Corpus allatum (CA) ablation results in juvenile hormone (JH) deficiency and pupal lethality in Drosophila. The fly CA produces and releases three sesquiterpenoid hormones: JH III bisepoxide (JHB3), JH III, and methyl farnesoate (MF). In the whole body extracts, MF is the most abundant sesquiterpenoid, followed by JHB3 and JH III. Knockout of JH acid methyl transferase (jhamt) did not result in lethality; it decreased biosynthesis of JHB3, but MF biosynthesis was not affected. RNAi-mediated reduction of 3-hydroxy-3-methylglutaryl CoA reductase (hmgcr) expression in the CA decreased biosynthesis and titers of the three sesquiterpenoids, resulting in partial lethality. Reducing hmgcr expression in the CA of the jhamt mutant further decreased MF titer to a very low level, and caused complete lethality. JH III, JHB3, and MF function through Met and Gce, the two JH receptors, and induce expression of Kr-h1, a JH primary-response gene. As well, a portion of MF is converted to JHB3 in the hemolymph or peripheral tissues. Topical application of JHB3, JH III, or MF precluded lethality in JH-deficient animals, but not in the Met gce double mutant. Taken together, these experiments show that MF is produced by the larval CA and released into the hemolymph, from where it exerts its anti-metamorphic effects indirectly after conversion to JHB3, as well as acting as a hormone itself through the two JH receptors, Met and Gce. Drosophila metamorphosis. MF is produced by the larval CA and released into the hemolymph, from where it exerted its anti-metamorphic effects indirectly after conversion to JHB3, as well as acting as a hormone itself through a direct interaction with Met and Gce, the two JH receptors.Methyl farnesoate (MF) is the immediate precursor of juvenile hormone (JH) III in the JH biosynthetic pathway, and lacks the epoxide moiety characteristic of JHs. The potential role of MF as a JH in arthropods has been an issue of a long-standing debate. In this report, comprehensive molecular genetics studies demonstrated that MF plays a dual role in regulating  Several forms of JH have been identified, including JH 0, JH I, 4-methyl JH I, JH II, JH III, JH bisepoxide (JHB3) and JH skipped bisepoxide. JH III is found in most insect orders, whereas JH 0, JH I, and JH II are exclusive to Lepidoptera . The, and MF , 18, 19.Drosophila. JH serves an equally important function, regulating various aspects of reproductive maturation in most insects  methionine  from Perkin-Elmer Life Sciences . Methyltransferase activity in the brain-RG complexes isolated from larvae at 3hAIW was measured with JHA and FA as substrates, as described previously [3H] methionine (2.92–3.70 TBq/mmol) was purchased from Perkin-Elmer Life Sciences and TLC plates (20×20 cm2 plastic plate coated with silica gel F254) from Merck KgaA (Germany). JH biosynthesis in the brain-RG complexes was detected using the radiochemical assay followed by thin layer chromatography analysis as reported previously [S-Adenosyl-L-methionine (SAM) was purchased from Sigma-Aldrich and S-Adenosyl-L-[methyl-eviously , 23, 33.eviously , 19, 37.eviously .t-test and ANOVA. t-test: *, p<0.05; **, p<0.01. ANOVA: the bars labeled with different lowercase letters are significantly different (p<0.05). Throughout the paper, values are represented as the mean ± standard deviation of at least five independent experiments.Experimental data were analyzed with the Student’s S1 Fig(TIF)Click here for additional data file.S2 Figw1118 and jhamt2. (C and C’) Topical applications of acetone (control) and methoprene  on newly eclosed females of w1118 and jhamt2, and measurements of the average number of eggs laid by each pair of flies per 24 hours (C) and the ovary size of 6-day-old virgins (C’). (D and D’) Comparisons of the average number of eggs laid by each pair of flies per 24 hours (D) and the ovary size of 6-day-old virgins (D’) among w1118, jhamt2, jhamt2/jhamt2; Aug21-GAL4>UAS-jhamt, and jhamt2/jhamt2; jhamt-GAL4>UAS-jhamt.(A and B) Measurements of the periods from egg laying to wandering (A) and the body weights at the white prepupal stage (B) of (TIF)Click here for additional data file.S3 FigCG10527. CG10527187 has an intragenic deletion of CG10527 compared to w1118 [CG10527-F/CG10527-R. The brown line indicates the PCR products (1968 bp length and ~600 bp length) obtained with the above primer pair using the genomic DNA extracted from w1118 and CG10527187 as templates, respectively. The blank region denotes the deletion region of CG10527 in CG10527187. (B and B’) Three lines of the jhamt2CG10527187 double mutants were confirmed by genomic DNA PCR. (B) The 1968-bp and ~600 bp PCR products were obtained with primer pair CG10527-F/CG10527-R (the black bars) from w1118 and CG10527187, respectively. (B’) The white PCR products of expected sizes with the primer pairs jhamt-1/jhamt-2 (the red bars) and jhamt-3/jhamt-4 (the green bars) (as shown in CG10527-F/CG10527-R (the black bars) were obtained in the 3 heterozygous jhamt2CG10527187 lines . In the following experiments, the number 1 homozygous jhamt2CG10527187 double mutant was used. (C) As detected by Western blot analysis, CG10527 was expressed in the brain-RG complexes of w1118 and jhamt2 but not those of CG10527187 and jhamt2CG10527187. Tubulin was used as the internal control. (D) Immunohistochemistry revealed no expression of JHAMT in the CA of jhamt2 and jhamt2CG10527187, while JHAMT was expressed in the CA of w1118 and CG10527187. Arrows indicate the CA.(A) Genomic structures of o w1118 . The bla(TIF)Click here for additional data file.S4 Figw1118, jhamt2, CG10527187, and jhamt2CG10527187 at 3h AIW using FA (A) or JHA (A’) as substrates. (B) Measurements of JH biosynthesis in the brain-RG complexes in w1118, jhamt2, CG10527187, and jhamt2CG10527187 at 3h AIW using the RCA-TLC method. (C-C”) Quantitative measurements of whole body titers of JHB3 (C), JH III (C’), and MF (C”) in w1118, jhamt2, CG10527187, and jhamt2CG10527187 at 3h AIW according to the HPLC-FD protocol. (D) qPCR measurements of the relative mRNA levels of Kr-h1 in the fat body tissues isolated from w1118, jhamt2, CG10527187, and jhamt2CG10527187 at 3h AIW. (E and E’) Comparisons of the average number of eggs laid by each pair of flies per 24 hours (E) and the ovary size of 6-day-old virgins (E’) among w1118, jhamt2, CG10527187, and jhamt2CG10527187.(A and A’) Measurements of methyltransferase activity of the brain-RG complexes in (TIF)Click here for additional data file.S5 FigAug-GAL4>UAS-hmgcr dsRNA and jhamt-GAL4>UAS-hmgcr dsRNA at 3h AIW. (B) Lethality of Aug21-GAL4>UAS-hmgcr dsRNA during the embryonic, larval, and pupal stages. Aug21-GAL4/+ and UAS-hmgcr dsRNA/+ were used as the controls. (C) Lethality of jhamt2/jhamt2; Aug21-GAL4>UAS-hmgcr dsRNA during the embryonic, larval, and pupal stages. jhamt2/+; Aug21-GAL4/+ and jhamt2/+; UAS-hmgcr dsRNA/+ were used as the controls.(A) RNAi efficiency of (TIF)Click here for additional data file.S6 FigUSP (A) and Kr-h1 (B) mRNA levels in Kc cells in which USP expression was reduced by RNAi (GFP RNAi and DMSO as a control) for 48 h, followed with treatments with MF (1×10-10~-6 M) for 30 min.qPCR measurements of fold-changes of relative (TIF)Click here for additional data file.S1 Table(PDF)Click here for additional data file.


Salmonella typhimurium A1-R were analyzed in vitro and in vivo on prostate cancer cells and experimental bone metastasis. Both ZOL and S. typhimurium A1-R inhibited the growth of PC-3 cells expressing red fluorescent protien in vitro. To investigate the efficacy of S. typhimurium A1-R on prostate cancer experimental bone metastasis, we established models of both early and advanced stage bone metastasis. The mice were treated with ZOL, S. typhimurium A1-R, and combination therapy of both ZOL and S. typhimurium A1-R. ZOL and S. typhimurium A1-R inhibited the growth of solitary bone metastases. S. typhimurium A1-R treatment significantly decreased bone metastasis and delayed the appearance of PC-3 bone metastases of multiple mouse models. Additionally, S. typhimurium A1-R treatment significantly improved the overall survival of the mice with multiple bone metastases. The results of the present study indicate that S. typhimurium A1-R is useful to prevent and inhibit prostate cancer bone metastasis and has potential for future clinical use in the adjuvant setting.Bone metastasis is a frequent occurrence in prostate cancer patients and often is lethal. Zoledronic acid (ZOL) is often used for bone metastasis with limited efficacy. More effective models and treatment methods are required to improve the outcome of prostate cancer patients. In the present study, the effects of tumor-targeting  Prostate cancer is diagnosed in more than 500,000 men worldwide . Bone meSalmonella typhimuium (S. typhimurium) A1-R, is a genetically-engineered strain of Salmonella that is able to specifically target cancer cells [S. typhimurium A1-R was able to inhibit or eradicate primary and metastatic tumors as monotherapy in nude mouse models of prostate [S. typhimurium A1-R, and vascular destruction appears to play a role in S. typhimurium A1-R antitumor efficacy [S. typhimurium A1-R in immunocompetent transgenic mice expressing the nestin-driven green fluorescent protein (ND-GFP), which is selectively expressed in nascent blood vessels [er cells . S. typhprostate , 4, breaprostate –7, lung prostate , 9, pancprostate –14, ovarprostate , 16, stoprostate  and cervprostate , as wellprostate –23 and gprostate , 25, all vessels .S. typhimurium A1-R and the combination of S. typhimurium A1-R and ZOL was assessed in nude mice models of solitary and multiple bone metastases of prostate cancer.In the present study, the efficacy of S. typhimurium A1-R for 1 h. The cells were observed with a Fluoview FV1000 confocal microscope . Fluorescence imaging demonstrated that S. typhimurium A1-R expressing GFP selectively invaded and replicated intracellularly and killed PC-3-RFP cells . One week after intracardiac injection, half of the mice were treated once a week for 3 weeks with an i.v. injection of S. typhimurium A1-R (5 × 107 CFU)  Figure . Time-cop Figure . S. typhe Figure , 3d.5) cells were injected into the intramedullary cavity of the tibia in nude mice   5 times a week for 4 weeks  for a total of 3 weeks. ZOL+ S. typhimurium A1-R-group mice were treated with both s.c. injection of ZOL and i.v. injection of S. typhimurium A1-R at the same dosages listed above. Fluorescence imaging was performed with an iBOX Scientia Imaging System  every week. The fluorescence images demonstrated that the control group had rapid growth of metastatic bone cancer, whereas the ZOL group, S. typhimurium A1-R group, and the ZOL+S. typhimurium A1-R group mice had reduced metastatic growth   were used in this study. Mice were maintained in a barrier facility of high efficiency particulate air-filtered racks. The animals were fed an autoclaved laboratory rodent diet. Animal experiments were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals under National Institutes of Health assurance number A3873-01.S. typhimurium A1-R bacteria  were grown overnight on LB medium  and then diluted 1:10 in LB medium. Bacteria were harvested at late-log phase, washed with PBS, and then diluted in PBS [GFP-expressing d in PBS , 5.2 cells/dish). S. typhimurium A1-R-GFP was added to the cancer cells (1 × 107 or 1 × 108 CFU/dish). After 1 h incubation at 37°C, the cells were rinsed and cultured in medium containing gentamycin sulfate (100 μg/ml) to kill external but not internal bacteria. Invasion and destruction of PC-3-RFP cells by S. typhimurium A1-R-GFP was visualized with a Fluoview FV1000 confocal microscope . Eight days after treatment with S. typhimurium A1-R, PC-3-RFP colonies were fixed in methanol and stained with 1% crystal violet as previously described [PC-3-RFP cells were plated in 35 mm dishes (5 × 10escribed . ImageJ 5) were injected intracardially in nude mice. One week after injection, mice (treatment group) were administered S. typhimurium A1-R  once a week for 3 weeks. The remaining mice (control group) were administered the same volume of PBS. To evaluate metastasis-free survival, GFP-expressing lesions were initially observed using the Illumatool imaging system every 2 days. Metastasis-free survival was defined as the time from intracardiac injection of cancer cells to the time of detection of bone metastases with the Illumatool.PC-3-GFP cells (5 × 105) were co-injected into the intramedullary cavity of the tibia with a 1.0 mL 28 G latex-free insulin syringe . The skin was closed with a 6-0 suture. One week after injection, fluorescence imaging was performed to confirm the RFP-expressing tumor was growing, using the iBOX Scientia . The study mice were randomly divided into the following groups: control group; ZOL group; S. typhimuium A1-R group; and ZOL+ S. typhimuium A1-R-group. In the ZOL group, mice were treated with subcutaneous injection of 120 mg/kg ZOL for 5 times a week for 4 weeks  once a week for 3 weeks. ZOL+ S. typhimuium A1-R-group mice were administered both treatments. Fluorescence imaging was performed on treated and untreated mice, and GFP-expressing areas were recorded every week for 5 weeks using the iBOX. At the end of the follow-up, the mice were sacrificed and the metastatic tumors were excised. Tumor weight was compared to evaluate the efficacy of ZOL and S. typhimuium A1-R.A midline skin incision (5 mm) was made just below the knee joint to expose the tibial tuberosity Figure . Matriges Figure . S. typhp < 0.05. Data are expressed as mean ± SE. Statistical analyses were performed with EZR .Data showing comparisons among 3 or more groups were assessed using analysis of variance (ANOVA). The Kaplan-Meier method was used for bone metastasis-free survival and overall survival. Log-rank tests were used for statistical significance of the difference between the two groups. Differences were considered significant when


Odorant receptors (ORs) control several aspects of cell fate in olfactory sensory neurons (OSNs), including singular gene choice and axonal identity. The mechanisms of OR-induced axon guidance have been suggested to principally rely on G-protein signalling. Here, we report that for a subset of OSNs, deleting G proteins or altering their levels of signalling does not affect axonal identity. Signalling-deficient ORs or surrogate receptors that are unable to couple to Gs/Golf still provide axons with distinct identities and the anterior–posterior targeting of axons does not correlate with the levels of cAMP produced by genetic modifications. In addition, we refine the models of negative feedback by showing that ectopic ORs can be robustly expressed without suppressing endogenous gene choice. In conclusion, our results uncover a new feature of ORs, showing that they can instruct axonal identity and regulate olfactory map formation independent of canonical G-protein signalling and cAMP production. To accommodate this broad recognition, the olfactory system employs a large repertoire of odorant receptors (ORs) ,2. OR geEvery olfactory sensory neuron (OSN) is thought to express only a single OR gene from a single allele, which is referred to as singular expression ,8. OSNs Axons from OSNs that express the same singular expressed OR coalesce into two of the approximately 1800 glomeruli, typically one on the medial and one on the lateral side of the olfactory bulb . The OR We now provide evidence for an extended model of OR-mediated axonal wiring. Importantly, our results indicate that ORs also regulate A-P targeting and axonal identity via cAMP-independent mechanisms. In addition, our findings also redefine concepts within the field of OR gene regulation.2.2.1.growth-associated protein 43 (Gap43) and these neurons are below mature neurons that are positive for olfactory marker protein (Omp). Simultaneous in situ hybridization (ISH) using Gnas (Gsα), Gap43 and Omp probes revealed detectable expression of Gnas in the basal stem cell layer of the developing olfactory epithelium in postnatal day (PD)6 animals that are fl/flGnas-E1 . Coexpression of Gnas was observed in only a fraction of immature neurons expressing Gap43, when ORs are first expressed and impart axonal identity to the axons . By contrast, Gnal (Golfα) expression colocalized with Omp, indicating that it was mainly expressed in mature neurons . To directly test the role of Gsα signalling in M71 OSNs, we sought to rely on the previously described Gnas-E1fl/fl mice to obtain a conditional knockout allele (cKO) for Gsα [fl/fl mice could be used to delete Gs in OSNs. To this end, we used a transgenic Cre line in which Cre expression is driven by the olfactory epithelium specific #123 promoter. The #123 promoter has been previously shown to be active from an early developmental stage [Sema3F expression in immature OSNs [Cre was expressed in all zones and within basal cells, immature and mature neurons  and reliably removed the stop fragment in ROSA26-Stop-tauGFP reporter mice; mRNA expression of the tauGFP reporter was now observed in basal cells, immature and mature neurons . All olfactory glomeruli appeared labelled by the ROSA26-tauGFP reporter after Cre recombination . We next analysed the loss of Gsα from the entire olfactory epithelium in the #123-Cre×Gnas-E1fl/fl (Gs cKO) mice. The efficient excision of Gsα was readily observed in the vomeronasal organ (VNO), where the ubiquitous expression seen in WT mice was lost in Gs cKO animals (figure 1c versus d). In the olfactory epithelium, Gnas expression was strongly reduced in Gap43+ OSNs and was mainly limited to the most basal cells . Thus, this shows that the Gnas-E1fl/fl mice can be used to eliminate Gsα expression in OSNs. Of note, in the #123-Cre × Gnas-E1fl/fl mice, Gnal expression was maintained in mature OSNs with no derepression in immature or basal cells . Remarkably, glomeruli appeared normal in the #123-Cre × Gnas-E1fl/fl mice  and, by using OR reporters, we observed that M72-LacZ and MOR23-LacZ glomeruli were identical in pattern in both Gs WT and #123-Cre-induced Gs cKO genetic backgrounds . Since deletion of Gs has been previously shown to affect axonal targeting of OSNs [figure 1i). No aberrations in axonal targeting were observed in the mutant population of axons ; all axons co-converged and coalesced. Finally, we did not observe any deficits in the glomerular formation of M71-GFP axons in Gnal−/− (Golf KO) animals  and no expression of Gsα protein was observable in Golf KO M71-GFP OSNs (data not shown).We aimed to investigate the role of Gsα in the axonal targeting of M71 OSNs. The olfactory epithelium is stratified such that the basal stem cells reside beneath immature neurons that are positive for  for Gsα , since fal stage  and #123ure OSNs . We obse of OSNs , this su2.2.Cnga2 gene in female mice [ROSA26 Mosaic or RoMo system, for mosaic analysis by combining stochastic Cre recombination [ROSA26 locus [a). When using cell-type specific promoters to drive Cre expression in RoMo-control mice, we observed a mosaic population of cells, expressing either Turquoise or Tomato (data not shown). This led us to construct a second mouse strain, RoMo-dnGs, where the expression of Tomato is now linked to the expression of a dnGs gene via an internal ribosomal entry site (IRES) sequence. Initially, we crossed RoMo-dnGs with OMP-Cre mice to induce recombination in all mature OSNs. Again a mosaic population of either Turquoise+ or Tomato-dnGs+ OSNs was observed in the VNO and main olfactory epithelium (MOE) (b). No apparent segregation of axons was observed in the olfactory glomeruli (data not shown), possibly because the effects of dnGs occurred too late in the development of OSNs. Therefore, to induce recombination in immature OSNs, we crossed RoMo-dnGs mice with our previously described #123-Cre mice. Mosaic expression was observed in the MOE, but both populations of axons converged and comingled into normal glomerular patterns (c). Finally, we sought to investigate how the expression of dnGs affects the identity and basal activity of M71 expressing OSNs by crossing RoMo-dnGs to M71-Cre mice. Mosaic expression was observed with axons that fully converged and comingled (d), indicating that expression of dnGs did not change axonal identity or the glomerular position of M71 OSNs. This suggests that either the inhibition of basal Gs activity did not affect M71 axonal identity or that the dnGs was not functional. It has been reported that spontaneous OSN spiking is fully dependent on the basal signalling of ORs [figure 2e; one-way ANOVA: F = 7.96065; p = 0.00123). The instantaneous firing frequency (IF) between these groups also differed , with M71-Cre/Tomato-dnGs OSNs having a significantly higher IF (19.2 ± 1.8 Hz) than M71-Cre/Tomato+ (14.2 ± 1.0 Hz) and M71-GFP neurons , indicating that M71-Cre/Tomato-dnGs OSNs have a higher tendency for bursting. The basal and IF firing rates of M71-Cre/Tomato+ and M71-GFP OSNs were not different. The reduction in spontaneous firing of M71-Cre/Tomato-dnGs OSNs strongly suggests that the dnGs inhibits basal, agonist-independent G-protein signalling and cAMP production. Since axons of Turquoise+ control OSNs and Tomato+ dnGs-affected OSNs coalesced in the dorsal bulb, this suggests that crippling basal G-protein signalling did not affect M71 axonal identity or glomerular positioning.To corroborate our Gs cKO and Golf KO results, we envisaged an alternative approach to inhibit basal levels of G-protein signalling by expressing a dominant-negative Gsα (dnGs) mutant that has been reported to efficiently inhibit Gsα signalling . This dnale mice ,31. Thisbination  with gen26 locus  (figure um (MOE) b. No apppatterns c. Finallomingled d, indicag of ORs . Therefog of ORs . M71-Cre2.3.M71 locus was engineered to coexpress M71 together with a constitutively active Gs mutant (caGs) and a GFP reporter via a tricistronic mRNA (a). In homozygous M71-caGs-GFP mice, 16 out 36 bulbs showed a lateral GFP+ glomerulus at an A-P position comparable to that of WT M71 . Remarkably, in the remainder of bulbs (55%), axons failed to converge into a glomerulus (figure 3c), indicating that glomerular formation was disrupted. Importantly, by crossing in the M71-RFP allele, we observed that M71-caGs-GFP axons converged with M71-RFP axons (figure 3e). This shows that when M71-caGs glomeruli were formed, they were not posterior to WT M71 glomeruli. This is not because of an intrinsic inability to shift M71 glomeruli posterior, since we have previously reported that the M71::GFP fusion protein induces a clear posterior shift [figure 3e), indicating a subtle change in axonal identity. Together this shows that caGs co-expression did not shift the position of the glomerulus, but did affect axonal identity and glomerular formation.We have already addressed the consequences of downregulating G-protein signalling. Now, we wished to assess how axon targeting is affected by an increase in G-protein signalling in M71 neurons. Therefore, we generated the M71-caGs-GFP strain, in which the nic mRNA a. In homof bulbs 5%, axonsM71 locus. By generating the M71-M71-GFP strain, which harbours another tricistronic mutation in the M71 locus (a), we show that this is unlikely. In M71-M71-GFP mice, where instead of caGs we inserted a second M71 coding region, the formation of GFP+ glomeruli was always observed (d). Interestingly, M71-M71-GFP and M71-RFP axons also often formed a compartmentalized glomerulus (f), suggesting that the change in M71 protein levels was sufficient to change the identity of the axons.The frequent failure to form M71-caGs-GFP glomeruli suggests that OR-independent signalling via the caGs mutant could disrupt glomerular formation. However, we cannot exclude that the aberrant wiring resulted from the tricistronic structure of the gene-targeted 71 locus a, we shoobserved d. Intereomerulus f, sugges2.4.a). In contrast to M71-LacZ axons , M71(RDY)-LacZ axons showed poor outgrowth, typically did not reach the cribiform plate and were not observed on the dorsal bulb . Furthermore, the number of M71(RDY)-LacZ neurons rapidly decreased over time  and by PD21 their numbers were 10-fold lower when compared with M71-LacZ neurons in age-matched animals . This dramatic phenotype indicates that G-protein signalling in M71 OSNs is necessary when competing with normal OSNs.Our results indicate that Gs/Golf signalling and cAMP production are not the main drivers of A-P targeting in M71 OSNs. To further corroborate this, we wished to set up an experimental system in which OSNs would express different ORs but have the same level of G-protein signalling. If such axons would not coalesce, this would be a clear indication of an OR-instructed identity, independent of the levels of G-protein signalling. To achieve this, we thought to compare M71 OSNs in which the coding sequence (CDS) was exchanged with distinct signalling-deficient ORs and where G-protein signalling was rescued via expression of the caGs mutant. To induce signalling deficiency in ORs, we chose to mutate the highly conserved DRY motif, which is critical for regulating GPCR conformational states . Mutatinfigure 4a). Furthermore, in the MOR23(RDY) → M71-caGs-Tomato strain, the M71 CDS is replaced by a DRY → RDY mutant of the MOR23 CDS. The M71(RDY)-caGs-GFP and MOR23(RDY) → M71-caGs-Tomato OSNs thus express different ORs—M71(RDY) versus MOR23(RDY)—but should have the same level of G-protein signalling (derived from the OR-independent caGs mutant). Coexpression of caGs rescued axonal projections to the bulb for both M71(RDY)-caGs-GFP and MOR23(RDY) → M71-caGs-Tomato OSNs . The number of caGs-rescued OSNs remained very low  and as a consequence glomerular formation was often inefficient [f,h). These findings are also in line with what was seen for M71-caGs mice, where expression of caGs often disrupted glomerular formation (c). Importantly, however, M71(RDY)-caGs and MOR23(RDY) → M71-caGs axons projected to fundamentally different A-P regions . The differential identities of M71(RDY)-caGs-GFP and MOR23(RDY) → M71-caGs-Tomato axons were confirmed in a mixed cross, where coalescence was never observed between GFP+ and Tomato+ axons, and where GFP+ axons extended more posterior (f). Remarkably, M71(RDY) axons projected to the same A-P region as WT M71 axons, as was seen in M71-RFP × M71(RDY)-caGs-GFP mice (g). Similarly, MOR23(RDY) → M71-caGs-Tomato axons projected very close to WT MOR23 → M71-LacZ axons, which was anterior to M71 (h). Together this shows that M71(RDY)-caGs and MOR23(RDY)-caGs axons project to distinct A-P regions on the bulb, very close to the targeting site of their respective WT receptors. It is important to note that for both populations the caGs is expressed from the same locus with a similar tricistronic strategy. This suggests that ORs can regulate axonal targeting via cAMP-independent mechanisms.Now we were in a position to test two additional mouse strains: M71(RDY)-caGs-GFP and MOR23(RDY) → M71-caGs-Tomato, in which the RDY mutant OR is coexpressed with a caGs to rescue G-protein signalling a. Furtheato OSNs d,e. The fficient . In miceormation c. Import regions d,e. The osterior f. RemarkGFP mice g. Similar to M71 h. Togeth2.5.Omp+ mature, Omp+Gap43+ intermediate and Gap43+ immature cells in normal and RDY strains of mice: M71-LacZ, M71(RDY)-LacZ, M71(RDY)-caGs-GFP and MOR23(RDY) → M71-caGs-Tomato OSNs (a). An almost complete absence of Omp+ mature OSNs was observed in the M71(RDY)-LacZ population (a). M71(RDY)-LacZ OSNs also failed to upregulate Adcy3 expression, a key component of the signalling machinery in mature OSNs (b). By contrast, Omp+ and Adcy3+ mature M71(RDY)-caGs and MOR23(RDY) → M71-caGs OSNs were observed, albeit at a lower percentage as compared with WT M71 . These results suggest that G-protein signalling may be a checkpoint in OSN maturation. Alternatively, the loss in G-protein signalling may render OSNs uncompetitive and they may be quickly eliminated as they mature, which would explain the rarely observed only Omp+ expressing M71(RDY)-LacZ OSNs.Are DRY → RDY mutations in ORs blocking G-protein signalling? We observed that M71(RDY)-LacZ cells resided very basal in the epithelium suggesting that these cells were not mature and might not contain necessary signal transduction components to assess G-protein signalling. Thus, we used ISH to determine the percentage of ato OSNs a. An almpulation a. M71. In addition, MOR23(RDY) → M71-caGs OSNs exhibited almost no spontaneous activity, which is clearly present in WT MOR23 or M71 OSNs . Four conclusions are delineated: (i) DRY → RDY mutations uncouple ORs from G-protein signalling; (ii) no other ORs were coexpressed in these cells as MOR23(RDY) → M71-caGs OSNs never responded to the mixture of odorants (gene choice remained intact); (iii) spontaneous OSN spiking is indeed fully dependent on the basal signalling of ORs; and (iv) caGs does not elicit spontaneous spiking, indicating that the rescue of MOR23(RDY) OSNs does not require electrical activity.We next wished to perform physiology to assess OR functionality. Since M71(RDY)-LacZ OSNs remained immature, they would not be suitable for recordings as their signalling machinery may not be functional. We therefore chose MOR23(RDY) → M71-caGs-Tomato OSNs, on which we performed perforated patch-clamp recordings. MOR23(RDY) → M71-caGs-Tomato neurons were stimulated with saturating concentrations of lyral (an MOR23 ligand), acetophenone (an M71 ligand), or a mixture of odorants that activates 66% of randomly patched OSNs in the MOE . In our figure 52.6.a priori possible to coexpress a second OR in all OSNs. In theory, OR-mediated negative feedback mechanisms would prevent endogenous gene expression or endogenous genes would silence the expression of the ectopic OR [Our results show that signalling-deficient ORs can regulate the identity and A-P targeting of M71 axons, suggesting that this did not rely on differences in OR-induced cAMP levels. However, it may be argued that the M71(RDY) and MOR23(RDY) coding sequences differentially affect mRNA stability, thereby resulting in different caGs protein levels in M71(RDY)-caGs compared to MOR23(RDY)-caGs OSNs. To resolve these issues, we reasoned that forcing the coexpression of a WT OR in all OSNs may also rescue the maturation and axonal projections of M71(RDY) neurons. Furthermore, this might uniformly increase G-protein signalling in all OSNs, allowing a direct comparison of the A-P targeting of M71(RDY) with other ORs. Based on previous experiments using ubiquitously activated ORs via the ttA/tetO system –43, it wtopic OR ,42.MOR23 cDNA . This was subsequently cloned into the O/E2 targeting vector (TV) [O/E2 gene via gene targeting. In the resulting O/E2-M71-GFP strain, expression of an M71-IRES-tauGFP cDNA is placed under the control of the native O/E2 promoter, which is prominently expressed in all OSNs [b–e). All OSNs appeared GFP+, as did the glomeruli, which were homogeneously labelled and showed a normal morphology (f). Importantly, M71 mRNA was robustly observed in the whole MOE and VNO of O/E2-M71-GFP mice . O/E2 expression is typically observed prior to Gap43, at a very immature neuronal stage of OSNs. M71 expression from O/E2-M71-GFP mice was also seen in this early OSN stage (figure 6g). The MOE of O/E2-M71-GFP mice had a normal multi-layered structure and thickness, comparable to that of O/E2 WT control animals . Interestingly, it was evident that expression of M71 via the O/E2 promoter did not suppress endogenous OR expression. ISH showed coexpression of M71 with other unrelated class II OR genes (figure 6i). Next, NanoString was used to assess the expression of a random selection of OR genes in the MOE of O/E2-M71-GFP(+/−) and O/E2-WT littermates (figure 6j). Of the 19 OR genes tested, only one transcript showed a significant but small downregulation, showing that the majority of ORs had comparable expression levels in the two groups. In addition, the zonal expression of endogenous ORs in the MOE was not altered in O/E2-M71-GFP mice, as illustrated by crossing the mice to the M71-LacZ, MOR23-LacZ and P2-LacZ strains (a–c). As reported previously, OSNs that choose an OR locus that does not contain an OR gene—such as the ΔM71-LacZ allele where the M71 CDS is deleted—coexpress additional OR genes [d). Interestingly, distributed axons entering multiple glomeruli were also observed in ΔM71-LacZ mice crossed to O/E2-M71-GFP (d).In an attempt to express an OR in all OSNs, we started from the 3'Δ, see ), where cassette a. This wtor (TV)  and usedall OSNs . A widesrphology f. Import strains a–c. As rOR genes . Therefo-M71-GFP d.Figuren = 10) and MOR23(RDY) → M71-caGs OSNs (n = 10) responded to acetophenone . While responses were heterogeneous, most O/E2-M71-GFP neurons responded to concentrations above 10−5 M acetophenone . Responses to acetophenone were not widespread in the normal WT MOE, as in O/E2 WT littermates only 1 in 10 patched OSNs responded (c). As a reference we also patched WT M71 OSNs in M71-GFP mice and plotted a threshold frequency histogram, which represents the percentage of cells that responded to a specific threshold concentration (f). This showed that O/E2-M71-GFP neurons were less sensitive to acetophenone than M71-GFP OSNs. Importantly, O/E2-M71-GFP expression also rescued the spontaneous firing of MOR23(RDY) → M71-caGs OSNs, which now exhibited mean and instantaneous firing frequencies in the same range as that of randomly patched OSNs . Since without O/E2-M71-GFP expression MOR23(RDY) → M71-caGs OSNs exhibit no spontaneous firing (e), this shows that the ectopic M71 contributes to basal, agonist-independent, spiking in OSNs, while caGs does not.To investigate whether the ectopic M71 in O/E2-M71-GFP mice is functional and contributes to signalling and neuronal activity, we performed patch-clamp recordings on the MOE of O/E2-M71-GFP mice. In addition, we also crossed in the MOR23(RDY) → M71-caGs-Tomato mutation. In the resulting cross, we patched the dendritic knobs of random OSNs that express an unknown endogenous OR (GFP+ Tomato−) or MOR23(RDY) → M71-caGs OSNs (GFP+ Tomato+) and measured their responses to varying concentrations of the M71 ligand acetophenone. All randomly patched OSNs . However, M71 axons took altered routes on the bulb and traversed through regions were they are normally excluded and often formed an additional glomerulus at the same A-P position .We wondered how the ectopic M71 expression would affect M71-LacZ or MOR23 → M71-LacZ axonal projections. In O/E2-M71-GFP mutants, M71-LacZ+ and MOR23 → M71-LacZ OSNs formed glomeruli in the correct A-P position a,b. Howed,f). An ectopic convergence point was observed in an anteromedial region of the dorsal bulb, where both medial and lateral projecting axons co-converged . The lateral M71(RDY) glomerulus was observed at the same A-P region as WT M71 glomeruli , which was clearly more posterior than MOR23 → M71-LacZ or MOR23(RDY) → M71-caGs-Tomato axons . Importantly, in O/E2-M71-GFP mice, M71(RDY)-LacZ OSNs have lower levels of basal G-protein signalling than MOR23 → M71-LacZ OSNs or MOR23(RDY) → M71-caGs-Tomato OSNs. Indeed, while in M71(RDY)-LacZ OSNs only the O/E2-derived ectopic M71 contributes to cAMP production, in the latter two populations this is combined with cAMP stemming from the WT MOR23 receptor or the caGs mutant, respectively. Therefore, despite having lower levels of cAMP, M71(RDY) axons project more posterior than MOR23 → M71-LacZ or MOR23(RDY) → M71-caGs-Tomato OSNs. This again highlights that in M71 or M71-swapped OSNs A-P targeting relies on the expressed OR but not on the levels of cAMP production.We next tested the consequence of adding M71 expression to signalling-deficient M71(RDY) OSNs. O/E2-M71-GFP expression rescued axon outgrowth of M71(RDY)-LacZ OSNs, which formed two points of coalescence on the dorsal bulb d,f. An eM71 CDS is replaced by the vomeronasal receptor V1rb2, axonal outgrowth and coalescence is very inefficient [g). This shows that upon O/E2-M71 rescue, V1rb2 can efficiently substitute for an OR and induce a unique axonal convergence. Importantly, V1rb2 → M71-LacZ axons had a different identity and a convergence point that was positioned much more anterior than M71(RDY)-LacZ axons. Since both M71(RDY) and V1rb2 are GPCRs that cannot couple to Gs/Golf, this confirms that their axonal identity and A-P positioning were shaped by OR-dependent, Gsα/Golfα-independent mechanisms.Can we ascertain that the axonal identity and glomerular targeting of O/E2-M71 × M71(RDY) OSNs is imparted by the M71(RDY) OR or could it be derived from the ectopic O/E2-M71 alone? We have previously reported that in V1rb2 → M71-LacZ mice, where the fficient . Interesneuropilin-1 (Nrp1), plexin-A1 (Plxna1) and semaphorin-3A (Sema3a) and that these molecules are the main drivers of A-P targeting [h). There were no changes in the expression levels of any of the neuropilin, semaphorin or plexin genes tested. Surprisingly, we observed a significant downregulation of Kirrel2 and Epha5 together with an increase of ephrin-A5 in O/E2-M71-GFP mice (h). Remarkably, both signalling impaired CNGA2 KO mice and activity reduced, naris occluded animals are known to have this same expression pattern [It has been suggested that the level of basal, agonist-independent G-protein signalling regulates the expression of argeting . To examGFP mice h. Remark pattern . This su3.Our results provide a set of profound insights into how the OR regulates the developmental pathway of OSNs, starting from regulation of gene choice, to OSN maturation and finally to axonal identity and glomerular formation.3.1.Forcing the widespread expression of an OR using transgenic approaches has not been straightforward, which has contributed to shaping the current models of OR-induced feedback. To achieve ectopic OR expression, several groups have used the tetO-TTA system using immature/mature neuronal TTA drivers –43. Thescis- and trans-acting enhancers [An explanation for the lack of suppression is that the O/E2-M71 expression levels do not reach a required threshold level to induce feedback. This would imply that robust expression levels that are sufficient to rescue axon outgrowth and neuronal maturation of OSNs are not sufficient to induce negative feedback. It has recently been proposed that stable OR gene expression requires the recruitment of many nhancers . It is tnhancers  have sho3.2.Omp+ Adcy3+ OSNs and axonal projections on the bulb. One explanation for these observations is activity-dependent competition [Our results indicate that OSNs expressing ORs with G-protein coupling mutations in the DRY motif remained in an immature state, were unable to send axons to the bulb and were gradually eliminated from the epithelium. Co-expression of a caGs was able to rescue OSN maturation, with the reappearance of petition , which wpetition  have shopetition . Since tThe rescue of OSNs by O/E2-M71 is also likely to involve a permissive signal that promotes OSN maturation or survival. Surprisingly, however, OE2-M71 OSNs also showed alterations in the expression of activity-dependent axon guidance molecules that are normally suppressed or enhanced in naris occlusion or CNG2A KO backgrounds . Thus, tGnas locus is not very efficient, then Gs may be deleted with some delay following Cre onset. In the absence of Gs, low levels of Golf protein may rescue G-protein signalling. However, Golf by itself was not critical, since Golf KO animals retained normal formation of M71 glomeruli in the dorsal bulb consistent with previous reports for P2 [We show that mosaic Gsα expression using M71-Cre does not result in a differential A-P targeting of WT and Gs cKO axons. This does not strictly mean that Gs is not involved in the development or axonal targeting of M71 OSNs, but rather that the levels of Gsα expression can vary in convergent populations of axons. It also suggests that deleting Gs shortly after the onset of OR expression does not lead to a differential A-P targeting or segregation of WT and cKO axons. It is possible that Gs deletion occurs too late, for example that Gs protein carried over from the basal stem state to immature OSNs is sufficient to support signalling. In this regard, there are indications that Gs mRNA may have a long half-life . Also, is for P2  and rI7 s for P2  glomeruls for P2  have sho3.3.The molecular mechanism of how ORs and other chemosensory receptors provide ‘self’ identity, thereby regulating axonal interactions and glomerular formation in the olfactory system, remains unclear. The state of OSN activity has often been ascribed as having a role in axonal wiring . One modIf A-P targeting would solely rely on differences in basal cAMP levels, then OSNs with the same level of basal G-protein signalling would project to the same A-P position, irrespective of the expressed OR. Since every OR is suggested to induce different basal activities, we devised a genetic swap experiment, where we expressed different signalling-deficient ORs from the same endogenous locus, and restored activity via receptor-independent G-protein signalling. Importantly, this showed that signalling-deficient M71(RDY) and MOR23(RDY) ORs induced distinct identities and projected axons to different A-P regions on the bulb, close to the cognate WT receptor. These data show that even in the absence of effective coupling of ORs to G proteins, M71(RDY) and MOR23(RDY) ORs were still regulating A-P targeting. Furthermore, M71(RDY) and MOR23(RDY) → M71 axons did not coalesce, showing that they also had different identities. This suggests that both A-P targeting and axonal identity can be regulated via cAMP-independent mechanisms. However, we cannot rule out small differences in mRNA stability, leading to differential caGs protein levels, or that the RDY mutants still possess some difficult to detect residual activity. In this regard, the expression of caGs may provide a permissive environment to allow OSN maturation and axon outgrowth. Under this condition, the coupling of the mutant ORs with the G proteins, albeit ineffective, may still affect glomerular positioning. However, it is questionable if the system would be sensitive enough to detect such small variations in signalling. Moreover, a striking observation, which cannot easily be explained by the aforementioned arguments, was that M71(RDY) and MOR23(RDY) ORs sent axons to the same A-P position as their respective WT receptors.M71 locus, while Imai and colleagues [MOR23 locus. Our results may suggest that the importance of G-protein signalling and cAMP production in axonal wiring may vary depending on the OSN cell type. Alternatively, the difference may simply be due to the transgenic versus gene-targeted approach. The transgenic approach is sensitive to variations in transcription levels, cell-type expression and RNA stability, which can also affect the expression levels of caGs.To further investigate how subtle changes in G-protein signalling would affect axonal identity and A-P targeting in M71 OSNs, we also modified cAMP production through the coexpression of a constitutively active or dominant-negative Gsα mutant. Using a mosaic readout, neither was found to induce a shift in the position of M71 glomeruli. However, one cautionary note is that we do not yet know to what extent the dnGs or caGs mutants modulate basal cAMP production in OSNs. In the case of the dnGs mutant, we used spontaneous electrical activity as a proxy for basal cAMP signalling, which is based on previous observations ,50. Howelleagues  use the Additional evidence showing that ORs can regulate A-P targeting via cAMP-independent mechanisms was provided by the O/E2-M71 rescue experiments. These showed that upon O/E2-M71 rescue, the A-P position of the lateral M71(RDY) glomeruli closely matched that of WT M71, which could not be explained by invoking the levels of G-protein signalling and cAMP production. We also show that upon O/E2-M71 rescue, the V1rb2 receptor can robustly provide for axonal identity and the coalescence of axons. Importantly, the M71(RDY) and V1rb2 glomeruli were in completely different regions of the bulb. This shows that in a permissive environment (via O/E2-M71 expression), the M71(RDY) and V1rb2 genes regulate A-P targeting and axonal coalescence. Since both receptors have a deficiency in coupling to Gs/Golf, this suggests the involvement of cAMP-independent mechanisms.An important recent discovery was the existence of a developmental critical period in olfactory map formation ,53. Work3.4.Kirrel2, Epha5 and ephrin-A5.Importantly, in an O/E2-M71 background, M71(RDY) axonal projections did not completely mirror those of WT M71. Indeed, the medial M71(RDY) glomeruli were in a new ectopic anterior position. Two independent groups have shown that in Adcy3(−/−) animals, M71 and M72 axons form, besides the expected glomeruli, an additional glomerulus in an anteromedial region ,55. StriFurthermore, we observed that M71 and M71(RDY)-caGs axons had distinct identities. It is known that even single amino acid substitutions can alter axonal identity . Importa4.In conclusion, our results suggest that, besides activity-dependent mechanisms, the A-P targeting and axonal identity of OSNs is also regulated by mechanisms that do not rely on cAMP production. That ORs can regulate axonal wiring via cAMP-independent mechanisms is yet another surprising feature of these highly pluralistic proteins. How do ORs, which are present in olfactory axons ,57, supp5.5.1.ROSA26 locus targeting vectors [Targeting constructs for M71(RDY)-lacZ, M71(RDY)-caGs-GFP, MOR23(RDY) → M71-caGs-Tomato and M71-M71-GFP were generated by modifying the described M71 TV . The O/E vectors .5.1.1.M71 coding region (GACCGC → CGCGAC), resulting in the D121R;R122D substitutions. For the MOR23(RDY) → M71 swap, the 930 bp M71 coding region was replaced with the 930 bp MOR23 coding region, which again harboured four point mutations (GATCGT → CGTGAT) resulting in the same D121R;R122D substitutions. An FseI and an AscI restriction site was inserted three nucleotides after the stop codon of the M71(RDY) or MOR23(RDY) CDS, and was followed by an ACNF cassette, which is a neo selection cassette, that is self-excised during transmission though the male germ line [M71 CDS. The LNL neo cassette was removed by crossing to EIIA-Cre mice [Starting from the 9.2 kb M71 genomic sequence , we firserm line . The IREerm line . The M71Cre mice .5.1.2.The O/E2-M71-GFP targeting construct was generated by ligating an AscI M71-IRES-tauGFP-ACNF fragment into the O/E2 TV . This As5.1.3.ROSA26-DV3 destination vector [galactosidase–neomycin fusion gene, followed by three pA sequences, that allows for selection in ES cells and is expressed in the absence of Cre recombination. The dominant-negative Gαs mutant (α3β5/G226A/A366S) is previously described [WPRE) was used to increase expression levels [To generate the RoMo-control and RoMo-dnGs TVs, we modified the previously described attL4-pCAGG-loxP-Bgeo-3xpA-loxP-attR1 Gateway-compatible entry clone (pCAGG entry clone) , which wn vector  providedescribed  and was escribed  was kindn levels .Gene targeting was performed in E14 ES cells as described . ES cellfl/fl mice [Previously described strains that were used in this study  are: M71-LacZ, M71-GFP, MOR23 → M71-LacZ, V1rb2 → M71-LacZ, M72-LacZ, ΔM71-LacZ , P2-LacZ/fl mice , R26-STO/fl mice  and Golf/fl mice  were kin5.2.mageJ software. Intrinsic fluorescence in wholemounts was acquired with an upright Zeiss LSM710 microscope. Images were collected as z-stacks followed by maximum intensity projection to a single image.Wholemount X-gal staining was performed as previously described . The A-P5.3.a,b, every 10th section was collected from anterior to posterior and 42 sections were analysed per mouse.Mice were anaesthetized and perfused with 4% PFA in PBS. Heads were post-fixed, decalcified, cryoprotected, frozen and sectioned at 12 µm, as described . IHC wasfigure 55.4.Animals were allowed access to food and water ad libitum and were kept on a 12 L : 12 D cycle, with a constant temperature.−1 and 10 mg kg−1 body weight, respectively), and then decapitated. The head was immersed in ice cold Ringer's solution, which contained (in millimolar): NaCl 124, KCl 3, MgSO4 1.3, CaCl2 2, NaHCO3 26, NaH2PO4 1.25, glucose 15; pH 7.6 and 305 mOsm. The pH was kept at 7.4 by bubbling with 95% O2 and 5% CO2. The nose was dissected out en bloc. The olfactory epithelium attached to the nasal septum and the dorsal recess was removed and kept in oxygenated Ringer. Right before starting the recording session, the entire epithelium was peeled away from the underlying bone and transferred to a recording chamber with the mucus layer facing up. Oxygenated Ringer was continuously perfused at room temperature.Patch-clamp recordings were performed as described earlier ,68. Brieatchmaster software . Perforated patch-clamp was performed on the dendritic knobs by including 260 µM nystatin in the recording pipette, which was filled with the following solution (in mM): KCl 70, KOH 53, methanesulfonic acid 30, EGTA 5, HEPES 10, sucrose 70; pH 7.2 (KOH) and 310 mOsm. The junction potential was approximately 9 mV and was corrected in all experiments off-line. For odorant-induced transduction currents, signals were sampled at 20 kHz. Under voltage-clamp mode, the signals were initially filtered at 10 kHz and then at 2.9 kHz.OSNs' dendritic knobs were visualized through an upright microscope equipped with an Olympus DP72 camera and a 40× water-immersion objective. An extra 2-to-4× magnification was achieved by a magnifying lens in the light path. The GFP+ or tdTomato+ labelled cells were visualized under fluorescent illumination. Superimposition of the fluorescent and bright field images allowed identification of the fluorescent cells under bright field, which directed the recording pipettes. Electrophysiological recordings were controlled by an EPC-10 USB amplifier combined with P−5 M for each odorant were prepared before each experiment by adding Ringer. Forskolin, an activator of adenylyl cyclase, was prepared as a 10 mM stock solution in DMSO. IBMX, an inhibitor of phosphodiesterases, was prepared as a 100 mM stock solution in DMSO. Final solution containing 200 µM of IBMX and 20 µM of forskolin was prepared before each experiment by adding Ringer.A seven-barrel pipette was used to deliver stimuli by pressure ejection through a picospritzer . The stimulus electrode was placed approximately 20 µm downstream from the recording site. Distance (approx. 20 µm) and pressure (20 psi) were adjusted in order to minimize mechanical responses . Single Unless specified, all chemicals were from Sigma-Aldrich . Lyral was provided as a generous gift from International Fragrances and Flavors .itmaster (HEKA). Maximum amplitude of the response and kinetics characteristics was measured. Dose–response curves were drafted and fitted using Origin software (OriginLabs). Statistical analysis  were performed using Origin software (OriginLabs).Data were analysed using F5.5.Omp (NM_011010.2), Gnal (NM_177137.4), Adcy3 (NM_001159537.1), Ano2 (NM_153589.2) and Cnga2 (NM_007724.2). For the list of examined ORs and axon guidance molecules, see the electronic supplementary material, table S2. The raw NanoString counts for the various genes are included in the electronic supplementary material.Total RNA extraction from whole olfactory mucosa was performed as described . One mic


We previously characterised C6, a new monoclonal antibody specific for human B-FN and we suggested that it reacts with the B-C loop of the type III repeat 8 which is masked in FN isoforms lacking ED-B and that the insertion of ED-B in FN molecules unmasked it. Here we have now consolidated and refined the characterization of this B-FN specific antibody demonstrating that the epitope recognized by C6 also includes loop E-F of ED-B.Fibronectin (FN) is a large multidomain molecule that is involved in many cellular processes. Different FN isoforms arise from alternative splicing of the pre-mRNA including, most notably, the FN isoform that contains the “extra-domain-B” (ED-B). The FN isoform containing ED-B (known as B-FN) is undetectable in healthy adult tissues but is present in large amounts in neoplastic and foetal tissues as well as on the blood vessels during angiogenesis. Thus, antibodies specific for B-FN can be useful for detecting and targeting neoplastic tissues We built the three dimensional model of the variable regions of the mAb C6 and of the FN fragment EDB-III8 and performed protein:protein docking simulation using the web server ClusPro2.0. To confirm the data obtained by protein:protein docking we generated mutant fragments of the recombinant FN fragment EDB-III8 and tested their reactivity with C6.The monoclonal antibody C6 reacts with an epitope formed by the B-C loop of domain III8 and the E-F loop of ED-B. Both loops are required for an immunological reaction, thus this monoclonal is strictly specific for B-FN but the part of the epitope on III8 confers the human specificity. Fibronectin (FN) is a multi-domain molecule present in the extracellular matrix (ECM) and in body fluids. It is a dimer of two subunits of about 220/250kDa, linked at the C-termini by two disulfide bonds and each monomer consists of three types of repeating units. FN is involved in many cellular processes and different FN isoforms arise from the alternative splicing of its pre-mRNA –2. In paThe FN isoform containing ED-B (B-FN) is undetectable in healthy adult tissues but its expression levels are highly increased in tumour tissues and it accumulates around neovasculature during angiogenesis. This makes it one of the oncologist’s best markers of angiogenesis and neoplastic tissues –8. The dWe have previously described C6, a monoclonal antibody specific for human B-FN . Using vIn immunohistochemistry experiments on human tissue, the mAb C6 behaves exactly as an antibody that reacts directly with the ED-B, as it shows no reaction with healthy adult human tissues but shows a strong reaction with cancer tissue. However, unlike other ED-B specific antibodies, C6 shows no reaction with mouse tumours  and also the loop E-F of ED-B (that confers the specificity for B-FN). In previous works the authors did not take into account the possibility that the epitope could consist of a part of ED-B and a part of III8 –18. ThisConsidering that the mAb C6 interacts at the EDB and III8 interface  to whichin vivo to selectively target tumours. BC-1 recognizes an epitope, localized on the repeat III7, which is hindered in FN molecules lacking ED-B and exposed in FN molecules containing ED-B. The results obtained with BC-1 prompted the generation of recombinant antibodies directly reacting with ED-B [There are other B-FN-specific antibodies, such as BC-1 that was generated over 25 years ago. BC-1  was exteith ED-B –13. Thesith ED-B –13. Howeith ED-B , probablThree dimensional model structures of the scFv C6 was built using the Phyre2 program  and furtEco restriction site, and primer reverse sb-18 (5’-aatgcccggcgccagccctgt-3’) allowing the substitution (E1329-A); 2. FN fragment corresponding to amino acids 1326–1447 with mutation (E1329—A) obtained with primer forward sb-17 (5’-acagggctggcgccgggcatt-3’), allowing the substitution (E1329-A) and complementary to primer sb-18, and the primer reverse sb-9 (5’-ctcgcggccgctcatcatgttttctgtcttcctct-3’) including the Not restriction site and two stop codons. In the second step the two cDNA fragments were PCR assembled with primers TI-147 and sb-9. The obtained cDNA fragment was digested Eco/Not and inserted into Eco/Not digested pProEX-1 . All PCR reactions were performed with high fidelity PWO DNA Polymerase  following the manufacturer’s instruction. The restriction enzymes were from Roche Diagnostics. The DNA construct was used to transform DH5α competent bacteria cells. All FN recombinant fragments were purified from the bacterial lysate on Ni-NTA columns  using the His6 tag at the N-termini of the FN fragments. The purified FN fragments were analyzed by SDS-PAGE as previously described. The reactivity of monoclonal C6 with the FN fragments was assessed by ELISA assay as previously described [The human recombinant FN fragments EDB-III8 as well as its mutant (D1385—E) were previously described . The cDN


Additionally, the mutation is predicted to result in a significant destabilization of helix H8, which should negatively affect glutamate binding. This prediction was tested in HEK293 cells overexpressing GS by dot-blot analysis: Structural stability of H8 was impaired through mutation of amino acids interacting with R341, as indicated by a loss of masking of an epitope in the glutamate binding pocket for a monoclonal anti-GS antibody by L-methionine-S-sulfoximine; in contrast, cells transfected with wild type GS showed the masking. Our analyses reveal complex molecular effects underlying impaired GS catalytic activity in three clinically relevant mutants. Our findings could stimulate the development of ATP binding-enhancing molecules by which the R324S mutant can be repaired extrinsically.Glutamine synthetase (GS) catalyzes ATP-dependent ligation of ammonia and glutamate to glutamine. Two mutations of human GS (R324C and R341C) were connected to congenital glutamine deficiency with severe brain malformations resulting in neonatal death. Another GS mutation (R324S) was identified in a neurologically compromised patient. However, the molecular mechanisms underlying the impairment of GS activity by these mutations have remained elusive. Molecular dynamics simulations, free energy calculations, and rigidity analyses suggest that all three mutations influence the first step of GS catalytic cycle. The R324S and R324C mutations deteriorate GS catalytic activity due to loss of direct interactions with ATP. As to R324S, indirect, water-mediated interactions reduce this effect, which may explain the suggested higher GS residual activity. The R341C mutation weakens ATP binding by destabilizing the interacting residue R340 in the  Glutamine synthetase (GS) catalyzes the ATP-dependent ligation of ammonia and glutamate to glutamine, which makes the enzyme essential for human nitrogen metabolism. Three mutations in human GS, R324C, R324S, and R341C, had been identified previously that lead to a glutamine deficiency, resulting in neonatal death in the case of R324C and R341C. However, the molecular mechanisms underlying this impairment of GS activity have remained elusive. Our results from computational biophysics approaches suggest that all three mutants influence the first step of GS’ catalytic cycle, namely ATP or glutamate binding. The analyses reveal a complex set of effects including the loss of direct interactions to substrates, the involvement of water-mediated interactions that alleviate part of the mutation effect, and long-range effects between the catalytic site and structural parts distant from it. As to the latter, experimental validation is in line with our prediction of a significant destabilization of helix H8 in the R341C mutant, which should negatively affect glutamate binding. Finally, our findings could stimulate the development of ATP-binding enhancing molecules for the R324S mutant, which has been suggested to have residual activity, that way extrinsically “repairing” the mutant. Glutamine synthetase  catalyzes the ATP-dependent ligation of glutamate and ammonia to glutamine –6. GlutaHuman GS belongs to class II of GS enzymes  and formResidue R324, which is mutated to cysteine (R324C) or serine (R324S), is located in the catalytic site and forms an ionic salt bridge with the β-phosphate group of ADP in the crystal structure  22]. It. It22]. In vitro studies on the GS mutant H281A-H284A-Y288A (HHY), predicted to mimic the loss of interactions in the R341C mutant, provide evidence for this influence. These results can semi-quantitatively explain the observed GS deficiencies linked to the three mutations [Here, we investigated changes in GS structure, dynamics, and energetics at the atomistic level due to the three GS mutations R324C, R324S, and R341C by molecular dynamics (MD) simulations, rigidity analysis , 26, andutations –19 and p2+).We performed molecular dynamics (MD) simulations of the wild type GS and the three GS mutants, R324C, R342S, and R341C. Coordinates of human GS were obtained from a crystal structure available from the Protein Data Bank (PDB)  as PDB eAPO), with bound ATP (GSATP), with bound ATP and glutamate (GSATP+GLU), and with bound ADP and GGP (GSADP+GGP). All states were modelled for wild type GS and the three GS mutants R324C, R342S, and R341C. Models of GS mutants were obtained by amino acid exchanges in the wild type dimer using the SwissPDBViewer [Using the dimeric model we investigated the influence of the three mutations on four different states according to the suggested mechanism of glutamine formation : GS withDBViewer . For allS-sulfoximine phosphate (MSO-P), manganese ions (Mn2+), chloride ions, and crystal water [ATP and GSATP+GLU states, ADP was changed to ATP by adding the missing atoms with the LEaP program [et al. [ADP+GGP, ADP coordinates were taken directly from the crystal structure, and hydrogen atoms were added according to the library of Meagher et al [ATP+GLU and GSADP+GGP, glutamate and GGP were manually modelled based on the coordinates of the structurally similar inhibitor MSO-P present in the crystal structure. Structurally bound Mn2+ ions were changed into Mg2+ ions, for which well-validated simulation parameters [2+ ions [APO, GSATP, GSATP+GLU, and GSADP+GGP because the absence of divalent cations leads to a “relaxed” and inactive variant of GS [2+ ion in the case of GSATP and GSATP+GLU because the additional phosphate group of ATP causes clashes in the starting structure. Protonation states of histidines were assigned according to the protonation that was found to be most likely by visually inspecting the histidine environment.The GS crystal structure contains non-covalently bound ADP, the inhibitor L-methionine-al water . For the program  of Amber program  accordin [et al. . In the er et al . To generameters  are avai[2+ ions . Magnesint of GS , 35. NonLEaP program [et al. [respgen of AmberTools 1.4 [et al. [The generated model systems were prepared for MD simulation with the  program  of Amber program . Sodium  program , leaving [et al. . Atomic  [et al. . Geometr [et al.  using thools 1.4 . Angle p [et al. . All oth [et al. , 41.sander module of Amber11 [-1·Å-2 were applied to all protein atoms, ligands, and structurally bound ions within the catalytic site while all other atoms were free to move (500 cycles steepest descent (SD) and 2000 cycles conjugate gradient (CG) minimization). Second, we reduced the harmonic restraints and applied a force constant of 1 kcal·mol-1·Å-2 (2000 cycles SD and 8000 cycles CG minimization). Finally, the positional restraints were removed completely, and all atoms were free to move (1000 cycles SD and 4000 cycles CG minimization).The systems were relaxed by three steps of energy minimization, performed with the  Amber11 . First, -1·Å-2 were applied to all protein atoms, ligands, and structurally bound ions within the catalytic site. Afterwards, the temperature was raised from 100 K to ~300 K during 50 ps of isobaric-isothermal (NPT) MD. Subsequently, the density was adjusted to 1 g·cm-3 during 200 ps of NPT-MD. Finally, the harmonic positional restraints were removed by gradually decreasing the force constant from 1 to 0 kcal·mol-1·Å-2 in six NVT-MD runs of 50 ps length each. In the MD simulations, the particle mesh Ewald (PME) method [The MD simulation procedure started by heating the respective system from 0 K to 100 K in a canonical (NVT) MD simulation of 50 ps length. During this heating step positional restraints of 1 kcal·mol) method –45 was e) method  was applptraj [t-test. P values < 0.05 were considered significant.The 20–100 ns interval of each production run was considered for analysis. The analysis of the MD trajectories was carried out with ptraj  of Amberptraj . The folptraj . We analptraj  using thADP+GGP state that was equilibrated at 300.1 K; in this interval, the RMSD of GS relative to the starting structure remained particularly stable on average (~ 2 Å). In addition, we extracted an ensemble of 400 equally distributed structures from the 20–100 ns interval of MD simulations of the decameric wild type GS in the GSADP+ADP state. Coordinates  were extracted by mm_pbsa.pl [mm_pbsa.pl. This led to two sets of coordinates for dimeric and decameric GS, respectively, that differed only in residue 341. With CNA, thermal unfolding simulations of wild type GS and R341A GS were then performed to identify differences in the GSs’ structural stability [-1 with steps of 0.1 kcal·mol-1 was used [i and j (rcij) vanishes during the thermal unfolding simulation [rcij(wild type GS)–rcij(R341A GS); differences with p < 0.05 according to a Welch test [The Constraint Network Analysis (CNA) approach allows linking biomacromolecular structure, flexibility, (thermo-)stability, and function . To anal_pbsa.pl  of Amber_pbsa.pl . Coordintability . For thiwas used . Stabiliwas used  were themulation . Finallylch test  were conmm_pbsa.pl script [Effective binding energies, i.e., the sum of gas-phase energies plus solvation free energies , 54, forl script  of Amberl script , using tl script , 41 as il script  for the l script , 60, as l script , to adeqATP and GSATP+GLU states of wild type GS and all three mutants R324C, R324S, and R341C. In the case of the GSATP+GLU state, effective binding energy calculations were performed considering glutamate as the ligand, whereas ATP was considered part of the receptor. The effective binding energies were averaged over the respective ensembles.Effective binding energies were computed according to the 1-trajectory MM-PBSA approach, in which snapshots of complex, receptor, and ligand are obtained from MD simulation of the complex . While tG) were calculated by subtracting the effective binding energy of the wild type (ΔGwild type) from the effective binding energy of the mutant ΔGmutant for trajectory {1, 2, 3}  was calculated by error propagation according to SEM{1,2,3} is the SEM for trajectory {1, 2, 3}. The SEM of the relative effective binding energy  and polyclonal antibodies raised against the C-terminus of glutamine synthetase were from Sigma . The monoclonal antibody directed against GS (clone 6) was from Beckton-Dickinson . The monoclonal antibody against GFP (green fluorescent protein), which cross-reacts with the YFP-variant (yellow fluorescent protein), was from Miltenyi-Biotech . Horseradish peroxidase-coupled goat anti-mouse IgG antibodies were from Bio-Rad International . Horseradish peroxidase-coupled goat anti-rabbit IgG antibodies were from Dako . The monoclonal antibody against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was from Biodesign International . Lipofectamine 2000 was from Life Technologies .L-methionine-S-sulfoximine  or were left untreated for 2 h.Human embryonic kidney 293 (HEK293) cells were cultured on Petrie dishes (diameter = 60 mm) in minimal essential medium (MEM) containing Earle´s salt, L-glutamine and 5% fetal bovine serum . HEK293 cells were grown to about 70% confluency before cDNA (2 μg/dish) was introduced by lipofection using Lipofectamin 2000 according to the manufacturer’s instructions. 24 h after transfection, cells were either treated with L-methionine-Human glutamine synthetase was cloned using human liver cDNA and the following primers GS-YFP-for: 5’-CGGAATTCATGACCACCTCAGCAAGTTC-3’ and GS-YFP-rev: 5’-CGGGATCCGCGTAATTTTTGTACTGGAAGG-3’. The forward primer contained an EcoRI restriction site, and the stop codon in the reverse primer was replaced by a BamHI site. The PCR product was cloned into the pEYFP-N1 vector . Mutations were introduced into WT-GS-YFP using the QuikChange Multi Site-Directed Mutagenesis Kit  and the following mutagenesis primers GS-R341A: 5’-GGTTACTTTGAAGATCGTGCCCCCTCTGCCAACTGCG-3’ and GS-SKR: 5’-GGCCATTGAGAAACTAGCCGCGGCGCACCAGTACCACATCC-3’. For the HHY variant the mutations were introduced sequentially using the following primers: GS-H281/84A-for: 5’-GAGAAACTAAGCAAGCGGGCCCAGTACGCCATCCGTGCCTATGATCC-3’ and GS-Y288A: 5’-CAGTACGCCATCCGTGCCGCTGATCCCAAGGGAGGCCTGG-3’. Successful cloning and mutagenesis was verified by sequencing (GenBank accession number: NM_002065).Western-blot analysis was performed as described recently . In brieDot-blot analysis was performed as described recently , 63 by sPeroxidase activity on the membranes was detected using Western-Lightning chemiluminescence reagent plus . Digital images were captured using the Kodak Image Station 4000MM. Signal intensities were measured by densitometric analysis using the Kodak Molecular Imaging Software.2). Cells were washed twice with phosphate-buffered saline before MatTek dishes were mounted on the LSM510-META and analyzed for YFP and Hoechst34580 immunofluorescence.Immunofluorescence analysis was performed by confocal laserscanning microscopy . HEK293 cells were seeded on MaTek dishes  and transfected with cDNA constructs as described above. At the end of the transfection procedure, cells were incubated with Hoechst34580  for 10 min at 37°C in an incubator . P values < 0.05 were considered significant.For statistical analysis, experiments were carried out with three separate HEK293 seedings. Results are expressed as mean values ± SEM and compared using a two-sided Student’s ADP+GGP state were subjected to MD simulations of 100 ns length at T = 300 K to probe the stability of the systems and to validate that the binding site structure does not deteriorate in the model system.In order to perform MD simulations more efficiently and, thus, improve conformational sampling of wild type GS and GS mutants, we established a model system consisting of two adjacent subunits of the GS decamer forming a single catalytic site . The dim90), which is comparable to the value for the GS decamer (see above).First, the RMSD, a measure of structural deviation along the MD trajectories, of all protein backbone atoms relative to the starting structure was analyzed . The RMSSecond, we focused our analysis on the binding site region, i.e., all residues that are within 4 Å of the bound ADP and GGP . These rIn summary, small structural deviations of similar magnitude are found for the core regions, the binding sites, and the ligands of both the dimeric model system and the GS decamer with respect to the starting structures, demonstrating that both systems remain structurally stable over the simulation time. The dimeric model will thus be used to investigate the effects of the GS mutations.APO state . In addition, time series of the distances over the course of the MD simulations did not show the formation of hydrogen bonds between S324 or C324 and the β-phosphate group of ADP or ATP, respectively, in any conformation  and strong (dcut = 2.8 Å) hydrogen bonds. Only hydrogen bonds with a distance < dcut were considered for analyses. The frequency of occurrence of water-mediated hydrogen bonds between R324S and the β- and γ-phosphate groups of ATP is significantly higher than for R324C for both weak ; ATP+GLU state, however ; ATP+GLU state ) as it does in the GSATP state ) ) . Again,  0.1 Å)) . The obs1 and SB2 . These findings suggest that D339 and R341 stabilize R340 as flanking residues. This stabilization counteracts the inherent flexibility of the loop region all three residues are located in (1 and SB2 between the carboxylate oxygens of D339 and the thiol function of C341 is > 4.5 Å (SEM < 0.1) in all states  larger in the R341C mutant (1.00 ± 0.03 Å) than the wild type GS (0.74 ± 0.03 Å)  . In contSADP+GGP , likely SADP+GGP . These rrcij [p < 0.05) destabilization of the C-terminus of the GS dimer model , where biomolecular structures are represented as molecular frameworks  and analrcij , which ssee ref.  for an esee ref.  reveals ATP state, as well as of an H281A/H284A/Y288A triple mutant in the same state (hereafter termed HHY mutant). The HHY mutant serves as a mimic of the R341C mutant because here, as in the R341C mutant, no hydrogen bonds between residue 341 and residues 281, 284, and 288, respectively, can be formed. We computed the probability that residues 266–288 form a loop in the course of the MD simulations  human GS were transiently expressed in HEK293 cells. Expression of human GS-YFP in HEK293 cells was monitored by confocal laser scanning microscopy  and veriGS was also detected in GS-YFP transfected cells by Western-blot using a monoclonal antibody formerly shown not to react with GS when arginine at position 341 was mutated to cysteine (R341C) (15). As shown in HEK293 cells also expressed GS endogenously . HoweverThe results show that wildtype-, as well as R341A-, HHY- and SKR-mutated GS-YFP is efficiently expressed in HEK293 cells by lipofection and that GS-YFP expression levels are not affected by MSO-treatment compared to the respective control. The results also show that wildtype-, HHY- and SKR-mutated but not R341A-mutated GS is recognized by a monoclonal antibody raised against GS  in Western-blot. This is explained by the specificity of a monoclonal antibody, which recognizes only a single epitope, and by the interaction of the paratope of an antibody with an epitope, which is mediated by only about 5 amino acids . TherefoMSO binds non-covalently to the glutamate-binding pocket of GS  thereby As shown by dot-blot analysis using native protein, anti-GS immunoreactivity was significantly diminished by about 50% after MSO-treatment in HEK293 cells transfected with wild type- or SKR-mutated GS but remained unchanged in HEK293 cells expressing HHY-mutated GS compared to untreated controls . In contThe results suggest that MSO binds to the glutamate-binding pocket of wildtype- and SKR-mutated-GS but not to the pocket of HHY-mutated GS.G, ATP state and for glutamate bound to R342S, R342C, and R341C in the GSATP+GLU state. The average drift in the effective binding energy ΔGwildtype of glutamate binding to the GSATP+GLU state over the last 80 ns of the MD simulations used for analysis is 0.04 kcal·mol-1·ns-1, as determined by the slope of the least-squares line of best fit from a correlation analysis  but not in R324S GS   GS mutant by ΔΔl mol-1) . Conside  of wild type GS and the three mutants, respectively. The replicate MD simulations allow probing for the influence of the starting conditions and determining the statistical significance of the computed results [Initially, we established a model system consisting of two adjacent subunits of the GS decamer forming a single catalytic site for performing MD simulations in a computationally efficient way. During 100 ns of MD simulations, small structural deviations  from the analysis because this loop was identified to be highly mobile in a previous study [2+ ion at the metal binding site n2, which interacts with the nucleotide [-1 with respect to wild type GS  less favorable for the R341C mutant than for wild type GS   . We theronstant) .MSO binds non-covalently to the glutamate binding pocket of GS and irreversibly inactivates the enzyme  thereby In order to validate that MSO masks an epitope recognized by the monoclonal anti-GS antibody, protein samples were denaturated, which was shown to release MSO from GS  and to r6-(6-aminohexyl)-ATP-agarose and anti-GS Western-blot analysis. Both endogenous and overexpressed wildtype- or HHY-mutated YFP-GS were detected in precipitates of ATP-binding proteins from HEK293 cell lysates Click here for additional data file.


Caenorhabditis elegans. The immune effects of Se(IV) were investigated by examining the responses of C. elegans to Pseudomonas aerugonisa PA14 strain.Selenium (Se) is an important nutrient that carries out many biological processes including maintaining optimal immune function. Here, inorganic selenite (Se(IV)) was evaluated for its pathogen resistance and potential-associated factors in C. elegans showed increased survival under PA14 infection compared with untreated controls. The significant pathogen resistance of Se(IV) on C. elegans might not be attributed to the effects of Se(IV) on PA14 as Se(IV) showed no effect on bacterial quorum-sensing and virulence factors of PA14. This study showed that Se(IV) enhanced the expression of a gene pivotal for the innate immunity in C. elegans. The study found that the pathogen-resistant phenotypes contributed by Se(IV) was absent from the skn-1 mutant worms. Moreover, Se(IV) influenced the subcellular distribution of SKN-1/Nrf in C. elegans upon PA14 infection. Furthermore, Se(IV) increased mRNA levels of SKN-1 target genes (gst-4 and gcs-1).Se(IV)-treated C. elegans against P. aeruginosa PA14 infection by exerting effects on the innate immunity of C. elegans that is likely mediated via regulation of a SKN-1-dependent signaling pathway.This study found evidence of Se(IV) protecting  Dietary selenium (Se), an essential trace mineral, is involved in several key metabolic activities via selenoproteins that are essential for human health to protect against oxidative damage and to maintain optimal immune function Dietary Se and selenoproteins are not only important for initiating or enhancing immunity, but also involved in immuno-regulation, which is crucial for preventing excessive responses that may lead to autoimmunity or chronic inflammation Pseudomonas aeruginosa, is an opportunistic human pathogen commonly causing nosocomial contamination in medical care facilities P. aeruginosa may also cause serious infection in immunocompromised, HIV, and cancer patients, resulting in morbidity and mortality P. aerugonisa is regulated by secreted virulence factors which include secondary metabolites  and bacterial enzymes such as elastase and alkaline protease P. aerugonisa also adopts the biofilm mode of growth to make the bacteria recalcitrant to antibiotic treatments and to increase pathogenesis P. aerugonisa produces both cell-associated biofilm and extracellular virulence factors regulated by two well-defined quorum-sensing systems, namely lasIR system and rhlIR system, which are cell-to-cell communication systems of the bacteria The ubiquitous bacterium, P. aeruginosa causes lethal infection not only to human, but also to the nematode C. elegans, a powerful model organism for studying developmental biology and host-pathogen interactions C. elegans can be infected with numerous human pathogens and has a high degree of conservation in innate immune signaling pathways of mammals; hence, it has been widely employed to examine virulence factors, host innate defense mechanism, and drug discovery P. aeruginosa strain PA14 is a clinical isolate that can infect C. elegans; and screening assays of this pathosystem have been established to identify novel virulence factors and innate immune mechanisms P. aeruginosa strain PA14-infected worms. Herein, the protective potential of Se(IV) on C. elegans under P. aeruginosa infection was investigated. In addition, the effects of Se(IV) on the secreted virulence factors, biofilm formation, and quorum sensing of P. aeruginosa were also examined Moreover, immune response gene regulation that may be responsible for the Se(IV)-induced protection of C. elegans against PA14 infection was explored. Finally, the possible regulatory mechanism in Se(IV)-elicited immune response in C. elegans was investigated.In view of the effects of Se on immune systems, it was hypothesized that pretreatment with Se(IV) can improve the survival of 2SeO3, Se(IV)) was purchased from Sigma-Aldrich . Strains used in this study were Bristol N2 (wild-type); EU1, skn-1 (zu67); LD1, IdIs7  pha-1 (e2123); and denEx2[lys-7::GFP + pha-1(+)] C. elegans strains used in this work were provided by the Caenorhabditis Genetics Center. C. elegans was normally maintained and assayed (unless otherwise stated) at 20°C on nematode growth medium (NGM) agar plates using E. coli OP50 bacteria as food source. Pseudomonas aeruginosa strain PA14 (referred hereafter as PA14) was grown with aeration at 37°C overnight in King's B broth (KB broth) Inorganic selenite  600 0.1 as the assay medium containing Se(IV) of various concentrations. The Se(IV)-containing media were then aliquoted 500 µl/ well in a 24 well plate. About 20 worms were transferred to each well and incubated at 20°C; and the survival of the infected C. elegans was scored at 1-day intervals. At least three biological and three technical replicates were performed for each experiment.For PA14 infection, the 600 nm at 24-h incubation. The protease activity was expressed as OD 600 per µg of protein Protease activity was examined by measuring the skimmed milk hydrolysis efficacy of the secreted protease in the bacterial culture 590 nm. At least three biological and three technical replicates were performed for each experiment.Assay of the biofilm-forming ability of the bacteria was adapted from previous studies C. elegans preparation, wild-type C. elegans was treated similar to that described in previous sections except that the nematodes were collected on the first day of PA14 infection. For bacteria preparation, PA14 was cultured in KB broth without or with Se(IV) (0.01 µM) overnight at 37°C. Subsequently, the overnight PA14 cultures were centrifuged for 10 min at 1,500×g to collect the pellet. Total RNA from C. elegans or PA14 pellet was isolated using TRIzol according to manufacturer's instructions . cDNA was synthesized using Super-Script III First-strand synthesis super-Mix for qRT-PCR according to manufacturer's instructions (Invitrogen). The qRT-PCR reaction was performed on a Step One real-time cycler  using a SYBR Green qPCR kit . mRNA levels were normalized to that of ACT-1 for C. elegans and 16S rRNA for PA14, respectively. Primer sequences used for qRT-PCR are listed in C. elegans samples or untreated PA14 sample. At least three biological and three technical replicates were performed for each experiment.For lys-7::GFP) was cultured as described in previous sections. SAL105 cultures without or with Se(IV) (0.01 µM) were fed with OP50 or PA14, respectively, at 20°C for 24 h. The expressions of LYS-7 were directly measured by observing the reporter green fluorescent protein (GFP) fluorescence. Fluorescence in 20 nematodes randomly selected from each set of treatments was analyzed using microscopy Synchronized L1-stage transgenic SAL105 (E. coli OP50 bacteria at 109 cells/mL without or with Se (IV) (0.01 µM) for 72 h at 20°C. Subsequently, LD1 was fed with OP50 or PA14 for 5 h Synchronized L1-stage transgenic LD1 (SKN-1::GFP) p<0.05 (*), p<0.01 (**) or p<0.001 (***) (see figures).Statistical analysis of data was performed using SPSS, version 17.0 . Results are expressed as the mean ± standard error of mean (SEM). The statistical significance of differences between the experimental groups was analyzed by one-way ANOVA and LSD post hoc test. Statistical differences were indicated at C. elegansC. elegans was further explored. To evaluate the effects of Se(IV), P. aerugonisa strain PA14 was used as the target pathogen. Wild-type N2 synchronized L1 larvae were pretreated with various concentrations of Se(IV) for 72 h at 20°C followed by PA14 infections for 72 h. C. elegans against P. aeruginosa PA14 infection.Previous research indicated that trace amount of Se(IV) exerts beneficial effects on development, reproduction, cholinergic signaling, neuroprotection, and oxidative stress defense in lasI, lasR, rhlI, and rhlR)  in the culture medium (P. aeruginosa PA14.A possible mechanism that contributes to decrease the death of wild-type N2 nematodes following PA14 exposure is the inhibition of virulence factors by Se(IV) treatment. Therefore, the effects of Se(IV) on the expression of quorum-sensing genes and virulence factor genes of PA14 were examined. In all concentrations tested (0.01–1 µM), no adverse effect on PA14 growth was observed (data not shown). qRT-PCR analysis showed that 0.01 µM Se(IV) did not affect the mRNA levels of quorum-sensing genes (nd rhlR) . Moreovee medium . These r600 nm. Se(IV) treatment slightly decreased the total protease activity of pathogen-secreted enzyme by about 10%  were examined. The effect of Se(IV) on biofilm formation was studied by crystal violet staining. The results showed that Se(IV) did not inhibit the biofilm formation of PA14 . Moreovebout 10% . Taken tC. elegans against PA14 pathogenicity was examined. Three early response genes: irg-1 (infection response gene 1), hsf-1 (heat shock factor 1), and C29F3.7 (CUB-like domain) lys-1 (lysozyme-like protein), spp-1 (saponin-like protein), and abf-1  E. coli OP50, the standard laboratory food for C. elegans, served as the uninfected control to investigate the effects of Se(IV) under normal diet. Under normal diet, the mRNA levels of all tested immune-related genes, except irg-1 and hsf-1, were not significantly altered by Se(IV) compared with those of the control fed with OP50 (Ctrl + OP50) (C29F3.7 (Ctrl + PA14), were significantly suppressed by 20% to 80% compared with that of the uninfected group (Ctrl + OP50) (C. elegans.Whether immune response gene regulation may be responsible for the Se(IV)-induced protection of  + OP50) . In addi + OP50) . Noticea + OP50) . Taken tlys-7::GFP) was employed to observe the effect of PA14 infection and Se(IV) on lys-7 expression. It has been reported that PA14 suppresses C elegans immunity by repressing the expression of the lysozyme-like LYS-7 Swietenia macrophylla on the expression of lys-7 was previously described C. elegans, indicating decreased expression of lys-7 compared with that in the uninfected C. elegans  on immune systems, transgenic strain SAL105 ( elegans . Furtherlys-7 expression was not affected by Se(IV) under normal diet (E. coli OP50) but Se(IV) prevented the decrease of lys-7 gene expression during PA14 infection  mutant in response to Se(IV) was examined. Unlike the wild-type N2 C. elegans  treatment for 3 days at 20°C followed by PA14 infection compared with no treatment -treated worms under OP50 diet -treated group compared with the untreated group under PA14 infection (C. elegans could be attributed to Se(IV)-triggered SKN-1 nuclei translocation in intestinal cells.SKN-1 is a transcription factor playing multiple essential roles; and localization of SKN-1 in nuclei is an essential prerequisite for activating transcription of target genes, such as P50 diet . WithoutP50 diet , indicatnfection . Taken tE. coli OP50 diet, Se(IV) did not significantly affect the mRNA levels of both gst-4 and gcs-1 (Ctrl + OP50 vs. Se + OP50)   (gst-4 and gcs-1 under PA14 pathogen infection.To elucidate whether the increase in PA14 pathogen resistance described above was due to Se(IV) regulating SKN-1-dependent gene expressions, the mRNA levels of glutathione-S-transferase (GST-4) and gamma-glutamine cysteine synthetase (GCS-1) in response to Se(IV) treatment and PA14 infection were examined. Under normal  + OP50) . Upon PAp<0.001) . Moreovep<0.001) . Taken tC. elegansC. elegans against P. aeruginosa infection was further investigated. The C. elegans/P. aeruginosa slow-killing infection model was employed to test the ability of Se(IV) to induce immune responses in C. elegans. In addition, antibacterial potential of Se(IV) against the pathogenic P. aeruginosa strain PA14 was examined. The results showed that trace amount of Se(IV) protects wild-type N2 C. elegans against P. aeruginosa PA14 infection  exerts multiple beneficial effects on development, reproduction, cholinergic signaling, neuroprotection, and oxidative stress resistance in nfection , suggestC. elegans under Se(IV) treatment (Ascophyllum nodosum) and water extract of red seaweed Chondrus crispus (CCWE) have been shown to enhance host C. elegans immunity by suppressing the mRNA levels of quorum sensing and the virulence factors of PA14 C. elegans survival by limiting P. aeruginosa pathogenicity through the repression of several quorum-sensing genes Syzygium aromaticum, Terminalia chebula Retz., and Haliclona spp.) was shown to improve protections of C. elegans against pathogen infections C. elegans and PA14 were examined in the present study. Se(IV) did not affect the growth of PA14 (data not shown). qRT-PCR analysis showed that 0.01 µM Se(IV) did not affect the mRNA levels of the tested quorum-sensing genes  and virulence factor genes  of PA14  treatment  (Ctrl + PA14) were significantly suppressed 20% to 80% compared with that of the uninfected group (Ctrl + OP50) (C. elegans were treated with 0.01 µM Se(IV) (C. elegans by enhancing the immune responses of C. elegans. These similar effects were observed in water extracts from the brown seaweed (Ascophyllum nodosum) and red seaweed Chondrus crispusC. elegans carrying lys-7::GFP compared with that of uninfected C. elegans, whereas Se(IV) treatment enhanced the GFP fluorescence intensity to a level comparable to that of uninfected C. elegans   without M Se(IV) , suggest elegans . This siC. elegans, several conserved signal transduction pathways including the mitogen-activated protein kinase (MAPK) pathways, insulin/IGF-like signaling (IIS), and TGF-β pathways are involved in the immune responses C. elegansC. elegans pathogen resistance, suggesting SKN-1 as a regulator of the innate immunity C. elegans, whether Se(IV) is linked to SKN-1 activity in immune responses was examined. C. elegans, the enhanced survival against PA14 infection by Se(IV) was not observed in skn-1 deletion mutant  significantly up-regulated the SKN-1 target genes: gst-4 and gcs-1 gene expression under PA14 infection  protects C. elegans from P. aeruginosa infection via SKN-1.To further validate that SKN-1 is necessary for the pathogen resistance induced by Se(IV), the effect of Se(IV) on accumulation of SKN-1 in intestinal nuclei was examined . Exposurnfection , suggestC. elegans TRXR-1, an ortholog of the human thioredoxin reductase-1, was shown not to directly protect C. elegans from oxidative stress C. elegans selenoprotein. A recent study indicated that TRXR-1 is involved in Se(IV) regulating oxidative stress resistance in C. elegansC. elegans requires further elucidation.There has been increasing evidence suggesting that selenoproteins play important roles in regulating inflammation and immunity, providing important insight into mechanisms by which Se influences inflammation and immunity C. elegans obtained evidence supporting a beneficial effect of Se(IV) in host immune regulation. The findings revealed Se(IV) protecting C. elegans against P. aeruginosa PA14 infection by exerting effects on the innate immunity of C. elegans but having no direct effects on bacterial quorum-sensing and virulence factors. Se(IV) was also found to enhance the expression of a gene pivotal for the innate immunity in C. elegans. Finally, mechanistic study indicated that the protective effects of Se(IV) is likely mediated via regulation of a SKN-1/Nrf-dependent signaling pathway by inducing the expression of the target genes (gst-4 and gcs-1), thereby enhancing immune resistance on C. elegans against P. aeruginosa PA14 infection. These findings advance the understanding of the regulatory mechanism of Se in immune systems of intact organisms.In conclusion, the present study using the model animal Table S1Sequences of primers used for real time PCR.(DOC)Click here for additional data file.