Activation of systemic acquired resistance in plants is associated with transcriptome reprogramming induced by the unstable coactivator NPR1. Immune-induced ubiquitination and proteasomal degradation of NPR1 are thought to facilitate continuous delivery of active NPR1 to target promoters, thereby maximising gene expression. Because of this potentially costly sacrificial process, we investigated if ubiquitination of NPR1 plays transcriptional roles prior to its proteasomal turnover. Here we show ubiquitination of NPR1 is a progressive event in which initial modification by a Cullin-RING E3 ligase promotes its chromatin association and expression of target genes. Only when polyubiquitination of NPR1 is enhanced by the E4 ligase, UBE4, it is targeted for proteasomal degradation. Conversely, ubiquitin ligase activities are opposed by UBP6/7, two proteasome-associated deubiquitinases that enhance NPR1 longevity. Thus, immune-induced transcriptome reprogramming requires sequential actions of E3 and E4 ligases balanced by opposing deubiquitinases that fine-tune activity of NPR1 without strict requirement for its sacrificial turnover. Plant diseases cause devastating crop losses around the world and threaten the food supply of millions of people. Over time, plants have developed various mechanisms for fighting off infections caused by pests and other pathogens such as viruses and bacteria. When plants become infected they kick their immune system into action by rapidly switching on and off certain genes. They do this by activating the protein NPR1 which regulates the plant’s immune genes. NPR1 is essential for fighting off infections and plants that do not have this protein are highly susceptible to disease.Peculiarly, once the plant has detected an infection it builds resistance by destroying the NPR1 protein. A previous study suggested that plants do this to replace old ‘inactive’ NPR1 with newer versions that can activate the genes needed to stop the disease developing. This process, however, requires a lot of energy that could be re-directed to other aspects of the immune response. Now, Skelly et al. – including one of the researchers involved in the previous study – have explored whether there may be other reasons for why plants destroy the NPR1 protein.Arabidopsis thaliana, Skelly et al. found that the length of ubiquitin chains attached to the NPR1 protein could fine-tune its level of activity: short ubiquitin chains activate NPR1, while longer chains lead to its destruction and shut down the protein. This suggests that the steps leading to the destruction of NPR1 regulate the immune genes needed to fight off disease.Plant cells target NPR1 for destruction by repeatedly tagging it with molecules called ubiquitin to form ubiquitin chains. The length of these chains determines whether a protein is stable and ready for action, or whether it is ready to be destroyed. In experiments with a commonly studied plant known as This work has uncovered important new components of how plants defend themselves from infection. If these findings translate to crop plants they could inform future agricultural strategies for enhancing the plant’s own defences to increase crop yields, which would provide more food for a rapidly growing population. PR) genes 1). The majority of SA-induced genes are NPR1 dependent, indicating NPR1 is a master regulator of plant immunity  that accumulates upon recognition of biotrophic pathogens. SA not only induces resistance in infected local tissues, it is also required for establishment of systemic acquired resistance (SAR), a form of induced resistance with broad-spectrum effectiveness that is long-lasting and protects the entire plant from future pathogen attack . Establiimmunity . ConsequSince NPR1 exerts its activity in the nucleus , controlUpon activation of SAR, NPR1 is subject to an array of post-translational modifications. A combination of alterations in redox-based modifications, phosphorylation and SUMOylation of NPR1 result in the formation of a transactivation complex that induces the transcription of immune-responsive target genes . SubsequWhile transcription-coupled degradation of unstable (co)activators is an attractive model for controlling transcriptional outputs in eukaryotes, it is potentially also a costly sacrificial process. Therefore we explored the alternative possibility that prior to degradation, ubiquitination itself might act as a transcriptional signal. As chains of four or more ubiquitin molecules are thought to be necessary for recruitment of most substrates to the proteasome , it is pube4/muse3 plants exhibited enhanced disease resistance but this phenotype could only be explained in part by the increased stability of an NLR receptor  and mutant ube4 plants, did result in bacterial proliferation in mutant ics1 plants. In agreement with the gene expression data, enhanced susceptibility was maintained in ube4 ics1 double mutants  that preferentially bind chains of four or more ubiquitin molecules   followed mutants , but ubiolecules . Compare mutants , indicatcul3a cul3b mutants that were compromised in SA-induced expression of NPR1 target genes, plants carrying two different ube4 mutant alleles exhibited elevated expression levels that were much higher than in WT  (1) genes that were regulated by SA in both WT and mutant ube4 plants, and (2) genes that did not make the ≥2 fold change cut-off in WT but were highly regulated by SA in ube4 mutants. The majority of SA-induced genes in category 1, including PR1 and WRKY marker genes, received a boost in expression when UBE4 was knocked out  . We sepacked out . This pocked out . Similared to WT . By cont4 plants , suggestcul3a/b and ube4 mutants  were dramatically upregulated in cul3a cul3b ube4 mutants to a level higher than in any of the other genotypes. This suggests that in absence of CRL3 and UBE4, these genes were activated through another pathway or were highly responsive to elevated homeostatic levels of NPR1 protein and may therefore not be suitable readouts for this particular epistatic analysis  plants  plants . By contArabidopsis genome is predicted to encode for at least 65 DUBs  and may provide another layer of regulation of NPR1 activity. The  65 DUBs  with hig 65 DUBs  and NSC6 65 DUBs  strongly 65 DUBs , suggestn levels . Thus, DNext we tested more selective inhibitors that more specifically target one or a few DUBs. First we treated WT seedlings with various DUB inhibitors and compared the cellular levels of global ubiquitin conjugates with control-, and MG132-treated seedlings. While NSC632839 and MG132 treatments dramatically enhanced accumulation of ubiquitin conjugates, especially in combination with SA treatment, all other inhibitors had little effect on cellular ubiquitination levels . We thenArabidopsis genome using BLASTp. The identified Arabidopsis DUBs included members of the ubiquitin-specific protease (UBP) and ubiquitin C-terminal hydrolase (UCH) multi-gene families , an ubiquitin mimic that cannot be hydrolysed upon irreversible binding to DUB active sites . HA-UbVSArabidopsis UBP6 displayed very little deubiquitination activity on free ubiquitin chains or di-ubiquitin of K48 and K63 linkage types  seedlings with WP1130 inhibitor increased the levels of SA-induced polyubiquitinated NPR1-GFP while reducing the unmodified amount of this protein  (The role of  humans) . Althoug humans) . Progres humans) . Althoug humans) , it prob humans) . Althoug humans) , progres humans) . This ty humans) .PR1 target promoter (i) like their yeast and mammalian counterparts (ii) UBP6 and UBP7 prevent inactivation of NPR1 by opposing the formation of long ubiquitin chains.The complexity of the ubiquitin-dependent transcriptional timer was further revealed by the identification of UBP6 and UBP7 that deubiquitinated NPR1, thereby regulating its transcriptional activity and lifetime  and 6. Spromoter . These dterparts , UBP6 anUBP6 showed a similar DUB activity as its mammalian homologue USP14 , in thatArabidopsis thaliana which encodes for >60 DUB enzymes, many of which cluster into related sub-groups activators in general.Arabidopsis plants used in this study were in the Columbia genetic background, with WT referring to wild-type Col-0 throughout. Plants were grown under long day conditions (16 hr photoperiod) on soil in controlled-environment growth chambers at 65% humidity and 22°C unless otherwise stated. Seeds were stratified at 4–8°C in darkness for 2 days before moving to growth chambers. Plants were grown in a soil mix composed of peat moss, vermiculite and sand at a ratio of 4:1:1 respectively, and illumination was provided by fluorescent tube lighting at an intensity of 70–100 μmol m−2sec−1. For experiments on seedlings, seeds were sterilised by washing in 100% ethanol for 2 mins before incubating in 50% household bleach for 20 mins. After removal of bleach, seeds were washed at least 3 times with sterile H2O before use. Sterilised seeds were spotted on Murashige and Skoog agar media and stratified before placing under lighting conditions as above. All T-DNA insertion mutants used were genotyped by PCR using standard conditions with gene specific primers in combination with left-border primers specific to each mutant collection (All llection .UBE4 (At5g15400) and UBP6 (At1g51710) genes were amplified using Phusion polymerase (NEB) from WT Arabidopsis cDNA with the addition of CACC at the 5’ end required for TOPO cloning. The PCR products were gel-purified and cloned in to the pENTR/D-TOPO vector (Invitrogen) according to manufacturers’ instructions. The active site residue of UBP6 was then mutagenised to serine (C113S) using QuikChange Site-Directed Mutagenesis Kit according to manufacturers’ instructions. Genes were then recombined into pEarleyGate 104 and 202 plasmids by LR reaction (Invitrogen) as described previously  constructs. These plasmids were used to transform protoplasts or to transform Agrobacterium tumifaciens strain GV3101 (pMP90) as described previously  and 1 ml of ice cold 96% ethanol was added before incubating for 1 hr at −20°C. The precipitate was then centrifuged at 13,000 rpm for 5 min at 4°C, the pellet was washed with ice cold 70% ethanol and resuspended in 50 μl of H2O. Before cDNA synthesis, RNA samples were quantified using a NanoDrop spectrophotometer (Thermo Scientific) and appropriate dilutions were made to ensure all samples contained equal amounts of RNA. Reverse transcription was then performed using SuperScript II reverse transcriptase (Invitrogen) according to the manufacturers’ instructions. qPCR was carried out on 20-fold diluted cDNA using Power SYBR Green (Life Technologies) and gene-specific primers  before addition of an equal volume of phenol/chloroform/isoamylalcohol (25:24:1). The homogenate was vortexed and centrifuged at 13,000 rpm for 5 min. The aqueous phase was transferred to an equal volume of 24:1 chloroform/isoamylalcohol, vortexed and then centrifuged at 13,000 rpm for 5 min. This step was repeated once before the aqueous layer was added to a 1/3 vol of 8 M LiCl and incubated overnight at 4°C. The extract was then centrifuged at 13,000 rpm for 5 min at 4°C. The resulting pellet was washed with ice cold 70% ethanol then rehydrated and dissolved in 400 μl H primers  on a SteArabidopsis thaliana TAIR10 genome using Bowtie. TopHat identified potential exon-exon splice junctions of the initial alignment. Strand NGS software in RNA-Seq workflow was used to quantify transcripts. Raw counts were normalised using DESeq with baseline transformation to the median of all samples. Data were then expressed as normalised signal values (i.e. log2[RPKM] where RPKM is read count per kilobase of exon model per million reads) for all statistical tests and plotting. Genes were then filtered by expression (20%–100%) and differentially expressed genes determined by Benjamini Hochberg FDR with 2-way ANOVA (p=0.05). Additionally, we required SA-induced genes to meet a ≥ 2 fold change cut-off, whereas NPR1-dependent genes required ≥1.5 fold change in Col-0 or ube4 plants when compared to npr1 mutants. RNA Seq data have been deposited in Array Express at EMBL-EBIunder accession code E-MTAB-7369.RNA was extracted from biological duplicate samples as described above and further purified using an RNeasy Mini Kit (Qiagen) according to the manufacturer’s instructions. qPCR was carried out to confirm appropriate induction of SA-responsive marker genes. RNA was then quantified and submitted to GATC Biotech/Eurofins  for RNA sequencing. The RNA-Seq reads were aligned to the Chromatin immunoprecipitation was performed on leaf tissue of 4 week-old soil-grown adult plants essentially as described  but withFor protein degradation assays and analysis of NPR1 levels, seedlings were frozen and ground to a fine powder in liquid nitrogen before homogenising in protein extraction buffer (PEB) (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 0.1% Triton X-100, 0.2% Nonidet P-40, and inhibitors: 50 μg/ml TPCK, 50 μg/ml TLCK, 0.6 mM PMSF) . For anaFor analysis of polyubiquitination with TUBEs, seedlings were ground to a fine powder in liquid nitrogen and homogenised in 1x PBS supplemented with 1% Triton X-100, 10 mM NEM, 40 µM MG132, 50 μg/ml TPCK, 50 μg/ml TLCK, 0.6 mM PMSF, and 0.2 mg/ml GST-TUBE . Homogen6-V5-S5aUIM protein immobilised on agarose. Agarose beads were washed 5 times with extraction buffer before elution at 80°C for 15 mins in 1X SDS-PAGE sample buffer including 50 mM DTT. NPR1-GFP was detected by immunoblotting with anti-GFP (Roche).For analysis of long chain polyubiquitination, seedlings were ground to a fine powder in liquid nitrogen and homogenised in 1X PBS, supplemented with 1% Triton X-100, 10 mM NEM, 80 μM MG115, 50 μg/ml TPCK, 50 μg/ml TLCK, 0.6 mM PMSF, 1X phosphatase inhibitor cocktail 3 (Sigma). Homogenates were centrifuged at 13,000 rpm at 4°C for 20 mins to remove cellular debris and filtered through 0.22 µm filters before overnight incubation with 300 μg HisFor proteasome co-immunoprecipitation with FLAG-UBP6, seedlings were frozen and ground to a fine powder in liquid nitrogen before homogenising in proteasome extraction buffer (50 mM Tris-HCl (pH 7.4), 25 mM NaCl, 2 mM MgCl2, 1 mM EDTA, 10 mM ATP, 5% glycerol, and inhibitors: 50 μg/ml TPCK, 50 μg/ml TLCK, 0.6 mM PMSF). Extracts were centrifuged at 13,000 rpm at 4°C for 20 mins to remove cellular debris and filtered through 0.22 µm filters. Anti-FLAG M2 affinity gel was washed with the above buffer before incubating with samples overnight with rotation at 4°C. The resin was washed 3 times with the same buffer before immunoprecipitated proteins were eluted by boiling in 1X SDS-PAGE sample buffer including 50 mM DTT. FLAG-UBP6 was detected using rabbit anti-FLAG antibodies while co-immunoprecipitating proteins were detected with indicated antibodies.E. coli cells with the addition of 1 mM IPTG and cultures were incubated for a further 4 hr at 28°C before collecting by centrifugation. Cells were then lysed in 1X PBS supplemented with 1 mg/ml lysozyme, 25 U/ml Benzonase nuclease, 0.1% Triton-X-100 and a protease inhibitor cocktail before GST-TUBE was purified using Protino Glutathione Agarose 4B according to the manufacturers’ instructions. Purified GST-TUBE was dialysed against 1X PBS and stored with the addition of 10% glycerol at −80°C until use.N-terminal GST-tagged TUBE was generated by cloning the coding sequence of hHR23A into pGEX-6P-1 using EcoRI and SalI restriction sites. Primers used are listed in E. coli into pET151/D-TOPO. The resulting His6-V5-S5aUIM protein was expressed in BL21(DE3) E. coli cells by addition of 1 mM IPTG and incubation for 24 hr at 28°C before collecting by centrifugation. Cells were then lysed in lysis buffer , 25 U/ml Benzonase nuclease, 50 μg/ml TPCK, 50 μg/ml TLCK and 0.5 mM PMSF). His6-UBP6 was then purified using HisPur cobalt resin (Thermo Fisher) according to manufacturers’ instructions. Purified His6-V5-S5aUIM was dialysed against 1X PBS and covalently coupled to NHS-activated agarose to a final concentration of approximately 10 μg/μl following the manufacturer’s instructions (Thermo Fisher).Recombinant S5aUIM protein was generated by synthesising residues 196–309 from human S5a with codon optimisation for 6-T7-tagged UBP6 was generated by cloning the coding sequence of Arabidopsis UBP6 in to the expression vector pET28a using EcoRI and SalI restriction sites. Primers used are listed in E. coli cells with the addition of 1 mM IPTG and cultures were incubated for a further 3 hr at 28°C before collecting by centrifugation. Cells were then lysed in lysis buffer . His6-UBP6 was then purified using HisPur cobalt resin (Thermo Fisher) according to manufacturers’ instructions. Purified His6-UBP6 was dialysed against 50 mM Tris-HCl pH 7.4, 5M NaCl and stored with the addition of 10% glycerol at −80°C until use.N-terminal HisRecombinant FLAG-UBE4 was produced using cell-free synthesis via two-step PCR  using thN’-SALAAAKKEKDSNNTAAVKL-Cys. Rabbits were subsequently bled and antibodies were enriched by affinity purification .The anti-NPR1 polyclonal antibody was generated by immunising rabbits with a synthetic peptide based on a region of the NPR1 protein with the sequence 2, 1 mM DTT and 1 mM ATP. Before labelling, 350 nM His6-T7-UBP6 was pre-incubated with 50 μM WP1130 or DMSO control for 10 mins before addition of 10 nM Ub-VS treated 26S proteasomes (Ubiquigent). Reactions were incubated for a further 20 mins before addition of 700 nM HA-UbVS and further incubation for 30 mins. All steps were carried out at room temperature. Labelling was terminated with the addition of SDS-PAGE sample buffer including 50 mM DTT. Samples were heated at 70°C for 10 mins before SDS-PAGE and immunoblot analyses.For HA-UbVS labelling, 10 μl reactions were prepared in 50 mM Tris-HCl pH 7.4, 5 mM MgCl2, 1 mM DTT, 5 mM ATP). Where indicated, 1.25 nM Ub-VS treated 26S proteasomes and 20 nM UBP6 were added. Di-ubiquitin and polyubiquitin chain substrates were included at 400 nM. Reactions were incubated at 30°C for the times indicated in figure legends before terminating with addition of SDS-PAGE sample buffer including 50 mM DTT. Samples were heated at 70°C for 10 mins before SDS-PAGE and immunoblot analyses.All in vitro deubiquitination assays were performed in DUB buffer  (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, 0.1% Triton X-100, 0.2% Nonidet P-40, and inhibitors: 50 μg/ml TPCK, 50 μg/ml TLCK, 0.6 mM PMSF). Extracts were centrifuged at 13,000 rpm at 4°C for 20 mins to remove cellular debris and filtered through 0.22 µm filters. GFP-Trap A agarose (Chromotek) was incubated with extracts for 2 hr with rotation at 4°C before washing 10 times with PEB (without inhibitors) then twice with DUB buffer. Supernatant was completely removed before DUB reactions were set up as described above but with NPR1-GFP immobilised on GFP-Trap A as the substrate. Proteins were eluted by boiling in 1X SDS-PAGE sample buffer including 50 mM DTT, before analysis by immunoblotting.n refers to sample size.For pathogen growth experiments confidence intervals that would allow acceptance or rejection of the null hypothesis were used to estimate sample size, while no statistical methods were used to predetermine sample sizes elsewhere, nor were any methods of randomization. All experiments were repeated a minimum of two times with similar results. For quantitative immunoblotting, band intensities were acquired using Image Studio software and a LI-COR Odyssey FC imaging device (LI-COR Biosciences). NPR1 signal was normalised to GAPDH signal for three replicates, each of which consisted of an independent pool of at least 50 seedlings. For qPCR and ChIP-qPCR experiments with adult plants, samples consisted of a pool of at least six different plants while for seedlings, samples consisted of a pool of at least 50 seedlings. qPCR data is shown for three technical replicates of a representative repeated experiment. For pathogen growth experiments, replicates represent eight separate plants. In all figure legends, the statistical tests applied are stated while  In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, one of whom is a member of our Board of Reviewing Editors, and the evaluation has been overseen by Christian Hardtke as the Senior Editor. The reviewers have opted to remain anonymous.Thank you for submitting your article "Dynamic ubiquitination determines transcriptional activity of the plant immune coactivator NPR1" for consideration by eLife after appropriate revision.The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission. We would like to emphasize that we are overall very positive about the work and would like to see it published in Summary:This work uncovers highly complex and dynamic ubiquitination of NPR1, the master regulator of the SA pathway. CUL3, UBE4, and UBP6/7 act together to regulate progressive ubiquitination of NPR1, thereby controlling NPR1 activity, in addition to previously known ubiquitin dependent degradation. The work is of great interest to the broad field of transcriptional regulation, and is overall well executed.Essential revisions:ube4 and ubp6 ubp7). Unless functional complementation is provided, this is not robust enough to come up with a solid conclusion. Please provide additional genetic evidence supporting the role of UBE4 and UBP6/7 in NPR1 ubiquitination using other alleles.1) In most cases, the authors have used only a single allele for T-DNA knock-out mutants . Have the authors analysed the transcript levels in these lines, and are they comparable?2) The role of UBE4 in promoting NPR1 degradation are not entirely convincing. To show that NPR1 stability is affected by UBE4, they generated two lines, one expressing NPR1-GFP in Col-0 or, the other in the ube4 background. Normalising the signal to the corresponding S5a would help to make the point. Also related, is S5a not degraded via the proteasome? If authors have the coomassie, it would be good if they would include it.3) Because Figure 2A is central to the manuscript, it would be good to have some quantification here. The loading control is not really equal, protein concentration seems to go down in the Col-0, while up in the 4) It will be necessary to test association between UBE4 and NPR1 in vivo on SA-treated plants to support a direct action of UBE4 on NPR1. Finally, the ability of UBE4 to elongate ubiquitin chains is not consistent with Figure 2D or 6C where the ubiquitin chains seem to have the same length (reaching the same size). To convince the reader, the authors must provide more resolutive blots for high molecular weight. The elongation or trimming activities of UBE4 and UBP6/7, respectively, should result in marked migration differences on a western blot.5) UBP6 is proposed to trim ubiquitin off NPR1 upon SA treatment, counteracting UBE4 . This conclusion would be more convincing if the authors performed the exact same experiment without SA treatment. One should expect not to see the 50kDa 6x ubiquitin band showing up if UBP6 acts only on UBE4-extended chains.ube4 mutant observed may be a combination of both NPR1 function and NLR function. Although the authors show that the elevated defense gene expression and increased disease resistance in ube4 mutant require ICS1 and NPR1, the same maybe true for NLR activated defenses. This will need to be thoroughly discussed.6) Since UBE4 is also required for the degradation of multiple NLRs. The elevated defense gene expression and disease resistance in the  Essential revisions:1) In most cases, the authors have used only a single allele for T-DNA knock-out mutants (most important for ube4 and ubp6 ubp7). Unless functional complementation is provided, this is not robust enough to come up with a solid conclusion. Please provide additional genetic evidence supporting the role of UBE4 and UBP6/7 in NPR1 ubiquitination using other alleles.ube4 and ubp6 ubp7, which further support our conclusions on their roles in controlling NPR1 ubiquitination:We have now provided additional genetic evidence for ube4-2, we used the previously described muse3 mutant allele of UBE4 . New Figure 2—figure supplement 1D and E show that both mutant alleles compromise degradation of the endogenous SA-induced NPR1 protein.a) In addition to the ube4-2 plants, SA-induced PR1 gene expression is enhanced in the muse3 mutant.Moreover, in new Figure 3—figure supplement 1A we show that like in ubp6 ubp7 mutants with a 35S::FLAG-UBP6 construct. We now show in new Figure 6—figure supplement 1E that enhanced NPR1 turnover observed in ubp6 ubp7 mutants is indeed alleviated by complementation with 35S::FLAG-UBP6. Furthermore, in Figure 6G and 6F we already demonstrate that impaired expression of NPR1 target genes in the ubp6 ubp7 mutant is rescued to near WT levels when FLAG-UBP6 is expressed.b) In the original manuscript we already complemented 2) The role of UBE4 in promoting NPR1 degradation are not entirely convincing. To show that NPR1 stability is affected by UBE4, they generated two lines, one expressing NPR1-GFP in Col-0 or, the other in the ube4 background. Unexpectedly, NPR1 either accumulates to lower, or similar levels in the mutant background, compared to WT . Have the authors analysed the transcript levels in these lines, and are they comparable?NPR1-GFP transcript levels were comparable . Nonetheless, accumulation of SA-induced NPR1-GFP protein is somewhat variable between experiments , which was mirrored by the endogenous NPR1 protein . Therefore we strictly used qualitative and quantitative cycloheximide (CHX) chase assays rather than steady-state levels to draw conclusions on the stability of NPR1. This well established proteostasis approach clearly shows that NPR1-GFP and endogenous NPR1 are both stabilised in ube4 mutants.Thank you for this suggestion. To accurately compare stability of the NPR1-GFP protein in different genetic backgrounds, we ensured that 3) Because Figure 2A is central to the manuscript, it would be good to have some quantification here. The loading control is not really equal, protein concentration seems to go down in the Col-0, while up in the ube4 background. Normalising the signal to the corresponding S5a would help to make the point. Also related, is S5a not degraded via the proteasome? If authors have the coomassie, it would be good if they would include it.Quantification of our data and improving loading controls are excellent suggestions. We have done the following:ube4 mutant background.a) New Figure 2—figure supplements 1C-E show the quantification (n=3) of NPR1-GFP and endogenous NPR1 protein levels in CHX chase assays. These data demonstrate that NPR1 protein is significantly more stable in the b) Rather than using the S5a protein, we have now used the more stable GAPDH protein to normalise the data. Thus, we repeated the experiment shown in new Figure 2A to include GAPDH as a loading control and we also utilised GAPDH to normalise the quantitative data on NPR1 stability shown in new Figure 2—figure supplements 1C-E.4) It will be necessary to test association between UBE4 and NPR1 in vivo on SA-treated plants to support a direct action of UBE4 on NPR1. Finally, the ability of UBE4 to elongate ubiquitin chains is not consistent with Figure 2D or 6C where the ubiquitin chains seem to have the same length (reaching the same size). To convince the reader, the authors must provide more resolutive blots for high molecular weight. The elongation or trimming activities of UBE4 and UBP6/7, respectively, should result in marked migration differences on a western blot.As an E4 ligase the UBE4 protein is not expected to participate in direct protein-protein interaction with NPR1 itself. Rather UBE4 would be expected to transiently bind to the ubiquitin chain that decorates NPR1. In accordance, we have found it difficult to repeatedly co-immunoprecipitate UBE4 and NPR1 in vivo, which is not unusual for highly transient enzyme-substrate interactions. Nonetheless, using highly sensitive mass spectrometry approaches we observed that in some but not all experiments UBE4 co-immunoprecipitated with NPR1-GFP in vivo. Encouraged by this result we then immunoprecipitated ubiquitinated NPR1-GFP from plants that were treated with SA and proteasome inhibitor. Ubiquitinated NPR1-GFP was then incubated with in vitro synthesised FLAG-UBE4 and interaction assessed by co-immunoprecipitation. To our delight new Figure 2F demonstrates that FLAG-UBE4 indeed associated with polyubiquitinated NPR1-GFP.Regarding ubiquitin chain length, it should be noted that protein ubiquitination results in a branched (rather than linear) polypeptide, the size of which by definition cannot be determined accurately by SDS-PAGE. Indeed, it is known that even unconjugated, non-linear polyubiquitin chains do not migrate at expected molecular size. Our immunoblots are already performed on low percentage acrylamide gels so further resolution cannot be obtained, nor would it be a reliable indicator of ubiquitin chain length. For this very reason we performed pull down experiments with recombinant S5a ubiquitin interacting motifs (S5aUIM) that preferentially bind chains of four or more ubiquitin molecules . This experiment shows that UBE4 promotes formation of long ubiquitin chains on NPR1 .Moreover, differences in ubiquitination patterns observed in Figures 2D and 6C are due to distinct detection methods. While Figure 2D was visualised with an α-GFP antibody to detect only polyubiquitinated NPR1-GFP protein, Figure 6C was visualised with an α-ubiquitin antibody and thus shows both conjugated and free ubiquitin chains.5) UBP6 is proposed to trim ubiquitin off NPR1 upon SA treatment, counteracting UBE4 . This conclusion would be more convincing if the authors performed the exact same experiment without SA treatment. One should expect not to see the 50kDa 6x ubiquitin band showing up if UBP6 acts only on UBE4-extended chains.This is a great suggestion. Indeed, new Figure 6—figure supplement 1Dshows that only after SA treatment UBP6 released smaller ubiquitin species, suggesting it primarily acts only UBE4 extended ubiquitin chains.6) Since UBE4 is also required for the degradation of multiple NLRs. The elevated defense gene expression and disease resistance in the ube4 mutant observed may be a combination of both NPR1 function and NLR function. Although the authors show that the elevated defense gene expression and increased disease resistance in ube4 mutant require ICS1 and NPR1, the same maybe true for NLR activated defenses. This will need to be thoroughly discussed.Good point. We have adjusted our conclusions from these data in the Results section. We now described that:Arabidopsis the E4 ligase UBE4/MUSE3 has been implicated in the degradation of NLR (nucleotide binding and leucine-rich repeat) immune receptors. […] Therefore we investigated if UBE4 is involved in downstream NPR1-dependent immune signalling by acquiring a loss-of-function T-DNA insertion mutant .”“In Our concluding remarks for this section have been altered to:“Collectively, these data suggest that in unchallenged plants UBE4 suppresses the expression of SA-mediated NPR1 target genes and prevents autoimmunity, conceivably by altering the stability of upstream NLR immune receptors as well as the downstream NPR1 coactivator.”


However, the effect of Rab26 on β2-adrenergic receptor (β2-AR) trafficking or/and Toll-like receptor 4 (TLR4) expression in human pulmonary microvascular endothelial cells (HPMECs) is still unclear. Here, we investigated the role of Rab26 in regulating the expression of β2-ARs and TLR4 in HPMECs and the effect of these receptors' imbalance on endothelial cell barrier function. The results showed that there was unbalance expression in these receptors, where β2-AR expression was remarkably reduced, and TLR4 was increased on the cell membrane after lipopolysaccharide (LPS) treatment. Furthermore, we found that Rab26 overexpression not only upregulated β2-ARs but also downregulated TLR4 expression on the cell membrane. Subsequently, the TLR4-related inflammatory response was greatly attenuated, and the hyperpermeability of HPMECs also was partially relived. Taken together, these data suggest that basal Rab26 maintains the balance between β2-ARs and TLR4 on the cell surface, and it might be a potential therapeutic target for diseases involving endothelial barrier dysfunction.Rab26 GTPase modulates the trafficking of cell surface receptors, such as G protein-coupled receptors including  Acute lung injury (ALI) carries high mortality rates worldwide due to the overwhelming pulmonary inflammation and loss of endothelial barrier function induced by cell hyperpermeability , 2. Humaα2-AR transport from the Golgi to the membrane [β-AR in HPMECs [β-AR is a prototypic G protein-coupled receptor (GPCR) and is involved in maintaining vascular function and barrier integrity [β2-AR in human dermal PMECs [β2-AR may play a role in LPS-induced cell hyperpermeability [β2-AR trafficking to regulate the permeability and inflammatory response of HPMECs remains unclear.Rab26 is a new member of the Rab GTPase family which controls cellular survival, homeostasis, and the stress response . In an emembrane , directimembrane  and indumembrane . Furthern HPMECs , 5. β-ARntegrity . Anotheral PMECs . Our preeability . Neverthβ2-AR) protect the endothelial barrier and cell structure and function against damage from LPS-induced hyperpermeability at the HPMEC surface [β2-AR and TLR4 involves Rab26; however, the mechanism by which Rab26 controls these two receptors and the molecular relationship between these proteins in HPMECs remain poorly understood.Although GPCRs  and its mutants Rab26 Q123L  and Rab26 N177I ; they were cloned into vector pDsRed-monomer-C1 and HA-pcDNA3.1- [β2-AR siRNA (5′-CUCAAGACGUUAGGCAUCATTUGAUGCCUAACGUCUUGAGTT-3′) and TLR4 siRNA (5′-GGGCUUAGAACAACUAGAATTUUCUAGUUGUUCUAAGCCCTT-3′) were synthesized by Sangon Biological Engineering . The primary HPMECs were obtained from CELLBIO . Endothelial cell medium (ECM), Opti-MEM, and endothelial cell growth supplement (ECGS) were obtained from ScienCell . The ViaFect™ transfection reagent was obtained from Promega , and the X-tremeGENE siRNA transfection reagent was purchased from Roche . Isoproterenol (ISO), lipopolysaccharide , Tween 20, 4% paraformaldehyde (PFA), and 0.1% Triton X-100 were obtained from Sigma . The M-PER Mammalian Protein Extraction Reagent and Membrane Protein Extraction kit were purchased from Thermo Fisher Scientific . Fetal bovine serum (FBS) and 0.25% trypsin were obtained from Life Technologies . Polyvinylidene difluoride (PVDF) membranes and the enhanced chemiluminescence (ECL) reagent were purchased from Millipore . 4′,6-Diamidino-2-phenylindole (DAPI) was obtained from Santa Cruz Biotechnology . Transwell inserts were purchased from Costar , and biotin-conjugated bovine serum albumin was purchased from Thermo Fisher Scientific . All of the reagents were commercially available and used as received. The primary antibodies used in this study were Rab26 [β2-AR [+-K+-ATPase [β2-AR obtained from Abcam [The following plasmids were gifted from Guangyu Wu : Rab26 wild type  and plasmid (1 μg) for 6 h, respectively. After 72 h, the cells were treated with 1 μg/mL LPS for 6 h. The cells were washed with PBS, permeabilized with 0.1% Triton X-100 for 10 min, fixed in 4% paraformaldehyde at 4°C for 30 min, and blocked with PBS containing 2% BSA for 1 h at room temperature. Subsequently, the cells were incubated with anti-Rab26 (1 : 200 dilutions) at 4°C overnight. The cells were incubated with secondary antibodies including Alexa Fluor 594 at 1 : 300 dilutions for 1 h at room temperature. These immunofluorescence images were captured by a confocal laser scanning microscope .HA-Tagged-Rab26 plasmids were prepared. The experiments were divided into five groups, namely, the control (Opti-MEM), mock (empty vector)/LPS, Rab26β2-AR or anti-human TLR4-PE for 30 min at 4°C. To measure the β2-AR levels, the cells were washed with FACS buffer again and labeled with FITC-labeled secondary antibody at 4°C for 10 min. Subsequently, the cells were washed with PBS and centrifuged, and the cell surface expression of receptors was measured via the FITC or PE fluorescence intensity on a flow cytometer .The materials and treatment are as described in μg/mL) or ISO (1 μM) or pretreated with LPS for 6 h followed by ISO for 1 h at 37°C. Finally, the cells were used in subsequent experiments.The experiments were divided into four groups, namely, blank, ISO, LPS, and ISO/LPS. The protocol was performed as previously described . The celβ2-AR siRNA, TLR4 siRNA, and β2-AR siRNA/TLR4 siRNA. The cells were seeded in 6-well plates at a density of 2 × 105 cells/well and precultured for 24 h. The siRNA (100 nM) and siRNA transfection reagent (2 μL) were diluted in 200 μL of Opti-MEM in separate tubes, following the protocol from a previous study [The experiments were divided into four groups, namely, negative control (NC), μg) was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the proteins in the gel were transferred onto PVDF membranes for 1 h. Then, the membranes were blocked with 5% nonfat milk for 60 min and were incubated overnight with antibodies against Rab26 (1 : 500 dilutions), β2-AR (1 : 500 dilutions), TLR4 (1 : 200 dilutions), GAPDH (1 : 1000 dilutions), or Na+-K+-ATPase (1 : 500 dilutions). The blots were washed with TBST (3 × 5 min) and incubated with a secondary HRP-conjugated antibody (goat anti-rabbit) at 1 : 5000 dilutions with 5% nonfat milk for 1 h at room temperature. Finally, the protein signals were detected with an ECL detection system, and the results were analyzed with the Quantity One software [+-K+-ATPase was used as the standard to verify equivalent loading, and the results were expressed as the percentage of GAPDH or Na+-K+-ATPase.Western blots were performed as previously described . Cells w3 cells each well) lined with polycarbonate filters (0.4 μm) for 24 h. After treatment, biotin-BSA (500 μg/mL) was added into the upper chamber wells for 30 min; then, 100 μL lower chamber medium was collected to detect the biotin-BSA concentration. Finally, the concentration of biotin-BSA in the media was measured by enzyme-linked immunosorbent assays (ELISA); the protocol was performed as previous study [The permeability of HPMEC monolayers was determined by diffusion of biotin-BSA, following previous description . Brieflyilters 0. μm for 2us study .α, IL-6, IL-8, and IL-17A with ELISA kits . The experiments were conducted according to the instructions of the ELISA kits.Cell supernatants were collected and used to measure TNF-t-test was used for experiments consisting of two groups, and one-way analysis of variance (ANOVA) was used for experiments consisting of more than two groups. The results were deemed statistically significant when p < 0.05.The data are presented as the mean ± SD, and each experiment was repeated at least three times. Two-tailed Student's p < 0.05)  .α (p < 0.05), but it did not significantly change IL-17A expression among these groups (p > 0.05)  improved adherens junction integrity and store cellular permeability by activating autophagy, and Rab26NI promoted endothelial barrier damage [Then, we observed the effect of Rab26 on inflammation and permeability. The results indicated that upregulation of Rab26 decreased the expression of the inflammatory mediators IL-6, IL-8, and TNF- Figures ; the posthe mock . The resr damage . These dβ2-AR and TLR4 expression at the cell surface, we sought to determine the effect of Rab26 on the membrane expression of β2-AR and TLR4. The result clearly indicated that the expression of β2-AR was obviously decreased, but that of TLR4 was markedly increased after treatment with LPS in mock, compared with the control; however, Rab26QL significantly promoted the expression of β2-AR on the plasma membrane and inhibited the TLR4 expression on the cell surface, compared to mock; on the contrary, Rab26NI reduced the β2-AR expression and enhanced TLR4 expression, compared to mock (p < 0.05)  . Consist < 0.05) . These dβ2-AR and TLR4, after treatment with ISO or LPS, the protein level of β2-AR was significantly decreased; the TLR4 protein levels were significantly decreased with ISO treatment and remarkably increased with LPS challenge. Furthermore, TLR4 was decreased after stimulating with ISO and LPS compared with LPS (p < 0.05) . These data demonstrate that ISO and LPS have opposite effects on β2-AR and TLR4 and that β2-AR and TLR4 have mutual negative regulation in LPS-induced receptor imbalance.To elucidate the relationship between  < 0.05) . In the  Figures . In contβ2-AR function to protect the endothelial barrier and inhibit the inflammatory reaction in LPS-induced injury. Based on these findings, we further explored the effect of siRNA on β2-AR and TLR4. The result revealed that the expression of cell surface β2-AR significantly increased, and TLR4 markedly decreased after silencing with TLR4 siRNA; in contrast, the expression of TLR4 significantly increased, and β2-AR markedly decreased after treatment with β2-AR siRNA, compared with the NC group (p < 0.05). Moreover, β2-AR and TLR4 expressions also were significantly decreased after treatment with β2-AR siRNA and TLR4 siRNA, compared with NC; however, β2-AR expression was slightly increased at cellular membrane silencing with β2-AR siRNA and TLR4 siRNA, compared to β2-AR siRNA-treated alone; similarly, TLR4 also was increased after silencing β2-AR and TLR4, compared to the TLR4 siRNA group (p < 0.05) (p < 0.05) , but IL-17A expression was not appreciably different among these groups (p > 0.05). Thus, these data confirmed that a receptor imbalance phenomenon exists between β2-AR and TLR4 in HPMECs.Our previous data demonstrated that an agonist (ISO) enhanced  < 0.05) . The res Figures . FurtherRab proteins are key regulators of vesicle trafficking. Rab GTPase function is regulated by exchange cycles between the GDP-bound inactive forms and GTP-bound active forms , 6, 23. β2-AR is one type of GPCRs that plays key roles in protecting the endothelial barrier. Rab1 coordinates β2-AR transport from the endoplasmic reticulum (ER) to the Golgi body in permeability regulation in PMECs [β-AR internalization to protect the endothelial barrier [β-AR remains poorly understood. Rab26 mediates the transport of α2-AR from the Golgi to the plasma membrane in H293 cells, thereby regulating a series of biological cellular processes [β2-AR on the plasma membrane, which provided preliminary evidence that Rab26 transports intracellular β2-AR to the cell membrane. In addition, the TLR4 signaling pathway, which mainly responds to LPS-induced inflammation, plays a crucial role in ALI [β-AR but negatively regulated TLR4. Thus, we strongly suspected a phenomenon of receptor imbalance because of the crosstalk between these two receptors.in PMECs ; Rab5 GTe in ALI . Similarβ2-AR and TLR4 via agonists (ISO or LPS) and siRNA-mediated depletion promoted the permeability of HPMECs and regulated the production of inflammatory cytokines. Indeed, the β-AR agonist salbutamol may stimulate epithelial repair in ARDS [β2-AR function and decreased TLR4 expression to protect the endothelial barrier and alleviate LPS-induced inflammation. Furthermore, TLR4 activation by LPS induces cell hyperpermeability and inflammation and weakens the protective effect of β2-AR on the endothelial barrier. These data were consistent with those in previous studies showing that the LPS-induced TLR4 signaling pathway triggers a systemic inflammatory response during endothelial cell apoptosis and increases cultured endothelial cell permeability and that LPS administration in various models induces profound vascular leakage in vivo [Increasing the crosstalk between  in ARDS , specifi in ARDS  and redu in ARDS . In the  in vivo –30.β2-AR activation or silencing TLR4 improved LPS-induced hyperpermeability in HPMECs. Another study also has shown that DTA0118, an inhibitor of TLR4, prevents the LPS-induced lung epithelial cell injury by inhibiting the secretion of proinflammatory cytokines, downregulating TLR4, upregulating IκBα, and preventing apoptosis [κB pathway [β2-AR siRNA increased the permeability and inflammation of HPMECs. This effect may be involving inhibition of β2-AR downstream MAPK, ERK1/2, and cAMP signal transduction pathways. Furthermore, 2-choloroethyl ethyl sulfide, an inhibitor of β2-AR, reduces the responsiveness of β2-AR, induces generation of ROS, upregulates TNF-α, enhances infiltration of inflammatory cells, and increases stress-related endogenous catecholamine in lung injury [β2-AR and TLR4 attenuated hyperpermeability and inflammatory mediator production compared to β2-AR knocking down alone in HPMECs. These data suggests that β2-AR activation or TLR4 inhibition is the strategy for protecting endothelial barrier from LPS-induced endothelium injury.Herein, we found that poptosis . Consist pathway , 16. On g injury . Interesβ2-AR and TLR4 is involved in LPS-induced endothelial barrier dysfunction. Basal Rab26 maintains the balance between the two receptors through modulating cell surface receptor expression, which may be a potential therapeutic target for preventing endothelial barrier disruption and vascular endothelial inflammation. Nevertheless, in the present research, we observed a phenomenon of receptor imbalance related to Rab26 on cell levels, but the mechanism how Rab26 regulates the balance between β2-AR and TLR4 and studies in an animal model need further investigation.This study found that the imbalance between β2-AR and TLR4. These studies suggest that Rab26 may be a potential therapeutic target for diseases involving endothelial barrier dysfunction.In conclusion, our studies provided evidence that Rab26 is involved in the regulation of LPS-induced endothelial barrier dysfunction. This function of Rab26 is likely mediated through maintaining the balance between the


Igf1 mRNA expression at pnd7. Interestingly, at pnd14, both pup groups showed high hepatic Igf1 mRNA expression but low IGF-1 serum levels, and this was inverted at pnd21. However, serum glucose levels were lower in the AR pups at pnd14 but reached the same levels as the MR pups at pnd21. Moreover, hepatomegaly and higher hepatic GH-receptor levels were observed in the AR pups at pnd21, which was in agreement with an absence of a solid food meal. During AR, the pups lost the maternal interaction-stimulated GH secretion, which correlated with lower IGF-1 serum levels during the first week of postnatal development. Later, the AR pups exhibited hepatic responses, in order to satisfy the metabolic demand for the normal weaning, with low carbohydrates levels in their meal.During the lactation period, rat pups are fed by the dam, and the patterns of mother-pup interaction change during this period. Additionally, there are changes in feeding; first, mother´s milk is the only food needed for sustenance, and later, it is combined with solid food and water. GH serum concentrations depend on both maternal-pup interaction and energy metabolism. In the artificial rearing (AR) procedure, pups are deprived of mother-pup interaction, and the feeding pattern is controlled. This rearing paradigm has been used in rats to analyze the effects of maternal deprivation on social behavior. In the present study, we analyzed the variation in GH, acylated ghrelin and IGF-1 serum concentrations throughout the lactation period in AR pups. At pnd7, the maternal rearing (MR) pups responded to a 4 h fast with a drop in GH serum concentration, which is a well-known response to maternal deprivation. GH serum levels in the AR pups did not change, suggesting an adaptation phenomenon. A dopamine inhibitory effect of GH secretion was observed in pnd7 cultured somatotropes, suggesting dopamine regulation of GH secretion at this age. Acylated ghrelin serum levels in the AR pups showed an inverted pattern compared to that in the MR pups, which was related to the artificial feeding pattern. IGF-1 serum levels were lower in the AR pups than in MR pups, which was associated with hepatic GH resistance and with low  At birth, growth hormone (GH)-expressing somatotropes are one of the most abundant types of pituitary secretory cells. Somatotropes-GH secretion is regulated by both central and peripheral components , 2. Periin vitro . After sample protein quantification by bicinchoninic acid (BCA) protein assay , aliquots of 45 μg of protein were resolved by 10% SDS-PAGE. The positive control samples were dissociated cells and membrane enrichment homogenate from adult rat liver cells, and the negative control sample was membrane enrichment homogenate of adult rat erythrocytes. The GH-receptor was determined by Western blotting using 1:1000 primary Polyclonal rabbit antibody against rat GH-receptor  and secondary antibody horseradish conjugated goat anti-rabbit antibody at 1:20,000 (Invitrogen). Actin was used as loading control, using a primary mouse monoclonal antibody  and a secondary antibody goat anti-mouse .To quantify the amount of GH receptor (GHR) in the liver of the MR and AR groups of pups, the following protocol was used: once we excised the liver of each individual, it was homogenized in RIPA buffer [150 mM NaCl, 50 mM Tris, 10% (v/v) glycerol, 0.1% (v/v) SDS, 1% (v/v), 23 mM deoxycocholic acid, 5 mM NaF, 2 mM Na4 cells seeded over cover-glasses previously covered with poly-D-Lysine and culture in standard conditions. After 24 h in culture, the cells were fixed in 3.5% paraformaldehyde in PBS for 30 min, and permeabilised with 0.1% Triton X-100 for 10 min. Non-specific binding was blocked with 1% albumin-IgG free for 30 min, continued by the incubation with the antibodies: guinea pig anti-rGH (1:4000) and rabbit anti-Prl  obtained from Dr. Parlow  and rabbit anti-hDA D2 receptor (1:1000) purchase from Millipore . Incubation with primary antibodies was all night at 4°C, followed by a 60-min incubation with secondary antibodies: TRITC-donkey anti-guinea pig and TRITC-donkey anti-rabbit , and Alexa Fluor 488-donkey anti-rabbit , for 2 h at room temperature. Cell nuclei were stained with DAPI . The stained cells were mounted with anti-fade medium . To define the hormonal cell type that expresses the DA D2 receptors, the cells were fixed and blocked a second time and processed to stain for GH or Prl. The GH-positive cell proportion in cultures, was obtained by counting the positive cells and the nucleus in an epifluorescence microscope  with a 40× objective, and 10 fields were counted. Localization of the DA D2 receptor was visualised using a confocal microscope  with an oil immersion 63x objective.Cultured cells obtained as mentioned above were stained for GH using an immuno-fluorescent assay. Aliquots of 3 × 10The liver was extracted, weighed and fixed in 10% formaldehyde in PBS (v/v). Tissue was dehydrated in ethanol at different concentrations and embedded in paraffin and cut in 5–10 μm sections. The sections were stained with haematoxylin/eosin and were photographed in a light microscope  with a digital camera , using the software Axio Vision real.4.8 (Zeiss) with an oil LCI plan-Neofluar 63x objective. After the image acquisition (double blinded), the cell area was measured using the ImageJ free software v1.47 The right tibial bone was dissected from each pups of the MR and AR groups from the three different ages, and dried overnight at 38°C in an oven. The dried tibial bones were weighted using an analytical balance. The tibial bone length was obtained with a Vernier calliper and the lineal density was obtained as the relationship between dry weight (mg) and length (cm).t-test was carried out for differences in GH serum concentrations between pups immediately sacrificed and those sacrificed after fasting . Statistical differences between groups were considered to be significant at p< 0.05.All of the values are expressed as the mean ± S.E.M from at least 3 independent experiments. Statistical analysis searched for variations between the groups MR and AR, and the different ages were conducted by a parametric two-way ANOVA followed by a Tukey post-test when comparisons were performed between ages and Sidak when comparisons were performed between MR and AR. For the experiments with cultured cells, the analysis between groups, in each ages by two-way ANOV, because bromocriptine treatment showed opposite effects over GH basal secretion at pnd7 and pnd21. Unpaired Student´s The comparison of the body weight gain in MR and AR pups along the lactation period  did not P<0.0001). In contrast, at pnd21, the serum GH concentration was higher in the group of MR pups (after 4h fasting), 5.4 fold more . Interestingly, no differences in the serum GH levels between the two groups were observed at pnd14. At least, but very important, the circulating GH levels in AR pups immediately separated from the rear system were lower than those of the pups reared by the dam and immediately sacrificed, 34.2 ± 7.4 ng/ml and 81.3 ± 21.0 ng/ml, respectively .With the purpose of knowing the GH serum concentrations in pups with a minimum of disturbance, animals at pnd7, pnd14 and pnd21 were picked up from the nest and immediately sacrificed (no-fasted pups). In cultures of pituitary cells from MR and AR pups at different ages, GH basal-secretion was observed with variations through the lactating period . CultureGh1 mRNA levels obtained from pup pituitaries were normalised with respect to male adult rat levels and, at the different ages, to the percent of GH-positive cells . No differences were observed in GHR, the monomer and the dimeric forms, in the MR pup livers , and the protein ratio  of the liver , the hepatic cell area , which regulate the cytokine signalling [Igf1mRNA, even with normal hepatic GHR expression. A decrease in the IGF-1 levels during the infantile period induces a decrease in the periosteal circumference and cortical thickness of large bones [Igf1mRNA. According to our observations, there is a negative control in the Igf1mRNA transduction and a stimulation cell hepatic proliferation. At the moment we do not know what is responsible for this phenomenon. Despite this limitation, regardless of the rearing type, at pnd21, the relation on the Ifg1mRNA/IGF-1 protein was inverted. In addition, the body weight showed a significant increment, where low transcript IGF-1 concentrations generate high protein concentrations. This finding could be explained with the acquisition of a mayor role of the GH-IGF-1 in the longitudinal growth after weaning. However, AR pups at this age showed hepatomegaly with hypertrophy cells and higher GHR concentrations. The explanation could be the lack of change in feeding in AR pups. Whereas the MR pups initiate their feeding with solid food and water at pnd14, the AR pups are fed only with milk. Rat milk has low carbohydrate content, 7.6%, while the standard rodent chow diet has 50.8% carbohydrate; then, the pups must change their metabolism to a diet rich in this macronutrient. Our observations in pups at this age showed that both groups of animals responded to fasting increasing their GH serum concentrations and they exhibited similar serum glucose levels. The measurement of liver TAGs content of AR pups at pnd21 showed higher levels than MR pups. Moreover, AR pups exhibited higher hepatic GHR concentration, and liver size than MR pups. One of the GH effects in liver is an induction of TGAs uptake [When we analysed the changes in IGF-1 serum concentration during the lactating period in the MR and AR pups, we observed differences related to the postnatal age of the animals. At pnd7, the circulating IGF-1 levels were high, and it decline with the age of the pups. However, pups of the group AR at pnd7 exhibited lower serum IGF-1, which correlates with lower GH serum levels. These results are in accordance to the well-known relation between the two hormones . When wegnalling . It has gnalling . During gnalling . In mothgnalling , 56. Witge bones , 58. In s uptake . In addis uptake . Accordis uptake , and ours uptake . Our datS1 Fig####P <0.0001. Statistical differences between MR and AR groups in GH secretion were analyzed by two way-ANOVA, followed by Sidak´s multiple comparisons posttest, * P <0.05, *** P <0.001, **** P <0.0001.Cell cultures were performed with anterior pituitaries of MR and AR pups: at pnd7 (pools of 6 pups), pnd14 (pools of 3 pups) and at pnd21 (pools of 2 pups), n = 3 cell cultures. Basal and bromocriptine-stimulated GH-secretion were normalized by the percent of GH-positive cells. Differences between basal and bromocriptine GH secretion were analysed by two-way ANOVA, followed by Sidak´s multiple comparisons posttest for differences between MR and AR groups, (TIF)Click here for additional data file.


Grandiose narcissism has been associated with poor ability to understand one’s own mental states and the mental states of others. In particular, two manifestations of Narcissistic Personality Disorder (NPD) can be explained by poor mindreading abilities: absence of symptomatic subjective distress and lack of empathy.We conducted two studies to investigate the relationships between mindreading capacity, symptomatic subjective distress and narcissistic personality. In the first study (N = 246), we compared mindreading capacities and symptomatic distress in three outpatient samples: narcissistic patients (NPD); patients with other Personality Disorders (PD); patients without PD. In the second study (N = 1357), we explored the relationships between symptomatic distress, mindreading and specific NPD criteria.In the first study, the NPD patients showed poorer mindreading than the patients without PD and comparable to patients with other PDs. Symptomatic subjective distress in the narcissistic group was less severe than in the other PDs group and comparable to the group without PDs. However, no relationship emerged between mindreading and symptomatic subjective distress. In the second study, taking the clinical sample as a whole, symptomatic distress appeared negatively linked to grandiosity traits, while mindreading scores were negatively linked to empathy.NPD showed specific mindreading impairments. However, mindreading capacity did not appear to be directly connected with subjective distress, but did appear to be connected with specific aspects of narcissistic pathology. Several clinicians have associated NPD with impaired mindreading. According to Dimaggio and colleagues [The fifth edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-5) has inherited from previous editions the description of Narcissistic Personality Disorder (NPD) as a pattern of grandiosity, pursuit of admiration and lack of empathy . Individlleagues , metacoglleagues  suggestelleagues  found adlleagues  with susDiamond and colleagues showed that patients with comorbid narcissistic and borderline personality disorder scored low on mentalizing . In a seHowever, other studies on the relationship between narcissism and alexithymia have produced contradictory results and conclusions. In one research study on patients with eating disorders, Lawson and colleagues found that the presence of narcissistic traits was linked to alexithymia , while oThere is in fact a certain amount of evidence indicating that narcissistic patients are hyper-sensitive to information that could cause them psychological distress but at the same time, on the level of conscious awareness, apparently oblivious to such information. Cascio and colleagues, for instance, found that under experimental conditions of social exclusion the narcissistic individuals showed hyperactivity of cerebral areas anatomically defined as a social pain network, but that this hyperactivation was not reflected in their subjective experience as referred in their self-reports . Analogoimpaired ability to recognize or identify with the feeling and the needs of others; excessively attuned to reactions of others, but only if perceived as relevant to self; over or underestimate of own affect on others" (p. 890) [A second aspect of narcissistic pathology which has been seen as linked to mindreading deficits is lack of empathy . Althoug(p. 890) . Empiric(p. 890) . In one (p. 890)  compared(p. 890) . Further(p. 890) . AccordiHowever, although such studies provide some evidence for the hypothesis that narcissistic persons may possess a normal capacity for understanding other minds, there are other considerations which suggest that caution may be warranted with regard to these findings. Marissen and colleagues  found thThe fact that it is difficult to demonstrate the presence of mindreading impairments in patients with NPD may depend upon a number of different confounding factors, which include the limitations of self-reports and of laboratory tasks, the complexity of mindreading abilities and the impact of the general severity of the personality pathology. Some recent data appear to validate to a certain extent the self-reports of narcissistic patients on their own pathological symptoms . In anotdecentred reading of what is going on or, to use Frith and De Vignemont’s expression, an allocentric interpretation), the second individual may remain within his own egocentric perspective and may simply conclude that Joe must be angry with him. It is possible that some studies may miss mindreading impairments because the mindreading aspects they are investigating are not those which are specifically impaired in the sample under consideration.Moreover, mindreading is a complex process which calls for the use of a variety of implicit and explicit abilities, with different domains and different levels of complexity , 46, 47.Another possible confounding factor may be the effect of general personality pathology as measured on the basis of the number of PD comorbidities or on the basis of numbers of criteria met . For exaFor the current study, in order to minimize the effect of general personality severity, we selected only subjects who met criteria for a single PD diagnosis. In order to limit bias in self-assessments of narcissistic subjects, we used both self-report measures of mindreading and semi-structured interviews. Additionally, the interview we used is constructed around a real life experience of the subject which he or she considers emotionally meaningful.Our aim was to explore the relationships between mindreading and narcissistic personality functioning. The study is divided into two parts. In the first part we investigated whether patients with grandiose NPD present with low subjective symptomatic distress and poor mindreading skills and whether there is a connection between these two elements. In order to do this, we selected from a large sample of participants a group of patients with NPD, and compared it with a heterogeneous group of patients with a variety of PD diagnoses and with a patient group without PD diagnoses. Consistently with other studies, we expect that the patients with grandiose NPD will show less subjective symptomatic distress than the other two groups. Moreover, on the basis of our previous research , we expeIn the second part, conducted on a larger sample, we focused on narcissistic criteria rather than on narcissistic diagnosis in order to explore their relationships with symptomatic distress and mindreading. Congruently with previous research studies which have found a relationship between avoidance of psychological distress and the maintenance of grandiosity, we expect to find an inverse relationship between grandiosity trait and symptomatic distress. Given that narcissists tend to undermine their own interpersonal aims through counterproductive behaviors , we alsoIn the first study we mainly aimed at investigating the differences in perceived symptoms severity and mindreading among three different groups: Narcissistic PD (NPD), patients with other personality disorders than narcissistic (Other PD), and patients with no personality disorder (No PD). Secondly, we aimed at investigating the relationship between subjective perceived distress and mindreading among NPD patients. Finally, we aimed to observe discrepancies between self-reported and observed measures, especially measures of mindreading, and to compare the incidence of such discrepancies in the three groups. In order to do this, we conceived alexithymia as an indirect measure of self-report mindreading.Participants were adult patients who attended an Italian outpatient clinic for consultation or treatment between 2011 and 2016. From a large sample of patients seeking treatment (N = 1346), we selected three groups for a total of 246 patients. The mean (SD) age of the selected sample was 34.90 (11.61) years (range 18–73 years). A total of 113 (46%) were male and 133 (54%) were female. The first group  included participants who met the DSM-IV criteria for Narcissistic Personality Disorder  and were below the cut-off values for all the other PDs. The second was Other PD group  and included patients who met the criteria for only one Personality Disorder diagnosis other than Narcissistic, while Narcissistic Personality Disorder criteria were kept equal to zero. This restriction was chosen in order to reduce the potentially confounding effect deriving from the overlap among different personality disorders. The third group was made up of No PD patients  including participants who met one or less criterion for a personality disorder, according to the Diagnostic and Statistical Manual of Mental Disorders. Patients with multiple Personality Disorder diagnoses, neurological disorders, psychotic disorders, and active substance dependence were not included in the selected sample. PD evaluation and diagnosis were administered by a clinical team of psychologists and psychiatrists from the Third Center of Cognitive Psychotherapy of Rome, Italy. All participants signed written consent forms before participating in the study. Following informed consent, all participants completed the questionnaires described below and were then interviewed in order to evaluate their metacognition levels. The protocol was approved by the Scientific and Research Ethic Committee at School of Cognitive Psychotherapy, Rome, Italy.The Structured Clinical Interview for DSM-IV axis I and II. The DSM-IV axis I and II diagnoses were obtained using the Structured Clinical Interview for DSM Axis I and II Disorders (SCID-I and SCID-II) [κ = 0.90) [SCID-II) , 51. Int = 0.90) .The Symptom Checklist-90-R. The Symptom Checklist-90-R (SCL90-R) [not at all to 4 = very much. The SCL-90-R measures nine primary symptom dimensions . For the purposes of this study, we used the GSI as a measure of perceived symptomatic distress.SCL90-R)  is a 90 The TAS-20 scale. Alexithymia was used here as a measure of self-reported mindreading and was measured by the Italian version of the Toronto Alexithymia Scale (TAS-20) [strongly disagree to 5 = strongly agree) and measures three facets: Difficulties Identifying Feelings; Difficulties Describing Feelings; and Externally Oriented Thinking, which refers to a specific tendency to focus on superficial matters and to avoid emotional thinking [(TAS-20) . The TASthinking . We usedThe Metacognition Assessment Interview (MAI) [Monitoring, Integration, Differentiation, and Decentration.ew (MAI) , 57 is aMonitoring refers to the ability to identify and label components of mental states: emotions, thoughts, motivations and desires. Individuals with poor monitoring ability find it difficult to describe their internal states and to explain the motivations underlying their behavior.Integration refers to the ability to reflect upon different mental states and to identify internal contradictions and potential patterns. Integration promotes behavioural coherence by allowing us to organize mental contents adaptively in terms of relative importance and subjective priorities. Conversely, individuals with poor integration ability find it difficult to maintain coherence between mental processes and behaviors.Differentiation refers to the ability to distinguish between internal psychological contents  and external reality. Individuals with poor differentiation ability find it difficult to establish and maintain a critical distance to their own subjective mental representations.Decentration refers to the ability to assume the perspectives of others and to make plausible hypotheses about their mental states. This is a form of perspective-taking similar to the concept of decentration as described by Piaget [y Piaget  and alsoy Piaget . Individy Piaget .In order to test our hypothesis, a series of univariate ANOVA were performed to compare the mean scores of the three groups  on alexithymia (TAS-20) and symptoms severity  and metacognition scores (MAI interview). Post-hoc tests with Least Significant Difference (LSD) procedure were computed to test the differences between the three groups, if the omnibus F test was significant. A bivariate correlation was performed in order to test the relationship between self-report measures of perceived psychological distress (GSI), alexithymia and mindreading. Statistical analyses of data were performed using SPSS 20.0.To maintain the rate of type I error close to the nominal .05 level, we performed a Multivariate analysis of variance (MANOVA) considering all the dependent variables  before approaching the univariate tests. The Multivariate effect turned out to be significant  = 3.67; p < .001).Difficulties in identifying feelings and Difficulties describing feelings, no significant difference between the No PD group and the NPD group was found, but both groups showed lower scores than patients with Other PD.The differences among the three groups were not significant for the TAS subscale Externally oriented thinking. Concerning the results on symptoms severity (GSI-SCL-90-R) [CL-90-R) , NPD shoConcerning our second hypothesis, NPD showed lower scores of mindreading compared to No PD, as hypothesized. The differences between the groups on the global score of MAI were significant. The post hoc performed showed NPD had significantly lower mean scores than the No PD group, while NPD did not differ from the Other PD group. Other PD and No PD differed significantly.Differentiation and Decentration dimensions. On the dimensions Monitoring and Integration, NPD showed lower scores than No PD, but equal to Other PD group.Concerning the subscales of MAI, NPD patients showed the lowest scores in the r = .23; p = .201), symptomatic distress and metacognition , and alexithymia and metacognition  in the NPD group. The association between symptomatic distress and alexithyimia turned out significant  in the Other PD group. In the same group, the correlation between symptomatic distress and metacognition was not significant , as well as the correlation between alexithymia and metacognition . In the No PD group, no significant correlation emerged between subjective symptomatic distress and alexithymia , symptomatic distress and metacognition , and alexithymia and metacognition . In an exploratory fashion, we compared the magnitude of the correlations (relying on r-to-z transformations) among symptomatic distress, metacognition and alexithymia across the three groups  to investigate further whether the association (or lack thereof) between mindreading and subjective distress was specific for the NPD group. Comparing the coefficients across pairs of groups failed to yield any significant difference in the magnitude of the associations across groups . On the whole, this investigation on the association between subjective distress and mindreading appears to falsify the hypothesis that the relative absence of subjective distress among NPD patients is associated with low mindreading.No significant correlation emerged between subjective symptomatic distress and alexithymia  in predicting subjective distress  and mindreading . From a theoretical point of view, we aimed to investigate the specific role of narcissistic dimensions in predicting specific features of mental functioning, independently of a personality disorder diagnosis. In other words, we are here assuming that narcissistic criteria, if present, and even if not corresponding to any categorical diagnosis, can be associated with certain specific forms of dysfunction. We predict that grandiosity will be linked to low levels of subjective distress, and that lack of empathy, independently of NPD diagnosis, will be linked to low levels of mindreading ability.In this second study, we used the whole sample from which we had selected the three patient groups participating in the previous study, without differentiating for any specific diagnosis. A total of 1357 patients took part in the study . A total of 545 patients had no PD diagnosis, while a total of 812 had at least one PD diagnosis. Patients with neurological disorders, psychotic disorders, and active substance dependence were not included in the sample.Measures and procedure corresponded to those of study 1.In order to test our explorative hypothesis, we conducted three linear regressions where the dependent variables  were regressed on the NPD criteria (entered simultaneously in each equation). Statistical analysis of data was performed using SPSS 20.0.grandiosity was negatively associated with symptoms severity and alexithymia, as hypothesized. Other negative associations resulted between the criteria sense of entitlement, lack of empathy and envy, and the global score of MAI. Positive links were observed between the criteria fantasies of unlimited success, lack of empathy, envy and perceived symptoms severity (GSI), and between lack of empathy and alexithymia.grandiosity, and the dimension integration. Significant negative associations were evidenced between sense of entitlement and integration, decentration and differentiation. Lack of empathy was negatively associated to each dimension of MAI. The criterion envy was negatively associated to monitoring and differentiation. Finally, arrogant attitude was negatively associated to the dimension differentiation.envy. One possible explanation for this is that heightened envy or heightened perceptions of being the object of envy may indicate that subjects are more aware of difficulties or may be involved in more serious interpersonal conflicts. Considering that previous research studies have highlighted that social impairment may function as a mediator between narcissism and personal distress [We explored the relationships between the various criteria for NPD and symptomatic distress, alexithymia and mindreading. As expected, symptomatic distress stands in inverse relationship to grandiosity. This data is coherent with clinical descriptions which suggest that higher levels of inflated self-esteem protect narcissists from subjective distress . Symptomdistress , envy maGrandiosity emerges not only as not linked to poor mindreading ability but as positively associated with all measures of MAI, reaching small but statistical significance with integration. This data is partially coherent with Kernberg’s model [’s model , which alack of empathy. In line with the definition of empathy in section III of the DSM-5, which also highlights the cognitive aspects of lack of empathy in NPD patients, we found that lack of empathy is linked to poor decentering. It is worth noting that the criterion also presents a significant inverse relationship with other typical self-domain aspects of metacognition such as monitoring and integration [decentering), but also an ability to read our own minds  and a consequent recognition of what we may have in common with others.A number of NPD criteria emerged in this study as linked to various types of metacognitive impairment, to a greater or lesser degree. The criterion which appears as most tightly linked to low mindreading ability is egration . This maentitlement, envy and arrogance. Taken together, these criteria describe problematic interpersonal attitudes, feelings and behaviours. In consequence, our data may suggest that poor mindreading ability is linked to the interpersonal aspects of narcissistic pathology. We are aware that the effects we found were small, albeit significant. This suggests that poor mindreading plays a role in aspects of narcissistic interpersonal functioning but that other factors should also be taken into consideration.The other criteria which are significantly linked to poor mindreading ability are The results have confirmed our hypotheses, although the limited dimension of the effect does not allow us to draw definitive conclusion. Although based on preliminary and exploratory data, this results may indicate some interesting directions for further investigations. In the first place, these data may serve to enhance understanding of the distinction between high-functioning and low-functioning narcissism. As Calgor and colleagues  have proTaking into account that social impairment functions as a mediator between narcissism and psychopathology, our data might suggest that mindreading could be one of the mediators between narcissism and social impairment. If this is the case, low levels of mindreading ability could constitute a negative prognostic factor which renders patients more vulnerable to social dysfunction and symptomatic deterioration.The role played by grandiosity remains an open question. Eminent clinicians have suggested that grandiosity appears to perform a protective role with regard both to the development of symptoms and to mental functioning , 16. ConSome limitations should be acknowledged. First of all, in the first study we chose to focus our analyses on patients diagnosed primarily with NPD, with no other comorbid diagnosis. This decision was taken in order to preclude eventual confusing effects arising from comorbid diagnoses. However, one consequence of this strategy was that the group we studied was smaller than would otherwise have been the case. Another consequence is that the level of severity in this sample was less significant than that usually encountered in clinical practice, where patients generally show a number of comorbidities.Moreover, this study observed a sample of narcissistic patients with overt grandiosity and it is unclear whether and to what extent our findings may be transferable to the vulnerable phenotype. Further research on the vulnerable phenotype of narcissistic functioning would be required to clarify this. In addition, the exploratory analyses we conducted in the second study produced no insight into the type of causal relationship which may exist between low levels of empathy and low levels of mindreading ability. It is conceivable, for instance, that both low empathy and poor mindreading could depend upon a common motivational factor, bypassing mentalization altogether .Finally, we used only the criteria of the categorical diagnosis. An investigation carried out with dimensional measurements, in line with the alternative model of personality dysfunction proposed in DSM-5, could provide a more exhaustive description of the relationships between dimensions of narcissism and mindreading.S1 FileAll the variables described in the reported studies are included in the database.(SAV)Click here for additional data file.


Similarly OS was significantly shorter when ER/PgR ratio ≥2 in primary tumors . The analysis of the 24 patients who had biomarker determinations both in primary tumors and metastasis showed a better OS when AR/ER ratio in the metastasis was ≥ 0.90 (p = 0.022). Patients with a high AR/ER ratio in primary tumor that remained high in the metastasis had better prognosis in terms of OS (p = 0.011). Despite we suggested that the ratios AR/ER and AR/PgR could be used to identify patients with different prognosis, their real value needs to be better clarified in different BC settings through prospective studies.Steroid nuclear receptors are known to be involved in the regulation of epithelial-mesenchymal transition process with important roles in invasion and metastasis initiation. Androgen receptor (AR) has been extensively studied, but its role in relation to breast cancer patient prognosis remains to be clarified. AR/ER ratio has been reported to be an unfavorable prognostic marker in early primary breast cancer, but its role in the patients with advanced disease has to be cleared. We retrospectively analyzed ER, PgR, and AR expression on a case series of 159 specimens of primary BC samples by using immunohistochemistry and 89 patients of these had luminal tumors for which AR and ER expression and survival data were available. For twenty-four patients both primary and metastatic tumors were available. A significantly shorter overall survival was observed in primary tumors with AR/PgR ratio ≥ 1.54 (HR = 2.27; 95% CI 1.30-3.97; Steroid nuclear receptors are known to have an important role in the regulation of epithelial-mesenchymal transition (EMT) process and in the tumor progression .multiverse” of factors, including hormone receptor signaling pathways are involved in metastasis establishment regulating cell plasticity and motility [Beyond specific EMT-inducing factors such as Snail and Slug, a “motility . Given tmotility .Androgen receptor (AR) may have EMT promoting effects through the suppression of E-cadherin promoter activated by dihydrotestosterone. This is in contrast with the fact that AR expression is associated with a good prognosis suggesting a tumor and EMT-suppressing effects .The possibility that a receptor for androgen is expressed in BC is fascinating given that the tumor is predominantly estrogen-dependent.Although the AR has been extensively studied, its role in this malignancy remains to be clarified as well as its relation with the other steroid nuclear receptor involved in BC biology, such as estrogen receptor (ER) or progesterone receptor (PgR). Researchers are currently trying to understand whether AR interferes with ER or PgR activity. AR is already a therapeutic target, and the availability of selective AR inhibitors  approved for the treatment of prostate cancer has opened up the possibility of their use in BC patients whose tumors express AR. However, things are not as simple as they seem as AR appears to have different functions according to the BC subtype, e.g., ER positive or triple negative BC. In ER-negative BC, studies on the prognostic effect of AR expression yielded conflicting results , 5 even  in vitro and in vivo studies. This study can be considered a seminal work because the authors introduce the concept of the AR/ER ratio for the first time. This new parameter could be useful in ER positive BC as the majority of these tumors (84-91%) are AR positive. Hence, the simple evaluation of AR expression may not be sufficient to further subdivide the ER positive subset into different prognostic groups. Cochrane et al. identified 2.0 as the best AR/ER cut-off value to classify patients according to disease-free survival (DFS). In their study of around 200 patients with primary BC, an AR/ER ratio ≥ 2.0 identified a subgroup with a four-fold higher risk of failure during adjuvant tamoxifen [A few years ago Cochrane et al. reported a worse prognosis when AR levels were high and ER levels were low, which resulted in a resistance to tamoxifen. The authors defined the relationship between AR and ER expression as the AR/ER ratio. They had been prompted to perform this study by the observation that, while BC is well differentiated and more indolent when AR is expressed, AR overexpression leads to tamoxifen resistance inumors 84-1% are ARMore recently, Rangel et al. explored the usefulness of the AR/ER ratio in a larger population of ER+/HER2- BC patients. A higher AR/ER ratio was associated with unfavorable features . Like Cochrane et al., the authors identified 2.0 as the best cut-off to distinguish between prognostic cohorts. Thus, an AR/ER ≥ 2.0 predicted poor survival in primary BC patients in terms of both disease-free interval (five-fold higher risk of relapse) and disease-specific survival (> eight-fold higher risk of relapse) .The results from the above 2 studies in which the AR/ER ratio was used to estimate prognosis in patients with primary luminal BC  raises the question as to whether this new parameter will become mandatory for prognostic classification in this BC subset. We previously showed that the AR/PgR ratio, but not the AR/ER ratio or AR expression alone, plays a prognostic role in metastatic BC patients who are treated with antiestrogen therapy . We can For the present work we retrospectively analyzed a case series of patients consecutively enrolled from 2000 to 2008 at the Breast Unit of Morgagni-Pierantoni Hospital in Forlì. Eligible criteria were ≥18 years old, histological diagnosis of invasive BC, and a follow-up of at least 5 years. All patients had to have experienced distant disease relapse/progression. At least one specimen from the primary tumor and/or one from a metastasis had to be available for patients to be considered. The total number of patients analyzed was 159; primary tumor samples were available for all patients, while 24 had also a metastatic sample. All patients had to have hormone receptor expression data available. The Ethics Committee of IRST and AVR (Area Vasta Romagna) reviewed and approved the study protocol and patients provided written informed consent according to Italian privacy law.The original hematoxylin and eosin stained specimens were reviewed by an expert pathologist to identify the most representative inclusion of tumor tissue for each patient. Neutral buffered formalin was used to fix tumor material obtained during surgery. Paraffin-embedded sections (four-micron thickness) were mounted on positive-charged slides for each sample . Biomarker determinations were performed according to European Quality Assurance guidelines. Immunostaining for conventional biomarkers and AR expression was performed using the Ventana Benchmark XT staining system  with the Optiview DAB Detection Kit . ER, PgR, , and AR  antibodies were used. For ER and PgR assessment, tissue sections were incubated for 60 minutes with antibodies diluted 1:80 and 1:40, respectively, in antibody diluents . AR antibody, prediluted by the supplier, was used. Finally, all the sections were automatically counterstained for 16 minutes with hematoxylin II . Biomarker expression was detected and semiquantitatively quantified as the percentage of immunopositive tumor cells on the total of tumor cells. Two independent observers evaluated all the samples and a discordance of >10% of positive cells was resolved by consensus after joint review using a multihead microscope. As clear guidelines for AR expression have not been available until now, we have not used a cut-off value. Given that we aimed to calculate the AR/ER and AR/PgR ratios, AR expression value was considered as a continuous variable (percentage of immunopositive tumor cells ranging 0-100%) and not dichotomous (positive/negative). As reported in our previous work, when ER or PgR were negative, the AR value was used instead of AR/ER and AR/PgR values .Overall survival (OS) was calculated as the time from the date of the start of first-line treatment for metastatic disease to the date of death from any cause or the date of the last follow-up visit. OS was estimated using the Kaplan-Meier method and compared with the log-rank test. Hazard ratios (HRs) and 95% confidence intervals (95% CI) were calculated using the Cox regression model. The optimal cut-off values were obtained from receiver operating characteristic (ROC) curve analysis at a median OS of 63 months. The concordance rate was calculated as the proportion of concordant cases with respect to the total number of patients. The two-sided exact binomial 95% confidence interval (95% CI) was estimated. All p values were based on two-sided testing and statistical analyses were performed using SAS statistical software version 9.4 .We considered 159 BC patients with available primary tumor samples and data on AR and ER expression, of whom 125 patients had luminal tumor . In order to calculate ratios we considered the 113 patients with AR and ER both ≥1%. Of these, survival data were available for 89 patients for whom we performed OS analyses. The patients' characteristics are reported in The median AR/ER ratio of the primary tumors in the luminal case series was 0.95 (range 0.06-95.00) while the median AR/PgR and ER/PgR ratios were 1.55 (range 0.06-95.00) and 1.60 (range 0.08-90.00) respectively . The optWe evaluated the impact of the AR/ER, AR/PgR, ER/PgR ratios on OS. Hazard ratios (HRs) and 95% confidence intervals (95% CI) were calculated using the Cox regression model.Median OS was longer for patients with AR/ER values < 0.95  in primary tumors . p = 0.004)  . p = 0.021)  . p = 0.022)  . In addi= 0.022) .p = 0.011) (Patients with a high AR/ER ratio in primary tumor that remained high in the metastasis had better prognosis in terms of OS (= 0.011) .Analysis of the AR/ER ratio showed a concordance between primary tumors and metastases of 45.83% (95% CI 25.90-65.76) using 0.90 as cut-off value, whereas a concordance of 41.67% (95% CI 21.95-61.39) was observed for the AR/PgR ratio when the cut-off value was set at 0.96.We report the immunohistochemical images of one patient for whom both primary tumor and metastasis specimens stained for AR, ER, and PgR were available. The AR/ER, AR/PgR, and ER/PgR ratios were calculated in each specimen to underline how they can change in different lesions from the same patient . p = 0.014). AR/ER and AR/PgR ratios did not predict OS  values for ER, PgR, and AR. Of these, 104 patients had data on survival. Median ratios of AR/ER, AR/PgR, and ER/PgR were 0.95, 1.59, and 1.35, respectively . OS was edict OS . in situ of the breast), independently of treatment  [We previously demonstrated an unfavorable prognostic role of the AR/ER ratio in different subsets of patients  –14. Simitherapy) , 10. Thetherapy)  and for Although our study was based on a small case series, it had the advantage of being able to compare primary tumor and metastatic samples from the same patients, which is fairly unusual in this setting. In conclusion, our findings indicate that a prospective study is needed to better clarify the role of AR/ER ratio in different BC settings . The relation between AR and PgR has to be better understood even if our findings suggest that a high AR/PgR ratio in luminal tumors is prognostically unfavorable and could be used as an additional risk-stratification marker. The study of steroid hormone receptors and their relation with EMT process could lead to a more precise therapeutic approach.


Better control of AR may be achievable by using an AR treatment which has a rapid onset of action and which effectively targets breakthrough symptoms. Indeed, AR patients report complete symptom relief, lack of breakthrough symptoms, rapid onset of action, safety and use on an ‘as needed’ basis as key targets for new nasal sprays. MP-AzeFlu comprises intranasal azelastine and fluticasone propionate (FP) in a novel formulation delivered in a single device. It is the first AR treatment to break the 5 min onset of action threshold and provides clinically relevant symptom relief in 15 min, much faster than that noted for FP + oral loratadine. MP-AzeFlu also significantly reduces nasal hyperresponsiveness (NHR) which may be responsible for the breakthrough symptoms frequently reported by AR patients. Mechanisms underlying MP-AzeFlu’s effect include inhibition of mast cell degranulation, stabilization of the mucosal barrier, synergistic inhibition of inflammatory cell recruitment and a unique desensitization of sensory neurons expressing the transient receptor potential A1 and V1 channels.AR remains under-diagnosed, under-estimated and under-treated. A key component in understanding the AR landscape has been the realization of a significant mismatch between how physicians instruct AR patients to manage their disease and what AR patients actually do in real life. Data from the With the most rapid onset of action and onset of clinically-relevant effect of any AR medication currently available, and proven efficacy in the treatment of NHR, MP-AzeFlu is an AR treatment which provides what patients want, and fits how patients manage their AR in real life. The aim of EUFOREA is to reduce preventable and avoidable burden of morbidity and disability due to chronic airway diseases using personalized medicine, so that affected populations reach the highest attainable standards of health and productivity at every age, with these diseases no longer representing a barrier to patients’ well-being or socioeconomic development [The European forum for research and education in allergy and airways diseases  represents the most common chronic airway disease in the world, affecting some 500 million individuals . When syhttps://www.rhinologyresearch.eu/), under the auspices of EUFOREA [The second European Rhinology Research Forum held in Brussels , and is included in the B3 action plan of the European Innovation Partnership on Active and Health Aging [Allergy Diary have enabled us for the first time to see how patients really treat their AR in real life and to recognize how this vastly differs from the RCT environment. The Allergy Diary data showed that In real-life patients (1) are poorly compliant with their AR treatment; (2) rapidly switch from one AR treatment to another if the desired response is not achieved; (3) often use multiple AR therapies; (4) fail to achieve sustained AR control on an intranasal corticosteroid (INS) monotherapy or anti-histamine + INS therapy; (5) achieve good control on more effective therapies, like MP-AzeFlu ; but (5) often switch back to less effective therapies when AR is well-controlled. Figure Allergy Diary data from one typical AR user,inputted between Aug and Dec 2015. As can be seen, treatment is neither consistent nor consecutive. This user started with a multiple treatment regimen (INS + anti-histamine), switching to MP-AzeFlu (or MP-AzeFlu + anti-histmanine) to achieve MACVIA-ARIA defined disease control (i.e. VAS score < 5/10 cm). AR control was lost upon swiching to INS monotherapy, and regained once again on MP-AzeFlu  have recommended a patient-centered, mobile-health and clinical decision support system (CDSS) approach to achieve AR control in real life, linking all stakeholders in AR management , regulators and guideline producers/implementers) . This hath Aging . Data obpatients  are poorpatients  are poorBetter AR control may be achieved by listening to patients, taking account of how they actually manage their AR and providing what they want. Based on the evidence presented here, patients want an AR therapy, which provides complete and long-lasting symptom relief (i.e. no breakthrouh symptoms) and which has a rapid onset of action.MP-AzeFlu, is a relatively new addition to the AR treatment armamentarium, comprising intranasal azelastine (AZE) and FP in a novel formulation delivered in a single device . Its onsAs can be seen from Fig. As many as 36.5% of patients currently on an AR treatment continue to experience poorly controlled AR (i.e. VAS score ≥ 5), with this number remaining essentially the same irrespective of the type of pharmacological treatment used, be it INS (38.5%), oral anti-histamine (34.9%) or multiple therapies (35.9%) . This siPatients with moderate/severe persistent AR and NHR  > 20% upon CDA provocation) were randomized 2:1 to receive either MP-AzeFlu (1 spray/nostril bd) or placebo. The effect of MP-AzeFlu on NHR was assessed by PNIF decrease in response to CDA, as well as on the severity of nasal symptoms, measured at baseline and after 7 and 28 days of treatment . Nasal s2+ increase, with repeated applications of AZE + FP resulting in desensitization of sensory neurons expressing the transient receptor potential (TRP) A1 and V1 channels [The results showed that MP-AzeFlu significantly (p < 0.0001) reduced NHR Fig. a, with achannels . Activatchannels , 51. MP-channels , 53, andDespite abundant treatment options, AR remains uncontrolled for many patients , 54, 55 AR control may be significantly improved by improving communication with patients and by prescribing a treatment which takes account of their preferences, expectations and treatment behaviour in real life. MACVIA ARIA has recognized the importance of these issues, by developing apps for AR patients and their physicians to improve communication , and by AR control may also be improved in real-life by recognizing that many AR patients also experience NHR , and by Taken together these data should change the way that AR is managed in real-life by encouraging frank and open communication with patients, as well as prescription of medication which takes patient preferences, disease phenotype and treatment behaviour into account. It’s time to take a more pragmatic approach to AR management in order to provide a personalized medicine, centred around the patient ; to reco