding the potential for contracting an ILI during their flight, 114 (87%) were Australian residents. Public health authorities were not alerted to the potential for passengers carrying pandemic (H1N1) 2009 virus on this flight until 6 days after arrival. They were alerted after mandatory notification of the virus infecting 1 passenger.Survey data showed that 1 passenger was identified as having symptoms consistent with an ILI before the flight and that symptoms developed in 2 additional passengers on the flight. The passenger who was symptomatic before the flight was not tested for pandemic (H1N1) 2009; of the passengers whose symptoms developed during the flight, 1 was not tested and 1 (adult in seat 33D) was tested 6 days after flight arrival. At that time, the test returned a negative result.Six passengers were identified from the survey as having ILI symptoms within 7 days after flight arrival . Of thes INSERT SHAPE Some clustering of cases was seen, with a potential spread of disease from passengers who were symptomatic during the flight . Transmission of disease between adult and child in 33D could have occurred before or during the flight. With lack of a confirmed index case-patient for pandemic (H1N1) 2009, the increased risk for disease was not calculated. No other passengers on this flight had positive test results for pandemic (H1N1) 2009 infection after triangulation methods with the notifiable diseases database.Of 2 long-haul flights entering Australia within the first month after declaration of a level 5 alert for pandemic (H1N1) 2009, a total of 45 (6%) of 738 passengers on 2 aircraft were identified as having the potential to spread an ILI into the local community. Follow-up confirmed 9 passengers with pandemic (H1N1) 2009 infection; 8 of these were from flight 1.Flight 1, originating from a destination with documented widespread community transmission of pandemic (H1N1) 2009, had the greatest potential for introducing the pandemic virus into the Australian community, with 2% of its tested passengers being confirmed positive for pandemic (H1N1) 2009. Spread of the virus from a region known to have sustained community transmission was to be expected and formed part of the case definition in Australia during the early phases of the pandemic  2009 noted on a long-haul flight to New Zealand in 2009  2009. These ILIs could be caused by different viruses, as seen by Follin et al.  2009 and other ILIs can be spread to a community by passengers who were symptomatic before boarding the aircraft. Four of 9 of passengers in whom pandemic (H1N1) 2009 was diagnosed displayed symptoms preflight. This finding is similar to that in a recent study looking at the travel patterns of patients with pandemic (H1N1) 2009 reported from Singapore, where 25% of patients had symptoms before boarding their flights  2009 varies; therefore, if a passenger did not return the survey or contact medical personnel or health authorities after the flight, some cases may have been missed (Spread of pandemic (H1N1) 2009 and other ILIs occurred in limited zones of the aircraft during international flights into Australia during May 2009. The time required to contact passengers postflight resulted in the potential spread of disease into the community despite guidelines and policies in place to reduce the risk for disease importation. Nonetheless, application of these policies by Australian authorities may have assisted in delaying the importation of identified pandemic (H1N1) 2009 cases during the first month of the recent pandemic. The findings of this investigation suggest that efforts to prevent importation of respiratory diseases into a community and protection of individuals from in-flight exposure to ILI may require changes in international policies of both exit screening of symptomatic passengers preflight and contact tracing of those exposed to an ILI in flight. Further research on transmission of ILI in aircraft and into the effects of exit screening at international airport hubs to restrict travel of passengers with symptoms before flying would be of particular interest for respiratory disease of greater severity than pandemic (H1N1) 2009.


Dear Editor,I read with great interest the article by Yusuf Yilmaz et al.  publisheAPRI=AST level/ Upper normal limit of AST/Platelet count ( 10(9) / L )×100The authors suggested that the APRI score can be used as an appropriate non-invasive marker because it showed acceptable accuracy for the assessment of liver fibrosis in patients with CHC and NAFLD, but not in those with CHB.We performed the same study but with a different method. The APRI score was calculated in 35 CHB patients, 14 CHC and 13 autoimmune hepatitis (AIH) patients and in 20 controls 7]8]. T[8]. T7][. T[8]. T


To the Editor: Establishment of viremia is a characteristic feature of infection with human parvovirus 4 (PARV4). In northern Europe, PARV4 (human partetravirus) is primarily transmitted by blood-borne routes  were tested. Specimens were collected during January–December 2004 during a trial of intermittent preventive malaria treatment in the rural Afigya Sekyere District, Ashanti Region, Ghana . All samples were analyzed by using 2 real-time PCRs and primers described elsewhere (4 copies/mL (2 test).PARV4 genotype 3 DNA was detected in plasma of 32 (8.9%) of 361 children. Viral load ranged from ≈200 copies/mL to 3.0 × 10opies/mL . Median 2–1.4 × 104 copies/mL) and 3 (3.8%) showed viremia at both time points and identical viral nucleotide sequences (time between bleedings 8.7 months for 2 children and 9.0 months for 1 child). However, only short genomic regions  could be amplified and sequenced because of low viral loads. Four children had positive results in the first sample, and 3 had positive results in the second sample.PARV4 viremia status was already known for 78 children 24 months of age , a finding similar to ours for the 3 children. However, negative IgM results in the person with hemophilia suggest that the sampling window might have missed the acute infection.PARV4 viremia was detected in a study in the United Kingdom among 110 PARV4-negative persons with hemophilia screened over 5 years for PARV4 viremia and seroconversion (IgG and IgM) (10, with the higher concentrations in the previous study analyzing EDTA whole blood. Whether these differences were caused by the relatively small number of children included or by the fact that whole-blood samples were compared with plasma samples remains to be clarified. However, our previous hypothesis that prenatal or perinatal transient infection was an unlikely mode of virus acquisition needs to be modified because PARV4 infection in newborns has recently been demonstrated (Comparison of results of our study with those of our previous study (Although we lacked IgM and IgG serologic data to interpret our findings, our study suggests that PARV4 genotype 3 infection might be characterized by viral persistence, reactivation, or reinfection. Additional longitudinal studies, including serologic testing for short intervals, are needed to determine the pathogenesis and potential public health role of PARV4 infection.


Hsp90b1 is an endoplasmic reticulum (ER) chaperone  (MGI:98817) contributing with Hspa5  (MGI:95835) to protein folding in ER compartment. Besides its high protein expression in mouse oocytes, little is known about Hsp90b1 during the transition from oocyte-to-embryo. Because the constitutive knockout of Hsp90b1 is responsible for peri-implantation embryonic lethality, it was not yet known whether Hsp90b1 is a functionally important maternal factor.floxHsp90b1, MGI:3700023) in combination with the transgenic mouse line expressing the cre recombinase under the control of zona pellucida 3 (ZP3) promoter . Altered expression of Hsp90b1 in growing oocytes provoked a limited, albeit significant reduction of the zona pellucida thickness but no obvious anomalies in follicular growth, meiotic maturation or fertilization. Interestingly, mutant zygotes obtained from oocytes lacking Hsp90b1 were unable to reach the 2-cell stage. They exhibited either a G2/M block or, more frequently an abnormal mitotic spindle leading to developmental arrest. Despite the fact that Hspa5 displayed a similar profile of expression as Hsp90b1, we found that HSPA5 and HSP90B1 did not fully colocalize in zygotes suggesting distinct function for the two chaperones. Consequently, even if HSPA5 was overexpressed in Hsp90b1 mutant embryos, it did not compensate for HSP90B1 deficiency. Finally, further characterization of ER compartment and cytoskeleton revealed a defective organization of the cytoplasmic region surrounding the mutant zygotic spindle.To circumvent embryonic lethality, we established an oocyte-specific conditional knockout line taking advantage of the more recently created floxed Hsp90b1 line (Our findings demonstrate that the maternal contribution of Hsp90b1 is critical for the development of murine zygotes. All together our data indicate that Hsp90b1 is involved in unique and specific aspects of the first mitosis, which brings together the maternal and paternal genomes on a single spindle. HSP90B1 is located in the endoplasmic reticulum (ER) where numerous proteins translocate and require adequate folding. If misfolded proteins accumulate in ER, this induces a so-called ER stress and triggers the unfolded protein response (UPR) which induces the expression of several genes including ER chaperones such Hsp90b1 or Hspa5 We took advantage of the line generated by Yang et al. (2007) HSP90b1 was reported to be one of the most abundant protein in oocytes We used RTqPCR to analyze the level of Hsp90b1 and Hspa5 transcripts because it was previously described in somatic or cancer cells that Hspa5, a related major ER chaperone is coordinately regulated with Hsp90b1 floxHsp90b1) From the high level of Hsp90b1 expression it could be hypothesized that HSP90b1 is needed during oogenesis and/or early embryonic development. To test this hypothesis, we generated an oocyte-specific deletion of Hsp90b1 using the transgenic mouse line expressing the cre recombinase under Zp3 promoter (Zp3-cre) and the floxed Hsp90b1 line . Ovary histomorphology appeared grossly normal in MT females with the presence of all the follicular stages and corpus luteum as in WT females  and mutant females  oocytes . The levThird we used fluorescent immunodetection  to determine HSP90B1 presence and localization in WT and MT ovary sections. Immunostaining revealed that this chaperone was normally expressed in oocytes as well as in granulosa cells . The strConsistent with the overall normal ovarian histology, the same number of fully-grown oocytes  . The pupTo better identify at which developmental step MT embryos were arrested, we analyzed the progression of the WT and MT embryos during the first cell cycle. This can be determined by the size and position of both pronuclei corresponding to PN1 to PN5 stages see  [12]. WeThen we collected MT embryos blocked at 1-cell stage at 42–44 hours post hCG and stained them to label DNA and tubulin in order to determine at which stage of the cell cycle they were arrested. As described above, Hsp90b1 and Hspa5 are known to be often coregulated and we found that they exhibited the same profile of expression during the transition from egg-to-embryo . TherefoHaving identified the developmental stage requiring HSP90B1, we were then interested in the localization of both ER chaperones to better understand ER function in zygotes. Very little is known about ER organization during the first mitosis of mouse embryos As it was reported that ER dynamics was depending on actin cytoskeleton in addition to microtubule network during mitosis All together, our findings indicated that ER organization and probably dynamics were modified in absence of HSP90B1.Chaperones are important and abundant cellular proteins which protect cells against the consequences of accumulating misfolded proteins. In order to better understand the specific function of some chaperone, gene targeting experiments have been performed  flox/floxHsp90b1) females produced oocytes in which the expression of Hsp90b1 was altered from the primary follicular stage. MT oocytes exhibited a thinner zona pellucida. This was probably due to some default in the trafficking of ZP proteins as revealed by the ZP2 positive structures we identified in oocytes from secondary follicles. Those structures were part of the ER network as indicated by a positive staining by HSPA5 antibodies (data not shown). Zona pellucida defects are important even before fertilization as demontrated by ZP1 or Mgat1 knockout which displayed abnormal folliculogenesis, ovulation and severely reduced fertility Mutant  Regarding the ER chaperones themselves, our data were also in agreement with previous work showing that Hspa5 and Hsp90b1 are coregulated In conclusion, our findings demonstrate that Hsp90b1maternal contribution is involved in ZP protein trafficking during oocyte growth but is not essential to obtain fully grown and MII fertilizable oocytes. In contrast we show that HSP90B1 is critical to complete the first mitosis in murine embryos as its absence provoked a severe disorganization of both the microtubular spindle and the actin–ER network surrounding the spindle. This provides new insights regarding additional HSP90B1 clients whose identification requires further work.All animal work has been conducted according to relevant national and international guidelines. In particular, protocols for animal breeding and experiments were approved by the Departmental Veterinary Office (Haute-Garonne) according to French legislation .flox/floxHsp90b1 mice were generated previously and are described elsewhere  flox/floxHsp90b1 mice were crossed with transgenic mice carrying zona pellucida 3 (Zp3) promoter-mediated Cre recombinase (Tg(Zp3-cre)93Knw, MGI:2176187). Zp3-cre transgene is specifically expressed in oocytes in primary and further developped follicles, starting on day 5 after birth flox/flox;; Hsp90b1 or Zp3-cre/+flox/del.; Hsp90b1 Control females are either wild-type (WT) or flox/floxHsp90b1.The Oocytes were collected either at the GV stage from ovarian antral follicles. Embryos were collected at the indicated time from the oviducts following superovulation or not, as indicated. Superovulation, oocytes, embryos collection and culture were performed as previously described For histology and immunohistochemistry, mouse ovaries were fixed with Bouin, and histological preparations were performed according to a classical procedure . Immunohistochemistry was performed as described before According to the experiment, oocytes or embryos were stained with the following fluorescent dyes: DNA ; actin ; ER glycoconjugates . Fluorescent staining and immunofluorescence protocols were adapted from previous works  dilution 1∶50; HSP90b1  dilution 1∶500 or , dilution 1∶50; FITC-conjugated alpha-tubulin , dilution 1∶100.For chromosome spread and DNA staining at the metaphase I stage, oocytes were fixed in 1% PFA Confocal microscopy and imaging analysis was performed as described previously GCTACCAGGGCCTTTGAGATGGA -3′.Samples preparation, reverse transcription and quantitative PCR were performed as described in Metchat et al. Data were collected from samples collected from at least three animals. The results are presented as means ± SEM as indicated. Student t-test and Chi square were used to perform statistical evaluation, with p<0.05 considered as significant.


CYP2E1 mRNA levels correlated with CBFβ-MYH11 transcript levels and with bone marrow blast counts in all cases. CYP2E1 over-expression correlated positively with NQO1 mRNA levels . By immunohistochemistry, CYP2E1 protein was more frequently expressed in AML with inv(16) compared with other types of AML (p < 0.001). We obtained serial bone marrow samples from two patients with AML with inv(16) before and after treatment. CYP2E1 mRNA expression levels decreased in parallel with CBFβ-MYH11 transcript levels and blast counts following chemotherapy. In contrast, CYP1A2 transcript levels did not change in either patient. This is the first study to demonstrate concurrent over-expression of CYP2E1 and NQO1 mRNA in AML with inv(16). These findings also suggest that a balance between CYP2E1 and NQO1 may be important in the pathogenesis of AML with inv(16). Environmental exposure to benzene occurs through cigarette smoke, unleaded gasoline and certain types of plastic. Benzene is converted to hematotoxic metabolites by the hepatic phase-I enzyme CYP2E1, and these metabolites are detoxified by the phase-II enzyme NQO1. The genes encoding these enzymes are highly polymorphic and studies of these polymorphisms have shown different pathogenic and prognostic features in various hematological malignancies. The potential role of different cytochrome p450 metabolizing enzymes in the pathogenesis of acute myeloid leukemia (AML) in an area of active interest. In this study, we demonstrate aberrant CYP2E1 mRNA over-expression by quantitative real-time polymerase chain reaction in 11 cases of  Benzene is a long established leukemogen implicated in the pathogenesis of myelodysplastic syndromes (MDS) and acute leukemias . AlthougCYP2E1 is a hepatic monooxygenase involved in xenobiotic metabolism. The CYP2E1 gene is associated with the metabolism of many carcinogens. Polymorphisms of CYP2E1 and their possible effects have been reported by others. The CYP2E1*5 allele is associated with a higher risk of de novo acute myeloid leukemia (AML) and acute lymphoblastic leukemia . InThis study provides corroborating evidence implicating benzene in the pathogenesis of AML with inv(16), confirming the findings of others . High leThe strong association between CYP2E1 and AML with inv(16) has implications for pathogenesis. First, this association allows one to infer that AML with inv(16) may be related to exposure to environmental toxins. The elevated expression of CYP2E1 alone would enhance the carcinogenic potential of environmental toxins. The general population is exposed to benzene and its toxic metabolites through gasoline, cigarette smoking, air pollution and dietary intestinal breakdown of proteins . ElevateThe explanation for the elevated levels of CYP2E1 specifically in most cases of AML with inv(16) remains unclear. AML with inv(16) is associated with a favorable outcome with long periods of complete remission ,18. WhilNQO1 gene is located on chromosome 16q22.1, some authors have proposed that inversion (16) may be a primary event leading to disruption of this gene, thereby increasing the likelihood of leukemia [There are limitations to this study, the most important being the lack of NQO1 protein levels to reinforce the mRNA levels in the myeloid system. The results, therefore, need to be confirmed in a larger subset of patients with well characterized polymorphisms in the CYP2E1 and NQO1 genes. Since the leukemia . Furtherin vitro experiments also provide direct evidence of induction of cytochrome p450 and NQO1 by benzene and its analogues/metabolites. Based on this data, we suggest that cases of AML, especially those types associated with recurrent genetic abnormalities, are uniquely associated with an increase in specific types of cytochrome p450 enzymes. This increase appears to be more than just a generalized increase in cytochrome p450 enzyme expression. The increase in specific types of cytochrome enzyme also appears to be unique to each type of leukemia as shown by others, such as specific induction of CYP1A1 in AML with t [The results from our  t  as well  t . It alsoIn conclusion, we have shown simultaneously increased mRNA expression levels of the benzene metabolizing enzymes, CYP2E1 and NQO1, in AML with inv(16). Elevated levels of CYP2E1 mRNA and protein occur more commonly in AML with inv(16) compared with other types of AML. We further suggest that the balance between CYP2E1 (toxin producing) and NQO1 (detoxifying) enzymes may determines the overall carcinogenic potential of the xenotoxins.


Clinical trials with the newer 2nd generation antihistamines in children under the age of 12 years have been performed previously but further studies are needed in order to show efficacy and safety in the most unfavourable clinical conditions such as persistent allergic rhinitis (PER). Rupatadine oral solution was developed for children with allergic rhinitis in view of its rapid onset of action and its lack of relevant side effects. These advantages were confirmed previously in a phase III study in children 6-11 years.To assess the efficacy and safety of rupatadine (RUP) oral solution in a subgroup of children between 6 and 11 years weighing ≥25 kg with PER.A subanalysis was performed from a previous placebo-controlled study carried out in patients between 6-11 years diagnosed as PER according to ARIA criteria. This analysis included patients with a positive prick test, weight ≥25 kg and basal nasal symptoms score  ≥24 obtained in 4 days throughout the 2-week screening period. Patients were allocated to treatment with either RUP oral solution (1 mg/ml) or placebo during 6 weeks. The dose was 5 ml of oral solution. The main efficacy endpoint was the change from baseline of the nasal (4TSS) and global symptoms (5TSS) score at 4 and 6 weeks of treatment. Furthermore the assessment of children's quality of life at 4, 6 weeks by means of PRQLQ was also evaluated.The subgroup analysed was a total of 266 randomized to rupatadine n=131) or placebo (n=135). Table 31 or plaRupatadine oral solution (1mg/ml) was significantly more effective than placebo in improving nasal symptoms (4TSS) at 4 and 6 weeks. This is the first clinical evidence of a H1-receptor antagonist efficacy in children between 6-11 years over 25 kg with PER.


Xenopus, we investigated the putative implication of the bHLH factor Ascl1 in this process. We found that in addition to its previously characterized proneural function, Ascl1 also contributes to the specification of the GABAergic phenotype. We showed that it is necessary for retinal GABAergic cell genesis and sufficient in overexpression experiments to bias a subset of retinal precursor cells towards a GABAergic fate. We also analysed the relationships between Ascl1 and a set of other bHLH factors using an in vivo ectopic neurogenic assay. We demonstrated that Ascl1 has unique features as a GABAergic inducer and is epistatic over factors endowed with glutamatergic potentialities such as Neurog2, NeuroD1 or Atoh7. This functional specificity is conferred by the basic DNA binding domain of Ascl1 and involves a specific genetic network, distinct from that underlying its previously demonstrated effects on catecholaminergic differentiation. Our data show that GABAergic inducing activity of Ascl1 requires the direct transcriptional regulation of Ptf1a, providing therefore a new piece of the network governing neurotransmitter subtype specification during retinogenesis.In contrast with the wealth of data involving bHLH and homeodomain transcription factors in retinal cell type determination, the molecular bases underlying neurotransmitter subtype specification is far less understood. Using both gain and loss of function analyses in  These genes are mostly expressed in complementary patterns in the murine central and peripheral nervous system where they drive the production of distinct neuronal populations Neurog2 and Ascl1 appear respectively as key regulators of glutamatergic (excitatory) versus GABAergic (inhibitory) neuronal fates during telencephalic development Ascl1 in GABAergic cell specification has however proved to be highly time and space dependent Ascl1 is also involved in the development of other neuronal subtypes such as hindbrain serotonergic neurons During development, neural specification leads to the emergence of a large diversity of neuronal subtypes that will serve distinct functions in the adult nervous system. A key issue concerns the nature and action of the molecular cues underlying the acquisition of both generic and specific characteristics of neurons. A small number of basic helix-loop-helix (bHLH) transcription factors are necessary and sufficient for progenitor cell commitment towards a neuronal lineage at the expense of a glial fate and have consequently been qualified as “proneural genes” Ascl1 may also have a more specialized role in the commitment of particular cell subtypes since its expression is restricted to subsets of neuronal progenitors, mostly distinct here again from the Neurog2-expressing ones In the retina, beside its proneural function Ptf1a (pancreas transcription factor 1a), drives inhibitory neuron commitment in the retina, at the expense of a glutamatergic destiny Ptf1a within the retina remain to be investigated. Considering the GABAergic instructive function of Ascl1 in various regions of the brain, and the fact that Ptf1a expression is decreased in the dorsal spinal cord and retina of −/−Ascl1 mice Ptf1a transcriptional network.Our previous studies and others highlighted that the bHLH gene Ascl1 function during Xenopus retinogenesis. We found that Ascl1 is required for GABAergic retinal neuron genesis and sufficient to bias a subset of retinal progenitors towards a GABAergic destiny. Then, we took advantage of the Xenopus ectopic neurogenesis induction paradigm to investigate which genetic interactions contribute to Ascl1 activity as an inducer of neurotransmitter phenotypes. We showed that distinct Ascl1-dependent transcriptional networks sustain the production of GABAergic and catecholaminergic neurons. Together, our data suggest that the GABAergic activity of Ascl1 requires Ptf1a, this latter being a direct transcriptional target of Ascl1.To address these issues, we first analysed pCS2-Ascl1  were obtained by conventional procedures of in vitro fertilization and staged according to Nieuwkoop and Faber GFP mRNA was co-injected as a tracer. Loss of function experiments were performed using already described and validated antisense oligonucleotides morpholinos: Ptf1a-Mo Ascl1-Mo  Immunohistochemistry was performed using rabbit polyclonal anti-GABA , mouse monoclonal anti-GFP  and anti-mouse or anti-rabbit fluorescent secondary antibodies . Cell nuclei were counterstained with Hoechst (Sigma).gad1, VGlut1THtubb2bBrudpysl3Ptf1aAscl1in situ hybridization in situ hybridizations on cryosections Digoxigenin-labeled antisens RNA probes for ornithine decarboxylase (ODC) using Biorad CFX Manager Software. Primers sequences are: tubb2b forward 5′cccgtgccatccttgtggatttt3′, tubb2b reverse 5′gcccagttattgccagcaccactt3′, Ptf1a forward 5′gccgctcaggaaccccaaca3′, Ptf1a reverse 5′ggcagcccgtagtctgggtca3′, ODC forward 5′catggcattctccctgaagtacaagaa3′ and ODC reverse 5′ggacagtggtaggggcaagctca3′.Stage 19 embryos were treated with cycloheximide  during 2.5 hours. Total RNA from 8 embryos was then isolated using the Nucleospin RNA XS kit (Macherey Nagel). Reverse transcription was performed using IscriptcDNA Synthesis Kit (Biorad). RNA quality was evaluated using Experion (BioRad). qPCR reactions were performed in triplicate using SsoFast Eva green Supermix (Biorad) on a C1000 Thermal Cycler . All values were normalized to the level of the reference gene Total protein lysates were prepared from stage 14 embryos and submitted to western blot analysis using an anti-Myc antibody (Sigma) as previously described in situ hybridization signal intensity in the eyes of whole embryos was quantified using Adobe Photoshop CS4.Shown in figures are representative data from one experiment that has been performed at least in duplicate. Fluorescent staining was visualized with a M2 Zeiss microscope. Images were captured with a digital camera AxioCam MRc and AxioVision Rel 7.8 software. The quantification of Xenopus experimentation (authorization 91-29 and 91-28), in accordance with French government policies. The study was conducted under an institutional license (number B 91-471-102 up to 2012 and C 91-471-102 since 2013). The study protocol was approved by the institutional animal care committee, the Direction Départementale de la Protection des Populations (license B/C 91-471-102).All animal procedures were conducted under the supervision of several licensed personnel, including the director of research of the CNRS and professor at the university Paris-Sud, with licenses to perform Ascl1 in GABAergic and glutamatergic phenotype acquisition, we performed morpholino (Mo) injections in two cell stage embryos. We examined the effect of Ascl1 loss-of-function by analysing the expression of gad1 (glutamic acid decarboxylase) and VGlut1 (vesicular glutamate transporter 1), which respectively encode the rate-limiting enzyme for GABA biosynthesis and a glutamate transporter expressed in neurons. The expression of a retinal pan-neuronal marker BruAscl1-Mo injected retinas compared to control ones   . Thus, iAscl1 involvement in the specification of neurotransmitter phenotypes, we turned to a gain of function strategy using a glucocorticoid-inducible Ascl1 construct (Ascl1-GR), which avoids affecting early nervous system development. We analysed the fate of Ascl1-overexpressing cells at stage 38. Contrasting with the Ptf1a gain of function, which results in a dramatic overproduction of amacrine and horizontal cells Ascl1 overexpression led to a typical neurogenic phenotype, with early born neurons being increased  expression in stage 24 embryos . We thus decided to pursue our comparative study by monitoring TH labelling after Ascl1, Neurog2, NeuroD1, Atoh7 or Ptf1a mRNA injection. In contrast to Ascl1, none of these genes were able to promote the production of TH-positive neurons, emphasizing again the specific role of Ascl1 in neuronal subtype determination  retained a neurogenic activity, as inferred by their ability to promote the expression of the pan-neuronal marker dpysl3 . This demonstrates that the functional specificity of these two proteins resides in their respective bHLH domains. In addition, we found that the presence of the Ascl1 basic domain in chimeric constructs was necessary and sufficient to trigger TH and gad1 ectopic expression (bN and NHA to NbA for gad1 and TH staining). Conversely, only the chimeric proteins containing the Neurog2 HLH domain had the ability to induce glutamatergic neurons (VGlut1 labelling for AbN and NHA versus NbA). In line with this observation, the two chimeric proteins containing both the basic domain of Ascl1 and the HLH of Neurog2 (AbN and NHA proteins) were able to simultaneously promote gad1, TH and VGlut1 expression. Altogether, these results suggest that the functional specificities of Ascl1 and Neurog2 in neurotransmitter subtype specification do not reside in the same protein domain. GABAergic and catecholaminergic inducing activity of Ascl1 primarily relies on its basic domain while glutamatergic inducing activity of Neurog2 is imparted by its HLH domain.We next examined which Ascl1 domains account for the protein activity in neuronal subtype specification. We used a series of chimeric nstructs  with intnjection  and all bNA by in vivo lipofection . In the absence of CHX, Ascl1 overexpression lead to a strong increase of both tubb2b and Ptf1a expression level, as assayed by RT-qPCR analysis  Ptf1a overexpression could rescue Ascl1 knockdown. We found indeed that gad1 expression was restored to a control level in the retina of Ascl1 morphant embryos overexpressing Ptf1a (Ascl1 acting upstream Ptf1a in the transcriptional network controlling GABAergic neuron genesis in the retina.In order to know whether such transcriptional interaction could also hold true in the retina, we first carefully compared their expression pattern during retinogenesis . Consistng Ptf1a . Altogetin vivo neurogenic assay in the Xenopus epidermis, we found that Ascl1 GABAergic determining activity is instructive, as inferred by its ability to counteract glutamatergic differentiation programs. Finally, we gained insights into downstream genetic networks underlying Ascl1 functions in neurotransmitter subtype specification by showing that its GABAergic activity involves the direct transcriptional regulation of Ptf1a (The molecular bases underlying neurotransmitter subtype determination in the retina remain largely unexplored. As previously shown in the brain and peripheral nervous system of Ptf1a .Ascl1 gain and loss of function analyses in the retina have so far uncovered phenotypes predominantly related to its proneural activity. Ascl1 has indeed been shown to be required for neuronal versus glial fate decisions during late neurogenesis Ascl1-expressing progenitors contribute to all major cell types of the retina with the exception of ganglion cells Ascl1 or other bHLH genes such as Neurog2 or Atoh7 results in apparently similar cell type distribution defects within the Xenopus retina Neurog2 nor Atoh7 exhibit this property. Importantly, our results are consistent with a previous study in chick reporting that, unlike Neurog2, NeuroD1 or Atoh7, Ascl1 misexpression promotes an overproduction of amacrine cells, the major retinal GABAergic cell type Most The strong context-dependency of Ascl1 function in neurotransmitter subtype specification relies on the presence of other regionally expressed transcription factors including bHLH ones Ascl1-dependent ectopic GABAergic neuron production was found to be enhanced by Neurog2 co-expression. This suggests that, in the few locations where both factors are co-expressed in the same cells Ascl1 and Neurog2 lineages are largely distinct, such progenitors expressing both genes have recently been described Neurog2 was identified as a direct transcriptional target of Ptf1a in the chick dorsal spinal cord and cerebellum Ptf1a, Ascl1 and Neurog2 expressions partially overlap Neurog2 might thus participate within these domains in the Ascl1/Ptf1a-dependent GABAergic program. Additionally, Ascl1 and Neurog2 were shown to heterodimerize and function in common transcriptional complexes Ascl1 interactions with other expressed bHLH genes in the retina will surely help understand the combinatorial codes governing neurotransmitter subtype determination.Unexpectedly, Ascl1 expression may have interfered with proper GABAergic differentiation. Our domain swapping experiments led to an opposite conclusion, highlighting the essential role of Ascl1 basic domain in both its catecholaminergic and GABAergic inducing activities. Interestingly, the same was not true for Neurog2, whose ability to induce glutamatergic neurons was found to be imparted by its HLH domain. Although we cannot exclude that Ascl1 or Neurog2 might act through different molecular mechanisms depending on the cellular context, our data suggest that distinct domains within these bHLH factors mediate their functional specificities in neurotransmitter phenotype specification.Previous reports proposed that the HLH domain of murine Ascl1 contains the information underlying its specific activity in neuronal subtype specification Ptf1a and Phox2a/Hand2, respectively. We found in particular that Ascl1 ability to promote gad1-positive neurons requires Ptf1a. Additionally, Ascl1 was able to activate Ptf1a transcription and to be necessary for its retinal expression. In line with such a positive transcriptional regulation, previous data showed that Ascl1 is required for the induction and/or maintenance of Ptf1a during the late phase of dorsal sensory interneuron development Ptf1a might constitute a direct transcriptional target of Ascl1. Consensus binding sites for Ascl1 have previously been described in two conserved Ptf1a enhancer regions but have not proved to be functional Ptf1a regulatory regions.We showed that Ascl1 exerts its GABAergic and catecholaminergic determining activities by controlling distinct genetic cascades involving Altogether, our work demonstrated for the first time a role for Ascl1 in the generation of GABAergic interneurons in the retina and provided insights into the regulatory circuits responsible for its activity in neurotransmitter subtype specification.


Second, we examined the requirement for a putative bilateral interaction by unilaterally ablating the dI1 population in cultured explants of chicken embryonic spinal cord. Surprisingly, we find no evidence for a bilateral dI1 axon interaction, rather dI1 axons appear to project independently of each other.Axons use temporal and directional guidance cues at intermediate targets to set the rate and direction of growth towards their synaptic targets. Our recent studies have shown that disrupting the temporal guidance process, by unilaterally accelerating the rate at which spinal dI1  axons grow, resulted in turning errors both in the ventral spinal cord and after crossing the floor plate. Here we investigate a mechanistic explanation for these defects: the accelerated dI1 axons arrive in the ventral spinal cord before necessary fasciculation cues from incoming dI1 axons from the opposite side of the spinal cord. The identification of such an interaction would support a model of selective fasciculation whereby the pioneering dI1 axons serve as guides for the processes of the bilaterally symmetrical population of dI1 neurons. To test this model, we first developed the ability to “double”  During development, axons extend along stereotyped pathways to form the precisely ordered neuronal networks critical for the nervous system to function + (ATOH1) progenitor-derived dI1 neurons, a class of dorsal sensory interneurons in the developing spinal cord + dI1 axons: 1) the well-described TAG1/axonin1+ commissural axons The role for temporal guidance cues was first demonstrated for the MATH1Altering the rate at which dI1 axons grow had significant consequences for the development of the dI1 neural circuit. In particular, accelerating dI1 axon growth resulted in turning errors when dI1 axons reached the ventral spinal cord in ovo electroporation of chicken embryos. In these embryos, 1) accelerated early born contralaterally-projecting dI1 axons would have no bilateral axon interactions in the FP and 2) accelerated later born ipsilaterally-projecting dI1 axons would not encounter the opposing population of earlier born contralaterally-projecting dI1 axons as they navigated the ventral spinal cord. We have thus explored whether these two putative bilateral axonal interactions have a critical role in the guidance of dI1 axons, using chicken embryos as a model system. We first developed a “double” electroporation technique to examine whether the previously observed defects after unilateral acceleration of one population of dI1 axons can be rescued by the bilateral acceleration of both populations of dI1 axons. Second, we used an in vitro tissue culture assay to assess the effect of unilaterally ablating dI1 neurons on the axon trajectory of the spared population of dI1 axons on the other side of the spinal cord. Neither experiment provided any evidence for a bilateral interaction between dI1 axons, suggesting that the two populations of axons project independently of each other.A second, largely unexplored, source of guidance cues in the ventral spinal cord could come from bilateral interactions between the two populations of dI1 axons projecting from either side of the spinal cord. Selective fasciculation between pioneer axons is common in invertebrates tomato, 0.25 mg/ml Math1::farnesylated (f) Gfp and 0.6 µg/µl Math1::BmprIIΔLim-Gfp. When two fluorophores are electroporated into dI1 neurons using the Math1 enhancer, there is close to a 100% overlap between the expression of the fluorophores. In no case, did double electroporation alter the number of LHX2/9 dI1 neurons.Hamburger-Hamilton (HH) stages 14/15 White Leghorn chicken embryos (McIntyre Poultry) were injected with two sequential solutions of different combinations of the following expression constructs: 0.25 µg/µl Math1::tandem dimer (td) DNA solution #1 was injected into the lumen of the spinal cord and an electric current passed across the embryo using a BTX Electro Square Porator (ECM 830) set at ten 50 ms second pulses of 30 V. The embryo was permitted to settle for 10–15 minutes and the positive and negative electrodes were inverted.DNA solution #2 was electroporated into the lumen of the spinal cord using five 50 ms pulses of 30 V. A few drops of 1× penicillin/streptomycin/glutamine  were applied to the embryo, and the egg was wrapped in parafilm (VWR) and incubated at 37°C for 2 days until HH stages 24/25.Gfp or 0.6 mg/ml Math1::BmprIIΔLim-Gfp into the developing spinal cord lumen using an electric current generated by five 50 ms pulses of 30 V. The electroporated egg was incubated overnight at 37°C and then dissected in L15 medium (CellGro) at HH stage 18, before any dI1 commissural axons have reached the FP HH stages 14/15 chicken embryos were electroporated with 0.25 mg/ml Math1::fAll spinal cords were processed to result in fixed “open book” explants embedded in collagen. Antibody staining was as previously described Rabbit: LHX2/9 (pan LH2A/B), 1∶1000 Mouse: GFP, 1∶1000 . Species appropriate Cyanine 3 and Fluorescein conjugated secondary antibodies were used (Jackson ImmunoResearch Laboratories).Antibodies against the following proteins were used. + axons, i.e. those that normally turn rostrally before reaching the FP, to the number of contralaterally projecting GFP+ axons, i.e. those that normally turn rostrally after crossing the FP. Fluorescent images were collected on Carl Zeiss LSM510 confocal and Axiovert 200 M microscopes. Images were processed using Adobe Photoshop CS4.These explants were quantified by comparing the number of ipsilaterally projecting GFP+ dI1 neurons, we developed a novel method of independently labeling axons on different sides of the spinal cord by “double” in ovo electroporating chicken embryos. In control experiments to establish this procedure, HH stages 14/15 chicken embryos were electroporated twice, first with Math1::tdtomato and then with a mixture of both Math1::tdtomato and Math1::fGfp, reversing the position of electrodes between the injections  ipsilateral dI1 axon population  into dI1 neurons results in their extending axons that grow 40% faster than axons expressing GFP alone in ovo electroporation procedure. To unilaterally accelerate dI1 axons, BMPRIIΔLim-GFP was introduced into only one population of dI1s, whereas tomato was introduced bilaterally in all dI1s . This experiment gave similar results to those observed previously + tomato+ ipsilaterally projecting dI1 axons  axon pathway in the spinal cord is guided in part by heterophilic interactions between CAMs in the axons themselves  and in the cells of the FP . TAG1 has a neurite promoting activity in vivo phenotypes observed after blocking axonin1 or NrCAM are only a consequence of interactions between heterophilic CAMs or whether there was also a homophilic component resulting from a bilateral interaction between the different populations of dI1 axons growing within the FP and the ventral spinal cord. The close proximity of dI1 commissural axons extending across the FP and the co-incidence of the ipsilateral and contralateral dI1 axons growing within the ventral spinal cord make such an interaction feasible + commissural axons entering the FP could provide a positive substrate for one another, while the rostral turn taken by the contralateral dI1 axons could provide a scaffold for the rostral turn of the later born ipsilateral dI1 axons (arrow, These studies focused on the contact-mediated interactions between the commissural axons and the cells of the FP and did not examine the role of putative interactions between the opposing populations of commissural axons. Thus, it remained unresolved whether the s arrow, .Nonetheless, we were unable to find any evidence for such bilateral interactions between the opposing populations of dI1 axons. Restoring the bilateral interaction was not sufficient to rescue the defects seen after unilaterally accelerating the rate of dI1 axon growth. Moreover, ablating one population of dI1 neurons did not affect the turning behaviors of the remaining dI1 axons. Taken together, these data suggest that the turning behaviors of dI1 axons in the FP and ventral spinal cord occur independently from one another.Our studies The mechanistic basis for these guidance errors remains unresolved. Temporal guidance cues could permit axons to encounter directional information at the right time in development, such that neural circuits develop in concert with the rest of the embryo. However, as discussed above, many of the key molecular guidance cues are present in the FP prior to dI1 commissural axons reaching the ventral spinal cord. A notable exception is WNT4, which is expressed in the ventral spinal cord by HH stages 27/28 In summary, these studies show a surprising absence of any interaction between the two opposing populations of dI1 axons in the spinal cord as they navigate the ventral spinal cord.