Mantle cell lymphoma (MCL) is an aggressive B-cell lymphoma characterized by the aberrant expression of several growth-regulating, oncogenic effectors. Exportin 1 (XPO1) mediates the nucleocytoplasmic transport of numerous molecules including oncogenic growth-regulating factors, RNAs, and ribosomal subunits. In MCL cells, the small molecule KPT-185 blocks XPO1 function and exerts anti-proliferative effects. In this study, we investigated the molecular mechanisms of this putative anti-tumor effect on MCL cells using cell growth/viability assays, immunoblotting, gene expression analysis, and absolute quantification proteomics. KPT-185 exhibited a p53-independent anti-lymphoma effect on MCL cells, by suppression of oncogenic mediators , repression of ribosomal biogenesis, and downregulation of translation/chaperone proteins  that are part of the translational/transcriptional network regulated by heat shock factor 1. These results elucidate a novel mechanism in which ribosomal biogenesis appears to be a key component through which XPO1 contributes to tumor cell survival. Thus, we propose that the blockade of XPO1 could be a promising, novel strategy for the treatment of MCL and other malignancies overexpressing XPO1. MCL is  disease . About 2 disease . Becausecyclin D1 mRNA using adaptor proteins such as eukaryotic translation initiation factor 4E (eIF4e) in human cells [Nuclear-cytoplasmic transport of numerous molecules, including tumor suppressor and growth regulatory proteins, certain RNA species, and ribosomal subunits is mediated by the karyopherin family of proteins . Exportian cells . Other ian cells . We havean cells , and MCLan cells .Small-molecule selective inhibitors of nuclear export (SINE) that discriminately block XPO1-dependent nuclear export have been developed. SINEs specifically and irreversibly bind to the Cys528 residue in the cargo-binding groove of XPO1. Significant anti-leukemia activity of SINEs with negligible toxicity towards normal hematopoietic cells has been reported . SINEs rp53, and the Jeko-1 and MINO cells harbor mutant p53 [The MCL cell lines Z138, JVM2, MINO, and Jeko-1 were used in this study . The Z13tant p53 . The celtant p53 .Cell viability was assessed by the Trypan blue dye exclusion method as described previously , and celCip1/WAF1 and p27KIP1 ; p53 ; BRCA1; Cdc2 ; PIM-1 and XPO1 (marketed as anti-CRM1) and p-HSF1Ser326 ; CDC25C, c-Myc, cyclin D1, Hsp70, 4E-BP1, phosphorylated- (p-) 4E-BP1Thr37/Thr46, p-RbSer780, S6 (S6K), p-S6 ribosomal protein (S6K)Ser235/Ser236, PUMA, HSF1, HSP70 and horseradish peroxidase–linked anti-mouse and anti-rabbit IgG .Immunoblotting was performed as described previously . The folIsobaric tags for relative and absolute quantification (iTRAQ), a chemical labeling mass spectrometry method, has been performed following the manufacturer’s protocol  , 20. ProPUMA, Hs00248075_m1; p21, Hs00355782_m1; GAPDH, Hs99999905_m1, Applied Biosystems, Foster City, CA) or Reverse transcription real time quantitative PCR (RQ-PCR)  on a 7900HT Fast Real-Time PCR System. Relative quantification between different samples was determined according to the 2–ΔΔCt. Cells were treated by KPT-185 for 18 h .Messenger RNA expression levels were quantified using TaqMan gene expression assays using TaqMan low-density array cards (TLDAs) (JVM2 cells transfected with control shRNA (shC JVM2) or p53-specific shRNA (shp53 JVM2) were left untreated or treated for 18 hr with 100 nM KPT-185, using 3 independent replicates for each shRNA and condition. Total RNA was extracted using the RNAqueous kit . After confirmation of RNA quality using a Bioanalyzer 2100 instrument (Agilent), 300 ng of total RNA was amplified and biotin-labeled through an Eberwine procedure using an Illumina TotalPrep RNA Amplification kit (Ambion) and hybridized to Illumina HT12 version 4 human whole-genome arrays. Processing of bead-level data was by methods previously described . In briet-test. Statistical significance was considered when the P-value was ≤ 0.05. Where indicated, the results are expressed as the mean ± standard deviation (SD) of triplicate samples.Statistical analyses were performed using a two-tailed Student’s p53 mutational status. Treatment with KPT-185 resulted in a dose- and time-dependent cell growth inhibition in all the MCL cells examined irrespective of their p53 mutational status. The most prominent cell growth inhibition by KPT-185 was observed in the blastoid-variant Z138 cells , and induced PUMA mRNA in all tested MCL cells irrespective of p53 status  as assessed by PCR using TLDAs. These results suggest that KPT-185 induces p53-independent effects as well as p53 signaling activation in wt-p53 MCL cells. No significant change of pro-apoptotic Bim and Bax proteins or anti-apoptotic Bcl-2 protein was observed after KPT-185 treatment. KPT-185 decreased XPO1 in all tested MCL lines  or p53-specific shRNA (shp53) to evaluate p53-independent multi-targeted activities of KPT-185. p53-shRNA reduced p53 protein levels in JVM-2 and Z-138 cells by ≥ 80% as determined by immunoblot analysis [p53 knockdown (p < 0.01), which was also observed in Z-138 cells transfected with shC or shp53 .We then utilized wt-analysis . As showp53 status were detected as described in Materials and Methods. A total of 2461 gene probes were altered by KPT-185 treatment (100 nM for 18 hours) by at least 0.5 log2 (~ 40%) in at least 3 of 6 total replicates of shC and shp53 JVM2 cells. More uniform changes, affecting both shC and shp53 JVM2 cells similarly in at least 5 of 6 replicates, were found in 337 genes  driving proliferation that are exported by KPT-185 and to identify the signaling pathways involved in this regulation, iTRAQ proteomics was employed for exhaustive protein expression analysis. Because XPO1 has multiple cargos including p53, and the clonal heterogeneity of MCL might reflect the functional differences , we utilIn Z138 and Jeko-1 cells, a total of 2252 and 2176 unique proteins were identified including 137 and 112 proteins significantly altered by KPT-185 treatment, respectively. As shown in Interestingly, all of these factors are targets of the multifaceted transcription factor heat shock factor 1 (HSF1), which has been reported to be a central transducer linking translational activity of ribosomal biogenesis and transcriptional regulation . We therAmong the 13 proteins that were consistently upregulated by KPT-185, the glucose metabolic kinases, ATP synthase and apoptosis related proteins such as histone H2, heat shock protein 60 (HSP60), and prohibitin were included. Along with down-regulation of ribosomal biogenesis, Metacore and KEGG GO analysis showed a consistent pathway alteration in Z138 and Jeko-1 cells after KPT185 treatment including down-regulation of translation initiation and up-regulation of glycolysis, gluconeogenesis and pyruvate metabolism . ConcordIt has been shown that overexpression of XPO1 overcomes p16INK4a mediated checkpoint control . Overexpc-Myc and PIM1 mRNAs use XPO1 and the adapter protein eIF4e for their transport into the cytoplasm, which facilitates their translation [KIP, a cyclin dependent kinase (CDK) inhibitor in all tested MCL cell lines except Jeko-1 which showed only minimal changes  [KIP stabilization, thus causing a block in cell-cycle progression regardless of p53 status [Although p53 has been recognized as a key player linking ribosomal biogenesis and cell-cycle repression , p53-ind (eIF4e) , 30. Int (eIF4e) . On the  (eIF4e)  and p27K3 status . iTRAQ p3 status , and is 3 status , the glo3 status , and the3 status . The p533 status , or NF-κ3 status , 45. Of KPT-185 decreased XPO1 in all tested MCL cells, which is concordant with the previous reports of MCL and lung cancer cells , 46, in Unexpectedly, pathway analysis also detected the significant upregulation of glycolysis and gluconeogenesis in KPT-185 treated MCL cells. The nuclear localization of the transcription factor carbohydrate responsive element binding protein (ChREBP) is required for glucose metabolism , 48, andThe first specific nuclear export inhibitor Leptomycin B (LMB)  has beenThe clonal heterogeneity of MCL might reflect the functional heterogeneity and complex pathogenesis of the disease . RecentlAn orally bioavailable SINE Selinexor (KPT-330) is the only NEI currently in Phase I/II human clinical trials in hematological and solid cancers , and hasS1 FileMean fold-change was determined from the average of gene expression changes of each of 3 independent experiments in JVM2 transfected with control shRNA (shC JVM2) or p53-specific shRNA (shp53 JVM2) comparing KPT-185-treated cells to untreated controls.(DOCX)Click here for additional data file.


Ras-induced senescence mediated through ASPP2 represents a barrier to tumour formation. It is initiated by ASPP2’s interaction with Ras at the plasma membrane, which stimulates the Raf/MEK/ERK signaling cascade. Ras to Raf signalling requires Ras to be organized in nanoscale signalling complexes, called nanocluster. We therefore wanted to investigate whether ASPP2 affects Ras nanoclustering. Here we show that ASPP2 increases the nanoscale clustering of all oncogenic Ras isoforms, H-ras, K-ras and N-ras. Structure-function analysis with ASPP2 truncation mutants suggests that the nanocluster scaffolding activity of ASPP2 converges on its α-helical domain. While ASPP2 increased effector recruitment and stimulated ERK and AKT phosphorylation, it did not increase colony formation of RasG12V transformed NIH/3T3 cells. By contrast, ASPP2 was able to suppress the transformation enhancing ability of the nanocluster scaffold Gal-1, by competing with the specific effect of Gal-1 on H-rasG12V- and K-rasG12V-nanoclustering, thus imposing ASPP2’s ERK and AKT signalling signature. Similarly, ASPP2 robustly induced senescence and strongly abrogated mammosphere formation irrespective of whether it was expressed alone or together with Gal-1, which by itself showed the opposite effect in Ras wt or H-ras mutant breast cancer cells. Our results suggest that Gal-1 and ASPP2 functionally compete in nanocluster for active Ras on the plasma membrane. ASPP2 dominates the biological outcome, thus switching from a Gal-1 supported growth-promoting setting to a senescence inducing and stemness suppressive program in cancer cells. Our results support Ras nanocluster as major integrators of tumour fate decision events. Cancer associated Ras isoforms K-ras4A, K-ras4B, H-ras and N-ras are membrane anchored small GTPases that relay extracellular signals. Ras signalling can influence opposing cellular events, such as cell proliferation or differentiation, as compared to senescence or apoptosis –3. The fHVR-instructed specific activities of Ras proteins are supported by a number of observations. For example, different isoforms activate the effectors to different extents –7. MoreoThe HVR furthermore directs Ras isoforms to laterally segregate in the plasma membrane into distinct, non-overlapping nanoscale signalling hubs called nanoclusters –12. The Nanocluster formation and signalling output can be regulated by nanocluster scaffolding proteins. Only a few Ras nanocluster scaffolds are known, including Galectin-3 and nucleophosmin specifically for K-ras ,18, as wRas isoforms are mutated in the 30% of human cancers and are major drivers of tumourigenesis . Cells hin vitro experiments indicated that ASPP2 can form homodimers [Of note, the Ras/MAPK pathway engages ASPP2 on several levels. ASPP2 was reported to interact via its N-terminal ubiquitin-like (Ubl) domain with GTP-H-ras , and incmodimers .Here, we show that ASPP2 promotes nanoclustering, effector recruitment and downstream signalling of constitutively active H-ras, K-ras and N-ras. This activity appears to reside in the α-helical domain of ASPP2. ASPP2 is therefore the first nanocluster scaffold of N-ras. Intriguingly, ASPP2 is able to antagonize the activity of Gal-1, which therefore abrogates Gal-1 promoted transformation of H-rasG12V-NIH/3T3 cells and mammosphere formation. These data suggest that ASPP2 competes on the nanoscale with Gal-1 to effectively switch the cell fate from proliferation to senescence.2. Transfections were performed with jetPRIME , unless stated otherwise.Human Embryonic Kidney 293-EBNA (HEK), BHK21, MCF-7, HS-578T and NIH/3T3 cells were cultured in Dulbecco´s modified Eagle´s medium . MDA-MB-231 cells were incubated in Roswell Park Memorial Instituted medium . Media were supplemented with 10% fetal bovine serum (not heat-inactivated in case of NIH/3T3 cells) and 1% L-glutamine. The cells were incubated at 37°C with 5% COPlasmids pmGFP-H-rasG12V, pmGFP-K-rasG12V, pmGFP-N-rasG12V, pcDNA3-Gal-1, pcDNA3-Gal-1-C3S,L5Q,V6D,A7S , and mRFP-RBD with the Ras binding domain (RBD) of C-Raf were described before ,15,19,32HEK cells were seeded on a 6-well plate with glass coverslips, and transfected with the donor alone (mGFP-tagged Ras constructs) in control samples, or together with the acceptor. Acceptors were mCherry-tagged Ras constructs in nanoclustering-FRET experiments , or mRFP-RBD in C-Raf-RBD-recruitment FRET experiments . The cells were further cotransfected with either 1.5 μg empty pcDNA (control) or pcDNA plasmids for expressing Gal-1, full length ASPP2, ASPP2(1–360) or ASPP2(123–1128) constructs, or the combination of Gal-1 or N-Gal-1 and either of the ASPP2 proteins . After 48 h of transfection, coverslips were fixed with 4% PFA/PBS for 15 min and then washed with PBS, and coverslips were mounted with Mowiol 4–88 (Sigma Aldrich) on microscope slides.app) was calculated using the measured lifetimes of each donor-acceptor pair (τDA) and the average lifetime of the donor only (τD) samples. The formula employed was Eapp = (1-τDA/ τD) x 100%.The mGFP fluorescence lifetime was measured using a fluorescence lifetime imaging attachment  on an inverted microscope  as previously described . Per con2 areas of the plasma membrane sheet were identified using ImageJ, and the  coordinates of the gold particles were determined as described. A minimum of 15 plasma membrane sheets were imaged and analysed for each condition. A bootstrap test constructed as previously described was then used to evaluate the statistical significance of differences between replicated point patterns [Immunoelectron microscopy spatial mapping was performed as described previously ,34. Apicpatterns .For SDS-PAGE analysis, HEK cells were harvested in a buffer containing 50 mM dithiothreitol, 2% sodium dodecyl sulfate, 10% glycerol, 0.1% bromophenol blue and 10 mM Tris-HCl, pH 6.8. Proteins were first separated using SDS polyacrylamide gels (10%) and then electroblotted on nitrocellulose membrane . Membranes were immunolabelled using primary antibodies from the following sources: antibodies to phospho-T202/Y204 ERK , ERK 1/2 (no. 9102) and AKT (no. 9272) were from Cell Signaling Technologies ; phospho-T308 AKT (no. MAB7419) was from R&D Systems ; Gal-1 (no. 500-P210) was from Peprotech ; GFP (no. 3999–100) was from Bio-Vision  and β-actin (no. A1978) from Sigma Aldrich. ASPP2 (LX50.13) primary antibody was a kind gift from Dr. Xin Lu . HorseraHEK cells were transfected with mGFP-RasG12V plasmid or/and the plasmids encoding ASPP2 proteins. After 48 hours, cells were fixed with 4% PFA/PBS for 15 min and immunostained, if required, using primary anti-ASPP2 antibody and a secondary Alexa-647 anti-mouse antibody. Cells were then mounted with Mowiol 4–88 on microscope slides. Cells were imaged using a Zeiss LSM 780 confocal microscope  with a 63x NA 1.2 water immersion objective, using a 488 nm wavelength argon laser  for mGFP (detection: 500–580 nm) and a 633 nm wavelength HeNe laser (detection: 638–758 nm). Bidirectional scanning and 8x line averaging was used on a 1024x1024 resolution with zoom adjusted according to the cell of interest. Images were analysed using ImageJ .To study the interaction between ASPP2 constructs and H-ras, HEK cells were cotransfected with plasmids encoding mGFP-H-rasG12V and full-length ASPP2, ASPP2(123–1128) or ASPP2(1–360) . After 48 h, cells were washed twice with PBS and incubated with 0.8 mM of the cross-linker dithiobis(succinimidyl-propionate) during 30 min. The reaction was stopped with incubation of 20 mM Tris in PBS pH 7.4 during 15 min. After two washes with PBS, cells were lysed in 200 μl of lysis buffer  during 30 min and collected for centrifugation at 20,000× g for 10 min at 4°C. The supernatant was mixed with pre-washed GFP beads  with dilution buffer . The mixture was incubated at 4°C for 2 h with gentle rolling and centrifuged at 2,500× g for 2 min. The supernatant was removed and the beads were washed twice with dilution buffer. The bound proteins were eluted with SDS-PAGE sample loading buffer  heated at 95°C for 10 min and loaded into 4–20% Tris–Glycine gels. After electroblotting, membranes were immunolabeled using primary-antibodies anti-ASPP2 and anti-GFP  and membranes developed as described under Western Blotting.NIH/3T3 cells stably expressing mGFP-H-rasG12V or mGFP-K-rasG12V were used . 3,000 cMCF-7, HS578T and MDA-MB-231 cells were first seeded in 6-well plates and transfected with either empty pcDNA (control) or with 1.5 μg plasmid expressing Gal-1, ASPP2 or the combination of both  using JetPrime for MCF7 cells or Lipofectamine 3000 (Thermo Fisher Scientific) for HS578T and MDA-MB-231 cells . 24 hourGal-1 and ASPP2 effect on MCF-7 cell senescence was studied by following senescence-associated (SA)-β-gal activity. Cells were transfected with plasmids encoding Gal-1, ASPP2 or the combination of both proteins. Cells were allowed to grow for 7 days, fixed and stained with the X-gal solution from the Senescence Cells Histochemical Staining Kit  according to the manufacturer’s protocol. Senescent cells were quantified under a light microscope Olympus BX60  by counting SA-β-gal positive cells.Unless otherwise stated, statistical differences were determined using an analysis of variance (ANOVA) complemented by Tukey’s honest significance difference test (Tukey’s HSD) performed in GraphPad PRISM software. Statistical significance levels are annotated as ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001.ASPP2 was reported to bind to GTP-H-ras via its Ubl domain, which has structural similarity to Ras association or Ras binding domains . This meIn order to test for any effect of ASPP2 on Ras isoform specific nanoclustering, we used our well-established nanoclustering-FRET readout  14,40,4,440,41. L(r)-r, was plotted against the cluster radius (r). L(r)-r values above the 99% confidence interval (99% C.I.) indicate statistically nanoclustering. As expected, ASPP2 significantly enhanced nanoclustering of all Ras isoforms -spatial mapping. Intact plasma membrane sheets of BHK cells expressing one of the mGFP-tagged Ras isoforms without/ with ASPP2 were attached to EM grids and immunolabeled with 4.5 nm gold nanoparticles coupled to an anti-GFP antibody. The spatial distribution of gold particles was analysed using Ripley’s K-function. In isoforms , entirelleft) as previously observed [center), but it had no effect on N-rasG12V associated signalling (right). Overall, for both ASPP2 and Gal-1, their effect on pERK levels correlated exactly with that on nanoclustering-FRET and effector-recruitment FRET.We previously established a cell-based Ras effector-recruitment FRET assay, using the mRFP-tagged Ras binding domain of C-Raf (RBD) and the mGFP-tagged RasG12V . Using tobserved . Moreovegnalling , right. In conclusion, these data suggest that ASPP2 is a novel, pan-Ras nanoclustering scaffold that enhances effector recruitment and activates downstream ERK and AKT signalling.In order to understand, which domain of ASPP2 is responsible for increasing Ras nanoclustering we examined two truncation mutants , S1B FigInterestingly, these truncations did not affect the ability of ASPP2 to act as a general nanocluster-promoting scaffold for active Ras proteins , while iGiven that Gal-1 promotes cell proliferation and transformation , while AAs before, we coexpressed in HEK cells our FRET pairs of mGFP- and mCherry-tagged H-rasG12V  or K-rasleft, ) or K-rasG12V (right) transfected HEK cells were the same as those of cells transfected with ASPP2 alone.In the case of K-rasG12V, both full-length and the truncated ASPP2 proteins were able to revert the inhibitory effect of Gal-1 on K-ras nanoclustering-FRET, thus acting dominantly to promote K-rasG12V clustering increase , S3B FigFig 4EIn the light of the different activities of Gal-1 and ASPP2 on cell growth ,27, we wASPP2 mediates oncogenic H-ras induced senescence . We therFinally, in view of the emerging role of the ASPP members in stem cell regulation , we examIn conclusion, ASPP2 dominates over the pro-tumourigenic effects of Gal-1, thus robustly inducing senescence and abrogating mammosphere formation. This dominance is particularly obvious in H-ras mutated or Ras wt breast cancer cell lines, where Gal-1 expression alone significantly promotes sphere formation.ASPP2 interacts with Ras and p53 proteins thus literally being at the crossroad of drivers and suppressors of tumour growth, respectively ,45. HereWe showed that ASPP2 increases nanoclustering of active H-ras, K-ras and N-ras thus being the first pan-Ras nanocluster scaffold. Our data with C- and N-terminal deletion mutants of ASPP2 suggest that the α-helical domain is important for this activity. Like Gal-1, a known nanocluster scaffold, ASPP2 potentiates effector recruitment to Ras and enhances Ras downstream signalling. Intriguingly, ASPP2 competes with the tumour promoting nanocluster scaffold Gal-1, which positively regulates GTP-H-ras nanocluster. Thus ASPP2 efficiently diverts Ras signalling to cellular senescence or blockage of stemness features of breast cancer cell lines. We therefore tentatively suggest that ASPP2 acts as a janus-faced gatekeeper of GTP-Ras, on the one hand promoting nanoclustering, effector recruitment, MAPK- and AKT-signalling, but on the other hand directly linking the overactivity of this pathway to apoptosis and senescence to keep its transforming and tumourigenic potential in check.Others previously reported that the Ubl domain of ASPP2 was required for Ras interaction ,39. HoweWhile both Gal-1 and ASPP2 increase nanoclustering  and effeFurthermore, we confirmed that ASPP2 can abrogate cell growth in 3D spheres even in p53-mutated cancer cells , consistOur data suggest a revised mechanism of how ASPP2 converts oncogenic Ras activity efficiently into tumour growth suppression. ASPP2 engages with any Ras isoform to enhance nanoscale signalling complexes thus promoting activation of the major Ras-signalling pathways, including the MAPK-pathway that is required for its translocation to the nucleus and engagement with p53 to execute apoptosis or senescence [S1 Fig(A) Representative Western blots (n>10) from HEK cells expressing ASPP2- or Gal-1-plasmids (indicated on top). Probing antibodies are shown to the left. (B) Representative anti-ASPP2 Western blot (n>10) from HEK cells transfected with pCMV-Sport6-ASPP2 (lane 2), pcDNA3-ASPP2(1–1128)-V5 (lane 3), pcDNA3-ASPP2(123–1128)-V5 (lane 4) and pcDNA3-ASPP2(1–360)-V5 (lane 5).(TIF)Click here for additional data file.S2 FigA) Confocal microscopic images of HEK cells transfected with mGFP-RasG12V (green) isoforms or the full-length and truncated ASPP2 (red). (B-C) Confocal fluorescence microscopy on HEK cells cotransfected with (B) mGFP-K-rasG12V or (C) mGFP-N-rasG12V (green) and full-length or truncated ASPP2 (red). (D) Nanoclustering-FRET analysis in HEK cells coexpressing mGFP- and mCherry-tagged wild-type H-ras. Cells were serum-starved for 6 hours after overexpression of Gal-1 or the ASPP2 proteins. Statistical significance of differences between control and treated samples was examined using one-way ANOVA .((TIF)Click here for additional data file.S3 FigA-B) Nanoclustering-FRET analysis in HEK cells coexpressing mGFP- and mCherry-tagged (A) H-rasG12V or (B) K-rasG12V. Cells were analysed after overexpression with the proteins as indicated . Statistical significance of differences between control and treated samples was examined using one-way ANOVA .((TIF)Click here for additional data file.


C. elegans, despite extensive work on the innate immune responses in this organism. We report an investigation of the expression, function and regulation of the six ilys (invertebrate-type lysozyme) genes of C. elegans. These genes exhibited a surprising variety of tissue-specific expression patterns and responses to starvation or bacterial infection. The most strongly expressed, ilys-3, was investigated in detail. ILYS-3 protein was expressed constitutively in the pharynx and coelomocytes, and dynamically in the intestine. Analysis of mutants showed that ILYS-3 was required for pharyngeal grinding  during normal growth and consequently it contributes to longevity, as well as being protective against bacterial pathogens. Both starvation and challenge with Gram-positive pathogens resulted in ERK-MAPK-dependent up-regulation of ilys-3 in the intestine. The intestinal induction by pathogens, but not starvation, was found to be dependent on MPK-1 activity in the pharynx rather than in the intestine, demonstrating unexpected communication between these two tissues. The coelomocyte expression appeared to contribute little to normal growth or immunity. Recombinant ILYS-3 protein was found to exhibit appropriate lytic activity against Gram-positive cell wall material.Little is known about the relative contributions and importance of antibacterial effectors in the nematode  C. elegans. The ilys genes exhibit distinct patterns of tissue-specific expression and response to pathogenic challenge and/or starvation. The most abundantly expressed, ilys-3, exhibits constitutive pharyngeal expression, which we show is essential for efficient disruption of bacteria under non-pathogenic growth conditions, and consequently it contributes to normal longevity. ilys-3 is also strongly up-regulated in intestinal cells after starvation or exposure to Gram-positive pathogens such as Microbacterium nematophilum and acts as a ‘slow-effector’ in limiting pathogenic damage from intestinal infections. We show that this induction by pathogens depends on the action of an ERK-MAPK cascade, which acts in pharyngeal rather than intestinal cells; this implies communication between pharynx and intestine. Tagged ILYS-3 protein was detected mainly in recycling endosomes of intestinal cells and in the intestinal lumen after starvation. ILYS-3 was also expressed in coelomocytes (scavenger cells) but we found that these cells make little or no contribution to defense. We examined the enzymatic properties of recombinant ILYS-3 protein, finding that it has lytic activity against M. nematophilum cell-walls.Innate immune defenses against bacterial pathogenesis depend on the activation of antibacterial factors. We examined the expression and relative importance of a gene family encoding six invertebrate-type lysozymes in the much-studied nematode  Most animal epithelia possess innate defense systems that sense pathogenic and toxic insults and transmit stranger/danger signals to activate appropriate counter measures. The efficacy with which animals can respond to pathogens determines whether organismal homeostasis can be maintained and microbial clearance achieved. In vertebrates, the transcriptional programs that control the epithelial production of antimicrobials and mucosal homeostasis both act via coordinated activation of common innate immune receptors: Toll-like receptors (TLR), Nod-like receptors (NLR) and the cytosolic helicases RIG-1 and MDA5. Some of these also function as sensors of endogenous or exogenous damage-associated molecular patterns N-acetylmiewed by . An addiC. elegans and B. thuringiensis  . The int than WT . We estiiciently . In addiC. elegans.We therefore concluded that ILYS-3 is required in the pharynx for efficient disruption of live bacteria and to reduce bacterial colonization in the intestine of E. coli OP50 is commonly used as food despite the fact that it can be mildly pathogenic and its accumulation in the intestine is responsible for much variability in the lifespan of C. elegans   that has six α-helices and two β-sheets.The hydrolytic activity by ILYS-3 is in agreement with its high sequence homology to the bivalve invertebrate lysozymes in n.org/)] . The terC. elegans ILYS-3 appears to be largely similar to the invertebrate lysozymes from bivalves , GS2643 . DA541 [gpb-2(ad541)]. LGIII: CB6148 [mpk-1(ku1)], DP38 [unc-119(ed3)]. LG IV: VC2493 [ilys-3(ok3222)], obtained from the C. elegans Gene Knockout Consortium, outcrossed three times and sequence verified prior to phenotypic characterization. LG V: DH1201 [rme-1(b1045)]. Unknown LG: CB6603 eIs102[egl-5p::GFP::LIN-45*], RT311 {unc-119(ed3); pwIs69[vha-6p::GFP::RAB-11 + Cbr-unc-119(+)]}, RT476 {unc-119(ed3); pwIs170[vha-6p::GFP::RAB-7 + Cbr-unc-119(+)]}, NP871 {unc-119(ed3) III; cdIs66[pcc1::GFP::RAB-7 + myo-2p::GFP + unc-119(+)]}. The strain DA2200 carrying the integrated adIs2200 [myo-2p::MPK-1::GFP] was a gift from Leon Avery, University of Texas Southwestern Medical Center, Dallas. NP717 strain carrying the unc-119(ed3); arls37(myo-3p::ssGFP); cdls32(pcc1::DT-A(E148D) + unc-119(+) myo-2p::GFP was a gift of H. Fares . New strains constructed specifically for this study are listed in E. coli OP50, E. coli HB101 (DE3) and E. coli::GFP (kind gift of Jonathan Ewbank), M. nematophilum CBX102 and UV336; M. luteus DMS20030; P. aeruginosa PAO1. Bacterial cells were grown at 37°C in LB and lawns prepared from exponential phase growth cultures.unc-119(ed3) mutants with the PCR products obtained from co-amplification of the promoters and the fluorescent proteins. The plasmid unc-119 (+) (pDP♯MM016) was used as a genetic marker for the transgenes  or into CB7007 [ilys-3(ok3222)] adult worms at a concentration of 20 ng/μl. For the injections into ilys-3(ok3222) the co-injection marker sur-5::GFP (pTG96) was used at 60 ng/μl. Transgenic lines eEx752, eEx753 and eEx754 contain extrachromosomal versions of pMGN47.To generate the translational fusion of GFP::ILYS-3 transgene driven by its own promoter the GFP vector pPD117.01 was used (Andy Fire). For this the 4.5 Kb ilys-3 promoter was PCR amplified with oligonucleotides containing XbaI and NotI specific sites. The resulting amplicon was subcloned into pJet1.2 to generate pMGN50. The ilys-3 promoter was then restriction digested and cloned into the XbaI NotI sites of pPD117.01 to generate the plasmid 4.5 Kb ilys-3p::GFP, pMGN52. The entire coding sequence of ILYS-3 containing its own 3'UTR was obtained from N2 genomic DNA and amplified with oligonucleotides containing NheI and ApaI specific sites. The amplicon was subcloned into pJet1.2 to give pMGN51, and then excised and directionally cloned into the NheI and ApaI sites, downstream of GFP, into plasmid pMGN52. To establish transgenic lines, 20 ng of the resulting plasmid, pMGN53 was microinjected into the unc-119 mutant along with the unc-119 co-injection marker. Transgenic line eEx779 contains extrachromosomal version of pMGN53 to give CB7210.To construct N-terminal mtl-2 to mpk-1 cDNA from plasmid pHN3e .Total number of progeny counted for individual wild-type (N2) or (TIF)Click here for additional data file.S8 Figilys-3 animals (at L1 stage) were grown on the two different diets as indicated on the x-axis, and scored after 48 hours.Plot represents developmental progression on live cells of OP50 and CBX102. Synchronized N2 and (TIF)Click here for additional data file.S9 Fig(A) ILYS-3::mCherry and BSA::Alexa 488 colocalization. (B) Vacuoles accumulating ILYS-3::mCherry in the cup-5(ar465) mutant. Green shows endocytosed GFP (myo-3p::ssGFP). (C) Deficient endocytosis does not block ILYS-3::mCherry in the coelomocytes in the rme-1(b1045) mutant. (D-F) Some vesicles ILYS-3::mCherry-labelled colocalize with the RAB-7 GFP-marked late endosomes and lysosomes. (D) Red channel. (E) Green channel. (F) Overlay of the images of the corresponding red and green channels.(TIF)Click here for additional data file.S10 Figilys-3(ok3222), NP717 (coelomocyte-depleted), CB7137  fed on M. nematophilum from day 0 after bleaching and raised at 20°C. P value vs control calculated with the Mantel-Cox log-rank test (95% CI). Results are the mean of 2 independent trials. N ranges from 220 to 150. (B) Table summarizing the details of lifespan analysis.(A) Lifespan analysis of N2, (TIF)Click here for additional data file.S11 Figilys-3p::GFP reporter fed on the control RNAi L4440. (B) mpk-1 RNAi blocks the green fluorescence of the ilys-3 reporter in the intestine but not in the pharynx (arrows).(A) Green fluorescence in transgenic worms carrying the (TIF)Click here for additional data file.S12 Figgpb-2; ilys-3p::GFP_OP50 vs ilys-3p::GFP_OP50 and gpb-2; ilys-3p::GFP_CBX102 vs ilys-3p::GFP _CBX102 differ significantly .ROI set to 40 μ diameter and 0.4 μ thickness. Graph is representative of two independent experiments. Asterisks indicate the results of Mann Whitney test of fluorescence values, 95% confidence interval. Fluorescence intensity for (TIF)Click here for additional data file.S13 Figilys-3 promoter activity and the effect of intestinal MPK-1 in int8.Fluorescence quantification of  The construct mtl-2p::MPK-1 drives MPK-1 expression in the intestine. In the mpk-1 mutants, ilys-3 expression was blocked. This phenotype was not rescued when MPK-1 is restored in the intestine. Asterisks indicate the results of a Mann–Whitney Unpaired test statistical comparisons of the fluorescence intensity for mpk-1(ku1); ilys-3p::GFP; mtl-2p::MPK-1 vs ilys-3p::GFP; mtl-2p::MPK-1 and mpk-1(ku1); ilys-3p::GFP vs ilys-3p::GFP (*** p = 0.0002), mpk-1(ku1); ilys-3p::GFP; mtl-2p::MPK-1 vs ilys-3p::GFP (** p = 0.0012), which all differ from their controls. Mean values for mpk-1 mutants with the double transgene were not significantly different (NS) from their sibling controls harbouring the ilys-3p::GFP reporter only (p = 0.7137). N ≥ 15/group.(TIF)Click here for additional data file.S14 Fig(A-B) Image of an adult showing the active LIN-45* driven by the 1.3 Kb egl-5 promoter fragment which expresses GFP in the B, K, F, U, and P12.pa rectal epithelial cells and in three posterior body wall muscles. (A) Fluorescent channel (B) Merge image of GFP and DIC. (C-D). Representative image of a transgenic animal expressing intestinal ilys-3p::GFP. (D). Overlay. (E-F). Representative image of an animal bearing the two transgenes, showing that intestinal ilys-3p::GFP expression is not enhanced by constitutively activating MAPK signalling in the rectal epithelium. (F) Merge image of GFP and DIC. All images correspond to 1-day-old adults.(TIF)Click here for additional data file.S15 Figilys-3 promoter activity and the effect of pharyngeal MPK-1 in int8 of single and double transgene reporter strains. The construct myo-2p::MPK-1 drives MPK-1 expression in the pharynx and rescued the intestinal ilys-3 expression in mpk-1 mutants. Data analyzed with Mann–Whitney Unpaired test, 95% confidence level. Fluorescence intensity for mpk-1(ku1); ilys-3p::GFP; myo-2p::MPK-1 vs ilys-3p::GFP; myo-2p::MPK-1 and mpk-1(ku1); ilys-3p::GFP; myo-2p::MPK-1 vs ilys-3p::GFP were not significantly different (ns). Mean values for mpk-1 mutants with the double transgene differ significantly from their sibling controls harbouring the ilys-3p::GFP reporter only (** p = 0.004). N = 10-15/ group.Fluorescence quantification of (TIF)Click here for additional data file.S16 Figilys-3 in mpk-1(ku1) mutants and control strains grown on OP50 or CBX102. Levels were normalized against WT (N2) on E. coli and to the endogenous control gene rla-1. Gene expression was analyzed using the comparative ΔΔCt method. Each bar represents the average relative mRNA level from three independent RNA isolations obtained from synchronization L1 in populations of worms exposed for 24 hours to M. nematophilum. Control strains were as follows: N2, ilys-3p::GFP; unc-119; unc-119 (+), mpk-1(ku1); unc-119(+) , and mtl-2p::MPK-1; unc-119; unc-119(+), myo-2p::MPK-1. Data were analyzed with two-way Anova, Holm-Sidak's multiple comparison tests, 99% confidence interval. Error bars represent SEM. Expression levels of ilys-3 were significantly higher in transgenic animals expressing the pharyngeal MPK-1 relative to N2 controls regardless of nature of the bacterial lawn (*** p < 0.0001). The mean values for mpk-1 mutants with the intestinal MPK-1 transgene were not significantly different from their sibling single mutant controls (NS: p > 0.9999). For multiple comparisons see RT-PCR quantification of relative levels of (TIF)Click here for additional data file.S17 Figilys-3p::GFP reporter in worms fasted for 24 hours. (A) ilys-3p::GFP control. (B) ilys-3p::GFP; mpk-1 mutants showed similar intestinal fluorescence intensity relative to control animals.Fluorescence images of (TIF)Click here for additional data file.S18 FigC elegans ILYS-3 inferred from the crystal structure from of the lysozyme isolated from M. lusoria (PDB No:3AB6A). This model was created using PyMOL molecular graphic software. Residues E18 D29 predicted to be involved in muramidase activity are shown in red. Residues predicted to be responsible for the isopeptidase activity are marked in green. Of these only H113 seemed to have been conserved in both lysozymes. The other residue is a substitution of the S77 to A. (B) Sequence logo showing the conservation of amino acids in i-type lysozymes of Meretrix lusoria: 3AB6, Tapes japonica: 2DQA and C. elegans ILYS-3, based on a multiple sequence alignment (CLUSTALW) WebLogo3 was used to generate the sequence logos. Signal peptides were predicted by SignalP 4.1 server and removed prior to alignment. Poorly aligning N- and C- terminus were also removed. Amino acids are colored by charge: blue: positive and red: negative. The two muramidase residues E and D are indicated by red arrows and the possible active residues responsible for isopeptidase activity are marked with blue arrowheads. In contrast to the Tapes japonica protein, endowed with both the Serine and Histidine residues that confer isopeptidase activity, the C. elegans ILYS-3 and the M. lusoria 3AB6A appeared to have replaced the active Serine (S77) with Alanine.(A) Predicted 3D homology model structure of the (TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 TableP value vs control calculated with the Mantel-Cox log-rank test (95% CI). Results are the mean of 3 independent trials.(DOCX)Click here for additional data file.S3 TableP value vs control calculated with the Mantel-Cox log-rank test (95% CI). Results are the mean of 3 independent trials.(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.S6 Table(DOCX)Click here for additional data file.S1 Video(MOV)Click here for additional data file.S2 Video(MOV)Click here for additional data file.


Dupilumab (DPL) is a fully human IL-4Rα monoclonal antibody that potently inhibits both IL-4 and IL-13 signaling, drivers of T-helper 2 (Th2) mediated inflammation. In early clinical trials, DPL has recently been evaluated in proof-of-concept studies in asthma and atopic dermatitis (AD).NEJM 2013;368:2455) and AD . In the asthma study, inhaled glucocorticoids and long-acting beta-agonist were withdrawn after week 4 [W4] of 12 weeks of qw DPL [300 mg] or placebo [PBO]. Asthma exacerbation occurrence was the primary endpoint. Fractional exhaled nitric oxide (FeNO) and blood eotaxin-3, TARC and IgE were measured as pharmacodynamic markers. Two AD studies in adults with moderate-to-severe AD evaluated DPL  or PBO qw for 4 weeks on itch (5D pruritus questionnaire), Eczema Area and Severity Index (EASI), TARC and total IgE.The clinical trial design and results in adult patients were previously reported for asthma  TARC , eotaxin-3 , total IgE  and FeNO . Improvement in FEV1 and the reduction in FeNO were significantly correlated. In AD, 300 mg DPL markedly suppressed TARC (mean -66% DPL vs. -8% PBO) at W4. IgE gradually declined . TARC levels significantly correlated with the 5D pruritus score at baseline and W4.The suppression by DPL of Th2 biomarkers in both asthma and AD concur with improvement in key clinical endpoints. Declines in TARC, as well as eotaxin-3 in asthma, suggest that DPL suppresses Th2-mediated secretion of chemotaxins that perpetuate inflammation. Declines in IgE indicate that DPL suppresses Th2-mediated polarization of Ig-producing cells. A reduction in FeNO demonstrated a reduction in airway inflammation, which correlated with improved lung function (FEV1). The correlation of pruritus with TARC suggests Th2 inflammation may, in part, mediate itch in AD.


EU Paediatric Regulation was adopted by the European Parliament and the Council in December 2006. First meeting of the Paediatric Committee took place in July 2007.1. Paediatric investigation Plans (PIPs)In 2007-2013 The European Medicines Agency (EMA) and its Paediatric Committee have agreed more than 600 Paediatric Investigation Plans (PIPs) with pharmaceutical companies, to provide data on the efficacy and safety of medicines for diseases of children.· For treatment of JIA PIP was agreed for the following substances: abatacept, adalimumab, anakinra, anti-BAFF antibody, anti-IL-6 antibody, anti-IL17 antibody, anti-IL-17A antibody, apremilast, baricitinib, canakinumab, certolizumab, denosumab, etanercept, givinostat, golimumab, olokizumab, sarilumab, tocilizumab, tofacitinib and ustekinumab.· For treatment of SLE in children PIP was agreed for anti-BAFF, belimumab and epratuzumab.· For treatment of CAPS PIP was agreed for anakinra, anti-IL-1beta antibody and canakinumab.2. Clinical trialsMore paediatric clinical trials were done :3. New authorised indications for children· 12/11/2009: canakinumab for treatment of CAPS.· 25/08/2008: adalimumab - treatment of active polyarticular juvenile idiopathic arthritis in adolescents from 13 to 17 years of age.· 20/01/2010 abatacept - treatment of moderate to severe active polyarticular juvenile idiopathic arthritis in paediatric patients 6 years of age and older.· 18/03/2011 adalimumab - treatment of active polyarticular juvenile idiopathic arthritis in the paediatric population aged from 4 to 12 years.· 01/08/2011: tocilizumab - treatment of active systemic juvenile idiopathic arthritis (sJIA) in patients 2 years of age and older.· 24/08/2011: etanercept - polyarticular juvenile idiopathic arthritis (JIA) from the age of 2 years.· 24/08/2012: etanercept - treatment of RF+ and RF- polyarthritis, extended oligoarthritis from 2 years of age, psoriatic arthritis and ERA from 12 years of age.· 17/01/2013 - adalimumab - treatment of active polyarticular juvenile idiopathic arthritis in the paediatric population aged from 2 to 4 years old.· 25/04/2013 - tocilizumab - treatment of polyarthritis  from 2 years old (EC decision pending at the time of abstract submission).4. A network of paediatric research networksEnpr-EMA works by fostering collaboration from within and outside the European Union (EU), including between members, patients associations, academia and the pharmaceutical industry.) PRINTO and JSWG of PRES are members of Enpr-EMA.5. Expert meetings at EMA:Paediatric rheumatology , Gastroenterology and rheumatology (2010).6. Development of scientific guidelines .7. PharmacovigilanceENCEPP - (PharmaChild protocol agreed).None declared.


MGA271 is a humanized IgG1 monoclonal antibody-targeting B7-H3 (CD276), a member of the B7 family. MGA271 has been Fc-engineered to enhance binding to activating FcγR (CD16A), decrease binding to inhibitory FcγR (CD32B), and potentiate ADCC. B7-H3 has limited expression in normal tissues but highly expressed in a broad range of tumors, making it an attractive target for cancer immunotherapy. We initiated a Phase I investigation of the safety, tolerability, pharmacokinetics, pharmacodynamics and antitumor activity of MGA271.This Phase I study included dose escalation (complete) and an ongoing two-component cohort expansion (NCT01391143). Dose escalation utilized single-patient, intra-patient escalation, followed by a conventional 3+3 design, using MGA271 doses of 0.01-15mg/kg. MGA271 was administered weekly on a 4-week on, 4-week off schedule during cycle 1, and 3 out of every 4 weeks in subsequent cycles. Initial expansion cohorts enrolled patients (N=15/cohort) with melanoma, prostate carcinoma and other B7-H3+ tumors. Major modifications were subsequently made to enhance the functionality of the study prior to enrolling additional expansion cohorts. MGA271 administration was changed to uninterrupted weekly dosing, corticosteroid infusion reaction prophylaxis was reduced and immune-related response criteria and management principles were implemented. The additional expansion cohorts (N=16/cohort) were initiated in patients with melanoma (post checkpoint inhibitor), squamous cell head and neck carcinoma, renal cell carcinoma, triple-negative breast carcinoma, and high-B7-H3 expressing tumors including bladder and lung carcinoma. Pharmacodynamic testing included serum cytokines and modulation of the lymphocyte phenotype and T cell repertoire in peripheral blood.MGA271 was well tolerated, with no dose-limiting toxicity and no maximum tolerated dose defined up to 15 mg/kg. Patients were frequently heavily pre-treated, with a median of 3 (0-7) prior therapies. Any grade treatment-related AEs occurred in 71% of patients, including fatigue (30%), infusion-related reaction (26%), nausea (19%), chills (17%) and vomiting (13%). Grade 3/4 drug related AEs were noted in 6% of patients. No drug-related AEs led to study drug discontinuation. MGA271 showed linear PK. As of 30 July 2015, 20 of 46 patients treated under the new study design continue on study drug. Patients experienced disease stabilization (>12 weeks) and tumor shrinkage (2-69%) across several tumor types. The study continues to enroll patients and generate more mature data.MGA271 has a favorable safety profile and initial evidence of anti-tumor activity across several tumor types. These data support continued evaluation of MGA271 monotherapy and in combination with other immune modulators, including checkpoint inhibitors.ClinicalTrials.gov Identifier NCT01391143


Drosophila model of tissue damage-induced nociceptive hypersensitivity. Tissue-specific knockdowns and genetic mutant analyses revealed that both Tachykinin and Tachykinin-like receptor (DTKR99D) are required for damage-induced thermal nociceptive sensitization. Electrophysiological recording showed that DTKR99D is required in nociceptive sensory neurons for temperature-dependent increases in firing frequency upon tissue damage. DTKR overexpression caused both behavioral and electrophysiological thermal nociceptive hypersensitivity. Hedgehog, another key regulator of nociceptive sensitization, was produced by nociceptive sensory neurons following tissue damage. Surprisingly, genetic epistasis analysis revealed that DTKR function was upstream of Hedgehog-dependent sensitization in nociceptive sensory neurons. Our results highlight a conserved role for Tachykinin signaling in regulating nociception and the power of Drosophila for genetic dissection of nociception.Pain signaling in vertebrates is modulated by neuropeptides like Substance P (SP). To determine whether such modulation is conserved and potentially uncover novel interactions between nociceptive signaling pathways we examined SP/Tachykinin signaling in a DOI:http://dx.doi.org/10.7554/eLife.10735.001 Injured animals from humans to insects become extra sensitive to sensations such as touch and heat. This hypersensitivity is thought to protect areas of injury or inflammation while they heal, but it is not clear how it comes about.Now, Im et al. have addressed this question by assessing pain in fruit flies after tissue damage. The experiments used ultraviolet radiation to essentially cause ‘localized sunburn’ to fruit fly larvae. Electrical impulses were then recorded from the larvae’s pain-detecting neurons and the larvae were analyzed for behaviors that indicate pain responses .Im et al. found that tissue injury lowers the threshold at which temperature causes pain in fruit fly larvae. Further experiments using mutant flies that lacked genes involved in two signaling pathways showed that a signaling molecule called Tachykinin and its receptor  are needed to regulate the observed threshold lowering. When the genes for either of these proteins were deleted, the larvae no longer showed the pain hypersensitivity following an injury.Further experiments then uncovered a genetic interaction between Tachykinin signaling and a second signaling pathway that also regulates pain sensitization . Im et al. found that Tachykinin acts upstream of Hedgehog in the pain-detecting neurons. Following on from these findings, the biggest outstanding questions are: how, when and where does tissue damage lead to the release of Tachykinin to sensitize neurons? Future studies could also ask whether the genetic interactions between Hedgehog and Tachykinin (or related proteins) are conserved in other animals such as humans and mice.DOI:http://dx.doi.org/10.7554/eLife.10735.002 Neuropeptides are key regulators of behavior. They can act as local neurotransmitters  or as toqα and Gsα signaling are activated leading to increases in intracellular Ca2+ and cAMP  gene encodes a prepro-Tachykinin that is processed into six mature Tachykinin peptides (DTKs)  . Two Droeceptors . All sixP levels . In Drosractions , enteroeractions , stress ractions , olfactiractions , locomotractions , aggressractions , and pheractions . WhetherDrosophila are useful for studying the genetic basis of nociception and nociceptive sensitization  and TrpA1, mediate baseline thermal nociception in larvae  channels act as direct molecular sensors of noxious thermal and mechanical stimuli across phyla . In partn larvae , as welln larvae  and thern larvae . When lan larvae . WhetherDrosophila Tachykinin and Tachykinin receptor (TkR99D or DTKR) in nociceptive sensitization. Both were required for UV-induced thermal allodynia: DTK from neurons likely within the central brain and DTKR within class IV peripheral neurons. Overexpression of DTKR in class IV neurons led to an ectopic hypersensitivity to subthreshold thermal stimuli that required specific downstream G protein signaling subunits. Electrophysiological analysis of class IV neurons revealed that when sensitized they display a DTKR-dependent increase in firing rates to allodynic temperatures. We also found that Tachykinin signaling acts upstream of smoothened in the regulation of thermal allodynia. Activation of DTKR resulted in a Dispatched-dependent production of Hh within class IV neurons. Further, this ligand was then required to relieve inhibition of Smoothened and lead to downstream engagement of Painless to mediate thermal allodynia. This study thus highlights an evolutionarily conserved modulatory function of Tachykinin signaling in regulating nociceptive sensitization, and uncovers a novel genetic interaction between Tachykinin and Hh pathways.In this study, we analyzed Leucopheae madurae tachykinin-related peptide 1 (anti-LemTRP-1) -GAL4 driver decreased DTK expression, except for a pair of large descending neuronal cell bodies in the protocerebrum  . DTK wasemTRP-1) . PreviouemTRP-1) . Indeed,emTRP-1) . Expresscerebrum  and theicerebrum  in homozfunction . TherefodTk loss of function affects nociceptive behaviors. We first tested baseline nociception in the absence of injury, where larvae were challenged with noxious thermal stimuli at 45°C or 48°C, the middle and upper end of their response range, respectively  (JF01818Tk and KK112227Tk) targeting Tachykinin reduced the allodynia response from 70% to about 20% compared to relevant GAL4 or UAS alone controls 24 hr after UV irradiation  . Two nonadiation . Consistadiation , class Illodynia . As genellodynia . By contllodynia . TherefoTkR99D or CG7887) recognizes all six DTKs (TkR86C or CG6515) binds DTK-6 and a tachykinin-related peptide, natalisin , Gβ5 (CG10763), and Gγ1 (CG8261) all significantly reduced thermal allodynia compared to GAL4 and UAS-alone controls , is required within class IV neurons for UV-induced thermal allodynia  pathway, Smoothened  that constitutively activates Smo and causes ectopic thermal allodynia (RNAiUAS-dtkr. This did not block the ectopic sensitization (smo did (RNAiUAS-engrailed), suggesting that dtkr does not function downstream of smo. In a converse experiment, we combined UAS-DTKR-GFP with a number of transgenes capable of interfering with Smo signal transduction. Inactivation of Smo signaling via expression of Patched (UAS-Ptc), or a dominant negative form of DNsmo (UAS-smo), or a dominant negative form of the transcriptional regulator DNCubitus interruptus (UAS-Ci), or an RNAi transgene targeting the downstream transcriptional target RNAiengrailed (UAS-en), all abolished the ectopic sensitization induced by overexpression of DTKR-GFP  together with an inactivating transgene of the other pathway. Reduced allodynia would indicate that the second pathway was acting downstream of the ectopically activated one  function could trap the ligand within the neurons. Disp is necessary to process and release active cholesterol-modified Hh (disp by itself (no UV) had no effect; however combining UV irradiation and expression of RNAiUAS-disp resulted in a drastic increase in intracellular Hh punctae  or enhance (overexpression) thermal allodynia. How do brain-derived Tachykinins reach DTKR expressed on the class IV neurons? The cell bodies and dendritic arbors of class IV neurons are located along the larval body wall (In heterologous cells synthetic Tachykinins (DTK1-5) can activate DTKR . Our immody wall , beneathody wall . Howeverody wall  in closeody wall , we specody wall . An alteody wall  as neuroody wall  followinody wall , suggest2+, strongly pointing to Gαq as a downstream signaling component  the combination of RNAiUAS-disp and UV irradiation caused accumulation of Hh punctae within class IV neurons. Disp is not canonically viewed as a downstream target of Smo and indeed, blocking disp did not attenuate DNUAS-Ptc-induced or UAS-TNF-induced allodynia, indicating that Disp is specifically required for Hh production between DTKR and Smo. Thus, Tachykinin signaling leads to Hh expression, Disp-mediated Hh release, or both  served as control strains for behavioral analysis and staining. dTk mutant alleles used: EY21074dTk, and Df(3R)Exel7312 (dTk deficiency line). d08303dTk and f03824dTk insertion alleles were used to generate a custom deletion mutant of dTk. Detailed information regarding the generation of Δ1CdTk can be found in Supplemental information. dtkr mutant alleles used: f02797, dtkrMB09356, dtkrs2222dtkr, and Df(3R)Exel6213 (dtkr deficiency line).All experimental crosses were performed at 25°C. Flies were raised on regular corn meal media. Δ1CdTk, a deletion allele of dTk, we followed FRT-mediated custom deletion methodology Exel7312 was used for dTk, Df(3R)Exel6213 was used for dtkr. The 3smo  (UAS-Ptc (76UAS-Ci (=DNUAS-Ci) ), v1372 (dtkr), v105485 (Gsα60A), v102887 (CG30054), v19124 (Goα47A), v105300 (Gα49B), v17054 (Gα73B), v107613 (CG17760), v52308 (CG17760), v101373 (CG3004), v100011 (Gβ13F), v104745 (Gβ76C), v108261 (Gβ5), v101733 (RSG7), v100140 (Klp54D), v102706 (Gγ30A), 8261R1 (Gγ1), v105678 (engrailed), v37249 (TrpA1), v39477 (painless), v10004 (dispatched), v42255 (CG17760) from Vienna RNAi center, and 25800 ), 31132 (concertina), 28310 (Gβ5), 34372 (Gγ1), 31510 (painless), and 44633 (dispatched) from the Bloomington stock center.The smo3  allele w neurons , A58-GALpidermis , Myosin1 for gut , nubbin-sc patch , and daupression . UAS-DTKDTKR-GFP  was usedS-SmoDN) , UAS-Ptc  were useAS-CiDN) . RNAi liPlease see the list 'Flies used in this study' for genotypes of larvae that were used in each figure in this studySequence of primers used in this studydTKdelta1C_A TACTAGGGTTAGTTCTATGGGdTKdelta1C_B TAAACTGCGACTTGAAGCGGdTKdelta1C_C CGTACAAATTGTGAAAGTGCCdTKdelta1C_D TTTCAGTTGTGGTACATCTACGdTKdelta1C_E TTGATTTAAGGTTACAGCTGTGdTKdelta1C_F ATGCTTTGACATTTGAGAGCCdTKdelta1C_G TGCCATTTTATCCCACCGTGdTKdelta1C_H GTTGTTGGTTCACATTGCGTCpain_P1 AGACGAGGAATCCAACTCGAGpain_P2 TCGTTGATGTCTACGCGATC2 for about 5 sec using spectrolinker XL-1000 UV crosslinker . During the exposure to UV, a hand-held UV spectrophotometer  was placed next to the specimen reading the given UV level, which usually ranges 11 – 14 mJ/cm2. Then mock or UV irradiated larvae were returned to regular fly food until thermal nociception assays were performed. The metal tip of a custom-built thermal probe, whose surface temperature is fine-tuned, touches the dorsal side of an early third instar larva in abdominal segments A4-A6. Temperature dose response curve assays were performed at a heat probe setting ranging from 38°C to 48°C with 2 degree increments. Baseline thermal nociception was assayed at heat probe settings of 45°C and 48°C in the absence of tissue damage. Thermal allodynia assays were performed at a heat probe setting of 38°C 24 hr following UV irradiation. Aversive withdrawal behavior was scored under a dissecting stereomicroscope. The corkscrew-like rolling behavior  was monitored and the latency recorded up to a 20 s cutoff. All thermal nociception assays were performed where the experimenter was blind with respect to genotype of the animals tested. For categorical data presentation, each larva was put into one of three groups: non-responders (>20 s), slow responders (6≤ x ≤20 s), and fast responders (≤5 s). The behavioral results were tested in triplicates or more of n = 30, and tested for statistical significance using Chi-square analysis in Graphpad Prism unless noted otherwise in the figure legends. For some experiments the data was plotted non-categorically in line graphs of the accumulated percent response on the Y-axis versus latency on the X-axis, and tested for statistical significance using Log-rank (Mantel-Cox) test in Graphpad Prism.UV-induced tissue damage and thermal nociception assays were performed as described previously , and a bppk1.9-GAL4, ppk-eGFP/+, 3D: RNAippk1.9-GAL4, ppk-eGFP/+ and UAS-dtkr/+; ppk1.9-GAL4, ppk-eGFP/+, 3F: ppk1.9-GAL4/+, 3G: UAS-DTKR-GFP/+; ppk1.9-GAL4/+. 96 hr AEL third instar larvae were dissected to make fillet preparations. Fillets were prepared in external saline solution composed of (in mM): NaCl 120, KCl 3, MgCl2 4, CaCl2 1.5, NaHCO3 10, trehalose 10, glucose 10, TES 5, sucrose 10, HEPES 10. The Osmolality was 305 mOsm kg−1 and the pH was 7.25. GFP-positive (C4da) neurons were located under a Zeiss D1 microscope with a 40X/1.0 NA water immersion objective lens. After digestion of muscles covering the C4da neurons by proteinase type XXIII , gentle negative pressure was applied to the C4da neuron to trap the soma in a recording pipette  filled with external saline solution. Recordings were performed with a 700A amplifier , and the data were acquired with Digidata 1322A (Molecular Devices) and Clampex 10.5 software (Molecular Devices). Extracellular recordings of action potentials were obtained in voltage clamp mode with a holding potential of 0 mV, a 2 kHz low-pass filter and a sampling frequency of 20 kHz. For temperature stimulation, a perfusion system delivered room temperature (RT) or pre-heated saline that flowed through the recording chamber and was removed via vacuum to maintain a constant volume. Saline was perfused at a rate of 3 mL per minute and the fillet temperature was monitored from 25–45˚C using a BAT-10 electronic thermometer coupled to an IT-21 implantable probe . For each recording, average firing frequency during a 3 min RT perfusion was subtracted from the average firing frequency over 1 degree bins to quantify the change in firing frequency for each temperature.Extracellular recording of C4da neuronal activity was performed as described before . UV treaThe primary antibodies used in this study are a guinea pig antiserum against DTK6 (a gift from David Anderson), a rabbit antiserum against the cockroach peptide LemTRP-1 (a gift from Dick Nassel), a mouse antiserum against GFP , and a rabbit antiserum against Hh (a gift from Suzanne Eaton). The secondary antibodies are a Cy3-conjugated goat antiserum against guinea pig IgG , a Cy3-conjugated goat antiserum against rabbit IgG (Jackson ImmunoResearch Laboratories), and an Alexa488-conjugated goat antiserum against mouse IgG . Third instar larval brains and larval fillet were dissected in ice-cold PBS, fixed for one hour in 4% paraformaldehyde, and blocked for one hour in 3% normal goat serum in PBS-Tx . Fixed larvae were incubated overnight at 4°C in primary antibody solutions , and following 5 times wash in PBS-Tx for 20 min then they were incubated overnight at 4°C in secondary antibodies solutions (1:500 dilution in PBS-Tx). After wash, stained samples were mounted in Vectashield. Images were obtained from an Olympus Fv1000 Confocal microscope. Identical settings for laser intensity and other image capture parameters were applied for comparison of Tachykinin staining in the control and mutant brains. Confocal stacks were then projected using ImageJ software, processed universally and equivalently in Photoshop.ppk-Gal4>UAS-mCD8-GFP-expressing larvae were dissected in Schneider’s medium to remove gut and fat body and washed three times in 1 ml of Rinalidini solution . Washed larvae were incubated in 0.5 mg/ml Collagenase I solution (Sigma) for one hour at room temperature, washed in Schneider’s medium, and then mechanically dissociated by repeated pipetting. Dissociated tissue was filtered through a 40 µm cell strainer and cells were incubated with anti-mCD8a antibody-conjugated magnetic beads  on ice for 30 min followed by PBS washes. Isolated class IV neurons were plated on Concanavalin A (Sigma) coated coverslips and immunostained with rabbit anti-Hh antibody (1:100). Images were obtained from an Olympus Fv1000 Confocal microscope. Identical settings for laser intensity and other image capture parameters were applied for comparison of Hh staining in the control and RNAiUAS-disp-expressing cells. Confocal stacks were projected using Image J, processed universally and equivalently in Photoshop and quantification was performed using the particle analysis/threshold tools in image J.For isolated class IV neuron immunostaining, the experimental procedure was modified from Eeger et al.,  and IyerPlease note the X chromosome genotype is simplified. The actual genotypes for the X chromosome could be mixed, depending on the source RNAi collection, and the sex of individual larvae as male and female progeny were pooled together in test populations.Figure panels – genotypes tested:1118;w1118;; dTkwΔ1C1118;; dTkEY21074welav-Gal4/+RNAielav-Gal4/+; UAS-dTk (v103662)/+1118w1118;; dTkEY21074w1118;; dTkΔ1Cw1118;; dTkEY21074/dTkΔ1Cw1118;; ppk1.9Gal4/+w1118; UAS-dTkRNAiw(v103662)/+; ppk1.9Gal4/+1118;; ppk1.9Gal4/UAS-dTkRNAiw (25800)elav-Gal4/+1118; UAS-dTkRNAi (v103662)/+w1118;; UAS-dTkRNAi (25800)/+wRNAielav-Gal4/+; UAS-dTk (v103662)/+RNAielav-Gal4/+;; UAS-dTk (25800)/+1118w1118;; dTkEY21074w/+1118;; dTkΔ1Cw/+1118;; dTkEY21074w1118;; dTkΔ1Cw1118;; dTkEY21074/dTkΔ1Cw1118;; Df(3R)Exel7312w/+1118;; dTkEY21074/Df(3R)Exel7312w1118;; dTkΔ1C/Df(3R)Exel7312w1118; ppk-eGFP/+welav-Gal4/+RNAielav-Gal4/+; UAS-dTk (v103662)/+1118w1118;; dTkΔ1Cw1118welav-Gal4/+1118; UAS-dTkRNAi (v103662)/+w1118;; UAS-dTkRNAi (25800)/+wRNAielav-Gal4/+; UAS-dTk (v103662)/+RNAielav-Gal4/+;; UAS-dTk (25800)/+1118w1118;; dTkEY21074w/+1118;; dTkΔ1Cw/+1118;; dTkEY21074w/Df(3R)Exel73121118;; dTkΔ1C/Df(3R)Exel7312w1118;; ppk1.9Gal4/+w1118;; ppk1.9Gal4/UAS-dtkrRNAiw(v1372)1118w1118;; dtkrf02797w1118;; dtkrMB09356w1118;; ppk1.9Gal4/+w1118;; UAS-dtkrRNAiw(v1372)/+1118;; ppk1.9Gal4/UAS-dtkrRNAiw(v1372)1118; UAS-DTKR-GFPw/+RNAi; ppk1.9Gal4/UAS-dtkr(v1372)1118w1118;; dtkrf02797/+w1118;; dtkrMB09356/+w1118;; dtkrs2222/+w1118;; Df(3R)Exel6213w/+1118;; dtkrf02797w1118;; dtkrMB09356w1118;; dtkrs2222/dtkrMB09356w1118;; dtkrf02797/Df(3R)Exel6213w1118;; dtkrMB09356/Df(3R)Exel6213w1118;; dtkrs2222/Df(3R)Exel6213w1118;; dtkrf02797/dtkrMB09356w1118;; ppk1.9Gal4, dtkrf02797/dtkrMB09356w1118; UAS-DTKR-GFP/+; dtkrf02797/dtkrMB09356w1118; UAS-DTKR-GFP/+; ppk1.9Gal4, dtkrf02797/dtkrMB09356w1118;; ppk1.9Gal4/+w1118; UAS-DTKR-GFP/+w1118; UAS-DTKR-GFP/+; ppk1.9Gal4/+w1118; UAS-DTKR-GFP/+; ppk1.9Gal4/+w1118;; ppk1.9Gal4/+w1118;; UAS-dtkrRNAiw(v1372)/+1118;; ppk1.9Gal4/UAS-dtkrRNAiw(v1372)1118; UAS-DTKR-GFPw/+RNAi; ppk1.9Gal4/UAS-dtkr(v1372)1118w1118;; dtkrf02797/dtkrMB09356w1118;; ppk1.9Gal4, dtkrf02797/dtkrMB09356w1118; UAS-DTKR-GFP/+; dtkrf02797/dtkrMB09356w1118; UAS-DTKR-GFP/+; ppk1.9Gal4, dtkrf02797/dtkrMB09356w1118;; ppk1.9Gal4, ppk-eGFP/+w1118;; ppk1.9Gal4, ppk-eGFP/UAS-dtkrRNAiw(v1372)1118;; dtkrf02797/dtkrMB09356w1118; UAS-DTKR-GFP/+; ppk1.9Gal4, dtkrf02797/dtkrMB09356w1118;; ppk1.9Gal4/+w1118; UAS-DTKR-GFP/+; ppk1.9Gal4/+w1118;; dtkrf02797/+wFigure 3 FS1 – 1118;; ppk1.9Gal4, dtkrf02797/dtkrMB09356w1118;; ppk1.9Gal4/+w1118; UAS-RNAi/+; ppk1.9Gal4/ +w (if RNAi is on the second) or1118;; ppk1.9Gal4/UAS-RNAiw (if RNAi is on the third)1118;; ppk1.9Gal4/+w1118; UAS-CG17760RNAiw(v107613)/+; ppk1.9Gal4/+1118;; ppk1.9Gal4/UAS-CG17760RNAiw(v52308)1118; UAS-Gbeta5RNAiw(v108261)/+; ppk1.9Gal4/+1118;; ppk1.9Gal4/UAS-Gbeta5RNAiw(28310)1118;; ppk1.9Gal4/UAS-Ggamma1RNAiw(8261R-1)1118;; ppk1.9Gal4/UAS-Ggamma1RNAiw(34372)1118;; ppk1.9Gal4/+w1118; UAS-YFP/+; ppk1.9Gal4/+w1118; UAS-DTKR-GFP/UAS-CG17760RNAiw(v107613); ppk1.9Gal4/+1118; UAS-DTKR-GFP/+; ppk1.9Gal4/UAS-CG17760RNAiw(v52308)1118; UAS-DTKR-GFP/UAS-Gbeta5RNAiw(v108261); ppk1.9Gal4/+1118; UAS-DTKR-GFP/+; ppk1.9Gal4/UAS-Gbeta5RNAiw(28310)1118; UAS-DTKR-GFP/+; ppk1.9Gal4/UAS-Ggamma1RNAiw(8261R-1)1118; UAS-DTKR-GFP/+; ppk1.9Gal4/UAS-Ggamma1RNAiw(34372)1118;; ppk1.9Gal4/+w1118; UAS-RNAi/+; ppk1.9Gal4/ +w (if RNAi is on the second) or1118;; ppk1.9Gal4/UAS-RNAiw (if RNAi is on the third)1118; UAS-CG17760RNAiw(v107613)/+1118;; UAS-CG17760RNAiw(v52308)/+1118; UAS-Gbeta5RNAiw(v108261)/+1118;; UAS-Gbeta5RNAiw(28310)/+1118;; UAS-Ggamma1RNAiw(8261R-1)/+1118;; UAS-Ggamma1RNAiw(34372)/+1118;; dTkΔ1Cw/+3 b1 pr1w; smo/+3 b1 pr1/+; dTkΔ1Cw; smo/+1118;; ppk1.9Gal4/+w1118; UAS-Ptc1130x/+; ppk1.9Gal4/+w1118; UAS-Ptc1130x/UAS-YFP; ppk1.9Gal4/+w1118; UAS-Ptc1130x/+; ppk1.9Gal4/UAS-dtkrRNAiw(v1372)1118; UAS-Ptc1130x/UAS-enRNAiw(v105678); ppk1.9Gal4/+1118;; ppk1.9Gal4/+w1118; UAS-DTKR-GFP/+w1118; UAS-DTKR-GFP/+; ppk1.9Gal4/+w1118; UAS-DTKR-GFP/UAS-YFP; ppk1.9Gal4/+w1118; UAS-DTKR-GFP/+; ppk1.9Gal4/UAS-Ptcw1118; UAS-DTKR-GFP/UAS-smo.5A; ppk1.9Gal4/+w1118; UAS-DTKR-GFP/+; ppk1.9Gal4/UAS-Ci76w1118; UAS- DTKR-GFP/UAS-enRNAiw(v105678); ppk1.9Gal4/+1118; UAS-DTKR-GFP/+; ppk1.9Gal4/+w1118; UAS-DTKR-GFP/UAS-YFP; ppk1.9Gal4/+w1118; UAS-DTKR-GFP/+; ppk1.9Gal4/UAS-painRNAiw(v39477)1118; UAS-DTKR-GFP/+; ppk1.9Gal4/UAS-painRNAiw(31510)1118; UAS-DTKR-GFP, pain70/pain70; ppk1.9Gal4/+w1118; UAS-DTKR-GFP, pain70/+; ppk1.9Gal4, /+w1118;; dTkΔ1Cw/+3 b1 pr1w; smo/+3 b1 pr1/+; dTkΔ1Cw; smo/+1118; UAS-DTKR-GFP/+; ppk1.9Gal4/+w1118; UAS-DTKR-GFP/+; ppk1.9Gal4/UAS-Ptcw1118; UAS-DTKR-GFP/UAS-smo.5A; ppk1.9Gal4/+w1118; UAS-DTKR-GFP/+; ppk1.9Gal4/UAS-Ci76w1118; UAS- DTKR-GFP/UAS-enRNAiw(v105678); ppk1.9Gal4/+1118; egrRegg1c/+; ppk1.9Gal4/+w1118; egrRegg1c/+; ppk1.9Gal4/ UAS-dtkrRNAiw(v1372)1118; UAS-DTKR-GFP/+; ppk1.9Gal4/+w1118; UAS-DTKR-GFP/UAS-wgnRNAi; ppk1.9Gal4/+w1118w1118; pain70w1118w1118; pain70w1118;; ppk1.9Gal4/+w1118;; UAS-painRNAiw(v39477)/+1118;; UAS-painRNAiw(31510)/+1118;; ppk1.9Gal4/UAS-painRNAiw(v39477)1118;; ppk1.9Gal4/UAS-painRNAiw(31510)1118; UAS-Hh/+w1118;; ppk1.9Gal4/+w1118; UAS-Hh/+; ppk1.9Gal4/+w1118;; A58-Gal4/+w1118; UAS-Hh/+; A58-Gal4/+w1118; Myosin1A-Gal4/+w1118; Myosin1A-Gal4/UAS-Hhw1118;; ppk1.9Gal4, UAS-mCD8-GFP/+w1118; UAS-dispRNAiw(v10004)/+; ppk1.9Gal4, UAS-mCD8-GFP/+1118;; ppk1.9Gal4/+w1118; UAS-dispRNAiw(v10004)/+1118;; UAS-dispRNAiw(44633)/+1118; UAS-dispRNAiw(v10004)/+; ppk1.9Gal4/+1118;; ppk1.9Gal4/UAS-dispRNAiw(44633)1118; UAS-Hh/+; ppk1.9Gal4/+w1118; UAS-Hh/UAS-YFP; ppk1.9Gal4/+w1118; UAS-Hh/UAS-dispRNAiw(v10004); ppk1.9Gal4/+1118; UAS-Ptc1130x/+; ppk1.9Gal4/+w1118; UAS-Ptc1130x/UAS-YFP; ppk1.9Gal4/+w1118; UAS-Ptc1130x/UAS-dispRNAiw(v10004); ppk1.9Gal4/+1118; UAS-DTKR-GFP/+; ppk1.9Gal4/+w1118; UAS-DTKR-GFP/UAS-YFP; ppk1.9Gal4/+w1118; UAS-DTKR-GFP/UAS-dispRNAiw(v10004); ppk1.9Gal4/+1118; nubbin-Gal4/+w1118; nubbin-Gal4/+; UAS-Ci76/+w1118; nubbin-Gal4/+; UAS-hhRNAi/+w (v1402)1118; nubbin-Gal4/+; UAS-hhRNAi/+w (v1403)1118; nubbin-Gal4/+; UAS-hhRNAi/+w (31042)1118; nubbin-Gal4/+; UAS-hhRNAi/+w (31475)1118; nubbin-Gal4/+; UAS-hhRNAi/+w (25794)1118; nubbin-Gal4/+; UAS-hhRNAi/+w (4637R-2)1118;; da-Gal4/UAS-hhRNAiw(4637R-2)1118;; ppk1.9Gal4/+w1118;; ppk1.9Gal4/UAS-hhRNAi(1)w(4637R-2)1118;; ppk1.9Gal4/UAS-hhRNAi(2)w(v1403)1118;; ppk1.9Gal4/UAS-hhRNAi(1+2)w(4637R-2 + v1403)1118;; ppk1.9Gal4, UAS-mCD8-GFP/+w1118; UAS-dispRNAiw(v10004)/+; ppk1.9Gal4, UAS-mCD8-GFP/+1118; egrRegg1c/+; ppk1.9Gal4/+w1118; egrRegg1c/UAS-dispRNAiw(v10004); ppk1.9Gal4/+ review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.eLife posts the editorial decision letter and author response on a selection of the published articles . An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent , Mani Ramaswami (Reviewing editor), and two reviewers.Thank you for submitting your work entitled "Tachykinin acts upstream of autocrine Hedgehog signaling during nociceptive sensitization in The reviewers have discussed the reviews with one another and the Reviewing editor has drafted this decision to help you prepare a revised submission.Drosophila. The manuscript by Im et al. investigates the molecular pathways leading from UV damage to allodynia in the Drosophila larvae. Previous work from this group has implicated the gene hedgehog as being necessary for the induction of allodynia. The current manuscript greatly extends this model. Based on their observations, the authors concluded that upon UV treatment, dTk is released from brain neurons, activates Hedgehog (Hh) signaling in class IV neurons through dTk receptor (DTKR), and leads to Painless-dependent nociceptive sensitization. DTKR activates G-coupled protein receptors and ultimately leads to the production of hedgehog and the dis-inhibition of smo. Smo regulates the gene, pain, and results in increased class IV nociceptive neuron activation and behavioral allodynia. The evidence for this proposed model comes from behavioral assays using RNAi transgenes, genetic mutants, and electrophysiology. The experiments are logical, clearly explained, and proper controls are generally used. The figures are well displayed and we particularly appreciated the easy-to-follow schematic of their working model in This is a very nice paper on the mechanism of pain sensitization in Overall, this is a carefully designed and conducted study . The results are clear and the findings are of considerable interest. I do not have serious concerns regarding methodology, experimental results and their interpretation. For the benefit of readers, however, some points need clarification and further justification. These are described below.Concerns that must be addressed:1) The current methodology has the experimenter press a probe on the larvae to heat the animal. How can the amount of force applied by the probe be controlled prior to or during heat application? Would this effect the latency to rolling in allodynia? This methodology appears to be fairly common in the field of pain research, but it can likely be improved moving forward. Finally, if the behavioral and electrophysiological experiments were conducted in a blinded manner, the authors need to explicitly state this in the Materials and methods. If the experiments were not blinded, then is there reason to worry that this combined with the intimate interaction between researcher and larvae and the categorical nature of the statistics could lead to large unintentional biases in data?RNAiUAS-disp does not block UAS-TNF-induced ectopic sensitization. Based on this observation, one would suspect that suppression of the DTK/DTKR/Hh pathway  alone does not block UV-induced allodynia, because the TNF-induced pathway is activated after UV irradiation. However, this study demonstrates that suppression of the DTK/DTKR/Hh pathway (e.g. loss-of-function of dTk or dtkr) blocks UV-induced allodynia even in the intact TNF-induced pathway. The authors should provide a comprehensive picture, by further discussing and justifying the relationship between two potentially parallel pathways (i.e. DTK/DTKR/Hh and TNF pathways) that are involved in UV-induced nociceptive sensitization.2) As shown in 3) A broad concern that the authors should think about and consider regards the quantification of the behavioral data, even if some of these analyses are typical in the field. It appears possible that categorizing the latency data into arbitrary groups of slow, fast, or non-responders could create large statistical differences depending on the latency cut-offs that might not exist if the raw latencies were examined. Ideally, one would simply report the latencies of each animal and perform their statistics to this data. . Even better would be an assay that requires far less interaction between the experimenter and the larvae to prevent potential sources of accidental bias. Concerns that must be addressed:1) The current methodology has the experimenter press a probe on the larvae to heat the animal. How can the amount of force applied by the probe be controlled prior to or during heat application? Would this effect the latency to rolling in allodynia? This methodology appears to be fairly common in the field of pain research, but it can likely be improved moving forward. Finally, if the behavioral and electrophysiological experiments were conducted in a blinded manner, the authors need to explicitly state this in the Methods. If the experiments were not blinded, then is there reason to worry that this combined with the intimate interaction between researcher and larvae and the categorical nature of the statistics could lead to large unintentional biases in data?The reviewers are correct on two fronts: that assays involving probes or plates that contact the test animal are currently standard in the field  and that they are certainly improvable moving forward. That said, our group has already made a number of improvements to the original heat probe assay published by Tracey et al. . The major one is the introduction of feedback control to the probe so that the setpoint temperature remains essentially constant throughout application. A minor one relevant here is the use of machine-rounded tips to minimize any sharpness that might provoke mechanical responses. One can imagine potentially more optimal ways of delivering heat (perhaps a focused laser?)  but these would need to be built in such a way (for precise sensitization experiments) that one could control the larval surface temperature predictably (and hold it constant) and so that the experimenter could track the laser on a moving larva. Such a device would eliminate the variable of touch/pressure that currently exists.Can the amount of force applied be controlled? While we do not measure the applied force directly in our assay we can provide the following reassurances. First, the applied force is not sufficient to provoke any noxious mechanical aversive response – which is the same rolling that is observed with heat . Probes set to 38 ºC and below (sub-noxious temperatures) do not provoke aversive rolling. Second, in our lab multiple independent probe users produce temperature versus behavior dose-response curves that are  remarkably similar – this data is now provided in A second question posed is whether variations in the applied force might affect the latency of the allodynia response? This is conceivable but somewhat unlikely. Because in our assay the applied force is sub-threshold and is not sufficient to provoke aversive behavior we doubt that it is making a major contribution to the observed latency responses. Indeed, the fact that the temperature versus aversive behavior dose-response data tracks remarkably tightly with temperature suggests that any variations in force of probe application can have a minimal effect on the behavioral output, at most.Finally, the reviewers ask us to address blinding. All behavioral experiments were performed where the investigator was blind with respect to the genotype of the larvae being tested and this is now noted in the methods section. Because of this methodology unintentional expectational bias would be difficult to arise. We address the categorical statistics further below.2) As shown in  UAS-dispRNAidoes not block UAS-TNF-induced ectopic sensitization. Based on this observation, one would suspect that suppression of the DTK/DTKR/Hh pathway  alone does not block UV-induced allodynia, because the TNF-induced pathway is activated after UV irradiation. However, this study demonstrates that suppression of the DTK/DTKR/Hh pathway (e.g. loss-of-function of dTk or dtkr) blocks UV-induced allodynia even in the intact TNF-induced pathway. The authors should provide a comprehensive picture, by further discussing and justifying the relationship between two potentially parallel pathways (i.e. DTK/DTKR/Hh and TNF pathways) that are involved in UV-induced nociceptive sensitization.RNAiUAS-wengen) is not sufficient to block DTKR-induced genetic thermal allodynia and knocking down RNAidtkr (UAS-dtkr) is not sufficient to block TNF signaling-induced genetic thermal allodynia. We have added in the Discussion a brief consideration of how these various epistasis results might come about.The data provided in 3) A broad concern that the authors should think about and consider regards the quantification of the behavioral data, even if some of these analyses are typical in the field. It appears possible that categorizing the latency data into arbitrary groups of slow, fast, or non-responders could create large statistical differences depending on the latency cut-offs that might not exist if the raw latencies were examined. Ideally, one would simply report the latencies of each animal and perform their statistics to this data. . Even better would be an assay that requires far less interaction between the experimenter and the larvae to prevent potential sources of accidental bias.Drosophila . At the high end we felt that the standard 10 s cut off discarded a fairly large number of responders, especially in the lower noxious temperature ranges. The boundaries of the fast/slow categories were admittedly chosen to generate the most attractive dose-response curve with control larvae. All boundary options work  – it just determines whether the dose-response curve is skewed towards the slow/non-noxious side or the fast/noxious side. Five seconds is aesthetically well-centered. The reviewers are correct that there are statistical ways to treat the non-responder class and we are thankful for prompting us to more thoroughly investigate these. Here, we present a way of doing this that converts the raw latency data into a curve that is similar conceptually to an inverted “survival” or “lifespan” curve. This has the virtue of capturing the precise latency information for every larva tested. At a number of points throughout the manuscript we have converted some of the categorical data to this form  are somewhat arbitrary. But not entirely. These were chosen when we were originally preparing our first manuscript describing sensitization in his form . In onlyt screen  analyzedReferencesWilson, S. G. & Mogil, J. S. Measuring pain in the (knockout) mouse: big challenges in a small mammal. Behav. Brain Res. 125, 65-73 (2001).Drosophila larvae. J Vis Exp, doi:10.3791/3837 (2012).Chattopadhyay, A., Gilstrap, A. V. & Galko, M. J. Local and global methods of assessing thermal nociception in


Antigen cross-presentation from tumor cells by dendritic cells (DC) is a likely dominant mechanism to achieve CD8+ T cell stimulation. We established a cross-presentation model in prostate cancer in which DC present a naturally expressed oncofoetal tumor antigen (5T4) from irradiated DU145 tumor cells to 5T4-specific T cells. Ionising radiation (12 Gy) caused G2/M cell cycle arrest and cell death, increased cellular 5T4 levels, induced passive release of high-mobility protein group-B1 (HMGB1) and upregulated surface calreticulin and Hsp70 expression in DU145 cells. Co-culture of DC with irradiated tumor cells lead to efficient phagocytosis of tumor cells and upregulation of CD86 and HLA-DR on DC. CD8+ 5T4-specific T cells, stimulated with these DC, proliferated and produced IFNγ. Inhibition of HMGB1 function decreased T cell stimulation but not DC activation, while TRIF/MyD88 inhibition only had a marginal effect on T cell stimulation. Unlike previous reports, we found no functional evidence that DC with Asp299Gly toll-like receptor-4 (TLR4) single nucleotide polymorphism had impaired ability to cross-present tumor antigen. However, we observed a highly significant and robust prevention of antigen cross-presentation when tumor cells were pre-treated with the novel Hsp70 inhibitor, VER155008. The inhibitor also prevented CD86 upregulation on DC co-cultured with irradiated tumor cells. Together, our study demonstrates that radiation induces immunologically relevant changes in tumor cells, which can trigger CD8+ T cell responses via a predominantly Hsp70-dependent antigen cross-presentation process.Immune responses contribute to the success of radiation therapy of solid tumors; however, the mechanism of triggering CD8