Human infections with avian influenza viruses (AIVs) have frequently raised global concerns of emerging, interspecies-transmissible viruses with pandemic potential. Waterfowl, the predominant reservoir of influenza viruses in nature, harbor precursors of different genetic lineages that have contributed to novel pandemic influenza viruses in the past.Two duck influenza H5N2 viruses, DV518 and DV413, isolated through virological surveillance at a live-poultry market in Taiwan, showed phylogenetic relatedness but exhibited different replication capabilities in mammalian Madin-Darby Canine Kidney (MDCK) cells. This study characterizes the replication properties of the two duck H5N2 viruses and the determinants involved.The DV518 virus replicated more efficiently than DV413 in both MDCK and chicken DF1 cells. Interestingly, the infection of MDCK cells by DV518 formed heterogeneous plaques with great differences in size [large (L) and small (S)], and the two viral strains (p518-L and p518-S) obtained from plaque purification exhibited distinguishable replication kinetics in MDCK cells. Nonetheless, both plaque-purified DV518 strains still maintained their growth advantages over the plaque-purified p413 strain. Moreover, three amino acid substitutions in PA (P224S), PB2 (E72D), and M1 (A128T) were identified in intra-duck variations (p518-L vs p518-S), whereas other changes in HA (N170D), NA (I56T), and NP (Y289H) were present in inter-duck variations (DV518 vs DV413). Both p518-L and p518-S strains had the N170D substitution in HA, which might be related to their greater binding to MDCK cells. Additionally, polymerase activity assays on 293T cells demonstrated the role of vRNP in modulating the replication capability of the duck p518-L viruses in mammalian cells.These results demonstrate that intra-host phenotypic variation occurs even within an individual duck. In view of recent human infections by low pathogenic AIVs, this study suggests possible determinants involved in the stepwise selection of virus variants from the duck influenza virus population which may facilitate inter-species transmission. Influenza has been a major concern for global health due to dynamic changes through continuous antigenic changes and occasional antigenic shifts resulting from the reassortment of viruses derived from different hosts . EmerginRoutine virological surveillance in live-poultry markets (LPMs) has become imperative since the outbreaks of H5N1 influenza virus occurred in Hong Kong in 1997 . Most imWaterfowl, the major influenza virus reservoir, harbors genes that might contribute to AIVs with the potential to infect humans , 13. RecHere we report that the two Taiwan duck influenza H5N2 isolates, DV518 and DV413 , with hiSeveral Taiwan duck LAPI H5N2 viruses with different gene constellations that were isolated from fecal droppings in the environment of a live-poultry market of the previous study before 2012  were exaMadin-Darby Canine Kidney epithelial cells (MDCK) and chicken embryo fibroblast cells (DF1)  were groin vitro from an M-gene-carrying plasmid, which was constructed as previously described [Viral RNA (vRNA) was extracted from the supernatant of infected cells by using the QIAamp Viral RNA Mini kit (Qiagen). The standard M-vRNA of influenza virus was transcribed escribed . The tesescribed  in paralCT] values of the diluted M-vRNA standard with known copy numbers.For quantitation, the tested samples were measured by quantitative RT-PCR for M vRNA. All cDNA samples were subjected to real-time PCR using the KAPA SYBR FAST qPCR kit (KAPA) with AMF (5’-GAGTCTTCTAACCGAGGTCGAAACGTA -3’) and FluA-R (5’-CAAAGCGTCTACGCTGCAGTCC-3’) primers . StandarThe viral stock was first titrated by plaque assay using MDCK cells. Briefly, MDCK cells were seeded on six-well plates and grown until confluent. Ten-fold serial dilutions of the tested samples were performed in duplicate. After one-hour adsorption at 37°C, the inoculum was then removed and a 1:1 mixture of 1% agarose gel and 2X serum-free DMEM with TPCK-trypsin at a final concentration of 2 μg/mL was immediately added as an overlay medium. After 72 hours’ incubation at 37°C, the infected cells were fixed with paraformaldehyde and stained with crystal violet. For plaque-purification, approximately 5–10 plaques were picked after 72 hours incubation without fixing the cells and then individually inoculated into SPF eggs. The p518-L and p518-S virus strains were isolated from large and small plaques, respectively through three rounds of plaque purification from the DV518 virus stock, whereas p413 virus was isolated through only one round of plaque purification from the DV413 virus stock for further virological characterization. Experiments for different virus strains were conducted independently to prevent possible cross-contamination. Plaque morphology was examined after each round of plaque purification; and the sizes and numbers of plaques in one well were analyzed by software Image J 1.45A 50 method was modified as previously described .This research used only cell lines to investigate viral properties. The sources of the three cell lines used in this study, including (1) chicken embryo fibroblast DF1 cells (ATCC CRL-12203), (2) Madin-Darby Canine Kidney epithelial (MDCK) cells (ATCC CCL-34), and (3) human kidney 293T cells (ATCC CRL-3216) were kindly provided by Dr. Ching-Ho Wang, Dr. Chuan-Liang Kao, and Dr. Shiou-Hwei Yeh at the National Taiwan University, respectively. They were all bought from the American Type Culture Collection (ATCC), Rockville, Maryland, USA  in the USA and the National Science Council  had no role in study design, data collection and analysis, decision to publish or preparation of the manuscript.in vitro, we first used quantitative RT-PCR to quantify the vRNAs in both mammalian and avian cell culture supernatants. 7.03 vs 106.54 copies/mL (p<0.05) at 48 h p.i. and 106.90 vs 106.01 copies/mL (p<0.05) at 72 h p.i.]. In addition, chicken DF1 cells infected with these two virus strains at an MOI of 0.1, also showed quite similar results, with DV518 producing a significantly higher level of vRNA at 48 h p.i. than DV413  (To investigate the replication levels of DV518 and DV413  p<0.05) .50 method. MDCK cells infected with DV518 at a MOI of 0.1 resulted in significantly higher viral yield at 36 and 48 h p.i. when compared with the same cells infected with DV413. [Mean: DV518 vs DV413: 102.51 vs 101.28 TCID50/mL (p<0.05) at 36 h p.i. and 103.32 vs 101.25 TCID50/mL (p<0.05) at 48 h p.i.]  and small plaques in DV518-infected MDCK cells  as well as the plaque in DV413-infected MDCK cells (named p413) and then compared their growth properties in vitro.In plaque assays of DV518 and DV413 using MDCK cells, different-sized plaques were observed, implying these duck isolates could be a mixture of heterogeneous viral sub-populations. To test this, we analyzed the distribution of viral plaque sizes of DV518 and DV413 in MDCK cells. The results revealed that the duck DV518 strain generated plaques that were larger and more heterogeneous in size than those generated by the DV413 strain . Fig 2 s2 versus 0.3 mm2). In addition, the results from the focus-forming assay also verified the above findings .Further analysis of plaque sizes of these purified viral strains  showed tfindings . However50 method to measure the virus titers produced in MDCK cells. As shown in 50 method. It should be noted that the TCID50 method measures the viruses that can cause a cytopathic effect in MDCK cells. Thus the TCID50 method gave much lower titers for those virus strains with weak multiplication efficiency (i.e. p413) than those results obtained from the quantitative RT-PCR and the hemagglutination methods, which measure the amount of virus directly.The growth kinetics of these three plaque-purified virus strains  were simultaneously examined in MDCK and DF1 cells using the hemagglutination method. As shown in in ovo system with 14-day-old embryonated SPF eggs to mimic selective pressure for AI viruses in chickens [50 units, and the virus yield in allantoic fluid at 48 h p.i. was examined. As shown in 7.25, 106.3, and 106.25 TCID50/mL, respectively), although the differences did not reach statistical significance. This result is consistent with the data shown in To verify the above findings from DF1 cells, we used an chickens , 28. TheTo gain more information on the mechanism involved in phenotypic variations of the plaque-purified virus strains, we sequenced p518-L, p518-S and p413 strains and compared the results with those obtained from the original viral isolates  possessing HA-170D mutation replicated more efficiently in MDCK cells, even though these viruses had receptor specificities similar to those of the parental duck virus, and exhibited the potential to infect human cells in vitro and ex vivo [By contrast, inter-host variations of the two Taiwan-H5N2 DV518 and DV413 viruses were isolated from fecal droppings between poultry ducks  where ducks came from Yilan and Yunlin counties). These two viruses involved another three amino acid changes  in HA, NA, and NP, respectively –45. DV51 ex vivo . The pos ex vivo   of plaque in a representative well for each strain was analyzed by software Image J, and was shown as mean ± standard deviations in the parentheses.MDCK cells were infected with DV413, DV518, p413, p518-S and p518-L virus strains. After incubation at 37°C for 72 h, cells were fixed by paraformaldehyde and stained with crystal violet. Mean plaque area (mm(PDF)Click here for additional data file.S2 FigMDCK cells were infected with p518-L or p518-S viruses. After incubation at 37°C for 48 h, infected cells were treated with a mixture of mouse anti-influenza M monoclonal antibody (Millipore) and mouse anti-influenza NS1 monoclonal antibody (Santa Cruz Biotechnology) after fixation. The HRP-conjugated anti-mouse secondary antibody (Jackson) was then added and the foci were subsequently developed by adding peroxidase substrate using a commercial kit (Vector Laboratories).(PDF)Click here for additional data file.S3 Fig50 method was used to examine the viral growths. The results are shown as means ± standard deviations of triplicate samples.MDCK cells were infected with p518-L, p518-S, and p413 virus strains at an MOI of 0.01. The supernatants were harvested at the indicated time points. The TCID(PDF)Click here for additional data file.S4 FigAbbreviations: 518, DV518 virus; 413, DV413 virus; S-P1, S-P2, and S-P3, small-plaque virus obtained by the first, second, and third round of plaque purification of DV518, respectively; L-P1, L-P2, and L-P3, large-plaque virus obtained by the first, second, and third round of plaque purification of DV518, respectively; 413-P1, virus obtained by first-round plaque purification of DV413.(PDF)Click here for additional data file.S5 FigA549 and DF1 cell monolayers were infected with p518-L, p518-S and p413 viruses at the same copy number and then incubated at 4°C for 1 h. After extensive wash, viral RNAs were extracted from the infected cells and influenza viral M RNAs were quantified by quantitative RT-PCR. The copy numbers of M RNA attached to the cells were normalized to that of DV30 in each set of data. Results are shown as means with standard deviations in three experiments.(PDF)Click here for additional data file.S6 FigSurface polarities of HA with positions of 170D (a) and 170N (b) are shown. Position 170 (or 158 in H3 numbering) of HA is at the top of the HA molecule (c). The orange region, HA 161–175, represents the residues changed to the amino acids possessed by DV518 or DV413 (d). All structures shown are based on the backbone of A/Viet Nam/1203 (pdb: 2FK0).(PDF)Click here for additional data file.


In multicellular organisms p53 maintains genomic integrity through activation of DNA repair, and apoptosis. EBNA3C can down regulate p53 transcriptional activity. Aurora kinase (AK) B phosphorylates p53, which leads to degradation of p53. Aberrant expression of AK-B is a hallmark of numerous human cancers. Therefore changes in the activities of p53 due to AK-B and EBNA3C expression is important for understanding EBV-mediated cell transformation. Here we show that the activities of p53 and its homolog p73 are dysregulated in EBV infected primary cells which can contribute to increased cell transformation. Further, we showed that the ETS-1 binding site is crucial for EBNA3C-mediated up-regulation of AK-B transcription. Further, we determined the Ser 215 residue of p53 is critical for functional regulation by AK-B and EBNA3C and that the kinase domain of AK-B which includes amino acid residues 106, 111 and 205 was important for p53 regulation. AK-B with a mutation at residue 207 was functionally similar to wild type AK-B in terms of its kinase activities and knockdown of AK-B led to enhanced p73 expression independent of p53. This study explores an additional mechanism by which p53 is regulated by AK-B and EBNA3C contributing to EBV-induced B-cell transformation. Epstein-Barr virus (EBV) is a γ-herpesvirus that establishes a lifetime infection in most of the adult human population . AlthougA growing number of studies have suggested an essential role for EBNA3C in viral associated-oncogenesis, specifically through direct interactions with regulator proteins responsible for maintaining cell-cycle checkpoints. Recently, we demonstrated that EBNA3C can block E2F1-mediated apoptosis induced by DNA damage, as well as facilitates the G1 to S transition by enhancing Cyclin D1 function , 5. FurtOne of the most important cellular proteins affected by EBNA3C is the p53 tumor suppressor. P53 maintains genomic integrity in multicellular organisms by activating DNA repair, arresting cells at the G1/S checkpoint, and inducing apoptosis . DeletioMore recently, we discovered that EBNA3C up-regulates and stabilizes AK-B, a serine/threonine kinase that plays multiple roles which are integral to accurate cell division . AK-B coPreviously, we had shown that EBNA3C up-regulates and stabilizes AK-B, leading to cell proliferation . We founTo determine if p53 in return can directly contribute to down-regulation of AK-B, we transfected BJAB and HEK-293 cells with a p53 expression plasmid in a dose-dependent manner and performed Western blots, probing for AK-B protein levels Fig. . EndogenWe previously showed that EBNA3C can bind to the AK-B promoter, and can directly up-regulate AK-B at both the transcription as well as protein levels . To furtIf the level of p53 was maintained at a constant level with a dose-dependent increase in levels of EBNA3C, the levels of AK-B as determined by Western blot increased Fig. . The assWe speculated that the ETS-binding domain, located between −68 and −71bp upstream of the AK-B TSS, may be responsible for recruiting ETS-1 to the AK-B promoter region to modulate p53 activity. To determine whether ETS-1 attenuates the effect of p53 on AK-B, we transfected Saos-2 cells with a constant dose of ETS-1 with increasing amounts of p53  suggesting that the S215 residue may be an important contributor to the transcription activity of p53. To determine the effect of p53, EBNA3C, and AK-B on cell growth, we performed colony formation assay (CFA) using MEF cells expressing EBNA3C and AK-B in combination with wild type p53 and its phospho-mutants. Strikingly, we found that there was no significant increase in colony formation when EBNA3C and AK-B were expressed with the p53 S215 mutant Fig. . The p53106D and D205A mutants were not effectively reduced, possibly by reduced ubiquitination or de-ubiquitination in the presence of EBNA3C. The wild type and K207R mutant showed much less ubiquitination in the presence of EBNA3C  cells with plasmids encoding short hairpin against AK-B to knock down the proto-oncogene. The transcript and protein levels were assessed by Western blot and RT-PCR analysis, respectively Fig. . We founAK-B and p53 are two crucial cellular regulators of the cell-cycle whose aberrant expressions are hallmarks of tumorigenesis. AK-B has multiple roles in mitosis such as the localization of centromeric proteins, the bi-orientation of chromosome attachments, and proper cytokinesis . Over-exEBNA3C is a major viral-encoded transcription factor found in EBV-positive cells expressing the Type III Latency program. EBNA3C is essential for the transformation of B cells leading to proliferating lymphoblastoid cell lines. EBNA3C derails cell-cycle activities by interacting with multiple checkpoint proteins , 50, 51.Previous studies have linked EBNA3C, p53 and AK-B through multiple processes involved in gene expression, protein-protein interactions as well as post-translational modifications , 28, 45.We have previously shown that EBNA3C can up-regulate and stabilize AK-B in stable cell lines as well as in EBV infected primary cells . FurtherAK-B expression is induced in a number of tumors where p53 is defective . In thisin vitro and in vivo to regulate gene expression [While p53 can suppress AK-B in LCLs, the levels of EBNA3C present are much lower compared to that expressed in newly infected or reactivated cells. We sought to investigate whether increasing EBNA3C levels could overcome p53-mediated down-regulation of AK-B, tipping the balance between the two proteins. The results of our luciferase assays demonstrated that EBNA3C can overcome the effects of p53 to up-regulate AK-B. The truncation of the AK-B promoter (−337) without the ETS-1 binding domain failed to activate more than the control in response to EBNA3C, suggesting that an ETS-1 transcription factor is also required. ETS-1 factors are classified by their highly-conserved DNA-binding (ETS) domain and are involved in multiple facets of cellular regulation . It has pression . We prop106 residue [106D mutant, which lacks the kinase activity, degraded more quickly in cyclohexamide stability assays and was not readily de-ubiquitinated by EBNA3C (15). We sought to determine if the kinase activity of AK-B was required for EBNA3C-mediated stability by comparing the behavior of the wild-type protein with that of several point mutants. Previously, Gully et al reported that the AK-B D205A mutant was essentially kinase-dead and that the K111M mutant displayed severely diminished kinase activity [111M mutant would have stability between that of the wild type AK-B and its kinase-dead mutants. Additionally, a mutation at K207R, the residue required for AK-B SUMOylation gives rise to similar phenotypic defects as the D205A mutant [207R mutant has uncompromised kinase activity, it was selected as a pseudo-control to compare with the D205A mutant. Our results revealed that the AK-B kinase activity is important for EBNA3C-mediated stabilization of p53, however K207R behave different than kinase mutants  as expected due to its pseudo-control nature of muation. Wild type AK-B and the K207R mutant were effectively stabilized through de-ubiquitination, but not the kinase dead mutants. Intriguingly, the changes in levels of kinase activity between the AK-B constructs were not particularly striking unless EBNA3C was present to stabilize those with active kinase domains. An earlier study from our group demonstrated that EBNA3C directly binds to p53 and inhibits its transcriptional activities [We showed previously that EBNA3C is able to stabilize AK-B, and that this mechanism was dependent on the AK-B K residue . Additio residue . The K10activity . We seleA mutant . Since ttivities , 45. EBNtivities . Phosphotivities . Furthertivities . Prior ttivities , and supOverall, we have provided strong evidence for a proposed mechanism whereby EBNA3C up-regulates AK-B which directly leads to p53 phosphorylation and degradation Fig. . The eviThe University of Pennsylvania CFAR Immunology Core provided human peripheral blood mononuclear cells (PBMCs). The donor gave written, IRB approved informed consent based on the declaration of Helsinki protocols. For our studies all samples were deidentified so that there were no links to any individual donor.106R mutant, cloned in pcDNA3/myc-his and GFP vectors, were gifts from Erich A. Nigg . The AK-B promoter regions, cloned in pGL3B vectors and used for luciferase assays, were gifts from Yukio Okano . Aurora-B mutants cloned in GFP vector  were kindly gifts from Marcos Malumbres .Wild type AK-B and the KThe following cell lines were cultured in RPMI 1640 medium and other culture conditions described previously . We usedThe cloning of EBNA3C (full length) in the pEGFP, dsRed, pGEX2TK and pA3F and pA3M vectors, as well as various truncations in pA3F and pA3M , and pGEX2TK  vectors were previously described , 52. RabShort-hairpin oligonucleotides were designed to knock-down EBNA3C, AK-B and p53  , 54. TheTransiently transfected cells were harvested, washed with PBS, and lysed in RIPA buffer . Cell processing were performed as described earlier . BradforFor Western blot analysis, the input and IP complexes were fractionated by SDS-PAGE gel (8-12%) electrophoresis, and transferred to nitrocellulose membranes. The membranes were blocked in 5% milk in 1X-PBS for 40 minutes, washed three times in TBST buffer, and probed with the appropriate primary antibodies overnight at 4°C. Afterwards, the membranes were washed two times in TBST and incubated with appropriate infrared-tagged secondary antibodies at room temperature for 60 minutes. Scanning and densitometry analyses were performed by Odyssey Infra-Red scanner and software, respectively .Reporter assays were performed as described previously . Briefly6) that were stably transfected with short-hairpin p53 or a control vector were cross-linked with formaldehyde, washed with 1X PBS, and lysed in cell lysis buffer . The nuclei were pelleted by centrifuging (5000 rpm for 5 mins). Next, nuclear lysis buffer  was added to the nuclei and the DNA was sonicated for 2.5 minutes at 30-second intervals, producing fragments of approximately 600bp. The samples were pre-cleared with ssDNA/Protein A agarose beads (1 hours), and 20% was used as input. The remaining samples were each divided into two portions. One was rotated with the A10 antibody while the other was rotated with non-specific mouse serum, serving as the negative control. Both were kept at 4°C overnight. The DNA-protein-antibody complexes were collected by rotation with ssDNA/Protein A agarose beads (2 hours). The beads were collected, washed with a series of buffers, and treated with elution buffer . The mixture was shaken for 30 minutes. The samples were centrifuged (15000 rpm for 3 minutes) and the supernatant was transferred to a new tube. The input and ChIP samples were both incubated with RNase and NaCl  for reverse formaldehyde cross-linking. The DNA was purified, pelleted and dissolved in 30μl of water. Finally, the samples were amplified by PCR with three sets of primers, each spanning a separate region of the AK-B promoter. The products were run and quantified on agarose gels.ChIP assays were carried out as described previously . LCL1 ce20 million cells were transfected by electroporation with the plasmids for each experiment, including HA-tagged ubiquitin plasmids. Following 36 hours of incubation, the cells were treated with 20μM MG132  for another 12 hours to inhibit proteasome activity. The following procedures were as described earlier .Ten million MEF cells were transiently transfected with plasmids which include p53 wt and mutants, AK-B and EBNA3C by electroporation. After 36 hours of incubation, G418 was added to the cells to select for those containing plasmids. The media and antibiotic were replaced every other day for two weeks. Afterwards, cells were fixed on the plates with 3% PFA, stained with 0.1% crystal violet, and rinsed with water to remove excess dye. The plates were scanned using the LiCor-Odyssey system and growth was determined quantitatively using the Odyssey software.oC and washed with 1x PBS. The cells were treated with 50 μg/ml propidium iodide  and 1 μg/ml RNase (1 hour at 4oC). Flow cytometric analysis was conducted using the FACS Calibur cytometer .Appropriate plasmids were transfected into MEF cells by electroporation. 24 hours post-transfection, the cells were incubated in 0.1% serum media for 12 hours and treated with 20 μM etoposide  for six hours to induce DNA damage. The cells were then harvested for propidium iodide staining and flow cytometry analysis. Prior to PI staining, cells were fixed with an equal ratio of acetone : methanol for two hours at 4oC. The cultures were pelleted by centrifuging (3000 RPM for 10 mins) and the pellet washed with STE buffer and resuspended in NETN buffer. Dithiothreitol (DTT) and 10% Sarkosyl in STE buffer were mixed into the solution, and the sample was sonicated to solubilized the proteins. The lysates were collected by centrifugation and the supernatant transferred and followed by addition of Triton X-100 in STE and Glutathione-Sepharose with rotation. The beads were collected by centrifugation  and washed with NETN buffer. 250μL of NETN was added to the washed beads, 20μL of which was resolved in an SDS-page gel and stained with coomassie blue to confirm expression of the fusion protein.GST-fusion proteins were purified essentially as described previously  with slioC with GST protein-bound beads to serve as a negative control.For GST pull-down assays, cells expressing AK-B was lysed using RIPA buffer. The lysates were pre-cleared with Glutathione-Sepharose beads, 10% was set aside as input, and the remainder was divided into two portions. One was rotated with the appropriate GST fusion protein-bound bead while the other was rotated overnight at 42 were previously reported [Transiently transfected cells were harvested and washed with ice-cold PBS. RNA extraction was performed using the TRIzol reagent (Invitrogen), and 2.5μg of each RNA sample was converted to cDNA using the Superscript II Reverse Transcriptase Kit . Quantitative PCR was conducted using the following pair of primers. Primer sequence for AK-B forward- GGAGAGCTTAAAATTGCAGATTTTG, reverse- TGCAGCTCTTCTGCAGCTCCT, EBNA3C forward- AGAAGGGGAGCGTGTGTTGT, reversed- GGCTCGTTTTTGACGTCGGC, and GAPDH forward- CTCCTCTGACTTCAACAGCG, reversed- GCCAAATTCGTTGTCATACCAG. GAPDH amplification levels served as the internal control. Primer sequences of p73, Apaf1, p53, BAX, Bclreported , 55. Thereported .10 million cells were transfected with the indicated plasmids. Cells were harvested and Myc-tagged AK-B was immunoprecipitated (IP) using an antibody specific for the Myc epitope (9E10). A detailed protocol has been published earlier . Radioac


Specifically, we performed parallel labeling experiments with four fully 13C-labeled tracers, [U-13C]valine, [U-13C]leucine, [U-13C]isoleucine and [U-13C]glutamine. We measured mass isotopomer distributions of fatty acids and intracellular metabolites by GC-MS and analyzed the data using the isotopomer spectral analysis (ISA) framework. We demonstrate that 3T3-L1 adipocytes accumulate significant amounts of even chain length  and odd chain length (C15:0 and C17:0) fatty acids under standard cell culture conditions. Using a novel GC-MS method, we demonstrate that propionyl-CoA acts as the primer on fatty acid synthase for the production of odd chain fatty acids. BCAA contributed significantly to the production of all fatty acids. Leucine and isoleucine contributed at least 25% to lipogenic acetyl-CoA pool, and valine and isoleucine contributed 100% to lipogenic propionyl-CoA pool. Our results further suggest that low activity of methylmalonyl-CoA mutase and mass action kinetics of propionyl-CoA on fatty acid synthase result in high rates of odd chain fatty acid synthesis in 3T3-L1 cells. Overall, this work provides important new insights into the connection between BCAA catabolism and fatty acid synthesis in adipocytes and underscores the high capacity of adipocytes for metabolizing BCAA.The branched chain amino acids (BCAA) valine, leucine and isoleucine have been implicated in a number of diseases including obesity, insulin resistance, and type 2 diabetes mellitus, although the mechanisms are still poorly understood. Adipose tissue plays an important role in BCAA homeostasis by actively metabolizing circulating BCAA. In this work, we have investigated the link between BCAA catabolism and fatty acid synthesis in 3T3-L1 adipocytes using parallel  The branched chain amino acids (BCAA) valine, leucine and isoleucine are essential amino acids that comprise about 15–25% of total protein intake ; howeverDue to poor catabolism of BCAA in the liver, BCAA can circulate through the bloodstream and act as signaling molecules of the nutritional state. BCAA, especially leucine, have been implicated as important sensor molecules that interact with the cell survival signaling pathway, i.e. mammalian target of rapamycin (mTOR), and the insulin signaling cascade ,4,5. EleAdipose tissue, an often underappreciated endocrine organ and regulator of energy homeostasis ,14, may in vitro model system for adipose tissue and are commonly used as this cell line has similar phenotypic behavior as adipose tissue. It has been well documented that BCAA catabolic enzyme activities and expression levels greatly increase over the course of 3T3-L1 differentiation valine, [U-13C]leucine, [U-13C]isoleucine and [U-13C]glutamine. While conducting this work, we noted a significant accumulation of odd chain length fatty acids in 3T3-L1 adipocytes. With the help of a novel GC-MS analysis technique we systematically elucidated the pathway of odd chain fatty acid synthesis. Overall, this work represents an important advance towards understanding the interconnection between BCAA and fatty acid metabolism in adipocytes. Moreover, this study demonstrates the power of using parallel 13C-labeling experiments for pathway quantification and highlights a novel GC-MS methodology for fatty acid analysis.In this work, we have quantified for the first time the contribution of BCAA to fatty acid synthesis in 3T3-L1 adipocytes. To this end, parallel isotopic labeling experiments were performed with four 13C]Valine (99 atom% 13C), [U-13C]leucine (98%), isoleucine (99% 13C), [U-13C]glutamine (98% 13C) and [U-13C]algal amino acids (97+% 13C) were purchased from Sigma-Aldrich . Free amino acids were purchased from Fisher Sci. . Amino acid standard H was purchased from Pierce Sci. . Stock solutions (40x) of natural and uniformly 13C-labeled valine, leucine, and isoleucine were prepared at 40mM in deionized water. Stock solutions (40x) of natural and [U-13C]glutamine were prepared at 160mM in deionized water. [U-13C]algal amino acids were solubilized at 10mg/mL in 0.1N HCl phosphate buffered saline. All amino acid solutions were stored at -85°C until use.Culture materials were purchased from Cellgro . valine (MPE ~45%); 2) [U-13C]leucine (MPE ~42%); 3) isoleucine (MPE ~54%); 4) [U-13C]glutamine (MPE ~97%). The molar percent enrichment (MPE) of the BCAA tracers is less than 100%, since the BCAA tracers were added in addition to the ~0.8mM already present in DMEM media. All isotopic tracer experiments were conducted with four biological replicates (n = 4) where the only difference was which amino acid was labeled. In addition to the 16 flasks for the labeling experiments, six flasks were used for cell counting. Furthermore, four flasks without tracer addition were used for quantifying media metabolites and 500μL of media was removed at 0, 6, 12 and 24 hours. At the end of the labeling experiment (day 7), tracer experiment flasks were harvested and cells were extracted as described below to obtain intracellular metabolites for analysis of 13C-labeling by GC-MS. All samples were stored at -85°C prior to analysis.Four stable isotope tracers were used in the study: 1) algal solution, 10 μL of 60 mM [U-13C]glutamine and 30 μL of 0.1N HCl algal solution were determined  was conducted on the measured mass isotopomer distributions (MIDs) of C14:0, C15:0, C16:0, C17:0 and C18:0 fatty acids  to deter13C-glutamine and 13C-BCAA tracers. For example, rather than assuming that [U-13C]glutamine and [U-13C]-labeled BCAA would produce M+2 labeled AcCoA, in the ISA model we allowed each [U-13C]-labeled tracer to produce M+1 and M+2 AcCoA; similarly, [U-13C]valine and [U-13C]isoleucine were allowed to produce M+1, M+2 and M+3 labeled PropCoA , the cells were chemically differentiated with insulin, dexamethasone and IBMX to achieve the mature adipocyte phenotype . Undiffe13C-tracer experiments, it was desirable to have noticeable accumulation of triglycerides over the duration of the tracer experiment. We determined that day 6 would be ideal for isotopic experiments, since the adipocyte phenotype has already been developed by that time glutamine experiments, medium glutamine was initially 100% M+5 labeled; however after 24 hours glutamine was only about 60% M+5 labeled  by the cells . Signifi glycine . SecretiIntracellular fatty acids were assayed by GC-MS after extraction of cellular triglycerides and subsequent transesterification with methanol. A representative total ion chromatogram for fatty acid methyl esters (FAMEs) is shown in 13C]valine, [U-13C]leucine, [U-13C]isoleucine, and [U-13C]glutamine as tracers. Mass isotopomer distributions of FAMEs of pentadecanoic (C15:0) and palmitic (C16:0) acid are shown in 13C]valine, C15:0 had a significant M+3 isotopomer, whereas C16:0 did not; 2) for [U-13C]isoleucine, C15:0 had a significant M+3 isotopomer and C16:0 did not; both fatty acids had M+2 isotopomers; 3) for [U-13C]leucine, both fatty acids had M+1 and M+2 isotopomers; 4) for [U-13C]glutamine, both fatty acids had M+2 and M+4 isotopomers.To investigate a potential link between BCAA catabolism and fatty acid synthesis, experiments were conducted with [U-13C]valine and [U-13C]isoleucine experiments. As expected, palmitic acid did not show M+3 incorporation, since even chain fatty acids are formed solely from condensation of AcCoA. Isoleucine degradation also contributes to AcCoA, which explains the M+2 isotopomers in [U-13C]isoleucine experiments. Leucine degrades only into AcCoA, which accounts for M+1 and M+2 isotopomers in the [U-13C]leucine experiment. Details regarding the formation of M+1 isotopomers from [U-13C]leucine are discussed in the next section. Glutamine also contributes to lipogenic AcCoA pool, which explains the M+2 and M+4 isotopomers for [U-13C]glutamine experiments.Catabolism of valine and isoleucine is known to produce propionyl-CoA (PropCoA), which can account for M+3 isotopomers in valine, this would result in pentadecanoic acid being labeled on the first three carbons from the ω-end, i.e. carbon atoms C13-15. By applying the picolinyl ester derivatization for the [U-13C]valine experiment, this hypothesis was tested by measuring labeling of fragments corresponding to the removal of carbon atoms C13, C14 and C15 from pentadecanoic acid , as well as the fraction of newly synthesized fatty acids . First, we demonstrated that 3T3-L1 cells accumulated both even and odd chain length fatty acids. Using a novel GC-MS method, we also demonstrated that PropCoA acts as the primer on fatty acid synthase for the production of odd chain fatty acids.In this study, we have investigated the connection between branched chain amino acid and fatty acid metabolism in 3T3-L1 adipocytes. Parallel labeling experiments were performed where the DMEM medium was supplemented with additional amino acids consisting of a mixture of valine, leucine, isoleucine and glutamine. Of the supplemented amino acids, one was uniformally 6 cells/h, or about 28% that of the glucose uptake rate. We determined that BCAA contributed significantly to lipogenic AcCoA and PropCoA pools. Based on ISA modeling results, we determined that ~25% of lipogenic AcCoA was derived from leucine and isoleucine. This value was consistent for all measured fatty acids, which included C14:0, C15:0, C16:0, C17:0 and C18:0. Despite the fact that there was no net consumption of glutamine, glutamine still contributed ~10% to AcCoA due to high exchange between medium glutamine and intracellular glutamine. Lipogenic PropCoA was derived solely from isoleucine and valine catabolism. From these results, it is evident that all three BCAA contribute a significant portion to overall lipid synthesis. It is important to note that the BCAA concentrations used in these experiments are several fold greater than observed in plasma and ~2-fold greater than DMEM media. While the concentrations are supra-physiological, these experiments do demonstrate the capacity of the 3T3-L1 adipocyte model to catabolize BCAA and the interconnection between fatty acid and BCAA metabolism.Previously, the contributions of glucose and acetate to palmitic acid synthesis in 3T3-L1 cells were determined . In addi13C]glutamine experiment was the transience of 13C-labeling in the medium. Initially, glutamine was 100% M+5 labeled; however, at the end of the 24-hour incubation, M+5 labeling was only about 60%. An assumption of ISA modeling is that the precursor pools are at steady state isotopic labeling, i.e. the labeling does not change with time. For the [U-13C]glutamine experiment this assumption is thus not valid. Since external glutamine labeling is changing with time, this will have a dynamic effect on the labeling of the resulting AcCoA pool as well. It is important to note, however, that the steady state assumption was valid for the other 13C-tracers used in this study. The observation of non-steady labeling of AcCoA for 13C-glutamine highlights one of the limitations of the ISA modeling technique. In light of this observation, it is important to discuss how it may affect the modeling results.One important observation from the [U-13C]glutamine labeling data is non-steady, the corresponding measured mass isotopomers will be a time-weighted average labeling and the total amount of 13C-labeling incorporation will be less than would be expected if [U-13C]glutamine labeling remained constant throughout the entire experiment. As a result, the g-parameter would be a time-averaged value. The value will be less than determined from [U-13C]leucine and [U-13C]isoleucine experiments. The discrepancy in g-values would not be an issue if modeling [U-13C]glutamine data alone; however, modeling the data with the other tracer experiments could pose a problem.Since [U-13C]valine experiments will set a hard constraint on the g-value, since it only contributes to the PropCoA pool and not AcCoA pool. This will cause the g-value to be fixed at the true value, although this value will be greater than would be predicted with [U-13C]glutamine. As a result, there will be too much 13C-labeling to explain the [U-13C]glutamine data and in order to compensate for this the model will decrease the amount of labeling, e.g. by decreasing the D-value for glutamine. The AcCoA and PropCoA precursor pools are interconnected due to isoleucine contributing to both pools. As a result, errors resulting in D-values for the AcCoA may cause indirect effects on the PropCoA pool (i.e. the low PropCoA contribution of M+2 from [U-13C]isoleucine). Based on this logic, we believe that the D-value estimates for AcCoA are underestimates. The glutamine dynamics does not invalidate our findings, but instead of determining the exact contributions, we deem the values to be the lower limits for this system.To gain more insight, consider the case of odd chain fatty acids. In this case, the valine experiments, we saw negligible labeling incorporation into TCA cycle intermediates  catabolism and fatty acid synthesis in adipocytes. We demonstrate that at least 25% of lipogenic acetyl-CoA is derived from BCAA catabolism in cultured 3T3-L1 adipocytes, and that propionyl-CoA, which serves as the precursor for odd chain fatty acid synthesis, is derived from catabolism of valine and isoleucine. Our results suggest that low activity of methylmalonyl-CoA mutase and mass action kinetics of propionyl-CoA on fatty acid synthase contribute to the high rates of odd chain fatty acid synthesis. Another important contribution of this work is in demonstrating the value of using parallel labeling experiments for quantitative pathway elucidation ,51, and S1 FileCell culture data, mass spectrometry data and modeling results.(DOCX)Click here for additional data file.


Proteobacteria possess the so-called nitrogen PTS (PTSNtr) that transfers a phosphate group from phosphoenolpyruvate (PEP) over enzyme INtr (EINtr) and NPr to enzyme IIANtr (EIIANtr). The PTSNtr lacks membrane-bound components and functions exclusively in a regulatory capacity. While EIIANtr has been implicated in a variety of cellular processes such as potassium homeostasis, phosphate starvation, nitrogen metabolism, carbon metabolism, regulation of ABC transporters and poly-β-hydroxybutyrate accumulation in many Proteobacteria, the only identified role of NPr is the regulation of biosynthesis of the lipopolysaccharide (LPS) layer by direct interaction with LpxD in Escherichia coli. In this study, we provide another phenotype related to NPr. Several lines of evidence demonstrate that E. coli strains with increased levels of dephosphorylated NPr are sensitive to envelope stresses, such as osmotic, ethanol and SDS stresses, and these phenotypes are independent of LpxD. The C-terminal region of NPr plays an important role in sensitivity to envelope stresses. Thus, our data suggest that the dephospho-form of NPr affects adaptation to envelope stresses through a C-terminus-dependent mechanism.Besides the canonical phosphoenolpyruvate-dependent phosphotransferase system (PTS) for carbohydrate transport, most  The ptslitensis , poly-β-allenged . HoweverNtr in various bacteria, the only identified role of NPr is the regulation of lipid A biosynthesis  was replaced by the ampicillin-resistance gene (AmpR). The ampicillin-resistance gene was amplified by PCR from the pRE1 plasmid with the following primers: forward primer, 5′-TGCTCCGAGCCTGTTCCACTGTTTGAGTGGGCAGGTTCTTAGGTGAAATTATGAGTATTCAACATTTCCG-3′ and reverse primer, 5′-ACCATGTACTGTTTCTCCTCACAACGTCTAAAAGAGACATTACCGAATAATTACCAATGCTTAATCAGTG-3′. The double mutants of the nitrogen PTS genes were constructed by P1 transduction of the antibiotic-resistant gene region. All plasmids were constructed using standard PCR-based cloning procedures and verified by sequencing. To construct pCR2HN, in which expression of NPr tagged with six histidines at its N terminus (His-NPr) is under the control of the pRE1-vector system, the pNPr plasmid was digested with NdeI and BamHI, and the fragment encoding ptsO was cloned into pRE1-His-Tag (NdeI site (underlined) in the ATG start codon (in boldface type) of the ptsO gene (5′-AACGTAACATATGACCGTCAAGCAAACTGT-3′) and a reverse primer with a synthetic BamHI site (underlined) downstream of six histidine codons (5′-AAAGTGAGGATCCTTAGTGGTGGTGGTGGTGGTGATCTTCATCAAAACC-3′) (the stop codon in bold type) were used to amplify the ptsO gene from MG1655 genomic DNA. After digestion, the NdeI–BamHI fragment was inserted into the corresponding sites of pRE1 . To construct pCR2HN85(H16A), which expresses His-NPr(H16A) truncated of the five C-terminal residues, the forward primer of the ptsO gene and a reverse primer with the synthetic BamHI site (underlined) (5′-ATTAATCGGATCCTTAAGAATTAAAGAGGG-3′) (a new stop codon in bold type) were used to amplify the truncated ptsO gene from the pNPr(H16A) plasmid. After digestion, the NdeI–BamHI fragment was inserted into the corresponding sites of pRE1-His-Tag (ptsO gene and a reverse primer (5′-TGAGGATCCTTACTCGAGTTCAGAATTAAA-3′), which has the three codons (CTCGAGTTC) encoding three residues (ELE) at the C-terminal end of HPr to replace the five C-terminal residues with the three residues of HPr. To construct pEINtr(H356A), a mutagenic primer pair covering the region coding for His356, a forward primer (5′-GGCGCAGCCAACTCCGCTGCTGCGATCATG-3′) and a reverse primer (5′-CATGATCGCAGCAGCGGAGTTGGCTGCGCC-3′) (changed bases underlined), was used. Similarly, plasmids for overexpression of point mutant proteins of five residues (GFDED) in the C terminus of NPr were generated using mutagenic primer pairs: D90A-F (5′-TCTGGTTTTGATGAAGCTTAATCTTCATCA-3′), D90A-R (5′-TGATGAAGATTAAGCTTCATCAAAACCAGA-3′), E89A-F (5′-TCTGGTTTTGATGCAGACTAGTCTTCATCA-3′), E89A-R (5′-TGATGAAGACTAGTCTGCATCAAAACCAGA-3′), D88A-F (5′-AATTCTGGTTTTGCTGAGGATTAATCTTCA-3′), D88A-R (5′-TGAAGATTAATCCTCAGCAAAACCAGAATT-3′), F87A-F (5′-CTCTTTAATTCTGGCGCCGATGAAGATTAA-3′), F87A-R (5′-TTAATCTTCATCGGCGCCAGAATTAAAGAG-3′), G86A-F (5′-GCCCTCTTTAATTCTGCTTTTGATGAAGAT-3′) and G86A-R (5′-ATCTTCATCAAAAGCAGAATTAAAGAGGGC-3′) (changed bases underlined). The expression vector pRE1-LpxD, for overproduction of LpxD, was generated using a primer pair: LpxD-F, 5′-TAAATAACATATGCCTTCAATTCGACTGGC-3′ (NdeI site underlined) and LpxD-R, 5′-GAACAAAGGATCCAACGTTAGTCTTGTTGA-3′ (BamHI site underlined). To remove the internal NdeI site within the lpxD gene, we designed an additional primer pair covering the internal NdeI site: LpxD-LK-F, 5′-CGTAATCAACGGGCTTATGGAAATATGCGA-3′ and LpxD-LK-R, 5′-AGCCCGTTGATTACGCTGGCTCCGCCGATC-3′ (changed bases underlined). We carried out the first PCRs to amplify the lpxD gene from MG1655 genomic DNA using the LpxD-F/LpxD-LK-R pair and the LpxD-R/LpxD-LK-F pair. The mixture of first PCR products was used as template for the second PCR using the LpxD-F/LpxD-R pair. The second PCR product was digested with NdeI and BamHI and the fragment encoding lpxD was cloned into pRE1.The bacterial strains and plasmids used in this study are listed in Table S1, available in the online Supplementary Material. Bacterial cells were grown as described previously  according to the manufacturer’s instructions from cells grown to mid-exponential phase in LB medium with or without 1 mM IPTG. The preparations were treated with RNase-free DNase (Promega) at 37 °C for at least 1 h to eliminate contaminating DNA. The absence of contaminating genomic DNA in RNA preparations was verified by PCR. The same amount of RNA from each culture was converted into cDNA using the cDNA EcoDry Premix (Clontech). The cDNAs were diluted 10-fold and subjected to RT-PCR analyses using lpxD-specific primers: forward primer, 5′-ATGCCTTCAATTCGACTGGCTGATTTAGCG-3′; reverse primer, 5′-GCAACCGGCACCGATAATCACGTTATCGCC-3′. The 16S rRNA transcript was used as a loading control. The amplification reactions were performed in a GeneAmp PCR System for 5 min at 94 °C, followed by 15 (16S rRNA) or 25 cycles (lpxD) of 94 °C for 20 s, 55 °C for 20 s and 72 °C for 1 min per kb, concluding with extension at 72 °C for 4 min. The transcript levels of ptsO were analysed by a similar manner using a ptsO-specific primer pair: forward primer, 5′-ATGACCGTCAAGCAAACTGTTGAAATCACA-3′; reverse primer, 5′-AAACCAGAATTAAAGAGGGCGATAACGGCG-3′.The transcript levels of To determine the intracellular levels of mutant NPrs, we made polyclonal antibodies against NPr using female ICR mice. Cells were grown in LB medium to mid-exponential phase and 0.4 ml of cell culture was collected. After boiling for 5 min, the samples were analysed with 15 % SDS-polyacrylamide gels. Immunoblotting was performed according to standard procedures using specific antibodies.2 and 2 % Triton X-100 for 15 min. After cooling at room temperature for 10 min, the mixture was centrifuged at 16 000 g at 25 °C for 15 min. LPS in the pellet was solubilized by incubating with shaking in a solution containing 50 mM EDTA and 2 % Triton X-100 at 37 °C for 4 h. The suspension was then centrifuged at 16 000 g at 37 °C for 15 min and the supernatant solution was transferred to a fresh tube. To reprecipitate LPS, the supernatant was mixed with MgCl2 , incubated at 37 °C for 3 h and centrifuged at 20 000 g for 90 min at 37 °C. The transparent LPS precipitate was resuspended in 2× SDS sample buffer and boiled for 5 min. Aliquots of the samples were analysed by SDS-PAGE and visualized by silver staining.LPS was isolated as described previously (Ntr (encoded by ptsP), the first enzyme in the phosphoryl-transfer cascade of the nitrogen PTS, significantly decreased salt tolerance of MG1655 cells in the LB medium containing 750 mM NaCl, whereas the deletion of the ptsO or ptsN gene hardly affected the sensitivity to salt stress  II which catalyses the first common step in the biosynthetic pathway of the three branched-chain amino acids. To clarify whether a growth defect of the ptsP mutant on salt stress was dependent on the ilvG genotype, we constructed a ptsP mutant strain in the ilvG+ genetic background and tested the effect on salt stress. As shown in Fig. S1, the ptsP mutant having a functional ilvG gene was also hypersensitive to salt stress, suggesting that this phenotype of the ptsP mutant was independent of the ilvG genotype.One of physiological roles of the nitrogen PTS is the maintenance of potassium homeostasis through regulating TrkA and KdpD . To knowt stress . HyperselvG gene . The ilvptsP mutant, we first checked whether the presence of the phospho-form of EINtr was necessary for the growth of E. coli cells in salt stress. We constructed a pRE1-based plasmid, pCR1(H356A), expressing a mutant form of EINtr in which the phosphorylatable His356 residue was mutated to Ala. Although the ptsP mutant transformed with pCR1 expressing wild-type EINtr could grow in the LB medium containing 750 mM NaCl to a level of WT cells, the growth defect of the ptsP mutant under salt stress could not be recovered by expression of EINtr(H356A)  . This inNtr→NPr→EIIANtr  were as sensitive to salt stress as the ptsP mutant  plasmid, expressing a mutant form of NPr which cannot be phosphorylated by EIP mutant . The samO-(R-3-hydroxymyristoyl)-glucosamine. Because an increase of dephosphorylated NPr can inhibit the LpxD activity, we assumed that sensitivity of the ptsP mutant to salt stress might be due to a decreased LpxD activity. To verify this assumption, we examined whether overexpression of LpxD could restore the growth of the ptsP mutant under salt stress conditions. The expression level of lpxD was significantly increased in the ptsP mutant cells harbouring the pET11d-based LpxD expression vector pDC015-1 . However, salt sensitivity of these cells was hardly affected by the LpxD level . Therefore, these data indicate that the sensitivity to salt stress caused by an increased level of dephosphorylated NPr was independent of NPr-mediated LpxD inhibition.In a previous report, it was shown that dephosphorylated NPr inhibits LpxD by direct interaction . In addition, the salt hypersensitivity of the dephosphorylated NPr-overproducing strain was also not suppressed by overexpression of the osmY gene. Therefore, these results suggested that the salt hypersensitivity of the ptsP mutant was independent of the expression level of osmY. Although the two genes within the rppH-ptsP operon are involved in the same phenotypic feature, their molecular mechanisms seem to be different. This conclusion is also supported by the fact that, despite the significantly increased expression level of osmY in the rppH mutant and the rppH ptsP double mutant, compared with WT and the ptsP mutant , the rppH ptsP double mutant was as sensitive to salt stress as the ptsP mutant . Phenotypes of the double mutant strains of the nitrogen PTS at sucrose stress were similar to those at salt stress . Additionally, cells expressing NPr(H16A) were extremely sensitive to sucrose stress, like the ptsP mutant , indicating that the dephospho-form of NPr regulates various osmotic stresses. The sensitivity to this stress was also independent of NPr-mediated LpxD inhibition .In addition to salt stress, the effect of other osmotic stress such as sucrose was investigated. The ptsP mutant under other stress conditions. We found that the ptsP mutant was also sensitive to ethanol and SDS stresses  was significantly insoluble, whereas NPr with six C-terminal histidines (NPr-His) was highly soluble (did not make an inclusion body) and was easier to purify than His-NPr (data not shown). These results implied that the intracellular feature of NPr-His may be considerably different from that of His-NPr. To explore this assumption, we constructed two pRE1-based plasmids, a pCR2HN(H16A) plasmid expressing His-NPr(H16A) and a pCR2HC(H16A) plasmid expressing NPr(H16A)-His. Like cells expressing NPr(H16A) without a His tag, the cells expressing His-NPr(H16A) were sensitive to envelope stresses . HoweverptsP mutant became extremely sensitive to salt stress, whereas ptsO and ptsN mutants exhibited normal growth under high salt conditions (ptsP mutant on salt stress was recovered by additional deletion of the ptsO gene, but not by additional deletion of the ptsN gene, suggesting that an increased level of dephosphorylated NPr in the ptsP mutant renders cells sensitive to salt stress (In this study, we investigated a novel cellular role of dephosphorylated NPr on envelope stresses. Phenotype analyses using single and double mutants of the nitrogen PTS genes provided us with a clue that the dephospho-form of NPr is involved in sensitivity to envelope stresses, such as osmotic, ethanol and SDS stress. The nditions . Growth t stress . Furthert stress  and S2. t stress  and S6.rpoE, it was suggested that ptsO and ptsN genes, together with the rpoN gene, are under the control of the extracytoplasmic stress sigma factor, σE (Ntr suppresses the essentiality of σE (Ntr reduced extracytoplasmic stress, whereas the dephosphorylated form of NPr increased envelope stress. The dephosphorylated form of EIIANtr regulates homeostasis of potassium (rpoN is involved in the envelope stress response remains obscure, a recent report using a comparative genome analysis suggested that the enigmatic sigma factor σ54 is a central controller of the bacterial exterior (rpoN operon including the nitrogen PTS and the envelope stress response needs to be investigated in more detail.Together with our results, several lines of evidence support the connection between the envelope stress response and the nitrogen PTS. From an accurate promoter prediction model and the upregulation upon overexpression of ty of σE . Notablyotassium , which iotassium . It was otassium . AlthougptsP mutant is caused by increased levels of the dephospho-form of NPr. Mutation of the ptsP gene in various bacteria affected various physiological processes, including virulence (ptsP mutant might be caused by the dephospho-form of NPr.From experiments using double deletion mutants of the nitrogen PTS genes, we discovered that the growth defect of the irulence , pyocyanThis study also demonstrated that the change in the C-terminal region of NPr abolishes its capability to negatively regulate the adaptation to envelope stresses. Because of the difficulty in overexpression and purification of wild-type NPr, the three-dimensional structure of NPr has been determined only in modified forms at the C terminus, a form fused with an intein sequence to a chitin-binding domain (


EGFR mutant selective TKIs are being explored to conquer this resistance. AZD9291  has been approved for treatment of the metastatic EGFR T790M mutation-positive non-small cell lung cancer. Resistance to AZD9291 has been described. C797S mutation was reported to be a major mechanism for resistance to T790M-targeting EGFR inhibitors. This review summarizes the latest development in identifying the C797S mutation and EAI045, the novel selective inhibitor overcoming the C797S mutant.T790M mutation is the most common mechanism for resistance to first- and second-generation tyrosine kinase inhibitors (TKI) for epidermal growth factor receptor (EGFR). Several third-generation  EGFR) tyrosine kinase inhibitors (TKI) [EGFR T790M-positive non-small cell lung cancer (NSCLC) patients [EGFR Cys797Ser (C797S) mutation, located within the tyrosine kinase domain, was recently reported to be a potential mechanism of resistance to irreversible EGFR inhibitors such as AZD9291, HM61713, WZ4002, and CO-1686 in T790M-positive patients [T790M mutation is the most common mechanism of resistance to the first- and second-generation of epidermal growth factor receptor (rs (TKI) . Clinicars (TKI) , 3. Thesrs (TKI) –10. AZD9patients , 10. HM6patients . Unfortupatients . EGFR Cypatients –16  leading to C797S amino acid mutation occurred in the same patients. Since only 6 out of 15 cases acquired C797S mutation, additional mechanisms of resistance to AZD9291 must be present.In the first-in-human phase I/II AURA trial of AZD9291, systemic progression in NSCLC patients was seen after treatment for a median of 9.6 months . CharactEGFR 19 deletion (del 19) and T790M at this point. She was enrolled in the phase 1 AURA study of AZD9291 (NCT01802632) and received AZD9291 for 9 months prior to disease progression. Tumor biopsy at this juncture showed the EGFR C797S mutation, in addition to the del 19 and T790M. Under the strong selective pressure of EGFR-TKIs, the tumor developed secondary T790M and tertiary C797S mutations in the EGFR gene to bypass the TKIs and maintain EGFR signaling.In a separate case report, a female non-smoker with widely metastatic lung adenocarcinoma progressed through first-line chemotherapy and second-line erlotinib . She wasHM61713 (BI 1482694) is another third-generation EGFR inhibitor and covalently binds to a cysteine residue near the kinase domain of mutant EGFR , 19. In EGFR del 19 [The first case report on resistance to HM61713 was on a 57-year-old female never-smoker with stage IV lung adenocarcinoma harboring EGFR gene, a group from Dana Farber Cancer Center utilized site-directed mutagenesis in EGFR mutant Ba/F3 cell lines harboring sensitizing mutations and/or T790M [EGFR L718Q, L844V, and C797S. All of the three mutations could cause resistance to both WZ4002 and CO-1686. Only C797S mutation confers AZD9291 resistance. Most interestingly, in the presence of del 19 or L858R and T790M, C797S mutation leads to resistance to all current EGFR inhibitors , but L858R/T790M/C797S mutant remains partially sensitive to cetuximab. It remains to be determined whether cetuximab or cetuximab-based combinations are effective clinically in NSCLC patients that develop the L858R/T790M/C797S mutant clone.To search for acquired resistance mutations in or T790M . The celcis) or in a different allele in the same cell  on the sensitivity to TKIs. It was clearly demonstrated in the in vitro system that del19/T790M was resistant to the second-generation TKIs, whereas del19/C797S was resistant to the third-generation inhibitors. When T790M and C797S were present in cis, the cells were resistant to all EGFR TKIs. Therefore, characterization of mutation status may guide clinical decision on therapeutic approaches.In a separate study, a cell line, MGH121, was established from pleural effusion of a NSCLC patient who became resistant to erlotinib . This ceHER2 amplification was identified without C797S mutation from the tumor biopsy.Since some patients who progressed on AZD9291 were negative for the C797S mutation, additional resistance mechanisms must be present. In a case report, a 54-year-old male with stage IV adenocarcinoma was found to have acquired T790M mutation after progression from second-line treatment with gefitinib . The disEGFR activating mutation in exon 21, L858R. She received erlotinib and gefitinib sequentially for 12 months. After disease progression, T790M was identified by NGS and she was treated with AZD9291 on the AURA trial [EGFR activating mutation and CMET amplification without T790M or C797S mutation. These two cases indicated that in refractory NSCLC without T790M or C797S mutations, additional gene mutations or amplifications of tyrosine kinases other than EGFR can be the mechanisms of resistance. Additional treatment targeting the mutations will be needed.The second patient from the same report was a 60-year-old female, a never-smoker, who was diagnosed with stage IV adenocarcinoma with pleural metastasis . MutatioRA trial . She hadUsing purified EGFR mutant kinase peptide containing L858R/T790M mutations, a library of approximately 2.5 million compounds were screened to search for selective inhibitors against the kinase mutant . EGFR alEGFR gene was found to confer resistance clinically to third-generation TKIs, AZD9291 and HM61713. This likely represents a tertiary acquired mutation that mediates resistance to all known third-generation EGFR TKIs. Some cases were found to harbor two independent clones of C797S mutation, while others even became T790M negative, indicating the heterogeneity of malignant cells. This remains a significant challenge for treatment of lung cancers.C797S mutation in the C481S mutation has been reported to mediate resistance to ibrutinib, the first-in-class irreversible Bruton tyrosine kinase (BTK) inhibitor –29. IbruSince more and more TKIs targeting EGFR and anaplastic lymphoma kinase (ALK) mutations are being used for NSCLC therapy –34, moleHER2 and MET amplification in C797S negative cases, were identified. Loss of T790M mutation was another mechanism of resistance to AZD9291 [With the availability of AZD9291 for clinical treatment of T790M-positive NSCLC, more and more resistant cases will appear. Additional mechanisms of resistance to third-generation TKIs, such as  AZD9291 . Due to  AZD9291 . AZD9291 AZD9291 –51. CombEGFR C797S mutation mediates resistance to third-generation TKIs, AZD9291 and HM61713. Additional mechanisms of resistance are being identified. Combination therapy of AZD9291 with other agents may be one way to overcome the acquired mutation. EAI045 represents a purposefully designed selective inhibitor overcoming EGFR C797S mutation.ALK, anaplastic lymphoma kinase; BTK, Bruton tyrosine kinase; cfDNA, cell-free DNA; EGFR, epidermal growth factor receptor; NGS, next-gene sequencing; NSCLC, non-small cell lung cancer


Four avian metapneumovirus (AMPV) subgroups (A–D) have been reported previously based on genetic and antigenic differences. However, until now full length sequences of the only known isolates of European subgroup C and subgroup D viruses  have been unavailable. These full length sequences were determined and compared with other full length AMPV and human metapneumoviruses (HMPV) sequences reported previously, using phylogenetics, comparisons of nucleic and amino acid sequences and study of codon usage bias. Results confirmed that subgroup C viruses were more closely related to HMPV than they were to the other AMPV subgroups in the study. This was consistent with previous findings using partial genome sequences. Closer relationships between AMPV-A, B and D were also evident throughout the majority of results. Three metapneumovirus “clusters” HMPV, AMPV-C and AMPV-A, B and D were further supported by codon bias and phylogenetics. The data presented here together with those of previous studies describing antigenic relationships also between AMPV-A, B and D and between AMPV-C and HMPV may call for a subclassification of metapneumoviruses similar to that used for avian paramyxoviruses, grouping AMPV-A, B and D as type I metapneumoviruses and AMPV-C and HMPV as type II. Metapneumovirus (MPV), in the family Paramyxoviridae, subfamily Pneumovirinae, includes globally important viruses in avian and human health. Avian MPV (AMPV) MetapneumovirusPneumovirus genus The Genus MPV have non segmented, single stranded, negative sense RNA genomes between 13.1 and 14.2 kb which are known to encode 9 proteins. MPV genomes are organized in the order 3′-leader-N-P-M-F-M2-SH-G-L-trailer-5′. Genetic and antigenic studies have revealed four AMPV subgroups (A to D) and two HMPV subgroups (A and B) with a high similarity between HMPV subgroup A (strain 001) and AMPV-C Determining full length sequences of viral genomes is an essential step towards studying the possible molecular basis for host tropism or pathogenicity, first by allowing the development of reverse genetics systems for the studied strains, and second by allowing genome wide sequence comparisons highlighting relevant regions to study using reverse genetics. Full length genome sequences of subgroup A and B viruses are available In combination with sequences described previously 104.20 and log105.00 TCID50/ml, respectively Fr-AMPV-C Muscovy duck/France/1999/99178 and Fr-AMPV-D Turkey/France/1985/85035  Nc = 2+9/F2+1/F3+5/F4+3/F6, F2 corresponds to the probability that two randomly chosen codons for an amino acid, possibly encoded by two distinct codons, are identical. F3 is the probability that three randomly chosen codons for an amino acid with three synonymous codons are identical and so on for F4 and F6. The Nc value was determined using CodonW 1.4.4 (http://codonw.souceforge.net) and was correlated to the percentage of G+C at the third position (GC3) as it has been shown previously to be a major factor influencing the synonymous codon usage pattern in the HMPV genome In the Nc value calculation formula: All available AMPV full-length genome sequences and one representative of each of the four HMPV sublineages were aligned using Clustal W. Alignments were also checked manually for a good correspondence of the common coding regions. Phylogenetic analysis was performed using MEGA 5.2 with the Neighbor-Joining method (1000 boostrap replicates) and the Kimura-2-parameter substitution model.The full length consensus sequences for Fr-AMPV-C and Fr-AMPV-D were 14152bp- and 13415bp-long, respectively. Three significant clusters were observed, one grouping all HMPVs, a second grouping AMPV-Cs and a third grouping the AMPV-A, B and D subgroups .Within the AMPV-C cluster, viruses isolated from Muscovy ducks  formed a separate sub-lineage from the others. This separation is potentially related to species rather than geographic origin as the Asian SO1, GDY, PL-1 and PL-2 isolates were split into different clusters  was identical to previously reported AMPV-C N ORFs regardless of host species and to those of all HMPV sublineages . High aaSubgroups A, B and D viruses were more closely related in terms of length and aa identity than they were to AMPV-Cs or HMPVs. Indeed, the length of the Fr-AMPV-D N protein (391aa) was identical to that of both AMPV-A and B and three amino acids shorter than AMPV-C and HMPV N proteins. Amino acid identities were high with A and B (89–90%) but lower with subgroup C and HMPV N proteins (71–74%). Localization of aa differences can be seen in The second main ORF in the AMPV genome encodes the P protein, which is also part of the polymerase complex. Consistent with N protein comparisons, the P ORFs of AMPV-Cs and HMPVs were more closely related than they were to AMPV-A, B and D. In the same respect, subgroups A, B and D also demonstrated closer relationships.The length of the Fr-AMPV-C, P ORF (294aa) was identical to other previously reported subgroup C P ORFs regardless of host species, and to those of all HMPV subgroups . The P OIn common with all subgroup C and HMPV P protein sequences analyzed previously The length of the Fr-AMPV-D P protein (278aa) was identical to subgroup A and just one aa shorter than the P protein of subgroup B. Amino acid identities were 71–72% conserved with subgroups A and B. In contrast to subgroup C and HMPV P proteins, subgroups A, B and D contained cysteine residues . CysteinThe third main ORF in the AMPV genome encodes the M protein which orchestrates the assembly of viral components at the plasma membrane, through interactions with the viral glycoproteins and nucleocapsid The fourth main ORF in the AMPV genome encodes the highly antigenic, type I membrane fusion protein F. In paramyxoviruses, F is synthesized as an inactive single precursor F0, which is directed to the endoplasmic reticulum by its N-terminal signal peptide. F0 is then cleaved at an arginine-rich cleavage site, mostly by host endoproteases such as furins, into functional F1–F2 subunits held together by disulfide bonds. The F1 subunit remains inserted into the virus membrane by its carboxy-terminal transmembrane domain Clv in The length of the Fr-AMPV-C, F ORF (537aa) was identical to previously reported AMPV-C F ORFs, one aa shorter than the F of AMPV-A, B and D, and two aa shorter than HMPV F . The aa The sequence of the signal peptide aa 1–18  at the N329RGD331  The F1 cytoplasmic tail also exhibited inter subgroup variation . Indeed,Finally, several regions in the F1 subunits of pneumoviruses and MPVs are important in the production of neutralizing antibodies The M2 gene contains two overlapping ORFs (M2.1 and M2.2). M2.1 is involved in virus synthesis and enhances the processivity of the viral polymerase whilst M2.2 has been suggested to alter the balance between transcription and replication In M2.2, a similar level of conservation was seen between subgroup C sequences (93–99%) but conservation was notably lower with the HMPV sublineages (54–58%) and with AMPV-A, B and D (20–24%) which was consistent with previous literature Three cysteine residues were conserved across all MPVs  and a further two conserved between AMPV-A, B and D (aa22 and 59). Cysteines 7, 16 and 22 fell within a region (aa0–25) identified in HMPV as critical to promote viral gene transcription SH is a small type II membrane glycoprotein protein localized in the endoplasmic reticulum, golgi and cell surface SH length (175aa) was conserved across all AMPVs with the exception of subgroup B (180aa). This was in contrast to the varying lengths seen in HMPV SH (177–183aa). A range of 83–100% aa identity was seen between all subgroup C SH sequences. SH conservation between AMPV-A, B and D was considerably lower (42–49% aa identity), and even lower between HMPVs and all APMVs (14–31%). In the SH transmembrane domain, subgroup-C sequences demonstrated a closer relationship with HMPVs (39–50%) than they did with subgroups A, B or D (19–30%). Subgroups A, B and D were more closely related in their transmembrane domains (70–86%).Further relationships between AMPV-C/HMPV or AMPV-A/B/D were evident in the conservation of cysteine residues. AMPV-A, B and D had fourteen (3 in the intracellular and 11 in the extra cellular domain) and AMPV-Cs and HMPVs had nine in the extracellular domain. Seven cysteines were conserved across all MPVs in the extracellular domain.B was introduced into a SH-deleted AMPV-A genome background. A similar result could not be achieved using SHCThese features make the SH protein the second most variable protein (after the G gene) in the MPV genome, with respect to inter-subgroup aa identity. Interestingly inter-subgroup differences did not prevent the restoration of a typical phenotype when SHG is a heavily glycosylated type II membrane protein, involved in, but not essential for virus attachment We have reported previously genetic analysis of the large G ORF in Fr-AMPV-C The final ORF of metapneumovirus genomes encode the large RNA-dependent RNA polymerase protein L. It is a major part of the polymerase complex responsible for most of the enzymatic processes involved in transcription and replication The length of Fr-AMPV-C L (2005aa) was identical to all other AMPV-C and HMPV sequences, one aa shorter than that of AMPV-A and B and two shorter than that of Fr-AMPV-D. Extremely high aa conservation was observed amongst subgroup-C viruses (98–100%), and amongst HMPV sublineages (94–99%). Closer relationships were observed between subgroup-C viruses and HMPVs (80–81%) than between these viruses and subgroups A, B, D (63–64%). Subgroup D again demonstrated a closer relationship with subgroups A and B (84–86%).NGDNQ. AMPV-A, B and D shared four or five amino acids in motif D that were not represented in the subgroup C viruses or HMPVs  have been identified in the L proteins of non segmented negative strand viruses or HMPVs . FurtherDifferent groups were revealed by the codon bias analysis i) HMPVs (Nc = 41.9 to 43.2) ii) AMPV-Cs (47.1 and 47.5) and iii) AMPV-A, B, D (51.4 to 52.7) . Data poThe numbers of nt between two consecutive ORFs  in Fr-AMPV-C were consistent with other subgroup Cs . The smaWe have previously described the 3′ and 5′ sequence extremities of Fr-AMPV-C This study provides the full length genome sequences for two new AMPV strains including the first full length sequence for AMPV subgroup D. Results supported previous reports that AMPV-C viruses are indeed more closely related with HMPVs than they are with other AMPV subgroups, and further demonstrate that AMPV-D is more closely related with the AMPV-A and B subgroups. Ideally, this study might be extended by sequencing more AMPV-D isolates. Unfortunately only two such isolates are currently available worldwide, both isolated in France, on the same date and within close proximity, consequently efforts to obtain new AMPV-D isolates should be continued. The three MPV “clusters” HMPV, AMPV-C and AMPV-ABD were also further supported based on phylogenetics, sequence comparisons and codon bias studies.These data combined with those of previous reports indicating antigenic relationships between subgroups A, B and D Table S1Partial nucleotide sequences previously released for Fr-AMPV-C and Fr-AMPV-D.(DOCX)Click here for additional data file.


H-Ras gene by alternative splicing, induces a G1/S phase delay, thereby maintaining cells in a reversible quiescence state. When P21 H-Ras is mutated in tumour cells, the alternative protein P19 H-Ras is also mutated. The H-Ras mutation Q61L is frequently detected in different tumours, which acts as constitutive activator of Ras functions and is considered to be a strong activating mutant. Additionally, a rare congenital disorder named Costello Syndrome, is described as a H-Ras disorder in children, mainly due to mutation G12S in p19 and p21 H-Ras proteins, which is present in 90 % of the Costello Syndrome patients. Our aim is to better understand the role of p19 and p21 H-Ras proteins in the cancer and Costello Syndrome development, concerning the miRNAs expression.P19 H-Ras, a second product derived from the Total miRNAs expression regulated by H-Ras proteins were first analyzed in human miRNA microarrays assays. Previously selected miRNAs, were further analyzed in developed cell lines containing H-Ras protein mutants, that included the G12S Costello Syndrome mutant, with PCR Real-Time Taq Man miRNA Assays primers.This study describes how p19 affects the RNA world and shows that: i) miR-342, miR-206, miR-330, miR-138 and miR-99b are upregulated by p19 but not by p19W164A mutant; ii) anti-miR-206 can restore the G2 phase in the presence of p19; iii) p19 and p21Q61L regulate their own alternative splicing; iv) miR-206 and miR-138 are differentially regulated by p19 and p21 H-Ras and v) P19G12S Costello mutants show a clear upregulation of miR-374, miR-126, miR-342, miR-330, miR-335 and let-7.H-Ras G12S mutation plays an important role in miRNA expression and open up a new line of study to understand the consequences of this mutation on Costello syndrome. Furthermore, they suggest that oncogenes may have a sufficiently important impact on miRNA expression to promote the development of numerous cancers.These results allow us to conclude that the The online version of this article (doi:10.1186/s12881-015-0184-z) contains supplementary material, which is available to authorized users. Ras, an important family of proto-oncogenes in humans, consists of three members  located on human chromosomes 1, 11 and 12 respectively [Ras gene mutations have been implicated in up to 30 % of all tumours tested. These mutations are different and depend on the tissues involved but most commonly result in pancreatic (90 %) and colon and thyroid tumours (50 %), and lung and myeloid leukemias (30 %). A point mutation in Ras codons converts these genes into active oncogenes as a result of decreased GTPase activity, thereby restricting the easy interchange of GDP to GTP and resulting in a constitutive activation of the downstream pathways, or loss of GAP function. Ras mutated proteins are indirectly involved in metastasic phenotype development as they promote the acquisition of cumulative alterations in cellular pathways which result in cytoskeletal rearrangements, loss of cell adhesion , tissue invasion, extravasation into lymphatic and blood vessels, and finally apoptosis evasion [ectively . Indeed, evasion .H-Ras gene codifies for two different proteins, namely p19 H-Ras and p21 H-Ras, by alternative splicing [et al. have shown that it stimulates p73β-induced apoptosis when both proteins are simultaneously overexpressed [The splicing –7. The msplicing –7. p19 msplicing . p19 H-Rsplicing , 7. Althxpressed . RNAi ofP19 was first described from in T24/EJ bladder carcinoma cell line that contains, in addition to G12V mutation, a small number of other nucleotide changes including an adenine (A) to guanine (G) mutation at position 2714. This latter region was showed to regulate the alternative splicing of H-Ras into two proteins, p21 and p19. The 2719 mutation decresed the p19 expression, and this mutation promotes 10-fold increase of p21 H-Ras levels and a corresponding increase of the transforming efficiency of structurally activated alleles , 10.H-Ras mutation is, present in more than 90 % of patients with Costello Syndrome (CS), a rare congenital disorder caused by germ-line activation of H-Ras oncogenes that affects both p19 and p21 H-Ras [The H-Ras mutation Q61L is frequently detected in different tumour cell lines, where it acts as a constitutive activator of the Ras-signalling pathway and is considered to be a strong activating mutant by decreasing GTPAse activity and increasing GDP/GTP exchange , 12. G1221 H-Ras . CS is c21 H-Ras –18.To better understand the role of p19 and p21 H-Ras proteins in the development of cancer, we transfected HeLa cells with p19 and p21 mutant sequences, which were reported in the literature to be commonly detected in tumour cell lines and in CS (G12S). We also evaluated the expression of selected miRNAs [−/− (KO) and double knock-out MEFs H-Ras−/− plus N-Ras−/− (DKO) were obtained from Dr. E. Santos’s laboratory [HeLa cells were cultured and transiently transfected as described elsewhere , 7. Knocboratory . KO and pEGFP-p19 and -p21, pRK5-p19 and pRK5-p19W164A have been described previously , therefomiRNAs were extracted using the miRVANA™ miRNA isolation kit from Ambion Inc. . Isolation was performed as described by the manufacturer’s protocol.miRNA microarrays were performed as described previously .cDNA was reverse-transcribed from enriched miRNA samples (miRVANA kit) using specific miRNA primers from the Taq Man MicroRNA Assay and reagents from the Taq Man MicroRNA RT kit (AB Applied Bio systems) according to the manufacturer’s instructions. Briefly, 1.33 μL of each resulting cDNA was amplified by PCR using Taq Man MicroRNA Assay primers with the Taq Man Universal Non amperase PCR Master Mix  and analyzed with a 7500 ABI PRISM Sequence Detector System according to the manufacturer’s instructions. miRNA expression was calculated from the relevant signals by normalization with respect to the signal for U18 (for HeLa cells) and U6 for MEFs. Stem-loop quantitative RT-PCR for mature miRNAs was performed as described previously using an Applied Biosystems ABI 7500 Real Time PCR system. All RT-PCR reactions were run in triplicate and gene expression, relative to U18, calculated using the 2-ΔΔCt method .5 HeLa cells using TRIZOL reagent , as described previously. cDNA was reverse-transcribed from total RNA samples using SuperScriptIII® from Invitrogen. The resulting cDNA was amplified by PCR using Taq Man Assay primers with the Taq Man Universal Non-amperase PCR Master Mix and analyzed with a 7500 ABI PRISM Sequence Detector System according to the manufacturer’s instructions. mRNA expression was calculated from the relevant signals by normalization with respect to the signal for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression. The assay numbers for exons E3-IDX p19 H-Ras (Hs00978053_g1), E4A-E4B H-Ras total (Hs00978051_g1) and E3-E4A p21 H-Ras (Hs00610483_m1) and for GAPDH (HS99999905_m1 housekeeping) were supplied by Applied Biosystems Gene Expression Assays (Applied Biosystems). Assays were run with Taqman Universal.Total RNA was extracted from 10 × 10These assays were performed as described previously , 7.No ethics approval was required for aspects of this study.versus RNAs from cells overexpressing empty vector. A second incubation was performed with RNAs from cells overexpressing p19mut in a similar way. Afterwards, both results were mathematically compared, and miRNAs that are upregulated by p19 overexpression but not by 19mut overexpression were selected  activate inclusion of the alternative exon IDX and subsequently p19 expression , 24. p68(−/−) KO MEFs and analyzed their effect on miR-206 and miR-138. Figure (−/−)/N-Ras(−/−) DKO MEFs whereas pEGFP-p19 does not. This finding is in accordance with our previous results, where we showed that p19 causes a G1/S delay in HeLa cells.In order to further understand how the alternative splicing of H-Ras alters miRNAs, we differentially expressed pEGFP-p19 or pEGFP-p21 in H-Raset al. [H-Ras gene. The most common mutation is found on codon 12 with the amino acid change G12S [H-Ras gene is mutated in CS patients, both these proteins p19 and p21, are present in their mutated forms. We obtained pRK5 plasmids containing p19(G12S) and determined how this mutant alters a selected group of miRNAs in HeLa cells. These miRNAs included miR-342 and miR-330, which are already known to be upregulated by p19 overexpression  induces a 10-fold increase in p21 H-Ras level and a corresponding increase in the transforming activity in a bladder carcinoma cell line [H-Ras knockout (KO) mice. These KO mice were found to be viable [H-Ras, thereby allowing the mice to survive, and that a strong homeostatic mechanism plays a role in controlling p21 and p19 levels, as p21 drives cell proliferation whereas p19 maintains a reversible cellular quiescence state [Although p21 H-Ras mutants have been extensively studied, this is not the case for specific p19 H-Ras mutants. However, it is known that a mutation in the 5′ splice-site of the IDX exon of ell line . p19 ovee viable  and fibre viable . These f(−/−) DKO H-Ras(−/−)/N-Ras(−/−) that stably express p21 or p19. Additionally, we obtained some preliminary results of miRNAs Taqman RT-PCR expression profile of G12S or G12A endogenous p19 and p21 proteins in fibroblasts cell lines established from CS tumour patients. Those results showed that miR-330, miR-335 and miR-374 are statiscaly and significally overexpressed in those CS patients cells, and thus they are putative cadidates to be miRNAs overexpressed in CS patients . Herein we have shown that combining p19 overexpression with a miR-206 inhibitor results in a partial decrease of the G1 phase with a clear recovery of the G2 phase, thus indicating that miR-206 is one of the factors contributing to the delay of the G1/S phase. Additionally, we have shown that miR-206 is regulated by the alternative splicing of H-Ras . miR-138, a miRNA that suppresses invasion and promotes apoptosis in some carcinoma cells [(−/−) mice that stably express pEGPP-19, whereas it is unaffected in KO H-Ras(−/−) mice that stably express pEGPP-21 /(−/−) mice that stably express pEGPP-19 (see Fig. et al. have indentified ERα as a direct miR-206 target, and further demonstrated that miR-206 inhibited the mRNA and protein expression of ERα in human ovarian cells [It may be significant that H-Ras is regulated by miRNAs, such as the let-7 miRNA family , 29, andRas Fig.  as the oma cells , was als-21 Fig. . This ob-21 Fig. . Finallyan cells . AdditioH-Ras mutants have been described as a potential marker for CS , 38, 39,Additional file