Mir-451 have key roles in erythropoiesis. MicroRNAs (miRNAs) are small endogenous non-coding regulatory RNAs thatcontrol mRNAs post-transcriptionally. Several mouse stem cells miRNAs are cloned differentially regulated in different hematopoietic lineages, suggesting their possible role inhematopoietic lineage differentiation. Recent studies have shown that specific miRNAssuch as pCDH-Mir-451. Erythroid differentiation was assessed based on the expression level of transcriptional factors  andhemoglobin chains  genes using quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and presence of erythroid surface antigens (TER-119and CD235a) using flow cytometery. Colony-forming unit (CFU) assay was also on days14thand 21thafter transduction. In this experimental study, murine embryonic stem cells (mESCs)were infected with lentiviruses containing Mir-451 expression level increased by 3.434-fold relative to the untreatedmESCs on day 4 after transduction (P<0.001). Mir-451 up-regulation correlated with theinduction of transcriptional factor  and hemoglobin chain  genesin mESCs (P<0.001) and also showed a strong correlation with presence of CD235a and Ter-119 markers in these cells  (P<0.05). Moreover,mESCs treated with pCDH-Mir-451 showed a significant raise in CFU-erythroid (CFU-E) colonies (5.2-fold) compared with untreated control group (P<0.05). Mature Mir-451 up-regulation strongly induces erythroid differentiation and maturation of mESCs. Overexpression of Mir-451 may have the potential to produceartificial red blood cells (RBCs) without the presence of any stimulatory cytokines.Our results showed that  Embryonic stem cells (ESCs) are multipotentstem cells derived from the inner cell mass of theblastocyst, an early-stage embryo -3. ESCs Erythropoiesis is the complex process throughwhich a fraction of primitive multipotent hematopoietic stem cells (HSCs) convert to the committed redcell lineage, undergoing differentiation to erythroidprogenitors [burst forming unit-erythroid (BFU-E)and colony-forming unit-erythroid (CFU-E)], normoblasts, erythroblasts, reticulocytes, and ultimatelymature erythrocytes . This prMiRNAs are small endogenous non-coding RNAmolecules 19 to 25 nts) that regulate gene expressionpost-transcriptionally  and are  nts thatMir-451 exists in mature circulating red bloodcells (Mir-451in murine erythroleukemia (MEL) cells promoted orimpaired erythrocyte differentiation, respectively  can induce proliferation of culturedchicken, mouse and human erythroid progenitors.Damaged signaling from the Epor not only affectsstress erythropoiesis, but also causes erythropoiesisdefects during normal development (β-globingene (Hbb)  .Mir-451 up-regulation could induceerythropoiesis differentiation from mESCs and beused as a replacement to the stimulatory cytokinesfor mESCs differentiation into erythroid cells. In this experimental study, we examined the earlystages of mESCs lineage commitment by investigating whether 2 accordingto the supplier’s instructions.Human embryonic kidney (HEK)-293T cellline was obtained from the National Cell Bank ofIran . The HEK-293T cellswere cultured in Dulbecco’s modified Eagle’s medium (DMEM), 10 % fetal bovine serum (FBS),100 U/ml penicillin, 2 mM L-glutamine and 100µl streptomycin . This cellline was kept at 37˚C in a humidified atmospherecontaining 95 % humidity and 5 % COpri-Mir-451 sequences into the XbaI /BamHI restrictionsites of the pCDH-CMV-MCS-EF1-copGFP vector . These fragmentswere elevated by polymerase chain reaction (PCR)reaction using following primers: pri-Mir-451 F:5′-GTC GTA TGC AGA GCA GGG TCC GAGGTA TTC GCA CTG CAT ACG ACA ACT CA3′ and R: 5′GTCGTATGCAGAGCAGGGTCCGAGGTATTCGCACTGCATACGACAACCTC-3′ on extracted genomic DNA. For lentivirusproduction; HEK-293T cells (3×103) were seededinto 10-cm plates containing DMEM medium supplemented with 10% FBS. The day after, pPAX2plasmid (containing gag and pol genes) and pMD2plasmid (containing vsv gene) were co-transfected with the pCDH-451 plasmid empty vector (pCDHempty backbone) as negative control into seededHEK-293T cells using the lipofectamin 2000 reagent according to the manufacturer’sprotocol. The supernatants containing generated lentiviruses were collected every 12 hours for 3 days aftertransfection and concentrated by ultracentrifugationat 40.000 g for 2 hours. Then for virus titration, HEK-293T cells were transduced with a different concen-tration of recombinant lentiviruses and the numberof viruses in the functional copy was detected usinggreen fluorescent protein (GFP) protein and fluorescent microscope forty-eight hours later.The pCDH-451 plasmid was produced by li-gating 250 bp fragments encompassing 2. ESC medium, which was exchanged daily, contained knockoutDMEM, 20% FBS-ES,1 mM sodium pyruvate , 2 mM Glutamine , 0.05mM b-mercaptoethanol, 1 mM non-essential aminoacids , 1,000 U/ml recombinant mouseleukemia inhibitory factor  and100 U/ml penicillin/streptomycin .Murine ESC (mESC) [E14Tg2A] lines were cultured on gelatin-coated tissue culture dishes  at an intensity of 40,000 cells/cmThe infection was done in three groups. Eachgroups had three samples. Embryonic bodies (EB)were cultured for 1 to 21 days under the followingconditions: i. Blank: EBs did not receive any treatment (untreated group), ii. pCDH-451 lentiviruses:EBs were transduced with pCDH-451 lentiviruses(pCDH-451 group) and iii. pCDH-empty lentiviruses: EBs were transduced with pCDH-emptylentiviruses (negative control group).Mir-451 upregulation in erythroid differentiation was monitored by analyzing expression of transcriptionalfactor  and hemoglobinchain  using quantitive reversetranscriptasePCR (qRT-PCR) and presence oferythroid cell surface markers (CD235a and Ter-119) using flow cytometry.After 14 and 21 days, the effect of Total RNA was extracted from test and controlgroups using the Trizol reagent , according to the manufacturer’s instructions. cDNAwas synthesized by Superscript II reverse transcription  and random hexamer primers,according to the manufacturer’s instructions.−ΔΔctmethod.Real-time RT-PCR quantification of miRNAswas undertaken using primers designed PrimerExpress version 2.0 . Briefly, first cDNA strand was synthesized through miRNA 1st-strand cDNA synthesiskit  and reverse transcribed intoqPCR-ready cDNA. After that, miRNA qRT-PCRwas carried out in triplicate on ABI PRISM 7500real time PCR System with the high-specifity miRNA qPCR core reagentkit  and normalized to U6 smallnuclear RNA (Snord47) as an endogenous control.Primer sequences are shown in Gata-1, Klf-) and hemoglobin chain genes was quantified using ABI PRISM 7500 real-time PCR System with the SYBR premix ExTaq kit according to the manufacturer’s instruction. TheqRT-PCR cycling conditions were done same asabove.Expression of transcriptional factor were collected for flow cytometry. The viability ofthe cells was examined by trypan blue exclusionand was always greater than 95%. They were immunostained with phycoerythrin (PE)–conjugatedanti-TER119 (1:200) and PE–conjugated anti-CD235a  antibodies. Propidium iodide was added toexclude dead cells from analysis. The cells werethen analyzed on flow cytometer PartecPAS III.3 mESCs werecultured in 35-mm plates with the medium containing 25% FBS, 2% bovine serum albumin,1.3% methyl cellulose, 0.05 mmol/L 2-mercaptoethanol, 3 U/mL erythropoietin (EPO), 2mmol/L L-glutamine, 50 ng/mL stem cell factor (SCF), 10 ng/mL IL-3 and 10 ng/mL granulocyte macrophage-colony-stimulating factorplus activin A (25 ng/mL). After incubation at37˚C, 5% CO2and 95% humidity for 12 days,the colony-forming unit-granulocyte, erythroid,macrophage, megakaryocyte (CFU-GEMMs),colony-forming unit-granulocyte, macrophage(CFU-GMs) and CFU-Es in every dishes wassorted and counted under a high-quality inverted microscope .Colony-forming cell (CFC) assay was carriedout in triplicate using methylcellulose completemedia  according to the manufacturer’s instructions. In all groups, 5×10All tests were repeated three times and data wereshown as mean ± SD. The comparison betweengroups was performed using the Student’s t test.P value less than 0.05 was considered statisticallysignificant.Mir-451was then generated.The pMD2G, psPAX2 and pCDH-451 plasmidswere co-transfected into HEK293T cells on a 10 cmplate using lipofectamin 2000 reagent  according to the manufacturer’s protocol(pCDH-451). Lentiviruses expressing Lentiviral vectors created from pCDH-emptyplasmids were used as negative control (pCDH-Neg). Transfection efficiency was confirmedeach time by fluorescent microscopy. Approximately 95% of cells in the pCDH-451 group and97% of cells in the pCDH-empty vector groupwith green fluorescence were distinguished 48and 72 hours after infection Figes, 2. No f7 to 7×107 viral particles per milliliter anddiverse multiplicities of infection were used tooptimize transduction conditions. Transductionefficiency was monitored each time by fluorescent microscopy and evaluated by flow cytometry for the GFP marker. GFP overexpressionof lentiviruses in mESCs was 60% of cells withpCDH-451 and 65% of cells with pCDH-emptyvectors and was distinguishable 96 hours afterinfection. No fluorescent-positive cell was detected in our control group  by qRT-PCR on day 14and 21, as an index of erythropoiesis. According toqRT-PCR results, the expression of these transcriptional factors distinguished the pCDH-451, indicating successful erythropoeisis  but was decreasedby 0.712and 2.454-fold, respectively on day 21(P<0.001). In this group, Epor expression was increased on day 14 and 21. Treatment of mESCswith pCDH-Mir-451 lentiviruses, led to the rise ofEpor expression by 23.183-fold relative to the untreated mESCs on day 21 (P<0.001).We then examined the effect of this up-regulationon the expression of erythroid specific markers compared with the untreated control groupon day 14 (P<0.05). On day 21, sharp increaseof accumulation of ε, α and β-globin transcriptswere detected in the pCDH-451 group  comparedwith the untreated control group (P<0.05). A moderate increase of ζ transcripts (by 2.035-fold) was seenin mESCs treated with pCDH-Mir-451 on day 21(P<0.05). The pattern of γ transcripts was decreasedin this group (by 0.742-fold) compared with the untreated control group (P>0.05). These results furtherconfirmed that Mir-451 may have a vital roles in theinduction of hemoglobinization ,respectively, on day 14 . Results on day 21 showedthe percentage of cells positive for TER119 andCD235a was 66.34 ± 2.81% and 46.38 ± 2.37%in Mir-451 treated mESCs and 5.07 ± 1.01% and3.48 ± 1.28% in untreated mESCs, respectively compared with theuntreated control group (P<0.05).On the 12or cells . The numMir-155, a lymphoid-restricted miRNA, have defective immune responses)  over a person’s lifetime ,36. The sponses) . In thise et al.  also shoGata-1, Epor, and Klf-1 transcription factors using qRT-PCR in all groups. Results revealed that these factors were expressed in mESCs transduced with lentiviral vector expressing pCDH-Mir-451. Gata-1 expression was decreased in all groups on day 21. Gata-1 reveals physiologically that occur during normal erythropoiesis (Gata-1 could obstruct cell proliferation (Gata-1 persuades G1 arrest during erythroid maturation and identified an extensive Gata-1-regulated network of gene activation and repression related to cell cycle control. Epor expression was increased in the pCDH-451 group on day 21. Erythropoietin (Epo) is a glycoprotein and a major regulator of the growth and differentiation of erythroid blood cells. Its biological influence is mediated through binding to the Epor on the cell surface (Klf-1 (and the related ubiquitously expressed protein Sp1) and Gata-1 are located in close proximity in cis-regulatory elements of erythroid-specific genes. In addition, both Sp1 and Klf-1 physically associate with the zinc finger region of Gata-1 and synergistically activate Gata-1 target genes in transiently expressed reporter constructs. Thus, protein-protein interactions between Gata-1 and Klf-1 may be implicated in facilitating the switch from fetal to adult globin expression. As markers of erythropoiesis, we examined the expression of opoiesis . During feration . Rylski feration  showed t surface . Klf1 ex surface  have shoMir-451 induced a significant rise in mESC hemoglobinization and similarly we detected a sharp increase of accumulation of αglobin and β-globin transcripts in the pCDH-451 group. Therefore, Mir-451 seems to have more effect on the progression of erythroid maturation that increasing expression level of α-globin and β-globin. These results are consistent with some previous studies indicating that Mir-451 has a strong positive correlation with the late stage of erythropoiesis (Mir-451 stimulated embryonic globin chains (ζ and ε) and γ-globin. In the first step of erythroid differentiation, expression level of γ-globin was at high level and at the late step of it, γ-globin expression was low , Portland I (ζ2γ2) and Portland II (ζ2β2)  . In addih et al.  showed ttitively ,55. Mir-451 and confirmed that they display stem cell properties based on CFC assays, consistent with similar findings obtained with HSCs  and reactivation of the αand β-globin chains has been shown to rescue the lethality of mice with αand β-thalassemia. Mir-451 and other miRNAs may be useful in designing effective therapeutic strategies for the possibility of reversing these abnormalities by gene therapy. We show that


JAZ7 T-DNA activation mutant confers increased JA-sensitivity, up-regulated defense and JA-mediated gene expression, and increased susceptibility to two pathogens that disrupt host JA-responses, Fusarium oxysporum and Pst DC3000.A  Fusarium oxysporum disease outcome. However, the roles of JASMONATE ZIM-domain (JAZ) proteins that repress JA-signaling have not been characterized in host resistance or susceptibility to this pathogen. Here, we found most JAZ genes are induced following F. oxysporum challenge, and screening T-DNA insertion lines in Arabidopsis JAZ family members identified a highly disease-susceptible JAZ7 mutant (jaz7-1D). This mutant exhibited constitutive JAZ7 expression and conferred increased JA-sensitivity, suggesting activation of JA-signaling. Unlike jaz7 loss-of-function alleles, jaz7-1D also had enhanced JA-responsive gene expression, altered development and increased susceptibility to the bacterial pathogen Pst DC3000 that also disrupts host JA-responses. We also demonstrate that JAZ7 interacts with transcription factors functioning as activators  or repressors (JAM1) of JA-signaling and contains a functional EAR repressor motif mediating transcriptional repression via the co-repressor TOPLESS (TPL). We propose through direct TPL recruitment, in wild-type plants JAZ7 functions as a repressor within the JA-response network and that in jaz7-1D plants, misregulated ectopic JAZ7 expression hyper-activates JA-signaling in part by disturbing finely-tuned COI1-JAZ-TPL-TF complexes.In Arabidopsis, jasmonate (JA)-signaling plays a key role in mediating  Received 5 November 2015; Revised 13 January 2016; Accepted 20 January 2016Fusarium oxysporum is responsible for vascular wilt disease in over 100 different plant species, including bananas (Musa spp.), cotton (Gossypium spp.), grain legumes and horticultural crops such as tomato (Lycopersicum esculentum)  where the pathogen-host interaction can be readily studied in a model system.The root-infecting fungal pathogen ulentum) . This paF. oxysporum have been proposed  . For example, mutation of MYC2, a key regulator of downstream JA-defense signaling, mutation of LBD20, a MYC2-regulated transcription factor, or overexpression of the Ethylene Response Factors ERF1 and AtERF2, activators of JA-defenses, results in up-regulated expression of a specific subset of JA-dependent defense genes and increased resistance to F. oxysporum (F. oxysporum infection but that non-defensive aspects have greater contribution to disease outcome (Contrasting roles for jasmonate (JA) signaling and JA-mediated defense in Arabidopsis resistance to xysporum . It is pCOI1-JAZ , bHLH013/JAM2 and bHLH017/JAM1, thereby interfering with the expression of JA-responsive genes. Upon increased JA levels, the ubiquitin-mediated degradation of JAZ proteins leads to the release of these transcription factors from repression , which together with JASMONATE ZIM DOMAIN (JAZ) proteins, perceives the JA-signal and forms part of the Skp1/Cullin/F-box (SCF) E3 ubiquitin ligase complex SCFBotrytis cinerea, Alternaria brassicicola, F. oxysporum with the coi1 mutant displaying a near-immune like resistance to this pathogen , herbivory and wounding (Pst (JAZ8 and the recently identified JAZ13 (both resulting in reduced JA-sensitivity) or T-DNA or RNAi knockdown lines of jaz1 or jaz10  , conferring activation of JA-signaling and increased JA-sensitivity. However, we demonstrate JAZ7 contains a functional EAR repressor motif, recruiting the co-repressor TPL and repressing transcriptional activation. Further, JAZ7 interacted with both transcriptional activators and repressors of JA-signaling. Based on these results, we propose the misregulated JAZ7 expression in jaz7-1D plants resulting from the JAZ7 T-DNA promoter insertion activates JA-signaling conferring increased JA-sensitivity through recruitment of TPL to specific transcriptional regulators, and disturbing the function of proteins acting within the multi-protein COI1-JAZ-TPL-TF complex.In this report, we examined the roles of JAZ family members during the Arabidopsis-A. thaliana Columbia-0 (Col-0) accession grown under a short daylight regime (8h light:16h dark) at 21ºC as described previously (coi1 (SALK_035548), jaz7-1D (SALK_040835), jaz7-1 (WiscDsLox7H11) and other jaz insertion lines (Supplementary Table S1 available at JXB online) were obtained from the Arabidopsis Biological Resource Centre (ABRC) or the Nottingham Arabidopsis Stock Centre (NASC). T-DNA mutants were confirmed for correct loci insert and homozygous state. Backcrossed, double or triple jaz insertion lines were all confirmed by PCR. For generation of JAZ7 overexpression (35S:JAZ7) lines, the JAZ7 CDS was amplified using JAZ7-HindIII-F and JAZ7-EcoRI-R, cloned into pKEN   and resulting T3 lines used in subsequent experiments. For generation of 35S:CUC1-JAZ7EAR plants the JAZ7EAR domain with an added stop codon was first cloned into pKEN  and syringe infiltrated into leaves at 5×106 cells ml−1. Infiltrated plants were incubated at 28°C (16h light/8h dark) under a clear plastic dome and bacterial growth quantified as previously described  or MeJA treatment . Three biological replicates were taken for all experiments comprising tissue pooled from 5–30 plants. RNA extraction, cDNA synthesis and Q-RT-PCR were conducted as described by β-actin 2, β-actin 7 and β-actin 8  were used for each cDNA sample using the equation: relative ratio gene of interest/actin=(Egene-Ct gene)/(Eactin-Ct actin) where Ct is the cycle threshold value. The gene specific primer sequences are listed in Supplementary Table S3.Quantitative-RT-PCR (qRT-PCR) experiments were performed on tissue collected after control, jaz7-1D plants were harvested 6h after mock or MeJA treatments. Treatment involved enclosing trays of 4-week-old soil-grown plants under clear plastic covers with a treated cotton ball attached to the inside of the cover, either 1ml of mock solution (100% ethanol) or 1ml of 5% MeJA dissolved in 100% ethanol, and sealing each tray in two layers of opaque plastic bags. Total RNA was extracted , then labeled, hybridized, washed and scanned by the Australian Genome Research Facility (AGRF)  onto 16 ATH1 GeneChip arrays and the resulting data analyzed using GenespringGX 7.3.1 (Agilent) as previously described (P<0.05) on the entire dataset with the inclusion of the Benjamini and Hochberg false discovery rate (FDR) (microarray data is deposited under accession number GSE61884 at the NCBI Gene Expression Omnibus). Gene Ontology (GO) term enrichment analysis was performed using agriGO v1.2 (P≤0.05) determined P-value significance. Functional annotations of genes and AGI symbols were sourced from TAIR9 datasets.Four independent biological replicates each consisting of shoot material from 20 wild-type and JAZ7, JAZ5, JAZ8, MYC2, MYC3, MYC4, TPL and JAM1 were PCR-amplified from Arabidopsis cDNA (accession Col-0) using the primers in Supplementary Table S2 followed by second amplification with pAttB1 and pAttB2, and cloned into the pDONRZeo plasmid (Invitrogen). The JAZ7mEAR motif was generated by mutating the conserved leucine residues of the EAR motif to alanine using the QuickChange II Site Directed Mutagenesis Kit (Agilent Technologies) following the manufacturer’s recommendations. The entry clones were then recombined with the Gateway-compatible yeast two-hybrid (Y2H) vectors derived from pGADT7 and pGBKT7 (Clontech). Y2H assays were performed using Clontech’s Matchmaker system with strain AH109.For Y2H experiments, JAZ7, JAZ7mEAR, JAZ5 and JAZ8 were constructed as described for Y2-H assays except JAZ7-R2, JAZ8-R2 or JAZ5-R2 (Supplementary Table S2) were used as reverse primers in order to remove stop codons and cloned into pDONRZeo plasmid (Invitrogen). The entry clones were recombined with the Gateway-compatible pEarleyGate 101 , and GFP cloned into pEarleyGate100 used as control (N. benthamiana plants were used for Agrobacterium tumefaciens-mediated transient expression of indicated constructs as described previously (eviously . Co-immuMYC3 and MYC4 were used to construct effector plasmids by fusing with the yeast GAL4 DNA-binding domain (GAL4DB)-coding region under the control of the CaMV35S promoter into the pGAL4DBGW vector . These results suggest some JAZ members may have roles in mediating responses to early stages of F. oxysporum infection, particularly in root tissues.To examine the role of culation  as JAZ eF. oxysporum disease symptoms become evident in the leaves of wild-type Col-0 plants at 7–10 d post-inoculation, we were also interested to determine the persistence of JAZ expression at later time-points. In this later time-course , fold-inductions were again typically higher in root tissues with JAZ6, JAZ7, JAZ9, JAZ10 and JAZ12 also highly induced in leaf tissues at 7 d post-inoculation . In summary, the differential expression patterns of multiple JAZ genes in response to F. oxysporum suggests that at least some members may have roles in modulating host responses to this pathogen.As culation . As withF. oxysporum resistance, we screened publically available JAZ T-DNA insertion lines with F. oxysporum. With the exception of JAZ1, JAZ3, JAZ6, JAZ7, JAZ8 and JAZ11, the remaining T-DNA insertions are located within the transcribed regions of the JAZ genes  displayed significantly  higher susceptibility to F. oxysporum than wild-type in three independent inoculation experiments .To investigate possible roles of JAZ proteins in eriments . As showeriments . No signF. oxysporum is known to trigger a leaf-chlorosis phenotype that is closely correlated with the F. oxysporum resistance/susceptibility phenotypes . Independently, JAZ7 activation mutant and with small stature. Progeny from two other separately isolated SALK_040835 lines also showed small rosette size and increased susceptibility to F. oxysporum.To determine how the T-DNA inserted into the promoter of JAZ7  in SALK_e plants . This suet al., 2014, unpublished) suggests SALK_040835 may contain other insertions, and this raises the possibility that these additional insertions, if confirmed, may contribute to the jaz7-1D phenotypes. One insertion is proposed to be located within the promoter of At2g47780 (rubber elongation factor protein), one in the coding sequence of At2g47790 (GIGANTUS), and the others in intergenic regions. We therefore screened SALK_040835/jaz7-1D plants by PCR for insertions in At2g47780 and At2g47790 but were unable to identify any insertion in At2g47790, while all plants were heterozygous for the At2g47780 insertion. We also examined the Col-0 and SALK_040835C RNA sequencing data of At2g47780 and At2g47790, and genes flanking the possible intergenic T-DNA insertions, but found no differential levels or truncated transcripts. Together, these results support the conclusion that the phenotypes observed in jaz7-1D are related to the JAZ7 promoter insertion.Recent re-sequencing of SALK T-DNA insertion lines  designated as jaz7-1, where the T-DNA is inserted into the second exon of the JAZ7 gene . In contrast, JAZ7 transcript levels were hypersensitive to induction by F. oxysporum in the activation tagged jaz7-1D mutant , jaz5 (SALK_053775) and jaz10 (SAIL_92_D08). Although further experiments need to be conducted to determine if JAZ transcript levels are affected in the remaining jaz insertion lines, none of these lines exhibited altered disease phenotypes compared to wild-type plants (data not shown).The finding that AZ7 gene . No deteD mutant . Comparee assays –E. The aJAZ7 expression in the jaz7-1D mutant correlated with increased susceptibility to F. oxysporum and JAZ proteins act as repressors in JA-signaling, we asked whether the Fusarium inducibility of JAZ7 requires COI1. As shown in F. oxysporum inducibility of JAZ7 was abolished in both roots and leaves of the coi1 mutant, suggesting that COI1 (or JA-sensing) is required for pathogen inducible JAZ7 expression.Given that increased F. oxysporum. For instance, as with F. oxysporum, JA-signaling promotes susceptibility to the bacterial pathogen Pst DC3000 , a JA-biosynthesis enzyme (e.g. LOX3) and JA-related defense proteins  were induced more strongly in the leaves of inoculated jaz7-1D plants than in jaz7-1 and wild-type plants at 4 dpi. Expression of senescence or oxidative stress associated transcripts  were also up-regulated in jaz7-1D. Furthermore, analysis of JAZ gene expression after F. oxysporum inoculations revealed that transcript levels of almost all JAZ genes were up-regulated in jaz7-1D while in jaz7-1 levels were either reduced or did not differ from wild-type levels  on the entire dataset with the inclusion of the Benjamini and Hochberg FDR. A comparison of differentially regulated genes by genotype identified 113 up-regulated and 25 down-regulated genes showing ≥2-fold in mock-treated jaz7-1D relative to mock-treated wild-type plants (Supplementary Tables S4–6). To gain insight into the functions of these genes, we performed GO term enrichment analysis. Significantly enriched biological processes from genes up-regulated in jaz7-1D were those involved in defense responses, multi-organism processes, and responses to stress, fungi, other organisms, biotic and abiotic stimulus, and organic substances, while those from the down-regulated dataset were enriched for genes involved in response secondary metabolic processes and response to stimulus (FDR<0.05). The majority of genes up-regulated in jaz7-1D are also associated with JA-signaling, plant defense and/or senescence including Thi2.1 which we previously identified as being up-regulated , an alpha-beta hydrolyase superfamily/lipase protein (AT2G42690) and a cyclic nucleotide-regulated ion channel (AT2G46450), respectively, have been identified as senescence markers in Arabidopsis (jaz7-1D encoding a N-acetyltransferase (AT2G39030/NATA1) is highly JA-inducible and linked to Pst susceptibility (DET2/DWF6) known to promote flowering . Again, this is consistent with the increased susceptibility phenotype of the jaz7-1D mutant as overexpression of methylesterase inhibitors in Arabidopsis provides increased pathogen resistance (To further dissect the effect of the egulated . For exajaz7-1D relative to MeJA-treated wild-type plants (Supplementary Tables S7–9). Genes in the up-regulated dataset were enriched for lipid biosynthetic and metabolic processes, response to external stimulus, localization and transport, while the down-regulated dataset were enriched for response to stimulus, stress, chemicals and organic substances. NATA1 was the highest up-regulated gene, as was it under control treatment. However, we noticed the MeJA inducibility of this gene and others in jaz7-1D over its control levels was nearly 2-fold less than its inducibility in wild-type plants, suggesting the primed expression of these genes prevents the same level of induction observed in wild-type plants . This included genes involved in JA-responses, defense and senescence such as the two defensins PDF1.2a (AT5G544420) and PDF1.2b (AT2G26020), LOX2 (lipoxygenase 2/AT3G45140), COR1 (coronatine-responsive protein/AT1G19670), a glucan endo-1,3-beta-glucosidase (AT4G16260) and DIN11 (DARK INDUCIBLE 11/AT3G49620). Overall, these results suggest the primed JA-response in jaz7-1D may limit further JA-mediated fold-induction and/or that JAZ7 may have a role in inhibition of JA-regulated responses.We next analyzed the two-way ANOVA data for genes differentially regulated by MeJA treatment and identified 56 up-regulated and 21 down-regulated ≥2-fold in MeJA-treated e plants . To dissJAZ7 and JA-regulated gene expression is up-regulated in the jaz7-1D mutant prompted us to generate JAZ7 overexpression lines. We generated three independent lines overexpressing JAZ7 under the control of the constitutive 35S promoter (JAZ7-OX) with expression ranging from 9-fold to 1800-fold over wild-type levels . Interestingly, the JAZ7-OX lines did not exhibit the small rosette size or reduced root length phenotypes of jaz7-1D under normal growing conditions, but did exhibit, although not significantly, increased basal expression of some but not all JA-marker genes tested . We also examined JA-sensitivity and Fusarium susceptibility in the overexpression lines and found only the lowest JAZ7 expression line JAZ7-OX1 (with JAZ7 levels comparable to jaz7-1D) displayed increased JA-sensitivity and increased Fusarium susceptibility, but only at early stages of infection .The finding that JAZ7-OX lines not phenocopying jaz7-1D may be jaz7-1D producing altered JAZ7 transcripts such as those harboring mutations, or formed as a result of altered splicing or altered transcription start sites (TSSs), or the presence of additional undetected T-DNA insertions in jaz7-1D. Therefore, we sequenced JAZ7 transcripts from Col-0, jaz7-1D and JAZ7-OX, but found no sequence variation. Further, inspection of RNA-seq data from JAZ7 transcripts  compared to wild-type Col-0. Next, to consider the possibility of additional insertions (not collated by SALK) in jaz7-1D affecting its phenotypes, we produced a backcrossed (to Col-0) line. The F2 progeny segregated 2:1 heterozygous jaz7-1D:Col-0 (confirmed via PCR) as suggestive of a dominant mutation, reiterating our previous results showing that homozygous lines of this insertion mutant may be lethal. The heterozygous progeny also conferred jaz7-1D phenotypes of short roots . If the JA-hypersensitive phenotypes in jaz7-1D were due to an additional T-DNA insertion we would expect to see this phenotype segregate, unless the insertion is closely linked. Therefore, combined with our JAZ7-OX results, it is possible that jaz7-1D JA-related phenotypes are a result of ectopic cell or tissue-specific JAZ7 expression as a consequence of the T-DNA insertion in the JAZ7 promoter and/or high levels of JAZ7 in jaz7-1D plants interfering within COI1-JAZ-TPL-TF multiprotein complexes.Possibilities for the jaz7-1D suggests a role for JAZ7 in activation of JA responses. However, JAZ proteins repress JA-responses through direct (EAR repressor motif) or indirect (NINJA-mediated) recruitment of co-repressor(s) and the EAR motif associated with gene repression is present in JAZ7  linked to the GUS gene (pGAL4UAS-GUS), together with CaMV35S expression constructs of MYC3 or MYC4 fused to the GAL4 DNA binding domain (GAL4BD) or GAL4BD alone, as well as empty vector, JAZ7, JAZ7mEAR or JAZ8 under CaMV35S promoter (LUC) gene was co-bombarded as a normalization control. The addition of the vector constructs expressing either MYC3- or MYC4-GAL4BD produced significantly higher transcription activity of the GUS reporter gene compared to the control effector plasmid (GAL4BD only) when co-bombarded with the empty vector. However, transcription activation abilities of the MYC3 and MYC4-GAL4BD fusion proteins were significantly reduced when co-bombarded with JAZ7 or JAZ8, but not with JAZ7mEAR. These experiments demonstrate JAZ7 and JAZ8, but not JAZ7mEAR, repress MYC3 and MYC4-mediated transcriptional activities.To determine whether JAZ7 has the capacity to repress these transcriptional activators we conducted transcriptional activation assays with JAZ7 against MYC3 and MYC4. In these experiments, we co-bombarded a reporter gene construct containing the GAL4 upstream activation sequence  bHLH003/JAM3 or bHLH013/JAM2 .jaz7-1D plants, we propose the misregulated expression of JAZ7 would obstruct the finely-tuned nature of the COI1-JAZ-TPL-TF multi-protein complex resulting in hyperactivation of JA-signaling.Combined, our results demonstrate through direct recruitment of TPL, in wild-type plants JAZ7 functions as a repressor within the JA-response network via its interaction with specific transcriptional regulators . In F. oxysporum (F. oxysporum resistance or susceptibility. We identified a highly susceptible T-DNA insertion line (jaz7-1D) with a promoter insertion resulting in constitutive JAZ7 expression and enhanced susceptibility to F. oxysporum. The jaz7-1D line also conferred increased JA-sensitivity, up-regulation of defense and JA-mediated gene expression, and increased susceptibility to the bacterial pathogen Pst DC3000. Both F. oxysporum and Pst DC3000 appear to target host JA- signaling to elicit disease, the first to hyperactivate JA-signaling and senescence processes, and the second to antagonistically suppress defense responses mediated by salicylic acid signaling. Thus the jaz7-1D line interferes with defense responses that integrate signals downstream of pathogens with two different virulence strategies.JA-signaling functions as a major determinant of disease outcome in Arabidopsis to the fungal pathogen xysporum . In thisJAZ genes were induced following F. oxysporum inoculation, with the largest inductions observed in root tissues for JAZ5 and JAZ10  NATA1 in the jaz7-1D mutant encodes a N-acetyltransferase, which acetylates ornithine to produce the defense-related metabolite Nδ-acetylornithine. jaz7-1D) and its levels are highly up-regulated over wild-type following MeJA treatment. NATA1 expression is highly responsive to JA, Pst and herbivory  are highly susceptible while coi1 plants with severely compromised Thi2.1 expression are highly resistant (jaz7-1D was Histone1-3 (HIS1-3). HIS1-3 encodes a linker histone which functions as a stabilizer of chromatin structure and its expression is highly drought inducible, suggestive of a role in stress tolerance (jaz7-1 (WiscDsLox7H11) knockout line, Yu and colleagues found senescence and H2O2-mediated responses and genes involved in these processes such as NATA1 and DIN11 were significantly up-regulated in jaz7-1 in darkness but not under light conditions. We found no alteration in Fusarium-induced senescence responses or oxidative stress responsive gene expression in jaz7-1 compared to wild-type plants  . The constitutive activation of JA-signaling in jaz7-1D may also be responsible for its small rosette phenotype and reduced root-length  also show stunted growth (jaz7-1D/SALK_040835C which may limit resources available for growth (JAZ7 overexpression lines (JAZ7-OX) that we generated was also increased, but not to the significantly high levels observed in jaz7-1D, and may account for why the JAZ7-OX lines did not exhibit the stunted jaz7-1D root and leaf phenotypes. To rule out the possibilities that altered JAZ7 transcripts  or other T-DNA insertions in jaz7-1D are responsible for its JA-hyperactivation phenotypes, we conducted several additional analyses and backcrossed jaz7-1D to wild-type plants. Our results suggest the T-DNA insertion within the JAZ7 promoter is associated with the jaz7-1D phenotypes. However we cannot exclude the possibility that undetected secondary mutations or possible chromosomal rearrangements resulting from T-DNA transformation may contribute.In addition to hyperactivation of JA-responses, the henotype . Links bt-length , 7C. ManJAZ8 and JAZ13 overexpressing lines  and repressors (e.g. JAM1) of JA-responses  and increased susceptibility to Pst DC3000  for G-box motifs (jaz7-1D (Supplementary Table S5) identified 19 to contain the CACGTG G-box motif, and 43 and 38 to contain the MYC2 binding variants CACATG and CACGTT, respectively (jaz7-1D (Supplementary Table S6) genes also contained these motifs . These findings suggest JAZ7 co-ordinates the expression of stress-responsive genes through its interaction with specific MYC or JAM transcription factors and their binding to G-box DNA motifs.JAM1, JAM2 and JAM3 bind the same DNA motif  as MYC2, MYC3 and MYC4 (JAZ7 in jaz7-1D plants confers increased JA-sensitivity (this work).The ZIM domain of JAZ proteins mediates their homo- or heterodimerization . AnotherF. oxysporum stimulates JA-signaling, resulting in increased JAZ7 expression and JAZ7-TPL-mediated repression contributing to the control of JA-responses and disease progression. Our characterization of the jaz7-1D mutant suggests the ectopic or non-wild-type high levels of JAZ7 in jaz7-1D is a major determinant of its phenotypes and that these abnormal levels may be detrimental to the normal COI1-JAZ-TPL-MYC/JAM regulatory network leading to hyperactivation of JA-signaling  and its interacting partners should reveal mechanistic details on how JAZ7 functions in wild-type plants.In summary, our results support a model in which ignaling . AdditioJXB online.Supplementary data are available at Fig. S1. Schematic representation of jaz T-DNA insertion lines. Fig. S2. Screening of jaz T-DNA insertion lines in F. oxysporum disease assays.Fig. S3. Detection of seed aborts in jaz7-1D and confirmation of jaz7-1.Fig. S4. Ectopic overexpression of JAZ7 in wild-type plants.Fig. S5. Backcrossed F2 jaz7-1D seedlings have short roots and are JA-hypersensitive. Table S1. jaz double and triple mutant lines screened in F. oxysporum disease assays.Table S2. Primers used for the generation of transgenic plants and Y2-H and Co-IP constructs.Table S3. Primers used for qRT-PCR.Table S4. List of genes differentially regulated by genotype from the microarray.Table S5. Genes differentially expressed ≥ 2-fold in the jaz7-1D line relative to wild-type.Table S6. Genes differentially expressed ≤ 2-fold in the jaz7-1D line relative to wild-type.Table S7. List of genes differentially regulated by MeJA treatment from the microarray.Table S8. Genes differentially expressed ≥ 2-fold in the jaz7-1D line relative to wild-type under MeJA treatment.Table S9. Genes differentially expressed ≤ 2-fold in the jaz7-1D line relative to wild-type under MeJA treatment.Table S10. Differentially regulated by MeJA treatment genes sorted by MeJA inducibility in wild-type plants.


Asthma management guidelines suggest little difference between EF and SP-ICS other than potency and therefore EF-ICS should be used at same dose as fluticasone (FP) and half the dose of SP-beclomethasone (BDP). Cohort studies suggest EF-BDP patients can achieve better asthma control than FP patients at lower doses. We compared baseline characteristics and first prescribed doses of patients initiating ciclesonide vs. SP-ICS.Data from the PHARMO Database Network  on patients (12-60 years old) with ≥2 prescriptions for asthma therapy (2005-2012) were compared over 1 year before initiating ciclesonide vs SP-ICS. Co-morbidities were evaluated over 1 year before and after ICS initiation. To avoid inclusion of potential COPD patients, those >60 years old and those using long-acting muscarinic antagonists were excluded. Sex and age at ICS initiation; initial ICS doses ; short-acting β2-agonists (SABA) use (year before initiation); prescriptions for acute oral steroids and overall asthma control  in the year prior and including initiation date; and prescriptions of drugs for treating co-morbidities (year before and after initiation) were compared using t-test/chi-square test (p<0.05).Of 4,064 patients, 34% initiated therapy as ciclesonide and 66% as SP-ICS, with same proportion of males (36%). Differences  for ciclesonide vs. SP-ICS were: mean(±SD) age (43±13 vs. 38±14 years); median(Inter Quartile Range) initial ICS doses 160(160-160) vs. 500(250-500) μg; proportion of patients not on SABA (72% vs. 57%) and on SABA daily dosage between 1-100 μg/day (21% vs. 29%), 101-200 μg/day (5% vs. 9%) and >200 μg/day (2% vs. 6%); proportion of patients not prescribed acute oral steroids  and with controlled asthma (87% vs. 82%); proportion of patients prescribed nasal (44% vs. 38%) and topical (31% vs. 28%) steroid preparations, proton-pump inhibitors (41% vs. 29%) and cardiac diseases or hypertension drugs (28% vs. 21%).For comparable asthma control and similar prevalence of co-morbidities, patients were prescribed triple the dose of SP-ICS versus ciclesonide. Further to this analysis, the effects on asthma control in the year following ICS initiation will be investigated.


Both CD11b+ myeloid cells populations have been proposed to infiltrate tissues during inflammation. While infiltration of Ly6C+ monocytes is an established pathogenic factor during hepatic inflammation, the role of Ly6C– monocytes remains elusive. Mice suffering experimental African trypanosome infection die from systemic inflammatory response syndrome (SIRS) that is initiated by phagocytosis of parasites by liver myeloid cells and culminates in apoptosis/necrosis of liver myeloid and parenchymal cells that reduces host survival. C57BL/6 mice are considered as trypanotolerant to Trypanosoma congolense infection. We have reported that in these animals, IL-10, produced among others by myeloid cells, limits the liver damage caused by pathogenic TNF-producing Ly6C+ monocytes, ensuring prolonged survival. Here, the heterogeneity and dynamics of liver myeloid cells in T. congolense-infected C57/BL6 mice was further dissected. Moreover, the contribution of Ly6C– monocytes to trypanotolerance was investigated. By using FACS analysis and adoptive transfer experiments, we found that the accumulation of Ly6C– monocytes and macrophages in the liver of infected mice coincided with a drop in the pool of Ly6C+ monocytes. Pathogenic TNF mainly originated from Ly6C+ monocytes while Ly6C– monocytes and macrophages were major and equipotent sources of IL-10 within myeloid cells. Moreover, Nr4a1 (Nur77) transcription factor-dependent Ly6C– monocytes exhibited IL-10-dependent and cell contact-dependent regulatory properties contributing to trypanotolerance by suppressing the production of TNF by Ly6C+ monocytes and by promoting the differentiation of the latter cells into macrophages. Thus, Ly6C– monocytes can dampen liver damage caused by an extensive Ly6C+ monocyte-associated inflammatory immune response in T. congolense trypanotolerant animals. In a more general context, Ly6C– or Ly6C+ monocyte targeting may represent a therapeutic approach in liver pathogenicity induced by chronic infection.Monocytes consist of two well-defined subsets, the Ly6C Trypanosoma congolense parasite. We established that infiltration of Ly6C+ monocyte subset initiated liver injury in infected mice. More importantly, we revealed that another myeloid cell subset for which the role in liver injury remained elusive, the Ly6C- monocyte subset, exerted hepatoprotective function in infected mice by secreting the anti-inflammatory cytokine IL-10 and by inducing, through cell-contact, the differentiation of pathogenic Ly6C+ monocytes into macrophages expressing genes coding for anti-inflammatory molecules. Thus, augmenting Ly6C- monocyte accumulation or functionality may represent a useful intervention strategy complementing anti-infective medication in conditions of liver injury due to chronic infections.The liver is not only a central organ for efficient metabolism of nutrients and for toxin clearance, but also for immune surveillance, including elimination of intravascular infections. However, excess of nutrients like fat or of toxins like alcohol and certain medications, as well as infections can trigger overactive immune responses which destroy the liver. Such chronic inflammations are major worldwide human health problem with often lethal consequences. Thus, understanding the particular function of various liver immune cells could provide original concepts to alleviate damages in this vital organ. Here, we dissected the heterogeneity, dynamics and function of the myeloid/monocytic cell compartment in the liver of mice infected with  Trypanosoma congolense infection, C57BL/6 mice are considered as "trypanotolerant", being resistant and tolerant to the disease. The resistance of these animals results from their capacity to develop IFN-γ and MyD88-dependent CD11b+ myeloid cells, i.e. M1-type myeloid cells, including CCR2-dependent Ly6C+ monocytes and macrophages that secrete trypanotoxic molecules like TNF and NO and exert phagocytic activity to control the parasitemia [+ monocyte subpopulation negatively affects the tolerance to T. congolense infection. Indeed, T. congolense-infected mice die from a systemic inflammatory response syndrome that is initiated by the engulfment of parasite components by liver myeloid cells, and culminates in apoptosis of liver myeloid cells and necrosis of liver parenchymal cells that reduce the host survival [+ monocytes have been shown to trigger liver cell apoptosis/necrosis, resulting in organ failure and early death by perpetuating a TNF-mediated pro-inflammatory immune response [T. congolense infection. This cytokine has been shown to down-regulate the Ly6C+ monocyte-induced pathogenicity and to induce regulatory, M2-type myeloid cells expressing a number of genes that could contribute to tissue healing, including maintenance of liver homeostasis. Both regulatory T cells and CD11b+ myeloid cells have been identified as sources of IL-10 during T. congolense infection in trypanotolerant animals [+ myeloid cell population, the subset responsible for the IL-10 mediated anti-inflammatory immune response, thus for trypanotolerance, remained to be identified. In this study, we reveal the mobilization of IL-10-expressing Ly6C- monocytes and macrophages after the control of the first peak of parasitemia when a M2-type regulatory immune response arises in the liver of T. congolense—infected mice. In particular, Ly6C- monocytes exerted regulatory functions through the suppression of TNF production by Ly6C+ monocytes and by promoting the differentiation of the latter monocytes into macrophages. Hereby, Ly6C- monocytes contributed to the trypanotolerance by dampening liver damage caused by T. congolense—induced pro-inflammatory immune response. Thus, while the liver protective function of IL-10-producing regulatory T cells is amply documented [- monocytes.Hosts can develop two different strategies to control pathogen infections, resistance and tolerance. During resistance, the host reduces the pathogen burden by activating and recruiting immune cells to the site of infection that mount a pro-inflammatory immune response. Tolerance refers to the action whereby the host repairs the tissue damage, i.e the pathogenicity, caused by the inflammatory immune cells that mediate the resistance , 2. Afriasitemia –9. This asitemia , 10. Howsurvival , 4. In tcumented –14, we h+ myeloid cells during parasite-induced liver inflammation was investigated in CX3CR1-GFP+/- C57BL/6 mice infected with T. congolense at day 7, 14 and 21 post infection (pi). Based on FACS analysis (+ 'inflammatory' monocytes (CX3CR1int CD11bhi CD115hi MHC-II- to int CD62Lhi F4/80int Mertk- CD64lo CD11c- Mar-1-), Ly6C- 'patrolling' monocytes (CX3CR1hi CD11bhi CD115hi MHC-II- to lo CD11ahi F4/80lo Mertk- CD64- CD11cint Mar-1-) and macrophages (Ly6C- CD11bint CX3CR1int F4/80hi Mertk+ MHC-IIhi CD115lo CD64hi CD11c- Mar-1-) [+ monocytes were found to be recruited predominantly at day 7 pi  . When adday 7 pi  when a May 21 pi , when a umulated . As comppression , suggesthe liver .+ and Ly6C- monocytes and their relationship with the macrophage pool was investigated. When BrdU was continuously administered in infected mice, blood Ly6C+ monocytes became more rapidly BrdU+ than liver Ly6C+ monocytes concomitant with the acquisition of Ki67 expression  were tr profile  represenhenotype , similarpanosome  affected+ monocytes at day 6 pi increased liver injury reflected by increased alanine aminotransferase (ALT) serum levels in T. congolense-infected mice  and macrophages (CX3CR1int CD11bint F4/80hi CD115lo CD64hi CD11c-) coupled with a decreased presence of Ly6C+ monocytes (CX3CR1int CD11bhi CD115hi CD62Lhi F4/80int CD64lo CD11c-). The Ly6C+ monocytes and the macrophages mobilized in the liver of infected mice were prominently MHC-II- to int and MHC-IIint to hi, while the MHC-II- to lo fraction of Ly6C- monocytes mounted up. The switch from Ly6C+ monocytes to Ly6C- monocytes and macrophages in the course of T. congolense infection reflected the change from a TNF, hepatodestructive to an IL-10, hepatoprotective milieu [+ monocytes were the main producers of TNF in infected mice while Ly6C- monocytes and macrophages contribute to IL-10 gene and protein expression. A similar sequential mobilization of TNF+ Ly6C+ and IL-10+ Ly6C- myeloid cell subsets in the acute and chronic phase of various inflammation models has been highlighted [As in other life-threatening liver inflammation models –28, liveid cells . Here, thlighted –33.+ and Ly6C- monocytes—depending on the pathogenic insult, their fate, timing of recruitment and location—exert tissue beneficial or detrimental functions. Hereby, Ly6C+ monocytes can infiltrate a variety of inflamed tissues, where they down-regulate their Ly6C expression and differentiate into M1- or M2-type macrophages that exert destructive or protective function in the initiation or resolving phase of inflammation, respectively [- monocytes can exert healing or damaging activity in different models of sterile and non-sterile inflammation [- monocytes can differentiate into macrophages or DCs in inflamed tissues [- monocytes are not bona fide monocytes but represent terminally differentiated "housekeeper" blood-resident macrophages crawling along the endothelium of the vessels [It is commonly agreed that circulating Ly6Cectively –37. Simiammation –41. Whil tissues , 38–43,  vessels , 44–47.+ monocytes mainly into Ly6C- F4/80hi Mertkhi MHC-II+ CD11c- Mar-1- macrophages in the liver of T. congolense trypanotolerant mice  provided mice receive a drug treatment that ameliorates the disease by decreasing inflammation [- monocyte population expanded during T. congolense infection and exerted a hepatoprotective/restorative function. Indeed, the absence of Ly6C- monocytes in infected Nr4a1-/- mice resulted in higher TNF production and increased liver damage. In contrast, treatment of infected WT or Nr4a1-/- mice with Ly6C- monocytes triggered the differentiation of Ly6C+ monocytes into macrophages, limited TNF production, resulting in hepatoprotection. The limiting effect of Ly6C- monocytes on TNF production by Ly6C+ monocytes was partially mediated by cell contact and by IL-10 , that we documented to exert anti-oxidant hepatoprotective function during infection [+ monocyte-derived macrophages developed a restorative activation program in vivo because the low amount of adoptively transferred cells homing in the liver of infected mice did not consent their purification. Of note, it has become evident in the recent years that upon sterile and non-sterile inflammation, monocyte subsets originating from bone marrow and their derived macrophages complement the long-lived tissue-resident macrophage pool that originate from embryonic precursors [T. congolense infection. However, no definitive reporter mice currently allow to discriminate the liver resident macrophages from the recruited monocyte-derived macrophages, hampering investigating their particular role, including M1/M2-type activation and proliferative status, in tissue pathogenicity.The Ly6Crophages . The disecursors , 61–64. ecursors –67. ThusT. congolense-infected mice Ly6C- monocytes and macrophages were major sources of the anti-pathogenic cytokine IL-10. Ly6C- monocytes exerted a hepatoprotective function through their IL-10 production and through cell-contact by limiting the TNF production by pathogenic Ly6C+ monocytes and by inducing the differentiation of the latter cells into restorative macrophages.In conclusion, we reveal that in the liver of Experiments, maintenance and care of mice complied with guidelines of the European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes (CETS n°123) and were approved by the Ethical Committee for Animal Experiments of the Vrije Universiteit Brussel, Brussels, Belgium .3CR1-GFP+/+ mice were bred in house with CD45.2 WT mice purchased from Janvier, France to obtain CX3CR1-GFP+/- mice. IL-10flox/flox mice kindly donated by Dr. A. Roers  were crossed with IL-10wt/wt LysM-cre+/+ mice  to generate IL-10flox/flox LysM- cre+/+ mice. LysM-GFP mice, kindly provided by Dr. T. Graf  and Nr4a1 (Nur77)-/- mice were bred in house. CD45.1 WT C57BL/6 mice were bred in-house.CD45.2 C57BL/6 mice: CXT. congolense variant antigen type 13  stored at -80°C were injected in cyclophosphamide-treated WT C57BL/6 mice. At day 4 post infection, mice were bled, parasites were purified by diethyl-aminoethyl-cellulose chromatography and used to infect experimental female, age-matched (8–14 weeks) mice .2) and livers were perfused through the portal vein with 10 ml of 120 U/ml collagenase type III  in HBSS (Gibco). Then, liver was disrupted mechanically and incubated in 10 ml of a 100 U/ml collagenase III solution in HBSS . The resulting cell suspension was passed through a 70-μm nylon mesh filter and washed by adding 40 ml of HBSS supplemented with 10% FCS and 2 mM EDTA (HBSS/FCS/EDTA) followed by centrifugation . After erythrocyte lysis , the pellet was resuspended in 10 ml of Lymphoprep (Lucron Bioproducts) and overlaid with 10 ml HBSS/FCS/EDTA. After centrifugation , the layer of low-density cells at the interface containing liver non-parenchymal cells was harvested, washed with HBSS containing 10% FCS (HBSS/FCS) and centrifuged . Cells were resuspended in HBSS/FCS, counted while excluding dead cells using trypan blue and diluted to working concentrations (5 x 106/ml). Liver immune cells and buffers were kept on ice during isolation protocols and subsequent analysis. CD11b+ Ly6C+ Ly6G+ granulocytes were not retained within the non-parenchymal fractions due to their high-density characteristic.Animals were euthanized (CO+ liver immune cells were isolated using magnetic separation columns according to the manufacturer’s protocol (Miltenyi Biotec). BD FACSAria II (BD Biosciences) was then used to sort from CD11b+ cells, Ly6C- monocytes from CX3CR1-GFP+/- (CD11bhi MHC-II- CX3CR1hi), Ly6C- monocytes from WT, IL-10wt/wt LysM-cre+/+ or IL-10flox/flox LysM-Cre+/+ mice (CD11bhi CD115+ MHC-II-) or Ly6C+ monocytes from CX3CR1-GFP+/-, WT or LysM-GFP mice (CD11bhi CD115+). Macrophages were sorted from CD11b+ cells of CX3CR1-GFP+/- mice as CD115+ F4/80hi Ly6C- CX3CR1int cells.At indicated time-points post infection, unfractionated CD11b- monocytes or Ly6C+ monocytes (8 x 105 cells/mouse). Control mice were treated with HBSS. One day after the last transfer, liver immune cells were isolated and intracellular spontaneous production of TNF in Ly6C+ CD11b+ monocytes and Ly6C- CD11b+ cells was analyzed by FACS. In addition, liver immune cells were cultured for 24 h and TNF concentration in supernatants was measured.For adoptive transfer experiments, recipient mice were treated iv with Ly6C6/ml, 24-well plates) were cultured in RPMI 1640 (Gibco) supplemented with 10% FCS, 100 U/ml penicillin, 100 mg/ml streptomycin, 0.1 mM non-essential amino acids, 2 mM L-glutamine . Alternatively, Ly6C+ and Ly6C- monocytes were cultured at indicated ratio in 200 μl (96-well plates). Cell supernatants were collected 1 day later. When required, cultures were treated with 3 μg/ml of anti-IL10R antibody  or control antibody . For in vitro macrophage differentiation test, liver sorted Ly6C+ monocytes (2 x 105) and GFP+ Ly6C- monocytes (2 x 105) were cultured without adding M-CSF in 200 μl medium. Cells and supernatants were harvested 7 days later. When required, Ly6C+ monocytes and GFP+ Ly6C- monocytes were plated in the bottom and upper compartments of transwell plates , respectively.At indicated time-points post infection, liver immune cells (2 x 10TNF and IL-10 was quantified in cell supernatants or in blood serum using specific sandwich ELISAs (R&D Systems), in accordance to the manufacturers’ protocols.6) were transferred in FACS tubes and stained for 20 min in ice-cold HBSS supplemented with 0.5% FCS using conventional protocols. Cells were pre-incubated with anti-FcγR antibody before adding commercial labeled surface marker antibodies  were cultured 4 h with brefeldin A (BD Biosciences), subsequently fixed and permeabilized following the manufacturer's instructions (BD Biosciences). Cells were then stained with PE-conjugated anti-TNF (clone MP6-XT22) or IgG1κ isotype control (R3-34) antibody and washed with ice-cold PBS. Cells were analyzed on a FACSCanto II (BD Biosciences). Delta-Median Fluorescence Intensity (dMFI) was calculated as difference between MFI of anti-TNF and isotype control antibody staining. Analyses were performed using FlowJo software.For extracellular stainings, liver immune cells (10RNA extracted using the RNeasy plus mini kit (Qiagen) was reverse-transcribed with oligo(dT) and SuperScript II RT (Invitrogen), following the manufacturer's instructions. Quantitative real-time PCR was performed in an iCycle, with iQ SYBR Green Supermix (Bio-Rad). Primer sequences are listed in S2 Table in ALT levels were measured in serum samples using a commercial kit (Boehringer).t test from GraphPad Prism 4.0 software.All comparisons were tested for statistical significance using the unpaired S1 TextS1 Table  and S2 Table  as supplement to Experimental procedures. The S1 Text contains also S1 Fig (Liver myeloid cells consist of 3 distinct populations in T. congolense-infected mice), S2 Fig (MHC-II expression on monocytes and macrophages from non-infected and infected mice), S3 Fig (BrdU and Ki67 labeling of monocytes and macrophages from infected mice), S4 Fig (Ly6C- monocytes do not differentiate into macrophages in infected mice), S5 Fig , S6 Fig (M2-type and M1-type gene expression levels in monocytes and macrophages from infected mice), S7 Fig , S8 Fig .Contains (PDF)Click here for additional data file.


In this work, we report a novel molecular function of the BDCP SPI in ribonucleoprotein particle formation. We show that SPI interacts with the P-body core component DECAPPING PROTEIN 1 (DCP1), associates to mRNA processing bodies (P-bodies), and regulates their assembly upon salt stress. The finding that spi mutants exhibit salt hypersensitivity suggests that the local function of SPI at P-bodies is of biological relevance. Transcriptome-wide analysis revealed qualitative differences in the salt stress-regulated transcriptional response of Col-0 and spi. We show that SPI regulates the salt stress-dependent post-transcriptional stabilization, cytoplasmic agglomeration, and localization to P-bodies of a subset of salt stress-regulated mRNAs. Finally, we show that the PH-BEACH domains of SPI and its human homolog FAN  interact with DCP1 isoforms from plants, mammals, and yeast, suggesting the evolutionary conservation of an association of BDCPs and P-bodies.Members of the highly conserved class of BEACH domain containing proteins (BDCPs) have been established as broad facilitators of protein–protein interactions and membrane dynamics in the context of human diseases like albinism, bleeding diathesis, impaired cellular immunity, cancer predisposition, and neurological dysfunctions. Also, the Arabidopsis thaliana BDCP SPIRRIG (SPI) is important for membrane integrity, as  Arabidopsis thaliana, the BEACH domain-containing protein SPIRRIG plays a role in the localization of transcripts and formation of mRNA granules, thereby regulating mRNA stability.During salt stress in the model plant  beigeand Chediak Higashi) domain containing proteins (BDCPs) are a highly conserved protein family in eukaryotes. BDCPs are known to be important for membrane dynamics such as vesicle transport, membrane fission and fusion events, and autophagy. Here we describe a new, membrane-independent molecular function of the Arabidopsis thaliana BDCP SPIRRIG (SPI) in the regulation of mRNA stability in P-bodies. We report that SPI associates with P-bodies—cytoplasmic mRNA or protein aggregations responsible for the posttranscriptional regulation of RNA storage and decay—in a salt stress-dependent manner. We show that spi mutants display salt hypersensitivity suggesting that SPI regulates the uptake of salt stress-regulated mRNAs to P-bodies concomitant with their stability. Finally, we found a direct interaction between the P-body core component DECAPPING PROTEIN1 (DCP1) and SPI and show that this interaction is evolutionarily conserved.BEACH ( F. Fwww.arrrection . Using bS1 Data(XLSX)Click here for additional data file.S1 FigN. benthamiana leafs. Left panel shows the corresponding transmission pictures. (C) BiFC interaction between VPS20.2-YFPN and VPS25-YFPC and (D) YFPC-AtMYC1 and YFPN-GL1 cotransfected with free RFP as a transformation control. Scale bar: 35 μm. (E) Percentage of cells showing YFP-gSPI in cytoplasmic dot-like structures in single or DCP1-mCHERRY cotransfected cells. Error bars represent standard deviations of three biological replicates (each 20 cells). Two-tailed student’s t tests were performed to compare single and double transfected cells (*** p < 0.001). Note that the data set for nonstress conditions also serves as a reference in part C and D of this Figure. (F) Recruitment of YFP-gSPI (left) to DCP1-mCHERRY-labeled P-bodies (middle) under nonstress conditions. Right picture shows the overlay. Scale bar: 50 μm. (G) Percentage of cells showing a relocalization of YFP-gSPI to P-bodies at increasing salt concentrations (½MS + indicated NaCl concentration for 10 h). Data denote the average from three independent biological replicates (each 20 cells). Error bars represent standard deviations. Two-tailed student’s t tests were performed to compare nonstress and salt stress conditions . (H) Relocalization of YFP-gSPI to P-bodies was quantified under nonstress (½MS) and osmotic stress conditions (½MS + 125 or 300 mM Mannitol (Man) for 10 h). Data denote the average from three independent biological replicates (each at least 20 cells). Error bars represent standard deviations. No significant changes between nonstress and osmotic stress conditions were observed (two-tailed student’s t tests). (I) Cytoplasmic distribution of free YFP under nonstress (½MS) and salt stress conditions (½MS + 140mM NaCl for 10 h) (n = 30 cells). Scale bar: 50 μm.(A) Cytoplasmic localization of VPS20.2-mCHERRY and (B) AtMYC1-CFP in transiently transformed (TIF)Click here for additional data file.S2 FigSPIRRIG T-DNA insertion lines. (B) T-DNA insertions in spi-2, spi-3, and spi-4 were confirmed by qualitative RT-PCR with a primer pair spanning the insertion side (I) and second pair covering the coding region before the insertion up to exon located T-DNA (T). cDNA from Col-0 plants and H2O were included as controls. M = 1 kb plus ladder (Invitrogen). (C) Change of P-body number (in %) after mock treatments (½MS supplemented with 0.5% DMSO for 90 min). Data denote the average from three independent biological replicates (each three whole leaf areas). Error bars represent standard deviations. No significant changes between P-body numbers before and after the treatment could be determined (two-tailed student’s t tests). (D) Change of P-body number (in %) in leaf epidermis cells transiently transfected with DCP2-mCHERRY after an incubation of 90 min in ½MS supplemented with 0.5 mM CHX. Data denote the average from three independent biological replicates (each three cells). Error bars represent standard deviations. Two-tailed student’s t tests were performed to compare cells before and after the treatment . (E) Representative images of YFP-gSPI (left) colocalizing with DCP1-mCHERRY labeled P-bodies (middle) 150 min after continuous CHX treatment. Right picture presents the overlay. Scale bar: 50 μm. (F) Change of P-body number (in %) in leaf epidermis cells transiently transfected with DCP1-YFP after simultaneous treatment with 0.5 mM and 140 mM NaCl for 90 min. Data denote the average from three independent biological replicates (each three cells). Error bars represent standard deviations. Two-tailed student’s t tests were performed to compare cells before and after the treatment (* p < 0.05).(A) Schematic presentation of (PDF)Click here for additional data file.S3 Figspi in red circles and salt stress-treated spi in red squares. The 1st dimension explains 76.4%, the 2nd dimension 6.5%. B) Mapman visualization of log2-fold changes comparing the transcriptional pattern between Col-0 and spi upon salt stress induction.A) Principle component analysis (PCA) presenting Col-0 in blue circles, salt stress-treated Col-0 in blue squares, (PDF)Click here for additional data file.S4 FigDarker colors in GO term categories represent higher q-values (BH-corrected).(PDF)Click here for additional data file.S5 FigDarker colors in GO term categories represent higher q-values (BH-corrected).(PDF)Click here for additional data file.S6 FigDarker colors in GO term categories represent higher q-values (BH-corrected).(PDF)Click here for additional data file.S7 FigDarker colors in GO term categories represent higher q-values (BH-corrected).(PDF)Click here for additional data file.S8 FigDarker colors in GO term categories represent higher q-values (BH-corrected).(PDF)Click here for additional data file.S9 Figspi mutants. (B) Venn diagram comparing the salt stress-dependent down-regulation of transcripts in Col-0 and spi.(A) Box Whisker Plot of the fold-change for 3,400 up-regulated genes (left) and 2,611 down-regulated genes (right) in salt-treated Col-0 and (PDF)Click here for additional data file.S10 Fign = 30 cells) and (C) TZF3 mRNA N-terminally fused to mCHERRY (n = 120 cells) under nonstress (½MS) conditions. Error bars represent standard deviations. (D) mRNA stabilities of ABF3 (in %) were determined 3 h and 6 h after application of Actinomycin D (ActD) relative to time point 0 under nonstress (½MS) and salt stress conditions (200 mM NaCl in ½MS liquid medium for 4 h). Data denote the average from three independent biological and two technical replicates. Error bars represent the standard error of the mean. No significant changes between wild-type and spi mutants could be determined (two-tailed student’s t tests). (E) Distribution of LambdaN22-mVENUS reporter coexpressed with gABF3 N-terminally fused to BoxB repeats and with (F) gABF3 N-terminally fused to mCHERRY under nonstress (½MS) and salt stress (NaCl) conditions.(A) Distribution of LambdaN22-mVENUS reporter coexpressed with the BoxB without any mRNA target under nonstress (½MS) and salt stress  conditions in transfected leaf epidermis cells. Data denote the average of 60 cells. Error bars represent standard deviations. Distribution of LambdaN22-mVENUS reporter coexpressed with (B) RD29B mRNA N-terminally fused to mCHERRY ((PDF)Click here for additional data file.S11 Fign = 65 cells), (B) 16BoxB-gTZF3 (n = 150 cells) under nonstress (-NaCl) and salt stress  conditions. Presented are the averages from four biological replicates of RD29B (n = 20 cells each) and seven biological replicates of TZF3 (n = 20 cells each). (C) Distribution of LambdaN22-mVENUS reporter before and after simultaneous treatments with 0.5mM CHX and 140 mM NaCl for 90 min in cells cotransfected with 16BoxB-gRD29B (n = 10 cells per replicate) and (D) 16B-gTZF3 (n = 15 cells per replicate). Presented are the averages from three biological replicates. Error bars represent standard deviations. Two-tailed student’s t tests were performed to compare nonstress and salt stress conditions (* p < 0.05).Data denote the average number of cells (in %) showing cytoplasmic accumulations of the LamdaN22-mVENUS reporter in cells coexpressing (A) 16BoxB-gRD29B ((PDF)Click here for additional data file.S12 FigResidues forming the aromatic triade in the hydrophobic cleft in the EVH1 domain (underlined with a dark grey bar) are in bold. Asterisks highlight positions that have a single, fully conserved residue. Colons indicate conservation between groups of strongly similar properties—scoring >0.5 in the Gonnet PAM 250 matrix. Periods represent conservation between groups of weakly similar properties—scoring <0.5 in the Gonnet PAM 250 matrix.(PDF)Click here for additional data file.S13 FigThe GRAM and PH domains of FAN and SPI are shaded in dark and light gray, respectively. The BEACH domain in both proteins is underlined. The region containing WD40 repeats is printed in bold; the part that is not included in the clone used in this study is in italic letters. Asterisks highlight positions that have a single, fully conserved residue. Colons indicate conservation between groups of strongly similar properties—scoring >0.5 in the Gonnet PAM 250 matrix. Periods represent conservation between groups of weakly similar properties—scoring <0.5 in the Gonnet PAM 250 matrix.(PDF)Click here for additional data file.S14 FigAnnotated drawing of Gateway®- compatible A) pCL112- and B) pCL113- vectors used for BiFC assays (donated by Joachim Uhrig).(PDF)Click here for additional data file.S1 TableTotal numbers of analyzed cells are provided.(DOCX)Click here for additional data file.S2 TableTotal numbers of analyzed cells are provided.(DOCX)Click here for additional data file.S3 Tablespi are highlighted by black boxes.Differentially enriched transcripts in Col-0 and (DOCX)Click here for additional data file.S4 Tablespi (mut) mutants under nonstress and salt stress (NaCl) conditions. Expression changes between control and salt stress (NaCl) conditions as well as Col-0 and spi are presented as log-fold change. Statistically significant changes between spi and Col-0 are highlighted by red boxes .Displayed are the mean expression values and for 19 salt stress-responsive genes in Col-0 (wt) and (DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S7 TablePresented are the numbers of total fragments, mapped fragments, uniquely mapped fragments, nonspecific mappings, unmapped fragments, mapped exons, spanned exon–exon borders, total exons, annotated introns, and the number of sequenced genes per sample.(DOCX)Click here for additional data file.S8 Tablespi under salt stress conditions; transcripts down-regulated in spi under salt stress conditions; transcripts down-regulated in nontreated spi in comparison to nontreated Col-0; transcripts up-regulated in nontreated spi in comparison to nontreated Col-0; transcripts down-regulated in salt-treated spi in comparison to salt-treated Col-0; transcripts up-regulated in salt-treated spi in comparison to salt-treated Col-0.Additional sheets present up-regulated in Col-0 under salt stress conditions; transcripts down-regulated in Col-0 under salt stress conditions; transcripts up-regulated in (XLSX)Click here for additional data file.S1 Textspi under nonstress conditions and salt stress-dependent transcriptional changes in Col-0 and spi are presented.General transcriptional changes between Col-0 and (DOCX)Click here for additional data file.S2 Text(TXT)Click here for additional data file.S3 Text(TXT)Click here for additional data file.


To the Editor: In 2011, enteroaggregative Escherichia coli (EAEC) O104:H4 strains that produce Shiga toxins (EAEC-STEC) caused an outbreak of hemorrhagic disease affecting nearly 4,000 patients in Europe (Escherichia coli). One phage genome (P13374) was sequenced (We investigated Stx-2 subunit A (Stx-2A) bacteriophages in a group of epidemiologically unrelated EAEC-STEC O104:H4 strains isolated from animals and food in Germany (collection of the National Reference Laboratory for orf15 and cIP13374, that were specific to Stx-2A bacteriophages found in EAEC-STEC O104:H4 strains (We identified 2 genes, –Our results provide experimental evidence that EAEC-STEC O104:H4 have evolved by uptake of a distinct type of Stx-2A bacteriophage. Bovine STEC harboring Stx-2A bacteriophages able to transduce Stx-2A genes to EAEC O104:H4 are found worldwide, and phage-mediated transfer of Stx-2A can occur in the environment (


Capicua Transcriptional Repressor (CIC) gene on chromosome 19q13.2. Here we show that endogenous long (CIC-L) and short (CIC-S) CIC proteins are predominantly localized to the nucleus or cytoplasm, respectively. Cytoplasmic CIC-S is found in close proximity to the mitochondria. To study wild type and mutant CIC function and motivated by the paucity of 1p19q co-deleted ODG lines, we created HEK293 and HOG stable cell lines ectopically co-expressing CIC and IDH1. Non-mutant lines displayed increased clonogenicity, but cells co-expressing the mutant IDH1-R132H with either CIC-S-R201W or -R1515H showed reduced clonogenicity in an additive manner, demonstrating cooperative effects in our assays. Expression of mutant CIC-R1515H increased cellular 2-Hydroxyglutarate (2HG) levels compared to wild type CIC in IDH1-R132H background. Levels of phosphorylated ATP-citrate Lyase (ACLY) were lower in cell lines expressing mutant CIC-S proteins compared to cells expressing wild type CIC-S, supporting a cytosolic citrate metabolism-related mechanism of reduced clonogenicity in our in vitro model systems. ACLY or phospho-ACLY were similarly reduced in CIC-mutant 1p19q co-deleted oligodendroglioma patient samples.The majority of oligodendrogliomas (ODGs) exhibit combined losses of chromosomes 1p and 19q and mutations of isocitrate dehydrogenase (IDH1-R132H or IDH2-R172K). Approximately 70% of ODGs with 1p19q co-deletions harbor somatic mutations in the  CIC [CIC mutations affected 53-69% of ODG cases, and included missense, nonsense, and out-of-frame insertions and deletions [CIC mutations were predicted to inactivate their encoded proteins [TP53 and FBXW7, but not in oncogenes [CIC DNA-binding HMG domain and the Groucho protein-protein interaction domain, respectively [Oligodendrogliomas (ODG)s are a histologically, therapeutically and cytogenetically distinct subgroup of gliomas . The majCIC , 5 locateletions , 5-7. Moectively , 5, 7, 8FUBP1) on chromosome 1p31.1 [TERT transcription. Exactly how these mutations contribute to oligodendrogliomagenesis has yet to be determined.Somatic mutations were also observed in the gene encoding far upstream element (FUSE) binding protein1  [undaries , 11, andundaries  in Drosotterning , 13 and tterning , CIC is feration . During dentity) , 11, witERM/ETV5 and ETV1 and overexpression of PEA3 family proteins was associated with invasive and metastatic phenotypes in breast and gastric cancers and in rhabdomyosarcoma [in vitro, such as ETV5 [in vitro and in vivo studies in mammalian systems are now required to elucidate the cellular functions of both CIC mutant and wild type proteins.Relatively few studies have addressed the role of CIC in human biology and disease. For example, CIC appears to repress the PEA3 family of ETS transcription factors in cancers. Rare cases of Ewing's sarcoma express a novel CIC–DUX4 fusion protein encoded by a recurrent chromosomal translocation t4;19) . This fuosarcoma . In HEK2osarcoma . This in as ETV5 . Finally as ETV5 , 19. An  as ETV5  and redude novo lipid synthesis and acetylation of histones in proliferating cancer cells [Alterations in glycolysis and citrate metabolism contribute to the survival of cancer cells including gliomas -22. In cer cells , 25. Cyter cells . Howeverer cells , 27. Inter cells . In glioer cells .CIC mutations co-occur with mutations in either IDH1 or IDH2 in approximately 53-69% of cases, but the functional consequences of this co-occurrence are unknown [In 1p19q co-deleted ODG,  unknown . Here weCIC using three different siRNAs (Methods) and quantitated CIC proteins using western blots and densitometry. Compared to scramble siRNA controls, CIC-siRNA1 or CIC-siRNAs1+2+3 significantly reduced both 230 KDa CIC-S and 400 KDa CIC-L protein bands to approximately 15% and 45% of control levels respectively  and BT088 (IDH1+/+/CICR1515H/−) [To further characterize the predominant endogenous CIC protein isoforms (CIC-L and CIC-S) in human cells , we prepy Figure . To confy Figure , confirmTo determine the subcellular localization of endogenous CIC-S and CIC-L isoforms in HEK cells, we fractionated cells, purified cytoplasmic and nuclear fractions, and detected proteins that were enriched in these fractions (Methods). To assess the distribution of CIC-S and CIC-L, we immunoprecipitated (IP) CIC proteins from cytoplasmic and nuclear fractions and analyzed the IP samples using western blots. In fractionated samples, CIC-S at 230 KDa and CIC-L at 400 KDa were enriched in the cytoplasmic (59+/5%) and nuclear fractions (89+/−5%), respectively Figure . In ODG To confirm the subcellular localization of CIC isoforms, we designed Multiple Reaction Monitoring (MRM) mass spectrometry (MS) assays for 10 peptides common to both isoforms and 9 peptides unique to the CIC-L isoform only Figure . CIC proDrosophila embryos, CIC acts as a transcriptional repressor [During terminal and dorsoventral patterning of epressor , yet in To determine whether CIC was localized to mitochondria, we examined subcellular fractions of HEK cells enriched for cytoplasmic organelles  or non-organelle cytosolic material. Control proteins ATP5A  and tubulin (cytosolic) were predominantly in the mitochondrial fraction and cytosolic fractions, respectively, as expected Figure . The CICTo explore the nature of the CIC-S association with mitochondria, we isolated the mitochondrial fraction from HEK cells and subjected it to increasing concentrations of Proteinase K (0-15 μg/ml) under isotonic conditions. Under these conditions, Proteinase K digests proteins exterior to the mitochondrial outer membrane. However, proteins embedded within the outer membrane and proteins located inside the membrane are protected from digestion. Mitochondrial proteins ATP5A (matrix), VDAC (embedded in the outer membrane), TOMM20  and RHAMM/HMMR, a protein loosely bound to mitochondria , served IDH1 (R132H) and wild type CIC alleles, BT088 lacks mutant IDH1 but carries a mutant CIC allele, encoding R1515H in the protein–protein interaction domain, and no wild type CIC allele [We next sought to determine the subcellular localization of both wild type and mutant endogenous CIC in ODG cell lines (BT054 and BT088) that carried 1p19q co-deletions in different IDH1 genetic backgrounds. While BT054 carries mutant IDH1 R13H and wilC allele . We perfIDH1 R1H and wilTo determine whether wild type and mutant CIC-S were detected in the mitochondrial fraction in ODG cells, we isolated cytosolic and mitochondrial fractions of both BT054 and BT088 and detected CIC using western blots. In both ODG cell lines, CIC-S was detected in both cytosolic and mitochondrial fractions Figure . CompareIDH and CIC genes. Since mutations in IDH1 and IDH2 increase the production of 2HG [In our previous study , 69% of n of 2HG  and endon of 2HG . To confn of 2HG , 29 in wNext, we used MRM-MS to measure the levels of 2HG and 2KG in lines stably transfected with wild type and mutant expressing IDH and CIC constructs. Comparison of cells expressing mutant IDH1-R132H-V5 to cells expressing wild type IDH1 showed significant increases in 2HG  [To assess whether our observations were specific to our HEK-derived lines, we derived stable HOG cell lines that ectopically expressed wild type CIC-S or mutant CIC-S (R1515H or R201W), in combination with wild type IDH1 or mutant IDH1-R132H. The HOG cell line was derived from a human glioma and expresses oligodendrocyte-specific proteins . This ceH2 genes , 36. We enes [+) . In agreExamining the expression levels of F-CIC, F-CIC-R1515H, and F-CIC-R201W over passages (passage #2-15) in HEK-derived stable cells lines showed gradually decreasing levels of F-CIC, but both mutant F-CIC-R1515H and F-CIC-R201W proteins were stable . AlthougTo further shed insight into CIC function, we sought to identify CIC interacting proteins in the cytoplasm. We performed three co-immunoprecipitations (Co-IP) of cytoplasmic fractions using an anti-CIC antibody and rabbit IgG IPs as controls. We also transiently expressed CIC-S proteins with N-terminal FLAG, Myc or GFP tags in HEK cells, extracted the cytoplasmic fractions and performed IPs using anti-FLAG, anti-Myc or anti-GFP antibodies. Cells transfected with empty FLAG, Myc or GFP vectors served as controls. Isolated protein complexes were characterized using liquid chromatography and tandem mass spectrometry (LC-MS/MS). Consistent with a previous report showing interaction of CIC with Ataxin family members , we deteSince ACLY is involved in cytosolic citrate metabolism and alterations in citrate metabolism are important in malignancies , we chosPhosphorylation of ACLY, including at Serine 454 (hereafter referred to as pACLY), contributes to increased ACLY stability and activity -40. ACLYWe next investigated whether IDH1 and CIC mutations could affect ACLY or pACLY levels. We used our HEK and HOG-derived stable cell lines expressing mutant CIC in wild type or mutant IDH1 backgrounds. In HEK stable lines, we saw significant differences in the levels of pACLY in cells expressing F-CIC-R1515H compared to F-CIC on both IDH1-V5 (p<0.004) and IDH1-R132H-V5 (p<0.02) backgrounds Figure . Cells e+/R132H/CIC+/−) and mutant CIC-R1515H . In agreement with the results we obtained using our stably transfected cell lines, reduced pACLY levels were observed in BT088 cells compared to BT054 cells, both in the cytoplasmic and mitochondrial fractions  showed elevated levels of either ACLY or pACLY compared to tumours with mutant CIC proteins . Compared to cells expressing IDH1-R132H-V5 or F-CIC-1515H alone, cells co-expressing IDH1-R132H-V5/F-CIC-1515H showed further reductions (36% and 28% respectively) in relative cell proliferation  under serum free conditions supplemented with 0.5% Fetal Bovine serum (FBS) and growth factors. Without 0.5% FBS, both HEK and HOG cells do not adhere and form spheres; however, with 0.5% FBS, HEK and HOG cells can adhere and proliferate. In HEK stable cells, ectopic co-expression of F-CIC with IDH1-V5 showed a 33% relative increase in proliferation compared to cells ectopically expressing IDH1-V5 alone  in our CIC wild type/mutant HEK-derived stable lines in combination with IDH1-R132H-V5. Cells expressing F-CIC-R1515H in either the IDH1-V5 or IDH1-R132H-V5 background showed significant increases Figure . Reductiin vitro , 44, 45.MRM mass spectrometry assays, we have confirmed the presence of two forms of endogenous CIC in HEK, oligodendroglioma (ODG) cells and tumour tissues  Glioma patients with IDH1-R132H or IDH2-R172K had slower tumour progression, longer survival and better response to radiation or Temozolomide treatment , 56; (b)utations , 58.TERT promoters in ODGs with 1p19q deletions and suggested that these mutations may promote a self-renewal capability in these tumours. It is possible that the mutations in IDH1 and CIC may in fact be moderately deleterious and reduce clonogenicity but may provide other timely survival advantages under certain conditions during oligodendroglioma disease progression.Although the reduced clonogenicity and cell proliferation of IDH1 and CIC mutations alone or in combination are in agreement with the slow growing nature of ODG with 1p19q co-deletions, the selective advantage that slow growth confers on malignant cells is unclear. One explanation may be that other oncogenic proteins or pathways may drive or be permissive for tumour progression of ODGs initially. In line with this hypothesis, a recent study by Killela and colleagues [humidified, 37°C, 5% CO2 incubator. Cell lysate preparations, cell fractionations and western blot analysis were performed using standard protocols and are described in detail in the HEK293A, MDA-MB-231, HOG, and HeLa cell lines were cultured in DMEM (Invitrogen) supplemented with 10% (v/v) heat inactivated FBS (Invitrogen). C643 and H522 cell lines were cultured in RPMI (Invitrogen) supplemented with 10% (v/v) heat inactivated FBS. Stable cell lines derived from HEK293A or HOG were cultured in DMEM+10% FBS with selection (G418 and zeocin). Unless otherwise indicated, experiments with stable cell lines were always performed between passages 8-14. Cells were cultured without selection for at least two passages before experiments. Cell culturing was performed in a oC for 5 minutes or 2X SDS sample buffer  at 98oC for 10 minutes  antibody-bound beads were prepared by incubating anti-CIC antibody, anti-myc antibody, normal rabbit-IgG, or mouse-IgG with protein G agarose beads (Roche) in IP buffer  at 4°C overnight. Cell lysates were first pre-cleared with Sepharose 4B beads (Sigma) at 4°C for 1 hour and the lysates were immunoprecipitated with the prepared protein-G beads at 4ºC for 4 hours. The captured proteins and protein complexes were released by boiling the beads in 2X LDS Sample Buffer (Invitrogen) at 95Cells were first plated in DMEM+10% FBS. The following day, media were replaced with Neurocult proliferation media supplemented with cytokines (Stem Cell Technologies) and growth factors EGF, FGF  and Heparin  and cells were grown for the time periods indicated. For caspase inhibition assays, cells were grown with or without the pan caspase inhibitor zVAD-FMK (BD Biosciences)4 cells per well in a 6-well plate. Cells were grown in ODG media for up to 2weeks. Colonies were fixed with 4% paraformaldehyde and stained with 10% crystal violet. Images of colonies were captured using a Bio-Rad imaging system. Retained crystal violet stain was resolubilized in 1ml 10% acetic acid by rocking for 15 min. Readings were taken at A590 using a plate reader (VersaMAX Microplate Reader).Stable cell lines were plated at 1×10For multiple comparisons of western blot results, 2HG/2KG levels and clonogenic crystal violet proliferation assays, one-way ANOVA with Dunnett's post-test were performed using Prism 6.0 Software (GraphPad Software Inc.).For further descriptions of Materials and Methods, please see the


SNHG1. The most abundant of these, sno-miR-28, directly targets the p53-stabilizing gene, TAF9B. Collectively, p53, SNHG1, sno-miR-28 and TAF9B form a regulatory loop which affects p53 stability and downstream p53-regulated pathways. In addition, SNHG1, SNORD28 and sno-miR-28 are all significantly upregulated in breast tumours and the overexpression of sno-miR-28 promotes breast epithelial cell proliferation. This research has broadened our knowledge of the crosstalk between small non-coding RNA pathways and roles of sno-miRNAs in p53 regulation.p53 is a master tumour repressor that participates in vast regulatory networks, including feedback loops involving microRNAs (miRNAs) that regulate p53 and that themselves are direct p53 transcriptional targets. We show here that a group of polycistronic miRNA-like non-coding RNAs derived from small nucleolar RNAs (sno-miRNAs) are transcriptionally repressed by p53 through their host gene,  TP53. Cellular insults such as DNA damage or aberrant oncogene expression engage the p53 pathway, resulting in rapid stabilization of p53 protein levels. Upon activation, p53 functions as a sequence-specific transcription factor to either activate or repress the expression of target genes. The global landscape of p53 transcriptional regulation is vast and complex, with gene expression profiling studies demonstrating that thousands of genes rapidly alter expression upon p53 activation -1H-imidazol-1-yl]carbonyl]-2-piperazinone) was purchased from Cayman Biochemicals , and Ponasterone A (PonA) was purchased from Invitrogen.Breast tumour and paired normal adjacent tissue samples were obtained from 26 breast cancer patients who have received surgery at the Royal Adelaide Hospital  between 2003 and 2011. Samples were stored in RNAlater solution (Ambion) for RNA stabilization before RNA extraction using miRNeasy kit (Qiagen) following the manufacturer’s protocol. RNA concentration was determined using a ND-1000 NanoDrop spectrometer . This research has been approved by the Institutional Review Boards of the University of Adelaide and SA Pathology. All samples were gathered according to the institutional review board-approved protocol and the written informed consent from each patient. Relevant clinical data was retrieved from patients’ records including human epidermal growth factor 2 (HER2), estrogen receptor (ER), progesterone receptor (PR) and proliferation index (MIB-1) status.5 cells/well were seeded in 6-well plates  or Western blot) and 1 x 105 cells/well were seeded in 24-well plates . Transfections were done 24 hours post-seeding with 50 nM Genepharma miRNA mimics  or negative control RNA (ncRNA) (Genepharma) using Lipofectamine RNAiMAX reagent (Invitrogen) following the manufacturer’s instructions. The cells were harvested 72 hours post-transfection for RT-PCR, Western blot, or luciferase assay.For miRNA overexpression studies, approximately 3 × 105 cells/well were seeded in six-well plates. The first transfection was done 24 hours post-seeding with 50 nM miRVana miRNA inhibitor (Ambion) or 50 nM miRVana miRNA inhibitor negative control RNA (ncRNA) (Ambion) using Lipofectamine RNAiMAX reagent (Invitrogen) following the manufacturer’s instructions. Cells were harvested 72 hours post-transfection for further experiments.For miRNA inhibition studies, approximately 3 × 10RNA interference of TAF9B was performed using siTAF9B (AGUAUGAACCAAGGGUUAUAA) (GenePharma). The transfection procedure using Lipofectamine RNAiMAX reagent (Invitrogen) is the same as described above for miRNA overexpression and inhibition.5 H1299 cells/well in 24-well plates. After 24 hours, in each well we co-transfected 0.4 ng/μL of either WT or MUT luciferase constructs with 8.33 nM of either sno-miR-28 mimics (Genepharma) or negative control RNA (ncRNA) (Genepharma), using Lipofectamine 2000 (Invitrogen) following the manufacturer’s instructions. Cells were harvested 72 hours post-transfection, and Renilla and Firefly luminescence were measured by a GloMax 20/20 Luminometer (Promega) following the manufacturer’s instructions. A ratio of Renilla/Firefly luminescence intensity was used to indicate the relative luciferase expression activity.For the luciferase assay, we cloned the TAF9B 3’-UTR (WT) downstream of the Renilla luciferase gene in the psiCHECK2 dual-luciferase vector (Promega) . Protein extracts were resolved using SDS PAGE electrophoresis on 10% polyacrylamide gels and electrotransferred to Hybond- C Extra nitrocellulose membranes .Cells were rinsed with PBS and lysed in lysis buffer ) on ice for 30 min. Insoluble components of cell lysates were removed by centrifugation for 10 min at 4°C, 12,000 WAF1 Ab-3 antibody ; Equal loading was confirmed by blotting of β-actin antibody . Chemiluminescent detection of protein was done using appropriate secondary antibodies conjugated with horseradish peroxidise  and the enhanced chemiluminescence kit  according to the manufacturer's instructions.For quantification, p53 protein was probed by mouse monoclonal p53 antibody (DO-1)  ; TAF9B was probed by rabbit monoclonal TAF9B antibody  ; p21 was probed by mouse monoclonal p21CDKN1A was used as a positive control  expression was normalized to PSMB4 (RNA (>250nt) extraction from cells was performed using the RNAeasy mini kit (Qiagen) using on-column RNase-free DNase digestion according to the manufacturer’s instructions. After extraction, RNA concentration was measured using a ND-1000 NanoDrop spectrometer (Thermo Scientific). Thereafter, 1μg of total RNA was reverse-transcribed into cDNA using Moloney Murine Leukaemia Virus (M-MLV) Reverse Transcriptase (Promega) with random 6’mer primers or oligo-dT primer (Promega) under the following the manufacturer’s instructions. Real-time PCR reaction was performed on a CFX Connect Real-Time PCR Detection System (Bio-Rad) using iTaq Universal SYBR Green Supermix (Bio-Rad). The RT-PCR program was: 95°C for 3min, then start cycles consisting 95°C for 10s and 57/60/61°C for 60s (depending on the primers used) for 40 cycles. After the reactions were complete, the Cto PSMB4 .Small RNAs (including miRNAs and sno-miRNAs) were extracted using the miRNeasy mini kit (Qiagen) following the manufacturer’s instructions. After extraction, total RNA concentration was determined using a ND-1000 NanoDrop spectrometer (Thermo Scientific). For quantification of small RNAs, TaqMan gene expression assays were obtained from Applied Biosystems , including TaqMan assays for miR-21, miR-155, miR-16, miR-24 and U6 snRNA, and Custom TaqMan assays for SNORD25, SNORD28, sno-miR-25, sno-miR-28. Reverse transcription was performed using a TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems) and specific Reverse Transcription stem-loop primers provided in the TaqMan RNA assays following the manufacturer’s instructions. Real-time PCR reaction was performed on a Rotor Gene 6000 Real-Time PCR Machine (Qiagen) using specific TaqMan RNA assays following the manufacturer’s instructions. Small RNA expression in cell samples was normalized to U6 snRNA, whereas small RNA expression in tissue samples was normalized to averaged relative expression level of a group of pooled normalizers: miR-24, U44, U48 and U6.T values were plotted to log10 of the miRNA concentrations respectively to create a standard curve  at 2000 cells/well and were incubated in normal culture conditions for 6 hours to attach. After that, these cells were processed at 0, 24, 48, 72, 96 hour time points using the CellTiter-Glo Luminescent Cell Viability Assay Kit (Promega) according to the manufacturer’s instructions. Fluorescence was measured using a LUMIstar Galaxy luminometer (BMG Labtech). The fluorescence readings in each experimental group were normalized against the 0 hour point.For colony formation, cells were re-plated in 6-well plates at 500/1000/2000 cells/well and were incubated in normal culturing conditions for 7 days. Then the cells were fixed in methanol for 15 minutes, and stained for 1 hour in 1:20 Giemsa Stain, Modified Solution (Sigma-Aldrich). Afterwards, colonies were photographed by a digital camera and counted.An Affymetrix Human Gene 1.0 ST array containing 234 annotated snoRNAs  was used to identify snoRNAs that were differentially expressed following the manufacturer’s instructions as previously described , 69. “FoResults are given as mean of at least three independent experiments ± standard deviation. Student’s t-test was performed using replicate values to indicate significance. Values of p<0.05 were considered statistically significant (as labelled with ** in figures), while values of p<0.1 were indicated by *.S1 Fig Standard curves were shown for absolute quantitation of endogenous sno-miR-28, sno-miR-25 using custom TaqMan assays with miR-155 included as a positive control. Synthesized sno-miR-28 and miR-155 were prepared at a series of known concentrations at 4nM, 0.4nM, 0.04nM, and 0.004 nM, while synthesized sno-miR-25 was prepared at 4pM, 0.4pM, 0.04pM, and 0.004 pM to match its low endogenous expression detected in tissues. RT-PCR was performed subsequently and CT values were plotted to log10 of the miRNA concentrations. The point representing 0.004pM of sno-miR-25 was not plotted because no CT value was detectable at this concentration. (D) sno-miR-28 TaqMan RT-PCR was performed using synthesized SNORD28 or sno-miR-28 as templates. RT-PCR readings are shown to evaluate the specificity of the sno-miR-28 TaqMan assay.(TIF)Click here for additional data file.S2 Fig(A) The standard curves in (TIF)Click here for additional data file.S3 FigBioinformatics analysis showed the binding of sno-miR-28 to BHLHE41 and TGFBR2 3’-UTR in a similar manner as other miRNAs binding to their targets. The schematic graphs are made to show the proposed binding sites for sno-miR-28. The seed-recognizing sites are marked in red; hypothesized duplexes formed by the interaction of the binding sites of the 3’-UTR of TAF9B and sno-miR-28 are illustrated, and the predicted free energy of the hybrids were indicated. The solid lines between two chains represent hydrogen bonds between adenine (A)-uracil (U) pairs and guanine (G)-cytosine (C) pairs, whereas dashed lines represent G-U pairings.(TIF)Click here for additional data file.S1 TableAs wild-type p53 was induced in H1299 cells, Affymetrix gene expression profiling identified a list of snoRNAs that were repressed. These snoRNAs are listed with their host genes.(PDF)Click here for additional data file.S2 TableWhen wild-type p53 was induced in WE-68 cells, Affymetrix gene expression profiling identified a list of snoRNAs that were repressed. These snoRNAs are shown with their host genes.(PDF)Click here for additional data file.S3 TablePrimers used in this study are listed by targets and usage.(DOC)Click here for additional data file.S4 TableAll genes significantly regulated in both H1299 and WE-68 cell lines upon p53 activation are listed with fold change and p values. “Fold change” refers to the ratio of expression in induced (H1299 treated with PonA and WE-68 treated with Nutlin) versus uninduced cells. Statistical criteria for microarray: fold change > 1.085 or <0.915, p<0.05.(XLSX)Click here for additional data file.