In high-density myoblast cultures S100B enhances basic fibroblast growth factor (bFGF) receptor 1 (FGFR1) signaling via binding to bFGF and blocks its canonical receptor, receptor for advanced glycation end-products (RAGE), thereby stimulating proliferation and inhibiting differentiation. Here we show that upon skeletal muscle injury S100B is released from myofibers with maximum release at day 1 post-injury in coincidence with satellite cell activation and the beginning of the myoblast proliferation phase, and declining release thereafter in coincidence with reduced myoblast proliferation and enhanced differentiation. By contrast, levels of released bFGF are remarkably low at day 1 post-injury, peak around day 5 and decline thereafter. We also show that in low-density myoblast cultures S100B binds RAGE, but not bFGF/FGFR1 thereby simultaneously stimulating proliferation via ERK1/2 and activating the myogenic program via p38 MAPK. Clearance of S100B after a 24-h treatment of low-density myoblasts results in enhanced myotube formation compared with controls as a result of increased cell numbers and activated myogenic program, whereas chronic treatment with S100B results in stimulation of proliferation and inhibition of differentiation due to a switch of the initial low-density culture to a high-density culture. However, at relatively high doses, S100B stimulates the mitogenic bFGF/FGFR1 signaling in low-density myoblasts, provided bFGF is present. We propose that S100B is a danger signal released from injured muscles that participates in skeletal muscle regeneration by activating the promyogenic RAGE or the mitogenic bFGF/FGFR1 depending on its own concentration, the absence or presence of bFGF, and myoblast density. The regenerative capacity of skeletal muscle tissue resides in satellite cells, the quiescent adult stem cells located between the sarcolemma of the myofiber and the basal lamina The regeneration of injured muscle tissue requires an ordered activation/inactivation of transcription factors, of which some are muscle-specific  while others  are not 2+-binding proteins of the EF-hand type that are only expressed in vertebrates, and exerts regulatory effects both as an intracellular factor and as an extracellular signal Rage results in delayed regeneration following injury, elevated satellite cell numbers, reduced myofiber numbers and myofiber hypertrophy S100B is a member of a multigenic family of CaThe process of mouse skeletal muscle regeneration after injury consists of sequential steps such as satellite cell activation, which starts a few hours after injury and proceeds for 24–36 h; proliferation of activated satellite cells, which starts during day 2 post-injury and proceeds for ∼3 days; myoblast differentiation, which starts at day 3 post-injury and proceeds for ∼2 weeks; and myofiber maturation, which starts at day 5 post-injury and is complete by day 21–28 post-injury We show here that injured muscle tissue releases S100B, bFGF and HMGB1 with different time courses: S100B is maximally released at day 1 post-injury, bFGF is minimally released at day 1 post-injury peaking at day 5, and HMGB1 is released starting at day 3 post-injury peaking between days 7 and 14. We also show that in low-density (LD) myoblast cultures and as long as the myoblast density is low, S100B (in the 0.1–1 nM range) interacts with and activates RAGE, but not bFGF/FGFR1 and simultaneously stimulates proliferation and activates the myogenic program; washing out S100B after 24 h of treatment results in a faster decrease in the proliferation rate and a higher level of differentiation during the next 24 h compared with S100B-untreated myoblasts. By contrast, prolonging the treatment of LD myoblasts with S100B for 3 or more days results in stimulation of proliferation and inhibition of differentiation as a result of a switch of the initial LD culture to an HD culture and consequent formation of the RAGE/S100B/bFGF/FGFR1 tetracomplex. However, raising the S100B dose to 100 nM in LD myoblast cultures results in activation of bFGF/FGFR1 and RAGE blockade, provided bFGF is present. Thus, S100B differentially activates RAGE and bFGF/FGFR1 depending on its own concentration, the presence or absence of bFGF, and myoblast density. We discuss these results in the context of the muscle regeneration process.+ lymphocytes +) cells after injury (To have a more detailed picture of leakage of S100B from injured muscle tissue we collected the conditioned medium (CM) from muscles at varying time points after injury and measured S100B therein. We extended our analyses to released bFGF and HMGB1. We found that levels of S100B in CM from injured muscles amounted to ∼21 ng/muscle at day 1 post-injury, i.e. in coincidence with satellite cell activation, remaining relative high  during the next 4 days post-injury, i.e. during the satellite cell proliferation and differentiation phases, and declining to ∼3 ng/muscle at day 7 post-injury, i.e. during late myoblast differentiation phase, and to control values by day 14 post-injury, i.e. by the end of the myoblast differentiation process . The higr injury . Also, Fr injury . Thus, ar injury .Rage−/− mice.Given the time-course of S100B, bFGF and HMGB1 release from injured muscles , the tim2) density in growth medium (GM) for 24 h and then switched the cultures to DM for 24 h plus or minus S100B and for another 24 h to DM without S100B. At the end of this treatment, S100B-treated LD cultures showed enhanced myotube formation   as expecThe different outcomes of short-term treatment of LD and HD myoblasts with S100B appeared to be dependent mostly on the different effect of the protein on p38 MAPK during the first 24 h of treatment: stimulation in LD cultures  and inhiIn LD myoblasts, neutralization of FGFR1 resulted in decreased basal ERK1/2 phosphorylation levels and increased basal p38 MAPK phosphorylation levels, and S100B activated both kinases akin to native conditions . In thisWhereas in HD myoblast cultures RAGE and FGFR1 co-immunoprecipitated with S100B As a long-term treatment of LD cultures with S100B caused inhibition of differentiation , we perfGiven the multiligand nature of RAGE, we asked whether another RAGE activator, i.e. HMGB1 Next we asked why S100B was unable to activate bFGF/FGFR1 in LD myoblast cultures at early differentiation stages. At 24 h after the switch to DM, LD culture media contained ∼3-times higher bFGF levels than HD culture media  pointingBy co-immunoprecipitation assay we found that upon treatment of LD myoblasts with 100 nM S100B all binding partners, i.e. S100B, bFGF, RAGE and FGFR1, were found in immunoprecipitates irrespective of whether anti-S100B, anti-bFGF, anti-RAGE or anti-FGFR1 antibody was used to generate the immunoprecipitates . Thus, aWe also found that a RAGE/S100B/bFGF/FGFR1 tetracomplex formed in LD cultures that had been scraped, suspended in DM containing 1 nM S100B, centrifuged (to increase the extent of cell-cell contacts), and subjected to co-immunoprecipitation using anti-S100B antibody . This reBy in situ PLA, no cells exhibiting S100B/RAGE or RAGE/FGFR1 complexes could be seen in uninjured skeletal muscle tissue . However+/myogenin−  satellite cells  satellite cells during the proliferation and differentiation phases, and is repressed at completion of regeneration; 2) maximum RAGE expression levels in injured muscle tissue are measured around day 5 post-injury , the inability of low S100B to interact with bFGF/FGFR1 in LD myoblasts is likely to depend on a higher binding affinity of the protein to RAGE than to bFGF/FGFR1. This conclusion is supported by the finding that, as investigated by co-immunoprecipitation assay and in situ PLA, increasing the S100B dose to 100 nM makes S100B capable of recruiting RAGE and bFGF/FGFR1 into a complex in LD in myoblasts at early differentiation stages resulting in stimulation of MEK-ERK1/2 and proliferation and inhibition of p38 MAPK and differentiation. Results obtained using C2C12 myoblasts pretreated with a RAGE neutralizing antibody and also see .Short-term treatment of LD C2C12 myoblasts with S100B results in activation of ERK1/2 and p38 MAPK. Simultaneous activation of ERK1/2 and p38 MAPK in myoblasts in DM is counterintuitive given the established role of ERK1/2 in stimulation of proliferation In the extracellular space, S100B forms oligomers RAGE activation by HMGB1 in HD myoblasts results in p38 MAPK-dependent stimulation of differentiation and inhibition of proliferation via p38 MAPK-dependent inhibition of ERK1/2 Rage−/− mice, RAGE signaling via p38 MAPK playing an important role in the induction of myogenin and myogenin-dependent repression of Pax7 expression RAGE expression in skeletal muscle tissue is developmentally regulated, RAGE being expressed up to a few postnatal days and repressed thereafter Recombinant bovine S100B, that is 97% identical to mouse S100B, was expressed and purified as reported 2 injection Tibialis anterior muscle of 8-wk old mice, under zolazepam/tiletamine anesthesia. Briefly, 50 µl of an aqueous 1.2% (w/v) BaCl2 solution were injected along the whole length of left Tibialis anterior muscles. Controlateral muscles were injected with vehicle and used as controls. After different recovery times (1–14 days) the animals were sacrificed and Tibialis anterior muscles were removed and incubated in phosphate-buffered saline (0.15 ml/muscle) for 2 h at 4°C under agitation Injury of C57BL/6 (Charles River) mouse muscles was performed by BaCl3 cells/cm2) or high density , were cultured for 24 h in Dulbecco's modified Eagle medium (Invitrogen) supplemented with 20% FBS (Invitrogen), 100 U/ml penicillin, and 100 µg/ml streptomycin (GM), in a H2O-saturated 5% CO2 atmosphere at 37°C and then shifted to Dulbecco's modified Eagle medium containing 0.5% horse serum (DM) to induce myoblast differentiation. C2C12 and primary myoblasts were cultivated in DM in the absence or presence of either S100B, bFGF (Peprotech) or HMGB1 plus or minus other additions as indicated in figure legends. Details of myoblast treatment after 24 h in DM are given in legends to pertinent figures.C2C12 and primary myoblasts seeded at a low density  incorporation assay. BrdU was added to cultures 2 h before fixation with cold methanol at −20°C and processing by immunofluorescence using a monoclonal anti-BrdU antibody . BrdUMyoblasts were lysed and cell lysates were subjected to Western blotting as described MyHC was detected by immunocytochemistry as described 5′-GCTGTATGAAACATCCCCCTA-3′ and 5′-CGCTGTGGGAGTTGCATT-3′ .Total RNA was extracted from C2C12 myoblasts using the TRIzol reagent (Invitrogen) according to the manufacturer's instructions. The following primers were used: murine myogenin 2, 10 mM NaF and 1 mM sodium orthovanadate in the presence of a mixture of protease inhibitors (Roche Applied Science). Solubilized proteins were subjected to immunoprecipitation using either a polyclonal anti-S100B antibody , a polyclonal anti-RAGE antibody , a monoclonal anti-FGFR1 antibody , a polyclonal anti-bFGF antibody  or non-immune IgG. Immunoprecipitates were probed with the following antibodies: polyclonal anti-S100B antibody , polyclonal anti-RAGE antibody , monoclonal anti-FGFR1 antibody , polyclonal anti-bFGF antibody , polyclonal anti-c-Met antibody .C2C12 and primary myoblasts cultivated for 30 min in DM in the absence or presence of S100B were lysed in buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1 mM CaClMyoblasts in DM were treated for 2 h with either polyclonal anti-RAGE antibody  or monoclonal anti-FGFR1 antibody  and cultivated for 24 h in the absence of additions or presence of S100B. In experiments of effects of neutralization of culture medium bFGF, monoclonal anti-bFGF antibody  was added to myoblasts in DM for 2 h followed by treatments as described in figure legends. Control samples received non-immune IgG.To investigate whether S100B interacts with bFGF, FGFR1 and/or RAGE we also used in situ proximity ligation assay (PLA), which allows the visualization of subcellular localization and protein-protein interactions in situ www.graphpad.com). Where appropriate data were represented as averages of 3 determinations ± SEM. Statistical significance was assumed when p<0.05.Each experiment was repeated at least three times. Representative experiments are shown unless stated otherwise. The data were subjected to analysis of variance (ANOVA) with SNK post hoc analysis using a statistical software package  LD primary myoblasts were cultivated in DM in the absence or presence of S100B for 24 h, washed and cultivated for another 24 h in DM with no additions. Myoblasts were lysed and cell lysates were subjected to Western blotting for detection of myogenin. (B) Same as in A except that LD myoblasts were viewed by phase-contrast at 48 h after washout. Bars = 250 µm (100 µm in insets).Short-term treatment of LD mouse primary myoblasts with S100B results in enhanced myogenin expression and myotube formation after S100B washout. ((TIF)Click here for additional data file.Figure S2A) Immunoprecipitation of S100B in LD myoblast cultures in the absence or presence of added S100B. C2C12 myoblasts cultivated for 30 min in DM in the absence (C) or presence (S100B) of 1 nM S100B were lysed in buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1 mM CaCl2, 10 mM NaF and 1 mM sodium orthovanadate in the presence of a mixture of protease inhibitors (Roche Applied Science). Solubilized proteins were subjected to immunoprecipitation using a polyclonal anti-S100B antibody . Inputs (In) and immunoprecipitates (IP) (20 µg protein each) were subjected to Western blotting using an anti-S100B antibody. (B) bFGF is found in culture media of LD myoblasts. C2C12 myoblasts were cultivated for 30 min in DM with no additions  or in the presence of added S100B (1 nM), and culture media were collected and trichloroacetic acid-precipitated. The pellets were resuspended in Laemmli buffer and subjected to Western blotting using anti-bFGF antibody. Purified bFGF (10 ng) was loaded on lane M.Detection of S100B and bFGF in culture media. ((TIF)Click here for additional data file.Figure S3A–C) During the first 24 h of treatment HMGB1 activated ERK1/2 and p38 MAPK (A), stimulated proliferation (B) and activated the differentiation program (C). (D) At 48 h after washout HMGB1-treated myoblasts expressed higher MyHC levels than untreated myoblasts. (E) Myoblasts were cultivated in DM in the presence of HMGB1 for 30 min and processed for immunoprecipitation using an anti-HMGB1 antibody. RAGE, but not FGFR1 co-immunoprecipitated with HMGB1.Short-term treatment of LD C2C12 myoblasts with HMGB1 results in stimulation of proliferation and activation of the myogenic program. Myoblasts were cultivated in DM in the absence or presence of HMGB1 for 24 h, washed and cultivated for another 24 h in DM with no additions. ((TIF)Click here for additional data file.Figure S4A) LD myoblasts were cultivated in DM for 24 h in the presence of increasing S100B concentrations and subjected to Western blotting using anti-FGFR1 or anti-RAGE antibody. (B) Myoblasts pretreated (2 h) with 10 µg/ml anti-RAGE antibody or 2 µg/ml anti-FGFR1 antibody were cultivated in DM for 24 h in the absence or presence of 1 nM S100B and subjected to Western blotting using anti-RAGE antibody or anti-FGFR1. Controls received 10 µg/ml non-immune IgG. *Significantly different from control (n = 3).S100B upregulates either RAGE or FGFR1 expression in LD myoblasts depending on the concentration. ((TIF)Click here for additional data file.Text S12, fixed in 4% formalin in tris-buffered saline (TBS) (pH 7.2), dehydrated and paraffin embedded. Paraffin sections were cut at 4 µm, deparaffinized with xylene, rehydrated in a graded ethanol series. Sections were washed with TBS, pH 7.4, incubated for 1 h with Blocking Buffer  and then probed with the following primary antibodies in BB (1∶20): goat polyclonal anti-RAGE (Santa Cruz Biotechnology), mouse monoclonal anti-Pax7 (R&D Systems). The secondary antibodies were donkey anti-mouse Alexa Fluor 488-conjugated (Invitrogen), donkey anti-goat Alexa Flour 594-conjugated (Invitrogen), and goat anti-rabbit rhodamine-conjugated (Sigma Aldrich). Western Blotting. Myoblasts were lysed and cell lysates were subjected to Western blotting as described (10). The following antibodies were used: monoclonal anti-MyHC , monoclonal anti-M-cadherin , polyclonal anti-caveolin-3 , monoclonal anti-α-tubulin , polyclonal anti-phosphorylated (Thr180/Tyr182) p38 MAPK , polyclonal anti-p38 MAPK , polyclonal anti-phosphorylated (Thr202/Tyr204) ERK1/2 , polyclonal anti-ERK1/2 , monoclonal anti-myogenin , anti-S100B , polyclonal anti-RAGE antibody , monoclonal anti-FGFR1 antibody , polyclonal anti-bFGF antibody , polyclonal anti-c-Met antibody , and monoclonal anti-p-Tyr (PY20) sc-508 . For analyses of culture medium bFGF, culture media were collected processed as described Supplementary materials and methods. Double Pax7/RAGE Immunofluorescence. Tibialis anterior muscles were removed from uninjured mice and from injured mice at day 3 after injection with BaCl(TIF)Click here for additional data file.


The splicing of the N exon in the pre-mRNA coding for the RE1-silencing transcription factor (REST) results in a truncated protein that modifies the expression pattern of some of its target genes. A weak 3'ss, three alternative 5'ss  and a variety of putative target sites for splicing regulatory proteins are found around the N exon; two GGGG codes (G2-G3) and a poly-Uridine tract (N-PU) are found in front of the N50-5'ss. In this work we analyzed some of the regulatory factors and elements involved in the preferred selection of the N50-5'ss (N50 activation) in the small cell lung cancer cell line H69. Wild type and mutant N exon/β-globin minigenes recapitulated N50 exon splicing in H69 cells, and showed that the N-PU and the G2-G3 elements are required for N50 exon splicing. Biochemical and knockdown experiments identified these elements as U2AF65 and hnRNP H targets, respectively, and that they are also required for N50 exon activation. Compared to normal MRC5 cells, and in keeping with N50 exon activation, U2AF65, hnRNP H and other splicing factors were highly expressed in H69 cells. CLIP experiments revealed that hnRNP H RNA-binding occurs first and is a prerequisite for U2AF65 RNA binding, and EMSA and CLIP experiments suggest that U2AF65-RNA recognition displaces hnRNP H and helps to recruit other splicing factors (at least U1 70K) to the N50-5'ss. Our results evidenced novel hnRNP H and U2AF65 functions: respectively, U2AF65-recruiting to a 5'ss in humans and the hnRNP H-displacing function from two juxtaposed GGGG codes. The alternative splicing mechanism generates vast numbers of protein isoforms amplifying the eukaryotic proteome and affecting human genetic diseases. Frequently, such diseases are caused by mutations in splicing regulatory sequences RE1-Silencing Transcription Factor) isoform . This is partly due to the expression of the truncated (t) REST  upstream of the 3'ss, by forming unique hydrogen bonds with the Uracil edges and promoting the U2 snRNP binding to the branch point site c-src N1 exon is regulated by the cooperative binding of hnRNP H and hnRNP F to an intronic splicing enhancer, localized downstream of the 5'ss in front of the exon Recently, an array composed of G-rich A sequence analysis of the N exon environment  revealedHere we investigated N50 exon activation in H69 cells. N exon/β-globin minigenes recapitulated N50 exon splicing when transfected into H69 cells, and mutant minigenes showed that the N-PU and the G2-G3 elements are required for N50 exon splicing. Biochemical and knockdown experiments showed that these elements are targets for U2AF65 and hnRNP H, respectively, and that they are also required for N50 exon activation. In agreement with the unique N50-5'ss selection in H69 cells and the absence of N exon activation in the proxy to normal MRC5 cells, U2AF65, hnRNP H and other splicing factors were highly expressed in H69 cells. CLIP experiments revealed that hnRNP H RNA-binding occurs first and is a prerequisite for U2AF65 RNA binding and EMSA experiments showed that binding of U2AF65 to the N-PU displaces hnRNP H from the G2-G3 codes; these hnRNP H and U2AF65 functions have not been previously described. Finally, U2AF65 appears to promote U1 snRNP 70K recruiting for N50-5'ss activation. Altogether, these findings are in agreement with the model in which in MRC5 cells the N exon could be repressed by hnRNP H binding to the GGGG codes, whereas in H69 cancer cells, such repression might be relieved by U2AF65 binding to the N-PU, whose recruiting appears to be facilitated by hnRNP H, leading to N exon activation.In H69 cells, the splicing of the N50 exon in the REST pre-mRNA originates the REST-N50 mRNA variant and its corresponding truncated protein Around the N exon, multiple putative targets of splicing factors were identified . A N-PU This complex array of elements suggest their possible involvement in the preferred N50- over N4- and N62-5'ss selection in H69 cells. To test this, N-PU mutant (UUUUUU to GAUAUC), G2-G3 mutant (GGGGGUGGGGG to CACGGUCACGG) and G2-G3∼N-PU double mutant minigenes were analyzed as before. We found that the single mutants rendered 20–30% less N50 exon inclusion, coordinated with a proportional increase in N exon-skipped and partially spliced products , lane 5.Given that there are no N exon sequence differences between H69 and MRC5 cell lines (not shown), N50 activation in H69 cells probably obeys to cellular context. Therefore, we investigated whether the nuclear factors U2AF65 and hnRNP H from H69 cells could differentially recognize the N exon RNA. Nuclear extracts (NEx) from H69 and MRC5 cells were UV cross-linked to synthetic flWT transcripts . Albeit In agreement with their reported functions, we expected that N50 activation and tREST expression could depend on the U2AF65 and hnRNP H splicing promoting activities. To test this possibility we monitored N50 inclusion and the expression of REST isoforms after U2AF65  and hnRNConfirming our previous findings, and in coordination with the REST-N50 mRNA expression, U2AF65 and hnRNP H expression was more robust in H69 cells . As expeSeveral biochemical approaches were used to test the interplay of the nuclear factors U2AF65 and hnRNP H with the N-PU and G2-G3 codes in the recognition of the N50-5'ss. Our previous experiments prompted the idea that RNA-bound hnRNP H might be a prerequisite for the recruitment of other nuclear factors to the N50-5'ss  and 4D. Therefore, similar experiments were carried out to explore the kinetics of hnRNP H and U2AF65 binding to the N exon wild type RNA. Given that additional G-rich and uridine-rich elements are present in the RNA and that the CLIP assays were carried out with whole NEx, to discriminate stable and direct (i.e. not mediated by other nuclear factors) protein/RNA interactions, the signals of mutant RNAs lacking the G2-G3 and N-PU elements were subtracted from those of the wild type RNA. The RNA immunoprecipitated with hnRNP H peaked between 4–8 min, and plummeted at 10 min . HoweverSince TIA-1 and TIA1-like proteins are able to bind to U-rich motifs downstream of 5'ss facilitating its recognition In view of the above results, and that binding of U2AF65 to pyrimidine-rich sequences in front of a 5'ss can activate the ss by means of U1 snRNP recruiting The activating role of U2AF65 was explored further. Mimicking H69 and MRC5 cellular contexts, U2AF65-depleted H69 NEx was not as efficient as whole NEx to recruit EMSA complexes with the G2-G3∼N-PU probe, and the complexes formed with the same probe and MRC5 NEx were supershifted with rU2AF65 . This suREST expression profiles in H69 cells directly affect the determination of the transformed phenotype. In fact, transfection of the N50-REST cDNA into MRC5 cells modifies the expression profiles of the REST target genes RRAD (Ras-related associated with diabetes) and TMS-1 (target of methylation induced silencing-1) . In thisAs previously demonstrated by Coulson and coworkers Previous works have identified multiple 5'ss surrounding the REST N exon, suggesting tissue-specific activation profiles The binding and in vivo splicing assays , and 6A Drosophila can promote U1 snRNP recruitment U1 snRNP is the first sub complex thought to bind the 5'ss during the spliceosome assembly. It is accepted that many auxiliary-splicing factors can promote weak 5'ss recognition by binding to enhancer sequences Collectively, our results suggest that the initial hnRNP H binding to the G2-G3 codes is a prerequisite for U2AF65 recruiting to the N-PU tract. Then U2AF65 along with other factors might help U1 snRNP recruitment to the N50-5'ss for N exon activation.We found here that N exon is a very versatile sequence with a complex array of splicing signals, and its activation in H69 cells depends on the relative amounts of hnRNP H and U2AF65. Although further studies are needed to unveil the activation of the N4- and N62-5'ss, we gained some insights in a novel-activating role of hnRNP H, being a prerequisite for U2AF65 recruiting to nearby sequences.The U2AF65 hnRNP H-displacing function evidenced here is new, and might have been acquired early in evolution. This particular function resembles that of SRSF1 and SRSF2 that bind to juxtaposed GGGG codes downstream of the chicken tropomyosin exon 6B displacing hnRNP A1 from these sites and promoting exon inclusion Finally, we show here precedents for the repressing and recruiting functions of hnRNP H (in a consecutive fashion) and U2AF65 (in a simultaneous manner) along the splicing mechanisms associated with cancer cells.The human H69 (SCLC), A549 , and MRC-5  cells were from ATCC . Cells were cultured with RPMI and DMEM media, respectively, both supplemented with 10% fetal bovine serum. Nuclear extracts were made according to the Dignam protocol H69 cells genomic DNA containing 593 nt around the N exon was amplified by PCR, using primers Seq1s and Seq1as , and 10 μg were resolved in 10% polyacrylamide gel. Antibodies against REST, hnRNP H, hnRNP F, pan-SRSF, U2AF65, U1 70K , and Actin (a gift from J.M. Hernández) were used for western blotting, CLIP, supershift, and pull-down assays. Digital image densitometries of PCR products and protein bands were carried out, from at least three experiments each, with the ImageJ software and expressed as Integrated Density (IntDen) or its percent.The pre-characterized U2AF65 (HSS117616), hnRNP H (HSS104905), and negative universal control (46-2001) siRNAs were purchased from Invitrogen. To construct the wt and mutant REST β-globin-N exon minigenes we first amplified the first two exons and intron of the β-globin gene with primers gloF and gloR. The fragment was cloned in the pCR2.1 plasmid. Next, the N exon sequencing fragment was used as template to amplify the wt, PU mut, G2-G3 mut and double mutant N exons using the forward primer 2REN/IVs and the reverse primers PU-rev, PU mut, G2G3m-as, and d-mut, respectively. Each fragment was inserted into the Bbs I site of the β-globin intron of the pCR2.1 clone, and each intermediary was subcloned into the pcDNA3 plasmid digested with Kpn I and Xho I. Clones were verified with Nsi I digests and sequencing. Plasmids were purified with Plasmid Midi Kit (QIAGEN), and transfected into human cells. The primers U2AF65F and U2AF65R were used to amplify de cDNA of U2AF65 by RT-PCR. The fragment was cloned into the Bam HI and Xho I sites of the pPROEX HTb plasmid. To maintain the RS domain of U2AF65, the Hind III-Sma I fragment was deleted by religation. Likewise, to maintain the SR domain of SRSF3, the Sma I-Xho I fragment was deleted from a cDNA SRSF3 clone in pGEX-2T. For overexpression experiments, the cDNA of U2AF65 was cloned in the plasmid pcDNA3.1. For transfection experiments, plasmids (2 μg) or siRNAs (20 nM) were transfected with Lipofectamine 2000 in six-well culture plates following the manufacturer's instructions (Invitrogen), and mRNA and protein expression were analyzed. Total RNA was prepared with RNasy Kit following the manufacturer's instructions (Qiagen). For reverse transcription, Superscript II (Invitrogen) was used. For H69 and MRC-5 whole-cell extracts, cells were disrupted in lysis buffer  as instructed by the manufacturer, RNAs were purified from a 5% denaturing acrylamyde gel using the RNA elution buffer . RNA was extracted with phenol:chloroform:isoamyl alcohol and ethanol precipitated.32P-labeled RNA for 30 min at 4°C in splicing buffer . After incubation RNA-bound NEx were UV irradiated for 20 min at a wavelength of 254 nm on ice, then incubated with 0.5 µg of RNAse A for 30 min at 37°C, and immunoprecipitated as described One µg of NEx was incubated with 200 fmole of 2, 20 mM spermidine, 0.1 mM DTT, 0.5 mM EDTA, 1.5 mM ATP and 20% Glycerol) containing 10 μg of nuclear extract and 0.4 μg/μL of tRNA, and were incubated for 15 minutes at room temperature (r/t). 2000 cpm of 32P-labelled substrate RNA was added and incubated for 15 minutes at r/t. Samples were loaded on a 4.0% native polyacrylamide gel, electrophoresed, and visualized by autoradiography. Proteins were purified from sliced RNA-protein complexes as previously described Splicing reactions were assembled in 10 μl of binding buffer  and induced with IPTG 0.2 mM during 4 hours. Proteins were isolated from bacterial pellets in denaturing conditions, as indicated by the manufacturer (the QIAexpressionistTM), and renatured by sequential dialysis in buffers with decreasing amounts of urea, and later incubated on ice overnight. Glycerol (20%) and protease inhibitors were added. Proteins were stored at −70°C until use.Plasmids pProEXHTb-U2AF65 (or the RS domain of U2AF65) were transformed into 9H2PO4.H2O, pH = 8.0) for 1 h at r/t. Beads were collected by centrifugation, washed three times with PBS and incubated 30 min in 300 µl of 10% Bovine Serum Albumin (BSA) at 4°C. The beads were then incubated overnight at 4°C with 200 µg of H69 NEx in 400 µl of PBS and unbound proteins were washed three times with PBS by centrifugation. Bound proteins were identified by western blot.Ten µg of 6xHis-tagged protein (U2AF65-RS domain or full-length) were bound to Ni-NTA beads in binding buffer : 80-8. The primers used to amplify the DNA templates for run-off transcription; sequencing and plasmid constructions are also shown. The PCR amplification conditions were preceded and followed by a denaturing cycle at 94°C for 5 min, an extension cycle at 72°C for 7 min, respectively.(PDF)Click here for additional data file.Figure S2The N-PU is a bona fide poly-Uridine tract recognized by U2AF65 from H69 cells. The wild type N-PU (lanes 1–4) or mutant N-Pn (lanes 5–8) RNAs (shown at the top) were used in EMSA experiments with 2 µg (lanes 2 and 6), 4 µg (lanes 3 and 7) or 6 µg (lanes 4 and 8) of rU2AF65; free probes were run in lanes 1 and 5, respectively. The two remaining uridines in the mutant RNA (UUUUUU to GAUAUC) account for the residual (roughly 20%) rU2AF65 complexes (r65 cx), suggesting that the N-PU is necessary for U2AF65 recruiting to the N exon alternative 5'ss. An empty arrowhead indicates a structured form of the mutant probe.(PDF)Click here for additional data file.Figure S3hnRNP H immunodepletion. NEx from H69 cells were depleted using antibodies against an unrelated antigen (Δur) or against hnRNP H (ΔH). hnRNP H antibodies were used to probe western blots after immunodepletion. The hnRNP H (P H) and unrelated proteins (P ur) bound to the pellets after immunodepletions were analyzed also. To control the hnRNP H depletion, the same membrane was stripped and incubated with antibodies against U2AF65 (lower panel). An asterisk indicates residual hnRNP H after stripping.(PDF)Click here for additional data file.Figure S4U2AF65 is necessary for complex recruiting at the N50-5'ss. The wild type probe  was used in EMSA with NEx from H69 cells (lane 2), or with H69 NEx depleted of U2AF65  or depleted of an unrelated antigen . Complex formation was reduced with the ΔU2AF65 NEx. Reciprocally, the probe was incubated with NEx from MRC5 cells alone (lane 6) or complemented with rU2AF65 (lane 7). rU2AF65 was required for shifting the mobility of the complex and the remaining probe, in a different manner than rU2AF65 alone (lane 8).(PDF)Click here for additional data file.Figure S5Transfection of the cDNA for tREST into MRC5 cells modifies RRAD and TMS-1 basal expression. (A) Putative binding sites for REST were localized close to the TATA box in the promoters of the human RRAD and TMS-1 genes. (B) RT-PCR showing the altered expression of RRAD and TMS-1 in cells transfected with the tREST cDNA (lane 2), compared to the MOCK transfections (lane 3), and no RT controls (lane 1). The GA3PDH amplicon was used for loading control.(PDF)Click here for additional data file.Figure S6cREST is readily degraded in H69 cells. (A) Western blots from emetine-treated  or untreated (-) H69 (upper panel) and MRC5 cells (lower panel). Incubation times were 1, 5 and 10 h. Canonical (cREST) and truncated (tREST) isoforms are indicated. (B) Nuclear extracts from H69 cells treated (+) or not  with the proteasome inhibitor MG132 at 10 μM final concentration during 4 h were probed with anti-REST antibody. REST (arrowhead in lane 1) and ubiquitinated-REST (brackets in lane 2) are shown.(PDF)Click here for additional data file.Figure S7hnRNP H from H69 cells is able to bind to all G codes. (A) Twenty fmole of a 32P-labeled RNA probe (shown on top) containing part of the N4-5ss only, the 3'ss, the Py and part of the upstream intron were incubated with 5 µg of each NEx from MRC5, A549 (other SCLC cell line) or H69 cells. The EMSA complexes were resolved in a 4% native polyacrylamide gel. Proteins from 20 complexes were purified and visualized in silver stained 10% SDS-polyacrylamide gels. Black arrows show the proteins that were differentially purified from NEx. (B) A sample of the purified complexes was electrophoresed and probed with anti-hnRNP H antibodies by western blot. A strong signal for hnRNP H was detected in MRC5 (lane 1) and H69 (lane 3) cells indicating that hnRNP H is recruited to the G1. A less intense hnRNP H signal was observed in A549 NEx. (C) Two different RNA probes (drawings at the bottom), with or without the G1 element, were labeled  and used in EMSA with H69 NEx. Both probes rendered complexes with the NEx (lanes 2 and 6) but that with the G1 element was supershifted more efficiently with the anti-hnRNP H antibodies (lane 3) as compared with the modest supershift obtained with the probe lacking the G1 element . Antibodies against hnRNP A did not supershifted the complexes formed with either probe.(PDF)Click here for additional data file.Figure S8Depletion of U1 snRNA or U2AF65 of H69 NEx inhibits complex recruiting around the N exon. The wild type probe (lane 1) was used in EMSA with NEx from H69 cells (lane 4), or with H69 NEx depleted of U2AF65  or with NEx in which the 5' end of U1 snRNA was degraded by RNase H-mediated digestion with a complementary oligonucleotide . Complex formation was not diminished when the U1 snRNA was degraded with a scrambled oligonucleotide as a control  and was partially recovered when the ΔU2AF65 extracts were supplemented with recombinant U2AF65 .(PDF)Click here for additional data file.


Anopheles minumus mosquito P450 isoforms CYP6AA3 and CYP6P7 are capable of metabolizing pyrethroid insecticides, however CYP6P8 lacks activity against this class of compounds.Cytochrome P450 enzymes (P450s) have been implicated in insecticide resistance. An. minimus P450 enzymes were constructed using the multiple template alignment method. The predicted enzyme model structures were compared and used for molecular docking with insecticides and compared with results of in vitro enzymatic assays. The three model structures comprise common P450 folds but differences in geometry of their active-site cavities and substrate access channels are prominent. The CYP6AA3 model has a large active site allowing it to accommodate multiple conformations of pyrethroids. The predicted CYP6P7 active site is more constrained and less accessible to binding of pyrethroids. Moreover the predicted hydrophobic interface in the active-site cavities of CYP6AA3 and CYP6P7 may contribute to their substrate selectivity. The absence of CYP6P8 activity toward pyrethroids appears to be due to its small substrate access channel and the presence of R114 and R216 that may prevent access of pyrethroids to the enzyme heme center.Homology models of the three Differences in active site topologies among CYPAA3, CYP6P7, and CYP6P8 enzymes may impact substrate binding and selectivity. Information obtained using homology models has the potential to enhance the understanding of pyrethroid metabolism and detoxification mediated by P450 enzymes. One of the major classes of insecticide detoxification enzymes is the heme-containing cytochrome P450 monooxygenases (P450s). These enzymes are responsible for the metabolism of endogenous and exogenous compounds and the expression of several P450s is increased in insecticide resistant insects . P450 en binding ,6,7.CYP6AA3, CYP6P7, and CYP6P8 in a laboratory-selected deltamethrin-resistant An. minimus mosquito, a major malaria vector in Thailand, relative to the parent susceptible strain [An. minimus CYP6AA3, CYP6P7, and CYP6P8 enzymes was conducted and molecular docking was performed with various insecticide compounds. Our analysis was able to predict the molecular basis of P450 activity against pyrethroid compounds and has the potential to assist in the investigation of new compounds that can bypass resistance due to P450 enzymes.We previously observed elevated expression of P450 isoforms AAN05727.1), CYP6P7 (GenBank: AAR88141.1), and CYP6P8 (GenBank: AAR88142.1) were aligned against protein structures deposited in Brookhaven Protein Data Bank (PDB) [1TQN) [1PQ2) [1OG2) [Amino acid sequences of CYP6AA3 (GenBank: nk (PDB)  using PS) [1TQN) , CYP2C8 ) [1PQ2) , and CYP) [1OG2)  were useComparative modeling of CYP6AA3, CYP6P7, and CYP6P8 was performed using a restrained-based approach implemented in MODELLER9v6 . Multiplhttp://chem.sis.nlm.nih.gov/chemidplus/ and used in docking of three target models. Partial charges of ligands and proteins were generated using Gasteiger method with the aid of AutoDockTools [6. Resulting docked conformations within 2.0 Å root mean square deviation (RMSD) tolerance were clustered and analyzed using AutoDockTools. Conformations with the lowest interaction energy and closest interaction to heme iron were selected. Residues showing interaction with docked ligands with less than 1.0 scaling factor of van der Waal radii were determined. Active sites and substrate access channels of enzyme models were calculated using the VOIDOO program [Three-dimensional structures of pyrethroids , alongside organophosphate (chlorpyrifos), and carbamate (propoxur) shown in Additional file ockTools . RestraiockTools  was usedockTools  to compaockTools . The secockTools  was empl program  with conHomology models of CYP6AA3, CYP6P7 and CYP6P8 were constructed based on crystal structures of CYP3A4, CYP2C8, and CYP2C9 human P450s that are involved in pyrethroid metabolism  using th//C loop, C-terminus of G-helix and N-terminus of I-helix. The putative CYP6P7 pw2b access channel is comprised of residues from B/B/loop and β1 sheet, while pw2e is observed in BC loop/B/helix and N-terminus of I-helix of CYP6P8 model. Differences in the geometry of the predicted active sites is remarkable with CYP6AA3 having an oval shape and a large volume, 245.69 Å3 in size  attacked by the enzyme. This prediction is in agreement with our results showing multiple products from CYP6AA3-mediated deltamethrin degradation [We have reported that CYP6AA3 and CYP6P7 can metabolize pyrethroids , but lacked activity against bioallethrin (pyrethroid), chlorpyrifos (organophosphate), and propoxur (carbamate) . Notablyradation . Multiplradation ,29-31. T/-phenoxybenzyl carbon of λ-cyhalothrin moving away from the CYP6P7 heme (4.17 Å) compared to that of cypermethrin  values ranging from 6.2-6.8. Since bioallethrin, chlorpyrifos, and propoxur  are more polar, they would be expected to have less favorable interactions with the active sites of CYP6AA3 and CYP6P7 resulting in a longer distance between these molecules and the heme iron (unpublished data). As a result both CYP6AA3 and CYP6P7 are predicted to be incapable of metabolizing bioallethrin, chlorpyrifos, and propoxur, consistent with our previous results [P of 1.62), temephos, and fenitrothion  to both of these enzymes is also predicted to be unfavorable (unpublished data).Residues in the CYP6AA3 cavity that interact with pyrethroids in our docking experiments Table  are geneIn the CYP6P8 model, the small access channel together with R114 and R216 are predicted to obstruct pyrethroid entry into the active-site cavity Figure , resultiThe model structures of CYP6AA3, CYP6P7, and CYP6P8 generated in this study have allowed us to better understand the different substrate preferences between these P450 enzymes and the predictions based on our docking studies are consistent with experimental results of pyrethroid metabolism mediated by these three enzymes. Variations in the predicted substrate channels and geometry of active sites appear to be responsible for their differences in binding to pyrethroids. Our findings indicate that differences in metabolic activities among P450 enzymes in insects can be attributed to structural differences that allows for selectivity in their activities against insecticides. These models have the potential to be used in the investigation of candidate P450 inhibitors or in the analysis of the binding and metabolism of insecticide compounds that have potential for use in the control of the mosquito vector.The authors declare that they have no competing interests.PL participated in design of the study, model building and data analysis, and drafting of the manuscript. EJ participated in design and data analysis of the study. PR participated in design and data analysis of the study, drafting and revising of the manuscript. All authors read and approved the final manuscript.Figure S1. Multiple sequence alignment of templates and target P450s. Template sequences of CYP3A4 (1TQN chain A), CYP2C9 (1OG2 chain A) and CYP2C8 (1PQN chain A) are aligned against target sequences CYP6AA3, CYP6P7 and CYP6P8. The first 25 residues on N-termini of target sequences are underlined and bolded. Residues mostly identical among templates and targets are marked yellow. Identical residues among all sequences are indicated by asterisks and yellow mark. Residues of target identical to CYP3A4 template are marked grey, whereas target residues identical to CYP2Cs are marked green. Predicted contact residues between targets and ligands are marked blue. Protruding arginine at the entry of substrate access channel in CYP6P8 model is highlighted red and arginine and phenylalanine in the channel are in violet. CYP6AA3 primary sequence comprises 505 residues, CYP6P7 509 residues and CYP6P8 506 residues.Click here for fileTable. Percent amino acid sequence similarity between crystallographic templates and target sequences.Click here for fileFigure S2. Insecticides used in docking study. Pyrethroids shown are cypermethrin (A), permethrin (B), deltamethrin (C) and λ-cyhalothrin (D). Non-substrate insecticides docked in this study are: propoxur, a type of carbamate insecticide (E); chlorpyrifos, a type of organophosphate insecticide (F); and bioallethrin pyrethroid insecticide (G). Geminal-dimethyl group, 5- and 4/-phenoxybenzyl carbons are indicated by arrows on cypermethrin.Click here for fileFigure S3. Docked deltamethrin conformations in oxyferryl state of CYP6AA3 (A-D) and CYP6P7 (E). Predicted metabolic sites are at geminal dimethyl group (A), 5-phenoxybenzyl carbon (B), 4/-phenoxybenzyl carbon (C), cyano group (D), and 4/-phenoxybenzyl carbon in CYP6P7 (E). Helices I of CYP6AA3 and CYP6P7 are shown in gold and silver cartoons, respectively. Oxyferryl heme is represented by grey stick, while deltamethrin is illustrated in green stick. Iron, oxygen, and nitrogen are colored orange, red, and blue, respectively.Click here for file


FT are key players in the floral transition in Arabidopsis. Here, we characterized the expression pattern and function of JMJ18, a novel JmjC domain-containing histone H3K4 demethylase gene in Arabidopsis. JMJ18 was dominantly expressed in companion cells; its temporal expression pattern was negatively and positively correlated with that of FLC and FT, respectively, during vegetative development. Mutations in JMJ18 resulted in a weak late-flowering phenotype, while JMJ18 overexpressors exhibited an obvious early-flowering phenotype. JMJ18 displayed demethylase activity toward H3K4me3 and H3K4me2, and bound FLC chromatin directly. The levels of H3K4me3 and H3K4me2 in chromatins of FLC clade genes and the expression of FLC clade genes were reduced, whereas FT expression was induced and the protein expression of FT increased in JMJ18 overexpressor lines. The early-flowering phenotype caused by the overexpression of JMJ18 was mainly dependent on the functional FT. Our findings suggest that the companion cell–dominant and developmentally regulated JMJ18 binds directly to the FLC locus, reducing the level of H3K4 methylation in FLC chromatin and repressing the expression of FLC, thereby promoting the expression of FT in companion cells to stimulate flowering.Flowering time relies on the integration of intrinsic developmental cues and environmental signals. FLC and its downstream target  FT work as repressor and activator, respectively, to regulate flowering time in Arabidopsis; thus the regulation of FLC and FT expression is the key for the control of floral transition. Epigenetic modifications are critical for transcription regulation. Here, we show that a novel JmjC domain-containing histone H3K4 demethylase, JMJ18, is a key regulator for the expression of FLC and FT in companion cells and flowering time. JMJ18 is dominantly expressed in vascular tissue; its temporal expression pattern was developmentally regulated, and negatively and positively correlated with FLC and FT, respectively. JMJ18 mutation leads to weak late-flowering, while JMJ18 overexpressor exhibited an obvious early-flowering phenotype. JMJ18 binds to chromatin of FLC, represses its expression, and releases expression of FT in companion cells. Our results suggest that JMJ18 is a developmentally regulated companion cell–dominantly expressed signal to control flowering time by binding to FLC—reducing level of H3K4 methylation in FLC and repressing expression of FLC—thereby promoting expression of FT in companion cells during vegetative development in Arabidopsis.Flowering is an important developmental transition during plant life cycle and the key process for production of the next generation. Flowering time is controlled by both intrinsic developmental and environmental signals. FLC and its target  DNA is packaged as chromatin in eukaryotic cells. Nucleosomes, which consist of an octamer of four histones wrapped around 146 base pairs of DNA, are the fundamental unit of chromatin In vivo, the lysine residues in histones display three distinct methylation states: mono- (me1), di- (me2), and tri-methylated (me3). These differences in methylation are important for the recognition of chromatin by chromatin modulators and for the recruitment of other modulators and regulators Arabidopsisin vivo is the latest to be discovered compared to other covalent forms of histone modification. The amine oxidase LSD1 was the first histone demethylase found to demethylate H3K4me2 and H3K4me1 through an FAD-dependent oxidation reaction HOX gene regulation, and germ cell development The methylation state of histone proteins is determined by the balance between methylation and demethylation, which is mediated by histone methyltransferases and demethylases, respectively Arabidopsis. They promote the floral transition by constitutively inhibiting the expression of FLCArabidopsis genome contains 21 JmjC family proteins in vitroFLD and LDLs are the homologs of human LSD1 in Flowering at the appropriate time is the most important factor in achieving reproductive success. To produce the next generation, plants rely on intricate signaling pathways involving a variety of intrinsic factors, including developmental stage and age, and environmental cues such as photoperiod, temperature, and light quantity FLOWERING LOCUS C (FLC) encodes a MADS-box transcription factor and floral repressor that regulates flowering time in a dosage-dependent manner by integrating the vernalization, autonomous, PAF1 complex, and H2B ubiquitination pathways FLC and the functional locus FRIGIDA (FRI) act together to produce winter-annual Arabidopsis accessions, which must be exposed to cold temperature for several weeks to repress FLC expression and promote flowering in the following spring FLC expression FLC antisense transcript affects the expression of sense transcript, thereby influencing flowering time in ArabidopsisFLC expression is predominant in the shoot apical meristem (SAM); however, it is also expressed in vascular tissue in young leaves and the root tip FLC expressed in the SAM and leaf vascular tissue contributes to the control of flowering time in ArabidopsisFLC chromatin, thereby repressing its expression in the SAM FLC expression in the SAM FLC expression in the SAM has been extensively studied, little is known about how FLC expression is regulated in leaf vascular tissue.FLC expression in Arabidopsis. In FLC chromatin, H3K4 hyper-tri-methylation and acetylation are associated with the activation of gene expression FLC chromatin leads to the repression of FLC expression and is required for maintenance of the repression of FLC expression in plants growing in the following spring after vernalization FLC transcription and early flowering Histone modification plays crucial roles in the regulation of FT expression is restricted to leaf phloem companion cells FLC-dependent pathways to control flowering time by regulating the expression of floral identity genes. It was shown previously that the companion cells specifically expressed FLC represses FT expression in leaf companion cells and delays the expression of its cofactor FD in the SAM FLC expression in companion cells for the control of floral development has not been characterized.FLOWERING LOCUS T (FT), which is a component of the photoperiod pathway, coordinates signals from the vernalization, autonomous, PAF1 complex, and photoperiod pathways to promote flowering in response to increase in day length FLC repressor in flowering time regulation FLC is not likely to be the direct target of REF6 FLC locus have yet been found.As mentioned above, several JmjC domain-containing proteins have been reported to be involved in flowering time control Arabidopsis. JMJ18 was predominant expressed in phloem companion cells, and its level of expression increased during vegetative development. JMJ18 was found to promote the floral transition in Arabidopsis by binding FLC chromatin and demethylating H3K4 methylation, leading to the repression of FLC and enhanced expression of the downstream flowering activator FT in companion cells.In this paper, we describe the expression pattern and function of JMJ18, a novel JmjC domain-containing histone H3K4 demethylase in ArabidosisJMJ18 (At1g30810) belongs to the evolutionarily conserved JARID1 family. Previous studies have shown that JARID1 family proteins demethylate H3K4 methylations in yeast, animal and To determine whether JMJ18 exhibits histone demethylase activity, we expressed recombinant His-tagged JMJ18 and purified the recombinant protein from insect cells . A MALDIin vitro. However, they were unable to demethylate H3K4me2 to H3K3me1 if H3K4me2 peptide was used as the substrate .We further observed that the absence of α-ketoglutarate (α-KG) or presence of EDTA completely abolished the enzyme's demethylase activity , while aJMJ18, we obtained three T-DNA insertion lines from the ABRC jmj18-1 (SALK_073442), jmj18-2 (GABI_649D05), and jmj18-3 (SAIL_861_F02)  . Reverseth lines . In the th lines , and fulth lines .jmj18-1 was complemented by JMJ18:JMJ18-GFP transformation (JMJ18 expression by using both double strand RNA interference (RNAi) and artificial micro-RNA (amiR) were generated, and these lines also exhibit weak late-flowering phenotype under LD conditions , and FT, by quantitative real-time PCR (qRT-PCR). No obvious change in expression compared to wild-type was detected at various vegetative developmental stages, but statistical significant for the expression of FLC and FT between wild-type and jmj18 mutants if any at later vegetative developmental stages  and short-day (SD) conditions . This laormation . Knock-dnditions . To addrl stages . This suArabidopsis, we examined its expression pattern using wild-type Arabidopsis plants carrying a JMJ18 promoter-fused GUS reporter construct. The whole intergenic region between JMJ18 and its upstream gene was first selected to be the promoter of JMJ18 , the companion cell-specific SUC2 promoter, and the constitutively expressed CaMV35S promoter of which is expressed in and outside of the companion cells in wild-type plants. All three types of overexpression lines exhibited earlier flowering compared to wild-type under inductive LD and non-inductive SD conditions : MJ18-GFP . Taking JMJ18 and FT spatio-temporal expression patterns  was echoed by that of 35S:JMJ18-GFP  transgenic plants exhibited a shorter life cycle than wild-type plants; however, the effect was weaker than that observed in 35S:JMJ18 plants (JMJ18 with a deletion in both the zinc-finger and FY-rich domains (35S:NC) (JMJ18 with a deletion in both the JmjN and JmjC domains displayed similar flowering time compared to wild-type plants (8 plants . Regardl(35S:NC) . Northere plants . Thus, t35S:T  transgenic plants also displayed an obviously earlier floral transition compared to wild-type plants  were also repressed to different extent in JMJ18 overexpressors . Thus, we propose that JMJ18 is a repressor of FLC.To analyze the temporal expression pattern of n day 15 . There wjmj18-1 flc-3 to test whether JMJ18 interacts genetically with FLC. The FLC mutation mainly blocks the late-flowering phenotype of jmj18-1   , indicatin vitro  was done to detect the level of H3K4 methylation across the entire FLC chromatin region. In wild-type plants, the regions around the transcription start site  had higher levels of H3K4me3 than the other regions , JMJ15 (at2g34880), JMJ16 (at1g08620), JMJ17 (at1g63490), JMJ18 (at1g30810), and JMJ19 (at2g38950). To examine whether Arabidopsis JMJ18 is a real histone demethylase, we characterized its histone demethylase activity in vitro and in vivo. We examined the histone demethylase activity of JMJ18 in vitro by purifying recombinant JMJ18 from insect cells and analyzing it by MALDI-TOF mass spectrometry. In that analysis, JMJ18 exhibited histone H3K4-specific demethylase activity  is expressed broader in root and leaf JMJ18 promoter-GUS fusion as Hong et al (2009) and transformed it into the wild-type plants. We observed that all 21 independent lines transformed with 1.7 kb length promoter-GUS we examined exhibits similar expression pattern, and the two versions of promoter (1.4 and 1.7 kb in length) exhibit very similar expression patterns  Cell- or tissue-specific expression is a significant part of enabling a gene to achieve its function in plant cell fate determination and development  jmj18-1 , it was pression  and S6.  flowers  and S6, minantly . In the minantly  and S6D.minantly  and S6, patterns  and S6. JMJ18:GFP in phloem indicated that JMJ18 is predominantly expressed in phloem companion cells  medium (Sigma-Aldrich) containing 1% sucrose and 0.3% phytagel (Sigma-Aldrich). Following ten days of growth under LD  or SD  conditions in a growth chamber  under a cool white fluorescent light (160 µmolm−2 s−1), the plants were transplanted to soil and grown in a growth room under LD  or SD  conditions at 50% relative humidity.The jmj18-1 (SALK_073442), jmj18-2 (GABI-649D05), jmj18-3 (SAIL-861-F02), ft-11 (GABI-290E08), and co-11 (SAIL_24_H04) were obtained from the ABRC. flc-3, fd-5, and FRI-Col were described previously JMJ18:GUS, 1,361 and 1667-bp genomic sequence upstream of the JMJ18 ATG were PCR-amplified and fused with GUS. The primers used for the two versions of promoter were forward for 1,361 bp 5′-CTGCAGACAAGACAGAAGAAGCAGGAAAC-3′, 1667 bp 5′-CTGCAGTGCGAGTTCTTCTTTCAATGTG-3′ and reverse for both, 5′-TCTAGAATGAATTGAAAAATCAATACTACTCAA-3′. The SUC2 promoter was cloned as described previously SUC2 promoter forward 5′-CTGCAGAAAATCTGGTTTCATATTAATTTCA-3′, and reverse 5′-TCTAGAATTTGACAAACCAAGAAAGTAAGA-3′.To construct JMJ18 CDS: JMJ18 forward 5′-TCTAGAATGGAAAATCCTCCATTAGAATC-3′, and reverse, 5′-GGATCCCCATCAAATCTACTCCGAAAAGTT-3′. GFP, RFP, or 6×c-myc was fused to the C-terminus of the JMJ18 CDS to produce tagged JMJ18. To construct 35S:NC, 35S:NCZ, 35S:T, 35S:JMJ18 and 35S:ZY, the following primers were used to amplify the truncated or full-length JMJ18 CDS: JMJ18 forward 5′-TCTAGAATGGAAAATCCTCCATTAGAATC-3′; JMJ18 NC reverse 5′-GAGCTCTTAATATAGTTCCACAGCGTTCTGTC-3′; JMJ18 NCZ reverse GAGCTCTTAACCTTCCTTTAACTTCTTCTCCTC-3′; JMJ18 T reverse, 5′-GAGCTCTTAAACTATAGAGGGTGAGAGGAAACC-3′; JMJ ZY forward, 5′-TCTAGAATGCAGAACGCTGTGGAACTATATAG-3′ and JMJ18 CDS reverse 5′-GAGCTCTTACATCAAATCTACTCCGAAAAGTT-3′.We used the following primers to amplify the full-length Agrobacterium tumefaciens strain GV3101. Plants were transformed using the floral dip method For plant transformation, the constructs were transformed into JMJ18Δ420–432 bp and JMJ18Δ420–474 bp) versions of JMJ18 were generated by PCR amplification from Arabidopsis cDNA and cloned into the pFastBac expression vector (Invitrogen). Baculoviruses expressing the various proteins were generated by transfecting the appropriate vector into High5 insect cells. The proteins were purified using Ni-NTA affinity resin (Qiagen). Select fractions were concentrated and purified by size-exclusion chromatography.Full-length and truncated . The reaction was stopped by freezing, and the mixture was diluted for mass spectrometry. The peptides used were: H3K4me3 ARTK(me3)QTARKS, H3K4me2 ARTK(me2)QTARKS, H3K36me3 ATGGVK(me3)KPHRYR, H3K36me2 ATGGVK(me2)KPHRYR, H3K9me3 ARTKQTARK(me3)STGGKAPRAQ, H3K9me2 ARTKQTARK(me2)STGGKAY, H3K27me3 ATKAARK(me3)SAPATG, and H3K27me2 ATKAARK(me2)SAPATG.The histone demethylase assay and MALDI-TOF mass spectrometry were performed as described previously JMJ18:GUS T2 lines were used for histochemical analysis, with at least ten individual plants observed for each line. Pictures were taken using a stereomicroscope (Leica MZ16) or light microscope (Zeiss Imager M1).GUS staining was performed as described previously JMJ18:JMJ18-GFP, 20 independent lines were observed, with at least ten individual plants analyzed per line. Images were collected using a Zeiss LSM 510 Meta confocal laser scanning microscope as described previously For the observation of GFP fluorescence, roots were cut from seedlings grown on MS medium under LD conditions for seven days. For JMJ18:JMJ18-RFP was constructed to pCAMBIA2300 and introduced into SUC2:JMJ18-GFP (in pCAMBIA1300) transgenic plants. The double transformants were selected on MS medium containing kanamycin and hygromycin. Transgenic lines expressing JMJ18-GFP and -RFP were selected and photographed using a Zeiss LSM 510 Meta confocal laser scanning microscope.Total RNA was isolated from the plant materials indicated in the text using RNAiso plus (Takara) and treated with RNA-free DNase (Promega) to remove all remaining DNA. Three micrograms of total RNA were used to synthesize first-strand cDNA with a reverse transcription kit (Fermentas). The cDNAs were diluted to 60 µl with sterilized water. One microliter of diluted cDNA was used for real-time PCR amplification with SYBR Premix Ex Taq (Takara). Three biological replicates were performed to calculate the mRNA abundance. Standard deviations were calculated from the replicates. The primers used were listed in Plant tissue (1.5 g) was collected after growth under LD conditions for eleven days. ChIP was performed as described previously with three biological replicates E. coli were used as antigens to immunize rabbits. Each rabbit was immunized once every fourteen days with 1 mg of protein. After four successive immunizations, the anti-serum was examined by ELISA and Western blotting using total protein from the mutant and overexpressor lines.Purified recombinant JMJ18 from insect cells and FT from Arabidopsis seedlings were ground to a powder in liquid nitrogen then homogenized in extraction buffer . The extracts were then centrifuged, the pellet removed, and the supernatant boiled in 6× SDS sample buffer. The proteins in the samples were separated by 8% SDS-PAGE, transferred to polyvinylidene difluoride membranes (Millipore), and detected using different antibodies. The antibody used for Tubulin detection was anti-α-Tubulin (Sigma-Aldrich T5168).For immunoblotting, JMJ18, a double strand RNA and artificial microRNA interference approaches were used. For a double strand RNA RNAi, a unique 616 bp JMJ18 sequence was amplified by PCR. The gene-specific primers were: forward 5′-GGGGTACCTCTAGAGGTGAAGGCTCTTTGGGAACTC-3′, reverse 5′-CGGGATCCGAGCTCCTCGACCGATACACCAAGGTTC-3′. The self-complementary hairpin RNA was constructed to pTCK303 as described by Wesley et al. (2001) To knock-down the expression of http://wmd2.weigelworld.org/cgi-bin/mirnatools.pl?page=1) for artificial microRNA interference assay. The primers are: amiR-s, 5′-GATGAGTCTTTAAAATGCAGGGCTCTCTCTTTTGTATTCC-3′, amiR-a, 5′-GAGCCCTGCATTTTAAAGACTCATCAAAGAGAATCAATGA-3′, amiR*s, 5′-GAGCACTGCATTTTATAGACTCTTCACAGGTCGTGATATG-3′, amiR*a, 5′-GAAGAGTCTATAAAATGCAGTGCTCTACATATATATTCCT-3′. The constructs for amiRNAi were established by following the protocols from Schwab et al.. The resulting three constructs were delivered to wild-type through agrobacterium–mediated transformation to generate JMJ18 amiRNA interference lines.Three primer pairs were designed through the web (Arabidopsis Genome Initiative database under the following accession numbers: ACTIN (At5g09810), CO (At5g15840), FLC (At5g10140), FT (At1g65480), FRI (At4g00650), JMJ18 (At1g30810), MAF1 (At1g77080), MAF2 (At5g65050), MAF3 (At5g65060), MAF4 (At5g65070), MAF5 (At5g65080), SUC2 (At1g22710) and TSF (At4g20370).Sequence data from this article can be found in the Figure S1in vitro.His-JMJ18 purification and characterization of its histone demethylase activity. (A) SDS-PAGE of purified recombinant His-JMJ18 from High5 insect cells. M, molecular standard; Total, total lysate; Input, cleared input. F1–F5, the fractions eluted from the nickel-affinity column. The numbers represent the molecular weights. (B) Purification of His-JMJ18 by size-exclusion chromatography. Top panel, UV absorbance; Bottom panel, results of the SDS-PAGE analysis of the purified and eluted recombinant His-JMJ18 fractions produced by size-exclusion chromatography. (C) Summary of the histone demethylase activity of His-JMJ18 (TIF)Click here for additional data file.Figure S2jmj18-1 by JMJ18:JMJ18-GFP transformation. (A) The late-flowering phenotype of jmj18-1 was complemented by JMJ18:JMJ18-GFP transformation. Twenty-eight-day-old plants were photographed. Bar = 2 cm. (B) and (C) Statistical analysis the phenotype of complementary plants by days to first flower open (B) and rosettle leaf number (C). At least 16 plants of each genotype were used for analysis. Asterisks indicate the significant differences analyzed by Student's t test (P<0.05).Complementation assay of (TIF)Click here for additional data file.Figure S3JMJ18 RNAi and amiR lines. The gene expression levels of JMJ18, FLC and FT in JMJ18 knock-down transgenic lines. RNAi: knock-down the expression of JMJ18 by double strands of RNA. amiR: knock-down the expression of JMJ18 by artificial microRNA. The numbers in parentheses indicate the amplification cycles.Characterization of (TIF)Click here for additional data file.Figure S4FLC and FT in jmj18 mutants at differential vegetative developmental stages. The expression levels of FLC at day 6 (A), 9 (C) and 15 (E), and FT at day 6 (B), 9 (D) and 15 (F) grown under long-day condition were measured. The eleven-day-old seedlings were collected at dusk at the day indicated. The expression level was normalized to that of ACTIN. Error bars indicate the standard deviation of three replicates. Asterisks indicate the significant difference between wild-type and jmj18 mutants analyzed by Student's t test (P<0.05).The expression levels of (TIF)Click here for additional data file.Figure S5JMJ18. (A) Two promoter regions selected for GUS constructs. The black filled boxes are shown exons, black lines for introns, and gray lines for intergenic sequence. (B) 7-day-old cotyledon. Bar = 1 mm. (C) 20-day-old rosette leaf. Bar = 1 mm. (D) 10-day-old root. Bar = 0.5 cm. (E)–(J) The marked regions of the root in (D). Bar = 0.1 mm. GUS staining in anther  and . 21 independent T2 lines were used for GUS staining. 1.7 kb:GUSJMJ18 transgenic plants were used in (A)–(L). All 21 independent lines we examined display the similar expression pattern for JMJ18. 1.4 kb:GUSJMJ18 transgenic plants were used in (M) and (N).The expression pattern of (TIF)Click here for additional data file.Figure S6JMJ18 in stem, hypocotyl and petal by using 1.4 kb:JMJ18-GFPJMJ18 and nuclear localization of JMJ18. JMJ18 was expressed in companion cells in different tissues. GFP fluorescence was detected in (A) the junction region between cotyledon and hypocotyl from 7-day-old seedling, where the vascular tissue branched, bar = 100 µm; (B) 7-day-old hypocotyl, bar = 50 µm and (C) petal, bar = 20 µm, respectively. (D) Nuclear localization of JMJ18. Bar = 20 µm.The expression pattern of (TIF)Click here for additional data file.Figure S735:JMJ18 transgenic plant was used to mark JMJ18 band, about 20% amount of the other samples was loaded for the sample from 35:JMJ18 transgenic plant. The arrows indicated the JMJ18-GFP or JMJ18 bands. The upper panel: the gel was exposure for 1 min; the lower panel: the gel was exposure for 20 min. Tubulin was used as a loading control.JMJ18 in JMJ18 overexpression and wild-type plants. The (TIF)Click here for additional data file.Figure S8TSF expression. qRT-PCR analysis of TSF mRNA expression in the transgenic plants. The expression level was normalized to that of ACTIN. Error bars indicate the standard deviation of three independent biological replicates. Asterisks indicate the significant difference between wild-type and transgenic plants analyzed by Student's t test (P<0.05).JMJ18 induced (TIF)Click here for additional data file.Figure S935S:ZY transgenic plants. (A) Schematic structures of full-length JMJ18 protein as well as JMJ18 ZY. (B) Flowering time distributions of wild-type and 35S:ZY at T1 generation measured by rosette leaf number. Thirty-two wild-type and 108 independent transgenic plants were analyzed. (C) Flowering time of wild-type and 35S:ZY transgenic plants counted by rosette leaf number. (D) JMJ18 ZY transcription levels in independent 35S:ZY transgenic line, and the rosette leaf number for each plant was indicated.Characterizing (TIF)Click here for additional data file.Figure S10ACTIN, AtMu1 and AtSN1 loci. The H3K4me3 modification levels were detected in wild-type and SUC2:JMJ18-GFP plants. The results were normalized to the input. The values are the mean ± standard deviation from three biological replicates. Asterisk indicates the significant difference between wild-type and transgenic plants analyzed by Student's t test (P<0.05).JMJ18 do not affect the H3K4me3 modification levels at (TIF)Click here for additional data file.Figure S11FLC/MAFs H3K4me3 levels. (A) Overexpression JMJ18 reduced H3K4me3 level at FLC locus in JMJ18 overexpression plant compared to wild-type plant. (B) ChIP analysis of H3K4me3 levels for other members of FLC clade genes. (C) The H3K4me3 levels were not obviously changed at ACTIN, AtMu1 or AtSN1 chromatin between wild-type and JMJ18 overexpression plants. The enrichments were normalized to total H3. The values are the mean ± standard deviation from three biological replicates.JMJ18 decreases (TIF)Click here for additional data file.Figure S12SUC2:JMJ18-myc line. (A) Twenty-four-day-old SUC2:JMJ18-myc plants grown under LD conditions were shown. Bar = 2 cm. Thirty-three out of 108 independent SUC2:JMJ18-myc lines displayed the early-flowering phenotype. The independent line shown was used to analyze FLC and FT expression and for ChIP. (B) Flowering time in wild-type plants and the SUC2:JMJ18-myc line based on the number of rosette leaves at flowering. The values are the mean ± standard deviation from 20 plants. qRT-PCR analysis of FLC (C) and FT (D) expression. The expression level was normalized to that of ACTIN. The levels of H3K4me3 (E) and H3K4me2 (F) across the FLC genomic region in wild-type and SUC2:JMJ18-myc plants were determined. The values in (C) to (F) are the mean and standard deviation from three technical replicates.Early-flowering phenotype of the (TIF)Click here for additional data file.Table S1JMJ18 expression leads to late-flowering phenotype. All the plants were grown in LD condition. The values are the mean ± standard deviation. n indicates the plants number scored for the analysis. Asterisks indicate the significant differences in the statistic analysis between wide type and mutants using Student's t test (P<0.05).Knock-down (DOC)Click here for additional data file.Table S2JMJ18 and FLC in flowering time control. All the plants were grown under long-day condition. The values are the mean ± standard deviation. n indicates the plant number scored for phenotype analysis.Genetic interaction between (DOC)Click here for additional data file.Table S3The primers used for detecting gene expression level.(DOC)Click here for additional data file.Table S4The primers used in ChIP assay.(DOC)Click here for additional data file.


Prevalence of these viruses increased during the 2008–09 season. To monitor oseltamivir-resistant influenza viruses A (H1N1) (ORVs) with H275Y in neuraminidase (NA) in Japan during 2 influenza seasons, we analyzed 3,216 clinical samples by NA sequencing and/or NA inhibition assay. The total frequency of ORVs was 2.6%  during the 2007–08 season and 99.7%  during the 2008–09 season, indicating a marked increase in ORVs in Japan during 1 influenza season. The NA gene of ORVs in the 2007–08 season fell into 2 distinct lineages by D354G substitution, whereas that of ORVs in the 2008–09 season fell into 1 lineage. NA inhibition assay and M2 sequencing showed that almost all the ORVs were sensitive to zanamivir and amantadine. The hemagglutination inhibition test showed that ORVs were antigenetically similar to the 2008–09 vaccine strain A/Brisbane/59/2007. Our data indicate that the current vaccine or zanamivir and amantadine are effective against recent ORVs, but continuous surveillance remains necessary. Influenza A and B viruses are major pathogens that represent a threat to public health with subsequent economic losses worldwide  (ORVs) was reported, mainly in countries in Europe where the prevalence varies, with the highest levels in Norway (67%) and France (47%)  and 1,482 samples of influenza A (H1N1) were isolated in the 2008–09 season (September 2008–April 2009). During the 2007–08 season, viruses were isolated primarily after December 2007. All influenza viruses A (H1N1) were subjected to full or partial (nt 615–1076) NA sequencing to detect H275Y substitution on the N1 NA protein. Representative influenza viruses A (H1N1), including ORVs and OSVs, were subjected to NA inhibition assay , full NA sequencing (891 and 83), hemagglutinin (HA) 1 sequencing (299 and 83), M2 sequencing (288 and 79), and hemagglutinin inhibition (HI) test (187 and 59).Influenza sentinel clinics send clinical specimens to local public health laboratories for virus isolation. Several culture tissues, including MDCK, Caco-2, and LLC-MK2, are used for virus isolation. Without successful viral isolation, clinical specimens are analyzed directly. Influenza viruses were collected from all 47 prefectures in Japan for this study; 1,734 samples of influenza A (H1N1) were isolated during the 2007–08 season  were calculated by a 4-parameter method using MikroWin 2000 version 4 software .The chemiluminescent NA inhibition assay was performed by using the NA Star Kit  with slight modifications of the instructions provided by the manufacturer. The final drug concentration ranged from 0.03 nmol/L to 6,500 nmol/L for oseltamivir and from 0.03 nmol/L to 12,500 nmol/L for zanamivir. Chemiluminescent light emission was measured by using an LB940 plate reader . Drug concentrations required to inhibit NA activity by 50%  and -sensitive viruses, box-and-whisker plots were used. The cutoff value was defined as upper quartile + 5.0×interquartile range from the 25th to 75th percentile. In this study, ORVs with H275Y were excluded from the overall population for statistical analysis. Outliers were excluded from the calculation of mean values and standard deviations for ICTo estimate the frequency of influenza A (H1N1) ORVs in each prefecture of Japan, 1,734 isolates during the 2007–08 season and 1,482 isolates during the 2008–09 season were collected from all prefectures and examined by NA sequencing to detect the H275Y mutation in NA protein. In the 2007–08 season, 45 viruses possessing H275Y mutation  during 2007–08 fell into either clade 2B, including the current vaccine strain A/Brisbane/59/2007, or clade 2C, and almost all influenza viruses A (H1N1) during 2008–09 fell into clade 2B. Most ORVs with H275Y belong to clade 2B, which can be further divided into 2 distinct lineages by an aspartic acid to glycine substitution at aa 354: a Northern-Eu lineage sharing 354G, which was first isolated from countries in northern Europe and now represents most ORVs worldwide; and a Hawaii lineage sharing 354D, which was first detected in Hawaii and was rarely isolated in a few countries during the 2007–08 season . In the In the Hawaii lineage, OSVs genetically close to ORVs were observed. The NA gene of some ORVs had only 1 nucleotide difference from that of OSV counterparts , and the other ORVs are also genetically close to OSVs from Japan . These H50 values were calculated from a normal sigmoid curve  tested by NA inhibition assay, 101 possessed a H275Y substitution. With the NA inhibition assay, although precise ICA curves , panel CA curves , panel D50 ± SD of 0.10 ± 0.05 nmol/L (range 0.01–0.35 nmol/L), and ORVs had a mean ± SD IC50 of 67.7 ± 44.1 nmol/L (range 26.1–239.2 nmol/L), showing a reduction of >260-fold in susceptibility to oseltamivir. One OSV identified as a statistical outlier  showed a D151E substitution on the NA protein , and 16 viruses (10 ORVs and 6 OSVs) were identified as outliers  (50 (41.89 nmol/L), and 19 of the other 22 viruses displayed an amino acid change G, N, or V at the D151 position  . Seven vposition . These dposition . We thusSusceptibility to M2 inhibitors was determined to find established-resistant markers by M2-sequencing. The 367 viruses  including 123 ORVs  and 244 OSVs  were tested. Viruses belonging to clade 2B were sensitive to M2 inhibitors, and viruses belonging to clade 2C were resistant to M2 inhibitors, so all ORVs except A/Yokohama/91/2007 were sensitive to M2 inhibitors. A/Yokohama/91/2007 belonged to clade 2C and was the only virus resistant to both oseltamivir and M2 inhibitors.To estimate the reactivity of ORVs and OSVs to ferret antiserum against 2008–09 vaccine strain A/Brisbane/59/2009, the HI test was performed. Good inhibition was achieved in 76% of OSVs and 69% of ORVs by A/Brisbane/59/2009 ferret antiserum, and 22% of OSVs and 28% of ORVs showed a 4-fold reduction in HI titer to the homologous titer, respectively . Only 2%Tottori Prefecture is located in the western part of the main island of Japan. Comprising 19 cities and geographically divided into 3 areas, this prefecture has the lowest population in Japan , panel BPhylogenetic analysis of NA genes showed that these ORVs formed 3 subclades , panel AA mapping study for ORVs showed that all ORVs in the Hawaii lineage were collected from Tottori city only, primarily at the end of January, whereas ORVs with the Northern-Eu lineage were collected from 4 cities, including Tottori city, during February and March. Genetically diverse ORVs belonging to T1-T3 were cocirculating only in Tottori city in the eastern area , panel BOur study demonstrated that ORVs dramatically increased in Japan from the 2007–08 season (2.6%) to the 2008–09 season (99.7%). All tested ORVs showed a reduction of >260-fold in susceptibility to oseltamivir by NA inhibition assay. On the other hand, almost all ORVs remained sensitive to the other antiviral-drugs, e.g., zanamivir, and M2 inhibitors. HI testing suggested that the current vaccine, A/Brisbane/59/2008, would be effective against recent ORVs. In addition, recent studies have reported that symptoms and hospitalization rates of patients infected with ORVs are no different from those seen with OSVs (Japan has the largest per capita use of oseltamivir (>70%) in the world , possessed a Q136K substitution, which reportedly confers resistance to zanamivir , supportBy sequencing of M2 gene, we confirmed that almost all Japanese ORVs belonging to clade 2B retained sensitive genotype to M2 inhibitors, consistent with previously reports that recent clade 2B viruses are sensitive to M2 inhibitors, but clade 2C viruses are resistant  (now known as pandemic [H1N1] 2009 virus) emerged in Mexico and the United States and spread rapidly to many countries, including Japan  A) neuraminidase (NA) genes and B) hemagglutinin (HA) (HA1 region) genes. Recent influenza viruses A (H1N1) fell into either clade 2B or clade 2C. Almost all oseltamivir-resistant viruses (ORVs) with H275Y belong to clade 2B and were further divided into 2 distinct lineages: Northern-EU lineage sharing 354G (pink shading); and Hawaii lineage sharing 354D. ORVs during 2008-09 shared A189T on HA, and formed 4 subclades: C-1 (HA: G185A and S141N); C-2 (HA: G185V); C-3 ; and C-4 (HA: G185S/N and N183S). OSVs during 2007-09, Japanese ORVs during 2007-08, Japanese ORVs during 2008-09, foreign ORVs during 2007-09 and 2008-09 current vaccine strains are indicated in black, blue, red, orange, and purple, respectively. Sampling month of each isolate is described after the strain name. Viruses resistant to zanamivir are marked with an asterisk. The phylogenetic tree of NA and HA1 genes was constructed by using neighbor-joining methods.


No homozygote DPP9S729A/S729A weaned mice were detected. DPP9S729A/S729A homozygote embryos, which were morphologically indistinguishable from their wild-type littermate embryos at embryonic day (ED) 12.5 to ED 17.5, were born live but these neonates died within 8 to 24 hours of birth. All neonates suckled and contained milk spots and were of similar body weight. No gender differences were seen. No histological or DPP9 immunostaining pattern differences were seen between genotypes in embryos and neonates. Mouse embryonic fibroblasts (MEFs) from DPP9S729A/S729A ED13.5 embryos and neonate DPP9S729A/S729A mouse livers collected within 6 hours after birth had levels of DPP9 protein and DPP9-related proteases that were similar to wild-type but had less DPP9/DPP8-derived activity. These data confirmed the absence of DPP9 enzymatic activity due to the presence of the serine to alanine mutation and no compensation from related proteases. These novel findings suggest that DPP9 enzymatic activity is essential for early neonatal survival in mice.Dipeptidyl Peptidase (DPP) 4 and related dipeptidyl peptidases are emerging as current and potential therapeutic targets. DPP9 is an intracellular protease that is regulated by redox status and by SUMO1. DPP9 can influence antigen processing, epidermal growth factor (EGF)-mediated signaling and tumor biology. We made the first gene knock-in (gki) mouse with a serine to alanine point mutation at the DPP9 active site (S729A). Weaned heterozygote DPP9 The DPP4 family of enzymes gained prominence with the success of oral DPP4 inhibitors as type 2 diabetes therapeutics 34-32DPP9 overexpression impairs cell adhesion While DPP4 and FAP are predominantly expressed on the cell surface, DPP9 and DPP8 are intracellular enzymes that are ubiquitously expressed in tissues and most cell lines in vitro and may cleave NPY inside cells in vivo are probably diverse such that the major role of DPP9 may be elusive.Little is known of the natural substrates of DPP9 but it can cleave the DPP4 substrates glucagon-like peptide (GLP)-1, GLP-2, peptide YY and NPY Until now, no lethal gene knockout (gko) mouse for a protein that possesses DPP activity has existed in vivoA report that certain DPP inhibitors that inhibit DPP9 and DPP8 activity are toxic Structurally, DPP9 is predominantly a dimer in vivo significance of DPP9 enzymatic activity, the first gki mouse was generated containing the S729A point mutation. This biological strategy is expected to mimic the effect of a non-toxic selective DPP9 inhibitor administered from the time of fertilization.DPP9 has an active-site catalytic triad that is conserved across the DPP4 protein family and can hydrolyze the post-proline bond two residues from the N-terminus of peptide substrates. The DPP4 family proteins are atypical serine proteases that have the catalytic triad in the reverse order to trypsin and are unusual in that the catalytic pocket is buried within the protein. In the 863 amino acid isoform (short form) of DPP9, the triad consists of Ser729, Asp807 and His839. We showed that mutation of the catalytic serine of DPP9 to alanine in amino acid position 729 of the short form of DPP9 (S729A) ablates enzymatic activity without affecting other characteristics of this protein The animal experiments were approved by the local Animal Ethics Committee of the University of Sydney  and conducted in accordance with all applicable laws and guidelines.Generation of DPP9 enzyme-inactive mice was performed by Ozgene  using standard techniques. The DPP9 targeting construct was generated by cloning polymerase chain reaction (PCR) products of the 5′ and 3′ homology arms, as well as a short loxP arm containing the S729A mutation , into thwt/flox ES cells. The clone chosen for generating the mutant mice, 1G6, was analysed by Southern blot and verified to be correctly targeted at each end of the construct (The targeting construct was electroporated into C57BL/6 Bruce 4 embryonic stem (ES) cells and clones were selected for neomycin resistance. Southern blot analyses , Table 1onstruct . Probingwt/flox ES clone was microinjected into BALB/c blastocysts which were implanted into pseudo-pregnant foster mothers. Chimeric male mice from the resultant litters were mated with C57BL/6 wild-type females and offspring with coat-colour transmission were genotyped for germline transmission of the S729A mutation by Southern blot of tail DNA  were mated with C57BL/6 mice for removal of the FLPe gene.The neo cassette was FLPe-deleted wt/S729A intercross progeny were genotyped by Southern Blot using the 5′ or 3′ probes  12.5 (ED12.5), ED13.5, ED15.5 and ED17.5 embryos were harvested from DPP9For post-natal studies, both observational and experimental studies were undertaken to define the phenotype. In observational studies, hourly litter examinations from birth ensured retrieval of non-surviving pups for genotyping and formalin fixation. All pups were closely observed for physical activity, breathing difficulties and unusual behaviours or appearance. In litters used for experimental studies, all pups were euthanized within six hours of birth, tails retained for genotyping and head and body fixed in 10% neutral buffered formalin.2O2. Bright-field imaging was performed using a Leica DM6000B microscope.To undertake histological observations, paraffin-embedded pups were sectioned at 5 µm and H&E stained. To immuno-localise DPP9 protein, the anti-DPP9 antibody  was used which bound to both the wild-type and S729A DPP9 proteins. Briefly, 5 µm sections were deparaffinized and rehydrated and then a pressure cooker and Universal Decloaker solution  was used to retrieve antigen. Sections were then covered with Background Sniper  for 10 mins, rinsed in PBS and incubated at room temperature for 1 h with primary antibody with 1% BSA in PBS and Renaissance Background Reducing Diluent . After thorough washing in PBS, sections were incubated for 30 mins with goat anti-rabbit conjugated to HRP , washed and then stained in 3,3-diaminobenzidine (DAB) with HDPP9-WT-EGFP and enzyme-inactive mutant DPP9-S729A-EGFP constructs et alIn litters used for detection of protein in urine, pups were euthanized within 6 hours of birth by decapitation, then urine collected and analyzed by a method modified from Putaala wt/S729A intercrosses were harvested under sterile conditions and MEFs prepared using standard protocols 2 as passage zero.ED13.5 embryos from DPP9Genotyping by PCR was performed on DNA isolated from embryonic head treated with proteinase K using the Wizard® Genomic DNA isolation kit .S729A/S729A littermates were infected with SV40 large T antigen-expressing lentivirus to generate immortal cell lines 5) plated out 24 h earlier were transduced in 6-well culture plates containing 2 mL complete growth medium and 8 µg/mL Polybrene , ‘spinoculated’ at 1,500 rpm for 1 h and then cultured for 4 days at 37°C. Puromycin selection (1 µg/mL) was added for 3 days to remove non-transduced cells. Colonies of immortalized MEFs emerged after 10 days.After the third passage, primary MEFs from wild-type and DPP94 MEF cells or 10 µg of liver sample lysate per well were added to black 96-well plates  in triplicate in the presence or absence of a selective DPP4 inhibitor, sitagliptin , at 1 µM and TE buffer to a volume of 50 µL per well. 50 µL of substrate was added to a final reaction concentration of 1 mM in TE buffer (pH 7.6) with 5% methanol. The fluorescence produced by substrate cleavage was monitored every 5 min over 1 h at 37°C in a Polarstar Omega microplate reader  with excitation at 355 nm and emission at 450 nm.DPP8/9 enzyme assay using the DPP fluorogenic substrate H-Gly-Pro-AMC  was adapted from described methods 2, 150 mM NaCl, 1% Triton-114, 10% glycerol, 1× Roche complete protease inhibitor cocktail  and stored at −20°C. Frozen neonate mouse liver samples were homogenized in lysis buffer using a bead-based homogenizer  at 4°C. Protein concentration was determined using the Micro BCA Protein Assay Kit  following the manufacturer's protocol.For immunoblot sample preparation, MEFs were harvested by trypsinization and washed with ice-cold PBS before re-suspension in ice-cold lysis buffer  were resolved on 4–12% Bis-Tris SDS-PAGE (Invitrogen) followed by immunoblotting with anti-DPP9 antibody (1∶2000) Total RNA was isolated using Trizol (Invitrogen) and cDNA synthesis was performed using SuperScript VILO cDNA Synthesis Kit (Invitrogen). qPCR was performed as described previously wt/S729A mice on a C57BL/6 background were generated and sequencing of purified PCR products verified the presence of the S729A mutation in the DPP9 allele  litters. Analysis of embryo genotype numbers (n = 41) confirmed that the DPP9 S729A allele was present in the expected Mendelian frequency (data not shown). Thus, it appeared that enzyme-active DPP9 is not essential for development of the mouse embryo to term.p>0.05) . Littermates were of a similar size and weight with no significant gender or genotype difference found for Day 1 neonates (p>0.05) . All appS729A/S729A or DPP9wt/S729A. This suggested a negative effect on survival to weaning for offspring carrying the S729A mutation.Investigating the mortality of newborn pups revealed that, of the 27 found dead in the first 24 hours, all were either DPP9wt/S729A intercross pups showed that the DPP9 S729A allele occurred in the expected Mendelian ratios. From 33 litters from DPP9wt/S729A breeding pairs, 317 pups were produced with 68 DPP9S729A/S729A, 167 DPP9wt/S729A and 82 DPP9wt/wt. Chi-square analysis confirmed no significant deviation from a 1∶2∶1 ratio (p>0.05). Similarly, a lack of significant gender bias shown in neonates precludes a gender effect by the DPP9 S729A allele on embryonic development , rather than an expected 2∶1 genotype ratio  and their DPP9wt/S729A (n = 2) and DPP9wt/wt (n = 4) littermates. Organs examined were gut, heart, lung, brain and skin (Light microscopic observations of H&E-stained whole neonate sections (Day 1) by a trained pathologist (DSS) revealed no histological differences between DPP9and skin  along wiwt/wt, DPP9wt/S729A or DPP9729A/S729AS on early whole neonate sections. Many neonate organs were strongly immuno-positive for DPP9 protein, most notably kidney, skin, liver  (qPCR results indicated that ild-type . When im130 kDa)  and conf130 kDa) . These rS729A/S729ADPP9 MEFs had significantly less DPP enzymatic activity than wild-type MEFs (S729A/S729ADPP9 neonatal liver contained significantly less DPP8/9-derived enzymatic activity than wild-type liver, which is consistent with a lack or large reduction of DPP9 enzymatic activity. Together with the Western blot and qPCR data, this provides evidence that DPP9-S729A enzyme-inactive protein was expressed at similar levels to DPP9-WT enzyme-active protein in MEF cell lines and neonatal livers.Enzyme activity derived from DPP9 and DPP8 can be measured in the presence of a DPP4 inhibitor and a substrate that is hydrolyzed by all three proteases ype MEFs . Moreoveype MEFs , DPP9S72in vitro, DPP9-overexpressing Huh7 cells and endogenous DPP9 in MEFs were examined by immunofluorescence staining and confocal microscopy. DPP9-S729A-EGFP had an intracellular distribution similar to DPP9-WT-EGFP (S729A/S729A MEFs was indistinguishable from DPP9 protein in wild-type MEFs (A point mutation in rat DPP4 -WT-EGFP . Similarype MEFs .In this study, we found that DPP9 enzymatic activity is essential for neonatal survival in the mouse. The mice express intact DPP9 protein but with the loss of enzymatic activity. This did not alter pre-natal development but resulted in neonatal deaths. Thus, the complete disruption of DPP9 expression was not necessary for early lethality, highlighting the biological importance of the enzyme activity of DPP9 at this stage of development. The lethality of lacking DPP9 activity indicates that DPP9 is unique such that no other enzyme can compensate for this enzyme at this developmental stage. The cause of death was not apparent. All neonates suckled and contained milk spots and males and females of each genotype were of similar body weight. No differences were seen by histological examination and no urinary proteinuria was observed.in vivo. The biology in this model is very different to previous studies that have treated adult animals with inhibitors of both DPP9 and DPP8 or of DPP9, DPP4 and DPP8 together The close structural similarity of DPP9 to DPP8 makes the development of selective DPP9 inhibitors challenging To verify the status of DPP9 protein in our mice, the MEF and neonatal liver data indicate that the S729A mutation produced a full-length DPP9 protein with no enzymatic activity. Comparable sizes and intracellular localizations of DPP9 intact protein and similar DPP mRNA levels were shown in DPP9 enzyme-inactive MEFs compared to MEFs and neonatal liver derived from wild-type littermates. In addition, related dipeptidyl peptidases were not upregulated in the DPP9 enzyme-inactive mouse to compensate for the absence of DPP9 activity. We similarly observed in our enzyme distribution studies on DPP4 gko mice Like DPP9, DPP8 has intracellular expression S729A/S729A pups survived parturition with no obvious gross anatomical and histological differences, it seems the resultant lethality in DPP9S729A/S729A mice is probably attributable to metabolic impairment. The literature reports dysfunctions in homeostasis due to failures in autophagy in vivo glucose homeostasis Many factors can contribute to neonate deaths but the time of death can provide clues to the potential causes


To the Editor: A novel swine-originated influenza A virus known as pandemic (H1N1) 2009 was first isolated from humans in Mexico in April 2009  belongs to the family In January 2011, an outbreak of severe respiratory problems occurred in pigs on a pig farm. Nine hundred growing and fattening pigs exhibited clinical signs of influenza, including fever, cough, runny nose, loss of appetite, lethargy, edema, watery eyes, conjunctivitis, diarrhea, and vomiting. The incidence rate was ≈80%, and the death rate was 22%. The outbreak lasted ≈2 weeks. However, no outbreak of respiratory disease occurred in other pig farms in the same area simultaneously, and no evidence of human-to-pig transmission was found.We collected lung samples from 3 dead pigs with underlying illness for reverse transcription PCR and virus isolation in 10-day-old specific pathogen–free embryonated chicken eggs. Viral RNA was extracted from the tissue suspension and allantoic fluids. Virus isolation was assessed by hemagglutination inhibition (HI) assay and neuraminidase inhibition assay by using a panel of reference serum samples . Later, 3 viruses were isolated. All of them had hemagglutination (HA) activity, and the HA titers ranged from 128–256. The HA-positive isolates were further identified as subtype H1N1. Subsequently, nucleotide sequences of the 8 viral genes were amplified and sequenced (GenBank accession nos. JN222372–JN222379). This analysis showed high identities to a pandemic strains A/California/04/2009 (H1N1), hemagglutinin (99.2%), neuraminidase (99.1%), matrix (99.3%), nucleoprotein (99.5%), nonstructural protein (98.5%), polymerase acidic protein (98.5%), polymerase basic protein 1 (99.7%), and polymerase basic protein 2 (99.6%) genes. Bacteria were cultured from spleen, liver, and heart-blood samples from 5 pigs. Four pigs were infected with porcine streptococci, and 1 pig with mild symptoms was negative for the bacterium.We sought to gain more insight into the epidemiology of pandemic (H1N1) 2009 virus in Guangxi Province and collected 600 bronchial swab samples and 200 blood serum samples when pigs were slaughtered every month from February through June 2011. The samples were used to isolate virus in 10-day-old specific pathogen–free embryonated chicken eggs, and then the virus isolates were subjected to sequencing of a partial genome of the HA gene. Overall, we obtained 10 strains of subtype H1N1 influenza virus, including 5 strains of classic swine H1N1, 3 strains of Eurasian avianlike H1N1, and 2 strains of pandemic (H1N1) 2009 virus, which were derived from 3,000 bronchial swab samples.In addition, a serologic survey was implemented by using HI testing with pandemic (H1N1) 2009 virus and SIV (H1N1) antigens. Serologic studies showed that 251 of the 1,000 samples tested had positive HI titers for pandemic (H1N1) 2009 virus, and 248 of these samples had positive HI titers for SIV (H1N1). Notably, cross-reactivity of pandemic (H1N1) 2009 virus between H1 subtype viruses has been reported recently in pigs (Our findings strengthened previous data by showing that growing and fattening pigs are susceptible to infection of pandemic (H1N1) 2009 virus (