Drosophila melanogaster and related species are known to be attracted to fermenting fruit for feeding and egg-laying, which poses the question of whether species-specific fly odours contribute to long-range premating communication.Mate finding and recognition in animals evolves during niche adaptation and involves social signals and habitat cues. D. melanogaster with a dual affinity to sex and food odorants. Female flies release a pheromone, (Z)-4-undecenal (Z4-11Al), that elicits flight attraction in both sexes. Its biosynthetic precursor is the cuticular hydrocarbon -7,11-heptacosadiene , which is known to afford reproductive isolation between the sibling species D. melanogaster and D. simulans during courtship. Twin olfactory receptors, Or69aB and Or69aA, are tuned to Z4-11Al and food odorants, respectively. They are co-expressed in the same olfactory sensory neurons, and feed into a neural circuit mediating species-specific, long-range communication; however, the close relative D. simulans, which shares food resources with D. melanogaster, does not respond to Z4-11Al.We have discovered an olfactory channel in The Or69aA and Or69aB isoforms have adopted dual olfactory traits. The underlying gene yields a collaboration between natural and sexual selection, which has the potential to drive speciation. Sexual communication subserves mate-finding and ultimately reproduction, which relies on finding mates and food for offspring. Volatile pheromones transmit species-specific messages over a distance to facilitate mate finding, which is particularly adaptive in short-lived insects , 2. FollPheromones are released into an atmosphere that is filled with environmental, habitat-related odorants, some of which manifest mating sites and food sources. The response to sex pheromones and food or habitat olfactory cues (kairomones) falls under sexual and natural selection, respectively. Pheromones and kairomones are always perceived as an ensemble in a natural context, leading to an interaction of sexual and natural selection during adaptive divergence of sexual signalling, which is thought to facilitate premating reproductive isolation –12.Drosophila melanogaster, but volatile pheromones that elicit flight attraction and encode species-specific mate recognition have not been found. Drosophila is attracted to yeast and fruit odorants for feeding, mating and egg-laying [Drosophila species, which raises the question of whether they account for species-specific communication [Olfactory sexual communication is studied at cellular and molecular resolution in the fruit fly g-laying –15 and tg-laying , 16. Forg-laying –20. Percg-laying , 21, 22.nication –26.D. melanogaster and D. simulans [Z,Z)-7,11-heptacosadiene , which is perceived through gustatory receptors [Drosophila uses, in addition, volatile pheromone signals that mediate specific mate recognition at a distance.Interspecific matings between the sibling species simulans , or otheeceptors –31. 7,11D. melanogaster, which greatly expands our understanding of Drosophila social communication. A pair of Ors, feeding into the same neural circuit, has developed a dual affinity to this pheromone and to environmental semiochemicals, encoding adult and larval food. A blend of this pheromone and a food odorant specifically attracts D. melanogaster, but not the close relative D. simulans. This becomes an excellent paradigm for studying the interaction of social signals and habitat olfactory cues in premating reproductive isolation and phylogenetic divergence.We have identified the first long-range, species-specific pheromone in Drosophila pheromone research has been on cuticular hydrocarbons and cVA, which are active during close-range courtship [D. melanogaster flies in a glass aeration apparatus and found 16 aliphatic aldehydes, according to chemical analysis by gas chromatography-mass spectrometry (GC-MS). Males and females shared saturated aldehydes with a carbon chain length of C7 to C18, but mono-unsaturated aldehydes were released by females only -4-undecenal (Z4-11Al) and synthesized.A focus in ourtship , 24. Ournly Fig. ; Table 1Z4-11Al was present also in cuticular extracts of females, albeit in lower amounts  than in headspace collections . Cuticular profiles of Drosophila flies have been investigated, but Z4-11Al or other aldehydes have not been reported [GC-MS analysis showed that reported , 32, 33.D. simulans did not release Z4-11Al, nor other monounsaturated aldehydes -7,11-heptacosadiene  [Z4-11Al and (Z)-4-eicosenal  , 29. Thinal Fig. . This wanogaster , 26 aldenogaster , 35.TablD. melanogaster . A weaker response from ab4A neurons to Z4-11Al probably reflects the sensitivity of Or7a (expressed in ab4A) to aldehydes such as the leaf volatile (E)-2-hexenal [Next, single sensillum electrophysiological recordings (SSR) from all basiconic, trichoid, coeloconic, and intermediate olfactory sensilla in ter Fig.  and gas ter Fig.  showed t-hexenal –38.Z4-11Al. In the D. melanogaster strains Canton-S and Zimbabwe, the monoterpene (R)-carvone elicited the strongest response from ab9A, although the response to Z4-11Al was not significantly different. In D. simulans, Z4-11Al elicited a significantly lower response than (R)-carvone  empty neurons [Z4-11Al.The Or69a gene encodes two proteins, Or69aA and Or69aB Fig. , in mostosophila , 41. Amisensilla . Heterol neurons  producedZ4-11Al seem structurally different at first glance, yet they share a carbonyl functional group with an equidistant double bond in position 4 as the structural motif -carvone, which elicited the strongest Or69aB response, at a strain energy cost of only 1.4 kcal/mol. Or69aA, on the other hand, is tuned to terpenoid alcohols and responded significantly less to Z4-11Al. The most active ligands (S)-α-terpineol and (S)- and (R)-linalool, which again share the functional group and a double bond in position 4, align at 3.0 kcal/mol -linalool [D. melanogaster and its sister species D. simulans co-occur in the same habitat and use partially overlapping food resources. The very rarely occurring hybrid matings are sterile; the antagonistic interaction between pheromone and food stimulus might therefore be adaptive and point towards a contributing role of Or69a in reproductive isolation.Moreover, an admixture of linalool   transgenic fly lines to verify that OSNs expressing Or69a sense ted Fig. .Z4-11Al is a powerful attractant that enables sexual communication and specific mate recognition at a distance. Its heterospecific role with respect to the sister species D. simulans was conserved in blends with the food attractant linalool.In summary, D. melanogaster, wherein twin Ors expressed in the same OSN population simultaneously sense pheromone and food odorants. Z4-11Al is the first species-specific, long-range sex pheromone of D. melanogaster. It is produced by females and perceived by Or69aB in both sexes. The precursor of Z4-11Al is the cuticular hydrocarbon 7,11-HD  of the fly brain, where third-order neurons partially overlap and integrate [Z4-11Al and linalool  and Dalby-HL   strains Z4-11Al. Canton-S/UAS TeTx (Bloomington #28838 and 28997) and Canton-S/Or69a-Gal4 (Bloomington #10000) were used as parental controls.We used the Or69a-Gal4/UAS TeTx, tetanus toxin knockout line to verify the role of Or69a in flight attraction to 2 and sexed under a dissecting microscope. Virgin flies were identified by the presence of meconium, and were kept together with flies of the same sex. Flies were kept in 30-mL Plexiglas vials with fresh food. Experiments were performed with 3- to 5-day-old flies.Flies were reared on a standard sugar-yeast-cornmeal diet at room temperature (19–22 °C) under a 16:8-h light:dark photoperiod. Newly emerged flies were anesthetized under COZ4-11Al and (E)-4-undecenal were synthesized (see below). Commercially available compounds were (R)-carvone , (S)-carvone , (S)-terpineol , (S)-linalool , (R)-linalool , citronellol , geraniol , 3-octanol , decanol , and undecanal .2O were obtained from a solvent purification system  and used in the reactions when dry conditions were needed. All other chemicals were used without purification. Reactions were performed under an Argon atmosphere unless otherwise stated. Flash chromatography was performed on straight-phase silica gel  employing a gradient technique with an increasing concentration (0–100%) of distilled ethyl acetate in distilled cyclohexane. In cases of very polar products, chromatography was continued with ethanol in ethyl acetate (0–20%). Thin-layer chromatography was performed to monitor the progress of the reaction on silica gel plates , using ethyl acetate (40%) in cyclohexane as an eluent, and plates were developed by means of spraying with vanillin in sulfuric acid and heating at 120 °C. The purity of the product was checked with GC analysis on a Varian 3300 GC instrument equipped with a flame ionization detector (FID) using a capillary column Equity-5  as the carrier gas and a split ratio of 1:20). The oven temperature was programmed at 50 °C for 5 min followed by a gradual increase of 10 °C/min to reach a final temperature of 300 °C. An Agilent 7890 GC equipped with a polar capillary column FactorFOUR vf-23 ms  was coupled to an Agilent 240 ion-trap MS detector for separation of some isomeric intermediates. The injector was operated in split mode (1:20) at 275 °C and a helium flow rate of 1 mL/min and a transfer line temperature of 280 °C. The analyses were performed in the external ionisation configuration. Electron ionisation spectra were recorded with a mass range of m/z 50–300 at fast scan rate. NMR spectra were recorded on a Bruker Avance 500  spectrometer using CDCl3 as solvent and internal standard.Dry THF and dry EtZ)-4-11Al was synthesized via a modified version of Wube et al. [Z-isomer and a 93% yield over two steps. Stereoisomeric purity was controlled with NMR and GC-FID by comparing the analysis for acid and ester, the appearance of a small quartet, in the NMR spectra, at 1.96 indicates the presence of E-isomer. Gas chromatographic separation on a polar Varian factorFOUR vf-23ms of Z- and E-ester proved that the stereochemistry was not affected by the acidic conditions during esterification. Methyl (Z)-4-undecenoate was purified on regular silica gel and on silver nitrate impregnated silica gel to obtain a stereoisomeric purity of 98.6%. Methyl (Z)-4-undecenoate was reduced to (Z)-4-undecenol with lithium aluminium hydride in diethylether and oxidized to Z4-11Al with Dess-Martin periodinane in dichloromethane. and the organic solvent was evaporated. The remaining water phase was extracted with Et2O (3 × 20 mL), the obtained organic phases discarded, and the basic aqueous phase was acidified with HCl (2 M) until pH 1 and extracted with Et2O (3 × 20 mL). The combined organic phases were dried over MgSO4 (anhydr.) and the solvent evaporated off. The obtained crude product was dissolved in pentane, cooled at –18 °C and filtered to remove the precipitated OPPh3 followed by evaporation of the solvent to result in 0.547 g of a yellow oil (87.5% yield). 1H-NMR: 5.52–5.30 , 2.35 , 2.04 , 1.96 , 1.37–1.19  and 0.89  ppm. The NMR data is in accordance with data previously reported [Z:E ratio of 80:20, which is supported by GC-MS analysis on a Varian factorFOUR vf-23ms column. The obtained crude product was used in the next step without further purification.NaHMDS  was added dropwise, over 30 min, to a suspension of (3-carboxypropyl)triphenylphosphonium bromide  in THF (25 mL). The mixture was stirred for 2 h then cooled to 0 °C on an ice/water bath, and heptanal  in THF (2.5 mL) was added slowly over 15 min. The mixture was stirred for 5 h at 0 °C then allowed to reach room temperature overnight. The reaction was quenched with Hreported , 67. TheZ)-4-11Al , as synthesized above, was dissolved in methanol (15 mL) and seven drops of concentrated H2SO4 were added followed by heating at 70 °C overnight. The mixture was allowed to reach room temperature and the methanol was evaporated and the remaining crude product was dissolved in Et2O (15 mL). The organic phase was washed with H2O (3 × 10 mL) and brine (2 × 10 mL), dried over Na2SO4 (anhydr.) and solvent evaporated, resulting in 0.547 g of a yellow oil (92.8% yield). GC-MS (FactorFour vf-23ms) showed a Z:E ratio of 80:20. 1H-NMR(CDCl3): 5.4 , 3.67 , 2.3 , 2.03 , 1.96 , 1.33–1.21  and 0.89  ppm (no data found in the literature). 13C-NMR(CDCl3): 134.2, 119.9, 32.3, 31.9, 29.5, 29.3, 27.43, 22.7 and 14.1 ppm; 13C-NMR data are in accordance with the literature [Z:E ratio between the diastereomers. Enrichment of the Z-isomer on AgNO3 (10%) impregnated silica resulted in 63 mg of a 98.6:1.4 Z:E ratio product according to GC-FID analysis on the vf-5 column as the diastereoisomeric purity was not possible to measure when using 1H-NMR.(terature . Proton Z)-4-undecenoate  was dissolved in Et2O (5 mL) and LiAlH4 (2 spatula tips) was added followed by stirring at room temperature for 30 min. HCl  was added to quench the reaction and the mixture was extracted with Et2O (2 × 3 mL), the combined organic layer was dried over MgSO4 (anhydr.) and solvent was evaporated. Purification with flash chromatography on SiO2 resulted in 37 mg. 1H-NMR(CDCl3): 5.43–5.32 , 3.67 , 2.16–2.10 , 2.08–2.02 , 1.69–1.60 , 1.39–1.22  and 0.89  ppm. NMR data were similar to that of Kim and Hong [Methyl (and Hong . DiasterZ)-4-Undecenol  in DCM (3 mL) was added to Dess–Martin periodinane  in DCM (0.5 mL). After 50 min, NaOH  was added to quench the reaction. The two layers were separated and the aqueous phase was extracted with Et2O (3 × 10 mL), the combined organic layers were washed with NaOH , dried over MgSO4 (anhydr.) and solvent was evaporated resulting in 30 mg of a yellow oil (81% yield). The crude product was purified with Kugelrohr distillation at boiling point , resulting in 17 mg. 1H-NMR(CDCl3): 9.77 , 5.48–5.22 , 2.47 , 2.37 , 2.04 , 1.37–1.23  and 0.88  ppm. 13C-NMR(CDCl3): 202.1, 131.8, 127.0, 43.9, 31.8, 29.5, 29.0, 27.2, 22.6, 20.1 and 14.1 ppm; both 1H- and 13C-NMR data were in accordance with published results [Z:E ratio, the E-isomer could not be detected by 1H-NMR.-4-undecenoic acid in a 56% yield (87.5% of the E-isomer). (E)-4-undecenoic acid was esterified under the same conditions as the (Z)-acid, without isomerisation of the double bond (according to GC-FID and 1H-NMR). The methyl-(E)-4-undecenoate was reduced to the alcohol with lithium aluminium hydride in diethyl ether and purified on silver nitrate-impregnated silica gel to obtain a purity of 99.8% of the (E)-isomer, which was oxidized with Dess-Martin periodinane in dichloromethane to obtain (E)-4-undecenal.A modified version of Virolleaud’s metathesis  was used2O (50 mL) and filtered through a short pad of silica gel. The product was purified with flash chromatography by gradient elution (0–100% EtOAc in c-hexane followed by 0–10% EtOH in EtOAC) resulting in 0.52 g of oil (56% yield). 1H-NMR(CDCl3): 5.51–5.33 , 2.41 , 2.32 , 2.04 , 1.97 , 1.37–1.22  and 0.88  ppm. The relation between the proton at 2.04 and 1.97 reveals a 87.5:12.5 E:Z ratio. The isolated product was used in the next step without further purification.4-Pentenoic acid  and 1-octene  were dissolved in DCM (50 mL), Grubbs II catalyst  was added and the reaction was refluxed. After 7 h, a second portion of Grubbs II catalyst  was added and the reaction refluxed for a further 16 h. The reaction was allowed to reach room temperature and the solvent was evaporated. The obtained crude product was dissolved in EtE)-4-undecenoic acid  was dissolved in methanol (25 mL), a catalytic amount H2SO4 was added and the mixture was refluxed overnight. After evaporation of the solvent, the crude product was dissolved in Et2O (10 mL) and washed with H2O (20 mL). The aqueous phase was extracted with Et2O (2 × 25 mL), the combined organic layer was washed with H2O (20 mL) and brine (20 mL), dried over MgSO4 (anhydr.) and evaporation of solvent resulted in 0.439 g (78% yield). 1H-NMR(CDCl3): 5.51–5.33 , 3.67 , 2.40–2.27 , 1.96 , 1.38–1.21  and 0.88  ppm. Purification with flash chromatography resulted in 0.401 g (71.7% yield). GC-FID showed the same stereoisomeric ratio as for the acid above. was added to methyl (E)-4-undecenoate  dissolved in Et2O (5 mL). After 30 minutes, HCl  was added to quench the reaction. The acidic water phase was extracted with Et2O (3 × 10 mL) and the combined organic layers were dried over MgSO4 (anhydr.) and evaporation of solvent resulted in 0.104 g (99% yield). Enrichment of the E-isomer with medium pressure liquid chromatography on AgNO3 (10% impregnated) silica resulted in 30 mg of a clear oil (>99.8% E). 1H-NMR(CDCl3): 5.43 , 3.65 , 2.08 , 1.97 , 1.63 , 1.35–1.21  and 0.88  ppm. 13C-NMR(CDCl3): 134.4, 131.3, 129.4, 62.6, 32.6, 32.5, 31.7, 29.6, 29.5, 28.9, 28.8, 22.6 and 14.1 ppm. All NMR data were in accordance with previously published data [LiAlHhed data .E)-4-undecen-1-ol  in DCM (4 mL). NaOH  was added after 1 h to quench the reaction. The aqueous phase was extracted with Et2O (3 × 10 mL) and the combined organic layers were dried over MgSO4 (anhydr.); evaporation of the solvent resulted in 30 mg (98% yield). Purification of the crude product with Kugelrohr distillation at 65 °C (2 mbar) resulted in 10 mg of product . 1H-NMR(CDCl3): 9.76 , 5.50–5.36 , 2.48 , 2.33 , 1.97 , 1.32–1.19  and 0.87 . 13C-NMR (CDCl3): 202.5, 132.2, 127.6, 43.6, 32.5, 31.7, 29.4, 28.8, 25.2, 22.6 and 14.1 ppm. The NMR data were in accordance with previously published data [Dess-Martin periodinane  was added to , D. melanogaster (Canton-S), or D. simulans, unmated females or unmated males (n = 5 for each) were placed in a glass aeration apparatus designed for collection of airborne pheromones (effluvia) [Groups of 20 flies, 3- to 5-day-old, ffluvia) . The fliD. melanogaster females for 5 min in 400 μL hexane containing 100 ng heptadecyl acetate. After 5 min, the extracts were transferred to Francke vials and concentrated to 10 μL before analysis. Fly extracts and volatile collections were stored at –20 °C.Cuticular extracts (n = 5) were obtained by placing 20 n = 3).Oxidation of 7,11-HD was analysed by dropping 100 ng of synthetic 7,11-HD into a 1.5-mL glass vial at 19 °C. Vials were rinsed with 10 μL of hexane, which contained 100 ng heptadecyl acetate as an internal standard, after 15, 30, 45, 60 and 75 min (f = 0.25 μm) or DB-wax , that were temperature-programmed from 30 °C to 225 °C at 8 °C/min. Helium was used as mobile phase at 35 cm/s. The MS operated in scanning mode over m/z range 29–400. Compounds were tentatively identified based on their mass spectra and Kovats retention indices, using custom and NIST (Agilent) libraries, followed by comparison with authentic standards. Each series of GC-MS runs is preceded by blank runs, including solvent, glassware and air filters.Samples were analysed by combined GC-MS . The samples (2 μL) were injected (injector temperature 225 °C) splitless (30 s) into the fused silica capillary columns (60 m × 0.25 mm) coated with HP-5MS UI -4-undecenal (released at 10 ng/min), (R)-linalool (10 ng/min) and the blend of (Z)-4-undecenal and (R)-linalool .Upwind flight behaviour was observed in a glass wind tunnel (30 × 30 × 100 cm). The flight tunnel was lit diffusely from above, at 13 lux, and the temperature ranged from 22 °C to 24 °C and relative humidity from 38% to 48%, and charcoal filtered air, at a velocity of 0.25 m/s, was produced by a fan . Compounds were delivered from the centre of the upwind end of the wind tunnel via a piezo-electric micro-sprayer , 76. Forn = 40).Mated 4-day-old males of the Or69aRNAi/OrcoGal4 line and the respective parental fly lines were used. Since the transgenic fly lines produced fewer offspring, all flies were used, instead of discarding individuals eclosing during the night, which may have mated. Unmated and mated wild-type flies did not show a significant difference in the response rate to 10 ng/min Z4-11Al  [Or69aA and Or69aB receptors were cloned from antennae of   . BrieflyOr69aA and Or69aB were amplified with unique 5’ primers and a common 3’ primer, reflective of genomic structure at this locus. These primers encompass the entire open reading frame of the receptor variants, and are located partially upstream and downstream of the start and stop codons. PCR amplicons were gel-purified and cloned into the pCR8/GW/Topo-TA Gateway entry vector  according to standard procedure, with vector inserts sequenced to confirm fidelity of Or sequence. Or inserts were subsequently transferred to pUAS.g-HA.attB  with LR D. melanogaster flies. Using the PhiC31 targeted genomic-integration system [Drosophila Stock Number: 24749). For expression of single receptor variants in the empty neuron system, Or69a transgenes were crossed into the Δhalo background to give genotype w; Δhalo/Cyo; UAS-DmelOr69a(A or B), and these flies were crossed to flies with genotype w; Δhalo/Cyo; DmelOr22a-Gal4, as described previously [Δhalo; UAS-DmelOr69a(A or B)/DmelOr22a-Gal4.Mini-prep purified pUAS.g-HA.attB plasmids with Or69aA or Or69aB insert were delivered to Best Gene Inc.  for generation of transgenic n system , vectorseviously . ExperimDrosophila Stock Number: 32233). The Or69aB transgene was crossed into the DmelOr22a-Gal4 line in Δhalo background to give genotype UAS-DmelOr69aB; Δhalo/Cyo; DmelOr22a-Gal4; these flies were crossed to flies with genotype w; Δhalo/Cyo; UAS-DmelOr69aA. Experimental electrophysiology assays were performed on flies with genotype UAS-DmelOr69aB/w; Δhalo; UAS-DmelOr69aA/DmelOr22a-Gal4.For co-expression of Or69aA and Or69aB in the same empty neurons, a second fly-line with Or69aB was generated with Or69aB present on the X-chromosome. The same UASg-HA.attB:Or69aB plasmid generated previously was injected into the fly strain y,w, P{CaryIP}su(Hw)attP8 -carvone, (S)-terpineol and (R)-linalool, using default settings . The assumed bioactive conformations of the conformationally more flexible compounds, Z4-11Al and (R)-linalool, were based on the position of the shared functional groups in the conformationally more restricted compounds, (R)-carvone and (S)-terpineol. The carbonyl and the double bond atoms were kept fixed during minimisation of the proposed bioactive conformation of Z4-11Al; the alcohol functional group and the double bond were kept fixed in (R)-linalool. Strain energies, the energy cost for adopting proposed bioactive conformations, were then calculated as the difference between the lowest energy conformations and the assumed bioactive conformation.MacroModel version 11.0  in the Maestro Version 10.4.017 were used to build, minimize and perform conformational analysis of D. melanogaster (Canton-S strain) males, under a binocular at 1000× magnification. Further recordings were made from small basiconic ab9 sensilla, in D. melanogaster (Canton-S and Zimbabwe strains) and in D. simulans males, and from large basiconic ab3 sensilla in mutant D. melanogaster, where Or69aA and Or69aB were heterologously expressed (see above).SSR were performed as described earlier . Unmated3 solution. The recording electrode was introduced with a DC-3 K micromanipulator equipped with a PM-10 piezo translator  at the base of the sensilla. The reference electrode was inserted into the eye. The signal from OSNs was amplified with a probe , digitally converted by an IDAC-4-USB (Syntech) interface, and analysed with Autospike software v. 3.4 (Syntech). Neuron activities were recorded during 10 s, starting 2 s before odour stimulation. Neuron responses were calculated from changes in spike frequency, during 500 ms before and after odour stimulation.Tungsten electrodes  were electrolytically sharpened with a saturated KNO2) and kept in Pasteur pipettes. The test panel contained the most active ligands known for Or69a [Odorants were diluted in redistilled hexane; 10 μg of test compounds in 10 μL hexane were applied to filter paper  between the flame ionization detector and the antenna. Towards the antenna, the GC effluent capillary passed through a Gerstel ODP-2 transfer line that tracked the GC oven temperature, into a glass tube (30 cm × 8 mm ID), where it was mixed with charcoal-filtered, humidified air .Generalized linear models with a Bernoulli binomial distribution were used to analyse wind tunnel data. Landing at source and sex were used as the target effects. Post hoc Wald pairwise comparison tests were used to identify differences between treatments. For all the electrophysiological tests, differences in spike activity derived from SSRs were analysed with the Kruskal–Wallis H test followed by pairwise comparisons with the Mann–Whitney U post hoc test. All statistical analyses were carried out using R (R Core Team 2013) and SPSS Version 22 (IBM Corp).


IA) subunit Kv4.2 expression and increases IA densities, in part by activating the insulin receptor signaling pathway. Molecular mechanisms of neuritin-induced Kv4.2 expression remain elusive. Here, we report that the Ca2+/calcineurin (CaN)/nuclear factor of activated T-cells (NFAT) c4 axis is required for neuritin-induced Kv4.2 transcriptional expression and potentiation of IA densities in cerebellum granule neurons. We found that neuritin elevates intracellular Ca2+ and increases Kv4.2 expression and IA densities; this effect was sensitive to CaN inhibition and was eliminated in Nfatc4−/− mice but not in Nfatc2−/− mice. Stimulation with neuritin significantly increased nuclear accumulation of NFATc4 in cerebellum granule cells and HeLa cells, which expressed IR. Furthermore, NFATc4 was recruited to the Kv4.2 gene promoter loci detected by luciferase reporter and chromatin immunoprecipitation assays. More importantly, data obtained from cortical neurons following adeno-associated virus-mediated overexpression of neuritin indicated that reduced neuronal excitability and increased formation of dendritic spines were abrogated in the Nfatc4−/− mice. Together, these data demonstrate an indispensable role for the CaN/NFATc4 signaling pathway in neuritin-regulated neuronal functions.Neuritin is an important neurotrophin that regulates neural development, synaptic plasticity, and neuronal survival. Elucidating the downstream molecular signaling is important for potential therapeutic applications of neuritin in neuronal dysfunctions. We previously showed that neuritin up-regulates transient potassium outward current ( IA) channels at both transcriptional and translational levels. However, the molecular mechanism underlying transcriptional induction of Kv4.2 by neuritin is unknown.Neuritin  is an important neurotrophin expressed during the development of the nervous system , 3. FurtIA channels play a critical role in dampening neuronal excitability and action potential (AP) firing frequency in a wide variety of neurons. In CGNs and hippocampal and cortical neurons, IA channels are encoded by pore-forming α-subunits of the Kv4 subfamily  and depolarization-induced nuclear accumulation of neuronal NFAT (A2 and GABRA4) by direct transcriptional co-regulation via binding to specific responsive elements in their promoters , consisting of NFATc1–4, is the key downstream effector of CaN signaling , 13. Upo16–nal NFAT , 19. Furromoters . Thus, iIA current density in mice CGNs. To test our hypothesis, Nfatc2−/− and Nfatc4−/− double knock-out (DKO) mice were used in this study. In addition, the subsequent effect of neuritin-induced Kv4.2 expression on cortical neuronal excitability was also explored with adeno-associated virus (AAV)-mediated overexpression of neuritin in Nfatc4−/− mice. Our study, for the first time, reveals a calcium/CaN/NFATc4 signaling pathway in neuritin-mediated up-regulation of neuronal Kv4.2 expression and examines its role in modulating neuronal excitability in mouse.The goal of this report was to investigate the effect of the calcium/CaN/NFATc4 pathway on neuritin-induced Kv4.2 expression and 2+/CaN/NFATc4 pathway plays a role in neuritin-induced IA densities, we first employed cyclosporin A (CsA) and FK520 to inhibit CaN, and CsA and FK520 were applied for 2 h prior to initiating treatment with neuritin and then remained in the culture during the entire duration (24 h) of neuritin treatment. IA was evoked by 200 ms of depolarization to +40 mV from a holding potential of −100 mV in the presence of 20 mm triethanolamine, which suppresses IK and permits better resolution of IA. The IA densities after incubation with 150 ng/ml neuritin for 24 h were significantly increased by 22.42 ± 1.39%  compared with the control group, whereas in the presence of 5 μm CsA or 0.2 μm FK520 (IA density was reduced to −8.37 ± 2.47% (n = 31) and 5.78 ± 5.63% (n = 37) for CsA and FK520, respectively (A). There was no significant difference compared with the corresponding control.To examine whether the Caμm FK520 , the neuectively A. There B and C). CsA at 5 μm or FK520 at 0.2 μm significantly reduced the induction of Kv4.2 mRNA from 31.54 ± 1.71% (n = 5) for neuritin alone to 5.94 ± 3.47% (n = 5) and 1.87 ± 3.28% (n = 5) for CsA and FK520, respectively (C). The induction in Kv4.2 protein was also significantly reduced from 38.40 ± 3.30% (n = 10) for neuritin alone to −6.07 ± 2.84% (n = 10) using CsA and to −4.45 ± 2.79% (n = 10) using FK520 (B). These data indicate that CaN contributes to neuritin induction of Kv4.2 expression and its subsequent potentiation of IA densities. By using the one-way ANOVA analysis, the data presented in Fig. 1C showed there was significant difference between control and CsA, but there was no significant difference between CsA and CsA with neuritin. Moreover, there was no significant difference between control and FK520. These phenomena suggested that CsA may affect the transcriptional expression of Kv4.2 under basal conditions by no calcineurin-dependent pathway.Inhibition of CaN with CsA and FK520 also abrogated the neuritin-induced increase in Kv4.2 mRNA and protein expression measured by RT-PCR and Western blotting, respectively , B and Cectively C. The inng FK520 B. These 2+ concentration and CaM activity is a prerequisite for CaN activation. Thus, we examined the intracellular Ca2+ ([Ca2+]i) level in CGNs following neuritin stimulation using the Ca2+-sensitive fluorescent dye fura-2 acetoxymethyl (AM). In vitro calcium imaging showed that neuritin increased the [Ca2+]i level in mouse CGNs at both basal and high K+ conditions (D). Under basal conditions, neuritin induced an increase in the Ca2+ concentration from 79.19 ± 1.32 nm (n = 34) to 108.40 ± 1.23 nm . Using a high K+ solution (27 mm KCl) to depolarize neurons and activate voltage-gated Ca2+ channels caused a rapid elevation in [Ca2+]i. Following incubation of CGNs with neuritin for 15 min, depolarization with high K+ induced an increase in the Ca2+ concentration that was enhanced from 115.70 ± 3.72 nm (n = 32) to 172.60 ± 1.85 nm . Similar to CaN inhibition, blocking CaM function using calmidazolium chloride (CMZ) also abrogated the induction of IA densities, Kv4.2 mRNA, and protein expression by neuritin (A–C). IA densities, Kv4.2 mRNA levels, and protein expression induced by neuritin were reduced from 22.42 ± 1.39% (n = 43), 31.54 ± 1.71% (n = 5) and 38.40 ± 3.30% (n = 8), respectively, to 0.32 ± 2.34% (n = 42), −7.18 ± 3.19% (n = 5), and −9.59 ± 5.27% (n = 8), respectively, in the presence of CMZ. Together, these data suggest that neuritin stimulation increases intracellular Ca2+ levels in CGNs, which activate CaN for the induction of Kv4.2 transcription and IA densities.Increases in intracellular Canditions D. Under neuritin , A–C. IA2+/CaN pathway  and 22.60 ± 5.12% , respectively, compared with the control group (A). By contrast, induction of Kv4.2 by neuritin was abolished in CGNs isolated from Nfatc4−/− or DKO mice (A). The expression of Kv4.2 protein in CGNs isolated from Nfatc4−/− or DKO mice was changed by only −7.05 ± 5.56% (n = 3) and −3.83 ± 4.56% (n = 3), respectively, with no significant difference compared with control. Based on these data, we thus exploited Nfatc4−/− mice to directly examine whether NFATc4 was required for the neuritin-induced increase in Kv4.2 expression and IA densities.Members of the transcription factor NFATc family, consisting of NFATc1–4, are the key downstream effectors in the Ca pathway . Because neurons , 23, we ol group A. By conDKO mice A. The exIA densities recorded by patch clamp and Kv4.2 mRNA levels measured by quantitative RT-PCR were not induced by neuritin in Nfatc4−/− mouse CGNs (B and C). In Nfatc4−/− mice, CGN IA densities and Kv4.2 mRNA levels after treatment with neuritin were changed minimally by −11.97 ± 7.13% (n = 26) and 7.14 ± 3.07% (n = 3), respectively. These data indicate that NFATc4 is required for the induction of Kv4.2 expression and increases in IA densities by neuritin. We simultaneously confirmed the lack of NFATc4 expression in the cerebellum and PFC of Nfatc4−/− mice by Western blotting. The results indicated that neither cerebellum nor PFC neurons in Nfatc4−/− mice expressed NFATc4 (D).Correspondingly, use CGNs , B and Cd NFATc4 D.168 and Ser170, and facilitates nuclear accumulation of NFATc4  compared with control cells (A). Meanwhile, either CaN inhibition or blocking neuritin signaling via the IR inhibitor hydroxy-2-naphthalenylmethyl phosphonic acid (HNMPA)  and −4.80 ± 4.46% (n = 3), respectively (A). These data indicate that activation of CaN by neuritin promotes dephosphorylation of NFATc4.CaN dephosphorylates key Ser residues in the NFAT homology domain of NFATc4, including Serf NFATc4 . Thus, wol cells A. Meanwh (HNMPA) , 25 abolectively A. These B). Static analysis indicated that the percentage of NFATc4 translocation in HeLa cells was significantly increased by 1125.99 ± 1.43%  following neuritin stimulation . In the presence of 5 μm CsA or 100 μm HNMPA, the percentage of NFATc4 nuclear translocation stimulated by neuritin was reduced to 13.07 ± 11.24% (n = 8) and 13.47 ± 16.62% (n = 9), respectively, which were not significantly different from the percentage of translocation when neuritin was applied alone.Next, we examined the localization of NFATc4 in HeLa cells by confocal microscopy because they express IR activated by neuritin  and haveFig. 3n = 4, p < 0.01), and cytoplasmic NFATc4 was decreased by 27.59 ± 4.40%  following neuritin stimulation, whereas this effect was blocked by CsA and HNMPA (D and E). In the presence of 5 μm CsA or 100 μm HNMPA, the percentage of NFATc4 translocation to nuclear stimulated by neuritin was reduced to −3.08 ± 5.68% (n = 4) or −11.8 ± 6.86% (n = 4), respectively. On the other hand, neuritin-induced decrease of cytoplasmid NFATc4 was reduced to 2.74 ± 5.73% (n = 4) or −1.43 ± 4.68% (n = 4), respectively, and this is not significantly different from the cells treated with neuritin alone. Together, these data indicate that neuritin activates and translocates NFATc4 into neuronal nuclei.Nuclear and cytoplasmic protein extraction and Western blotting analysis in mice CGNs also indicated that nuclear NFATc4 was increased significantly by 35.89 ± 6.33% . Induction of Kv4.2 promoter activity by neuritin, however, was abrogated by 5 μm CsA or 100 μm HNMPA, reducing expression to 15.90 ± 3.61% (n = 6) and −3.57 ± 1.61% (n = 6), respectively, which were not significantly different from corresponding controls. In addition, deletional mutagenesis of the NFAT enhancer to −1513 bp on the Kv4.2 promoter (promoter Δ) also abolished neuritin-induced Kv4.2 promoter activity . These data indicate that neuritin-induced Kv4.2 transcription is NFAT-dependent.A study showed that NFAT plays an important role in electroexcitability in the heart by regulating Kv4.2 transcription in cardiomyocytes . Indeed,B). Kv4.2 promoter DNA encoding the NFAT enhancers was detected in NFATc4 precipitates, whereas neither downstream NFAT enhancers nor the 5′-untranslated region of the Kv4.2 gene was detected. Together, these data confirm that neuritin regulates Kv4.2 gene transcription via the CaN/NFATc4 pathway.We further ascertained recruitment of NFATc4 to the Kv4.2 gene loci by ChIP assays B. Kv4.2 IA channels, which dampen neuronal excitability and action potential firing frequency  and 24.94 ± 7.27% , respectively, after incubation of cortical neurons with 150 ng/ml neuritin for 24 h (A and B). Next, we employed AAV9-mediated transduction to express exogenous neuritin following injection into the prefrontal cortex (PFC) (0.2 μl/mouse). Western blotting analysis indicated that expression of neuritin was increased by 117 ± 3.89%  after viral expression of exogenous neuritin in the PFC for 2 weeks (C).To test this hypothesis, we first ascertained whether neuritin induces similar potentiation of for 24 h , A and B 2 weeks C.n = 45, p < 0.001; Fig. 6A), whereas the latency of the first AP, the injected current to evoke the first AP, and the interevent interval increased significantly by 182.38 ± 22.62% , 47.94 ± 4.24% , and 89.34 ± 5.24% , respectively, in native control mice (B–D). Changes in neuronal excitability following neuritin overexpression, however, were abrogated in the Nfatc4−/− mice. The AP firing frequency, the latency of the first AP, the injected current to evoke the first AP, and the interevent interval did not differ significantly in Nfatc4−/− mice with and without neuritin overexpression (A–D). These data indicate that neuritin modulation of neuronal excitability in the mouse PFC is NFATc4-dependent.We then measured the AP from layer II–III of pyramidal neurons in mouse PFC. Overexpression of neuritin significantly decreased AP firing frequency by 57.17 ± 6.22%  in native control mice upon neuritin expression (A and B). Cumulative frequency distributions also showed significantly increased spine lengths after neuritin treatment  analyzed using a one-way Kruskal-Wallis test. The median and quartiles in the control group and the neuritin-treated group were 0.992  and 0.957 , respectively (C). However, the effect of neuritin overexpression on the increase in spine density and its length was abrogated in the Nfatc4−/− mice (A–C), in which the median and quartiles of Nfatc4−/− mice treated with neuritin were 1.00 . In addition, the effect of neuritin on the number of neurite branches and lengths of the first order and second order apical dendrites in native control and Nfatc4−/− mice was analyzed with Sholl and two-way ANOVA analysis. Neuritin increased the number of neurite branches neither in the control mice nor in Nfatc4−/− mice (D), and the dendrite lengths of the first order and second order were not significantly different between the four groups (E and F). Taken together, these data indicate that neuritin signals through the Ca2+/CaN/NFATc4 pathway and modulates neuronal excitability and formation of dendritic spines in vivo.We also examined whether the increased formation of dendritic spines by neuritin reported previously  requirespression , A and Bectively C. Howeve−/− mice , A–C, in−/− mice D, and thr groups , E and FIA in rat CGNs by increasing mRNA and protein expression of Kv4.2 via the IR pathway , and CMZ were obtained from Sigma. FBS, DMEM, and antibiotic-antimycotic solution were purchased from Invitrogen, Thermo Fisher Scientific. HNMPA was purchased from Santa Cruz Biotechnology .Recombinant human neuritin was purchased from Pepro Tech . Poly-l-lysine (10 μg/ml) or on coverslips in Petri dishes coated with poly-l-lysine (50 μg/ml) at a density of 105 cells/cm2. Cultured cells were incubated at 37 °C under 5% CO2 in DMEM supplemented with 10% FBS, insulin (5 μg/ml), KCl (5 mm), and 1% antibiotic-antimycotic solution. Cytosine β-d-arabinofuranoside (5 μm) was added to the culture medium 24 h after seeding to inhibit the proliferation of non-neuronal cells. The cells were used for experiments after 2–3 days in culture unless otherwise indicated. HeLa cells were maintained as a monolayer in high glucose DMEM, supplemented with 10% FBS and 1% antibiotic-antimycotic solution.CGNs were derived from cerebella of 7-day-old C57BL/6 mouse pups  as described previously . IsolateIA current recording, the culture medium was replaced with a bath solution containing 125 mm NaCl, 2.5 mm KCl, 10 mm HEPES (pH 7.4), 1 mm MgCl2, 0.001 mm tetrodotoxin, 20 mm triethanolamine, and 10 mm glucose. Soft glass recording pipettes were filled with an internal solution containing 135 mm K+-gluconate, 10 mm KCl, 10 mm HEPES (pH 7.3), 1 mm CaCl2, 1 mm MgCl2, 10 mm EGTA, 1 mm ATP, and 0.1 mm GTP. The pipette resistance was 4–6 MΩ after filling with internal solution.Whole cell currents of CGNs were recorded at room temperature using a conventional patch clamping technique . Prior t2 and 5% CO2) artificial cerebral spinal fluid containing 125.0 mm NaCl, l 2.5 mm KC, 2.5 mm CaCl2, 1.5 mm MgSO4, 26.2 mm NaHCO3, 1.0 mm NaH2PO4, and 11.0 mm glucose at room temperature for at least 1 h. Layers II/III of medial pyramidal neurons in the PFC were visualized using infrared differential interference contrast video microscopy with a 40× water immersion objective, and images were captured with a charge-coupled device camera. Patch pipettes (5–8 MΩ) were filled with 150.0 mm K+-gluconate, 0.4 mm EGTA, 8.0 mm NaCl, 2.0 mm ATP, 0.1 mm GTP-NH3, and 10.0 mm HEPES (pH 7.3–7.4) at 280 ± 5 mOsm for the recording.Coronal brain slices (200 μm) containing PFC were prepared using standard methods . The sliMice were anesthetized with pentobarbital sodium (50 mg/kg) and decapitated. The brains were removed immediately. Coronal sections (200 μm) were made at the level of the prefrontal cortex by vibratome . The prefrontal region was then subdissected with a dissecting microscope. After subdissection, the prefrontal tissues were moved into ice-cold Eppendorf tubes and homogenized with a grinding rod on ice.m HEPES, 150 mm NaCl, 0.5% Nonidet P-40, 10% glycerol, 2 mm EDTA, 100 μm Na3VO4, 50 mm NaF, pH 7.5, and 1% protease inhibitor mixture) on ice for 30 min. After centrifugation at 13,800 × g for 15 min, the supernatant was mixed with 2× SDS loading buffer and boiled for 5 min. A nuclear and cytoplasmic protein extraction kit  was used for nuclear and cytoplasmic protein extraction following the instructions of the kit as previously described . The Kv4.2 primer sequence was 5′-TGTCAGGAAGTCATAGAGGCAGCGTG-3′ (forward) and 5′-GGGGTGGTTACTGGAGGTGTTGGAAT-3′ (reverse). The sequence of housekeeping gene cyclophilin D, used as a control to exclude sampling errors, was 5′-GGACGTCTGTCTTCGAGTCC-3′ (forward) and 5′-AACAGACCGTGGAGATTTGG-3′ (reverse). The annealing temperature was set at 58 °C for Kv4.2 and 61 °C for cyclophilin D, with 38 amplification cycles for each product. The absolute mRNA levels in each sample were calculated according to a standard curve set up using serial dilutions of known amounts of specific templates against corresponding cycle threshold (Ct) values. The normalized ratio of Kv4.2 to cyclophilin D in each group was presented. The specificity of the primers was verified by both gel electrophoresis and sequencing of the PCR products.As previously described , TIANGEN2+ levels were measured by using single cell fura-2 acetoxymethyl (AM) as described by Grynkiewicz et al.  at 37 °C for 45 min in darkness. CGNs were then rinsed three times in fura-2 AM-free Hanks' balanced salt solution at room temperature. The coverslip was mounted on an open slide chamber , and the chamber was put on an inverted Epi-fluorescence microscope . The excitation wavelengths for fura-2 AM were 340/380 nm with emission at 505 nm. Baseline [Ca2+]i was determined for 60 s prior to the addition of K+ solution (27 mm KCl). The data were collected at 4-s intervals throughout the experiment. Quantification of the fluorescence intensity was performed using MetaFluor software .Intracellular Caz et al. . Brieflyin vitro using a calcium calibration buffer kit (Invitrogen). The calibration results were plotted by double-log of the equation [Ca2+]free = dK × [R − Rmin]/[Rmax − R] × Fmax380/ Fmin380, where R indicates the ratio of 505-nm emission intensity with excitation at 340–505-nm emission intensity with excitation at 380 nm; Rmin indicates the same ratio at zero-free Ca2+; Rmax indicates the ratio at saturating Ca2+; Fmax380 is the fluorescence intensity with excitation at 380 nm for zero-free Ca2+; and Fmin380 is the fluorescence intensity at saturating free Ca2+. After linear fit, dK was acquired from the resulting straight line, and the Ca2+ concentration corresponding to R was calculated by the equation.Calibration of calcium imaging was done m PBS for 15 min. Fixed cells were washed twice in ice-cold PBS and permeabilized with 0.25% Triton X-100 for 10 min. The cells were then washed three times in PBS for 5 min each and blocked in 1% BSA for 30 min. The labeling experiments were performed by incubating cells with antibody against NFATc4  or actin  at 4 °C overnight. After vigorous washing in PBS, the cells were incubated with the corresponding FITC-conjugated goat anti-rabbit IgG  or Cy3-conjugated goat anti-mouse IgG  for 1 h at room temperature. Fluorescence-labeled cells were visualized with a SP2 confocal laser scanning microscope  using a 40× objective lens.Cultured cells were fixed in fresh 4% paraformaldehyde in 0.1 Mouse Kv4.2 promoter (−2181 to +191 bp) and promoter Δ (−1513 to +191 bp) were subcloned into pGL3-Basic luciferase reporter plasmid. HeLa cells were co-transfected with Kv4.2 promoter and β-gal reporter plasmids. Luciferase activity prepared from transfected cell lysates was determined using a luciferase assay system . Luciferase activity was presented as the ratio of luciferase to β-gal activity.ChIP was conducted according to the manufacturer's instructions (Beyotime). Sheared chromatin DNA was immunoprecipitated with the NFATc4 antibody or normal IgG. The antibody-protein-DNA complex was precipitated with protein A+G-agarose and collected for reverse cross-linking after washing. The DNA recovered was subjected to PCR amplification using primers as indicated.Nfatc2−/− and Nfatc4−/− mice . The protocol was approved by the Committee on the Ethics of Animal Experiments of Fudan University (permit 20090614-001). Previously described −/− mice  were matin virus . Virus pin virus , or GolgIdentification of neurons was performed as described previously using Golgi staining . A homogt test for comparison of two samples or between one sample and the hypothetical mean and one-way ANOVA with Fisher's post hoc test for comparisons between multiple groups. Cumulative frequency distributions of spine length were analyzed using a one-way Kruskal-Wallis test followed by the Kolmogorov-Smirnov test for post hoc comparisons. Intersection numbers were analyzed by two-way ANOVA with distance from soma and group as two factors. Fisher's post hoc test was used for mean comparison. The data were presented as individual data points using a scatter plot with n as the number of neurons for electrophysiological recordings, the number of imaging experiments, or the number of replicates for molecular biology experiments. For electrophysiological experiments, data were collected from at least four different batches of neurons prepared at different dates, thereby minimizing bias resulting from culture conditions. A p value of ≤0.05 was considered statistically significant.The results were analyzed using Student's J.-J. Y. and Q.-R. Z. designed, performed, and analyzed the experiments; D.-D. L. provided technical assistance and contributed to the preparation of the figures; and C.-W. C. and Y.-A. M. designed the study and wrote the paper. All authors reviewed the results and approved the final version of the manuscript.


GFP mice, in conjunction with immunohistochemical markers, were utilized to define the spatiotemporal relationship of PDGFRα+ fibroblasts to endothelial, stromal and epithelial cells in both the proximal and distal acinar lung. Complimentary analysis with flow cytometry was employed to determine changes in cellular proliferation, define lipofibroblast and myofibroblast populations via the presence of intracellular lipid or alpha smooth muscle actin (αSMA), and evaluate the expression of CD34, CD29, and Sca-1. Finally, PDGFRα+ cells isolated at each stage of acinar lung development were subjected to RNA-Seq analysis, data was subjected to Bayesian timeline analysis and transcriptional factor promoter enrichment analysis.The following data are derived from key stages of acinar lung development and define the developmental role of lung interstitial fibroblasts expressing platelet-derived growth factor alpha (PDGFRα). This dataset is related to the research article entitled “Temporal, spatial, and phenotypical changes of PDGFRα expressing fibroblasts during late lung development”   •Spatiotemporal and transcriptional analysis of PDGFRα-expressing fibroblasts during acinar lung development.•+ fibroblasts in lung development and disease.The RNA-Seq data provide a vast source to study PDGFRα•+ fibroblast function.The transcription factor promoter enrichment analysis identify specific targets of PDGFRα Specifications Table1GFP E18.5 at  and lipofibroblasts (LipidTOX+) within each population is presented . Cells expressing PDGFRα were analyzed at E16.5, E18.5, P7 and P28. The spatiotemporal localization of PDGFRαE18.5 at  demonstrresented . Fig. 4 elopment , individroblasts , and charoblasts . Additioroblasts , ChIP-Seroblasts , and pro22.1PDGFRα(EGFP)Sortm11/J mouse-line herein designated PDGFRαGFP+ cell RNA-Seq analysis.B6.129S4-2.2Lung tissues were harvested, fixed with 4% PFA in PBS and frozen. Tissue was sectioned into 200 μm slices and stained with anti-αSMA , Pro-SPC and chicken polyclonal anti-GFP antibody . Data was analyzed by Imaris software, version 7.6.2.3GFP mice was harvested, processed into single cell suspension as previously described 2PO4, 8 g/L NaCl, and 1.15 g/L Na2HPO4) containing 0.5 mM EDTA·4Na and 3 mg/L Phenol Red).Lung tissue from PDGFRα2.4RNA-Seq data was quantitated using TopHat and Cufflinks 2.5+ fibroblast RNA-Seq profiles identified three candidate transcription factors: NRSF/REST, CTCF, and MAX. ChIP-Seq has been performed in the following mouse cell lines, and the data available in the public domain:http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSM912908MAX: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSM918744CTCF: https://www.encodeproject.org/experiments/ENCSR000AIS/NRSF/REST: Transcription factor promoter enrichment analysis of PDGFRα+ fibroblasts regulated by NRSF/REST, CTCF, or MAX during acinar lung development, we cross-referenced the dynamically regulated genes identified by our present RNA-Seq analysis with the above, previously-published gene sets To identify potential candidate genes in PDGFRα


Leishmania (V.) braziliensis and Leishmania(L.) amazonensis are the most pathogenic agents of American Cutaneous Leishmaniasis in Brazil, causing a wide spectrum of clinical and immunopathological manifestations, including: localized cutaneous leishmaniasis (LCLDTH+/++), borderline disseminated cutaneous leishmaniasis (BDCLDTH±), anergic diffuse cutaneous leishmaniasis (ADCLDTH-), and mucosal leishmaniasis (MLDTH++++). It has recently been demonstrated, however, that while L. (V.) braziliensis shows a clear potential to advance the infection from central LCL (a moderate T-cell hypersensitivity form) towards ML (the highest T-cell hypersensitivity pole), L. (L.) amazonensis drives the infection in the opposite direction to ADCL (the lowest T-cell hypersensitivity pole). This study evaluated by immunohistochemistry the expression of Toll-like receptors (TLRs) 2, 4, and 9 and their relationships with CD4 and CD8 T-cells, and TNF-α, IL-10, and TGF-β cytokines in that disease spectrum. Biopsies of skin and mucosal lesions from 43 patients were examined: 6 cases of ADCL, 5 of BDCL, and 11 of LCL caused byL. (L.) amazonensis; as well as 10 cases of LCL, 4 of BDCL, and 6 of ML caused byL. (V.) braziliensis. CD4+ T-cells demonstrated their highest expression in ML and, in contrast, their lowest in ADCL. CD8+ T-cells also showed their lowest expression in ADCL as compared to the other forms of the disease. TNF-α+showed increased expression from ADCL to ML, while IL-10+and TGF-β+ showed increased expression in the opposite direction, from ML to ADCL. With regards to TLR2, 4, and 9 expressions, strong interactions of TLR2 and 4 with clinical forms associated with L. (V.) braziliensis were observed, while TLR9, in contrast, showed a strong interaction with clinical forms linked to L. (L.) amazonensis. These findings strongly suggest the ability of L. (V.) braziliensis and L. (L.) amazonensis to interact with those TLRs to promote a dichotomous T-cell immune response in ACL. Leishmania that are widely distributed throughout Latin America ..3].®, Nestle) for 30 min. at room temperature. The slides were subsequently incubated overnight (at 4 °C) with the primary antibodies anti-TNF-α, anti-IL-10, and anti-TGF-β diluted in 1% BSA. The application reaction was performed using a LSAB-HRP kit , and the visualization reactions as well as subsequent steps were performed as previously described  and BDCL [532 ± 65.0 cells/mm2]), and were also greater (p< 0.05) than the forms of the disease caused by L. (L.) amazonensis: LCL, BDCL, and ADCL. It should also be noted that TNF-α+ cell densities in the LCL form (1154 ± 134.0 cells/mm2) caused by L. (V.) braziliensis were greater (p< 0.05) than the BDCL form (532 ± 65.0 cells/mm2) caused by that same species, as well as those of the LCL, BDCL and ADCL forms caused by L. (L.) amazonensis ..L. (L.) L. (L.) amazonensis showed that the TGF-β+ cell densities of the polar ADCL form (1595 ± 189.1 cells/mm2) were not only greater than (p< 0.05) the subpolar BDCL (1098 ± 96.5 cells/mm2) and central LCL (991 ± 165.0 cells/mm2) forms, but were also greater than the three forms of the spectrum of L. (V.) braziliensis: the central LCL (820 ± 69.1 cells/mm2), subpolar BDCL (958 ± 67.9 cells/mm2), and polar ML (708 ± 98.8 cells/mm2) forms. As such, it can be concluded that the polar ADCL/L.a form shows greater (p< 0.05) TGF-β+ cell density than those of the other clinical forms of ACL. Additionally, it should be noted that although there were no significant differences (p> 0.05) between the other two clinical forms caused by L. (L.) amazonensis  forms, both demonstrated greater expressions (p< 0.05) than the centralLCL (820 ± 69.1 cells/mm2) and polar ML (708 ± 98.8 cells/mm2) forms caused by L. (V.) braziliensis. Finally, it should also be observed that the subpolar BDCL form (958 ± 67.9 cells/mm2) caused by L. (V.) braziliensis demonstrated a greater TGF-β+ celldensity (p< 0.05) than the neighboring polar ML form (708 ± 98.8 cells/mm2)  -Fig 2)..L. (L.) L. (L.) amazonensis, the profiles of immunostained cell densities (IL-10+) were very similar to those of TGF-β+; that is, the polar form ADCL (1945± 160.2 cells/mm2) demonstrated greater expression (p< 0.05) than the neighboring subpolar BDCL (1172 ± 79.2 cells/mm2) and central LCL(1136 ± 100.0 cells/mm2) forms, as well as greater expression than the three other forms caused by L. (V.) braziliensis . It can therefore be concluded that, in the same way as seen with TGF-β+, the ADCL form showed the greatest (p< 0.05) IL-10+ cell density when compared to the other clinical forms of ACL. It was also observed that, while no significant differences (p> 0.05) were noted between the BDCL (1172 ± 79.2 cells/mm2) and LCL(1136 ± 100.0 cells/mm2) forms caused by L. (L.) amazonensis, both demonstrated greater (p< 0.05) IL-10+ cell densities than seen in the LCL (735 ± 150.9 cells/mm2) and ML (479 ± 117 cells/mm2) forms caused by L. (V.) braziliensis. However, the BDCL (992 ± 117.1 cells/mm2) form caused by L. (V.) braziliensis showed greater (p< 0.05) IL-10+ cell densities than the congenerous LCL (735 ± 150.9 cells/mm2) and ML (479 ± 117 cells/mm2) forms. Lastly, it is important to emphasize that the ML/L.b form demonstrated the lowest (p< 0.05) IL-10+ cell density among all of the clinical forms of ACL ..L. (L.) +, TGF-β+, and IL-10+ in the different clinical forms of ACL caused by L. (V.) braziliensis and L. (L.) amazonensis showed greater (p< 0.05) expressions of TGF-β+ andIL-10+ cytokines in the clinical forms associated with L. (L.) amazonensis , while TNF-α+ demonstrated greater (p< 0.05) expression in the clinical forms associated with L. (V.) braziliensis  .L. (L.) amazonensis, no differences were observed (p> 0.05) in TLR2+ expression between the central LCL (197 ± 24.6 cells/mm2) and subpolar BDCL (136 ± 45.4 cells/mm2) forms, with only the central LCLform showing greater expression (p< 0.05) than the polar ADCL form (96 ± 19.2 cells/mm2). In terms of the clinical forms caused by L. (V.) braziliensis, on the other hand, it was observed that while the central LCL (433 ± 102 cells/mm2) and subpolar BDCL (310 ± 41.9 cells/mm2) forms demonstrated no significant differences (p> 0.05) in their TLR2+ expressions, both forms showed greater expressions (p< 0.05) than the polar MLform (110 ± 133.0 cells/mm2). More significant than this, however, was the fact that the central LCL/L.b form showed greater expression (p< 0.05) than any of the forms caused by L. (L.) amazonensis , while the subpolar BDCL/L.b form was only greater (p< 0.05) than the ADCL form (In terms of the relative spectrum of ACL caused by DCL form .+) cell densities expressing TLR4+in the different clinical forms of ACL revealed expression profiles very similar to those of TLR2+, showing only some differences in the relative spectrum of ACL caused by L. (L.) amazonensis, with no differences (p> 0.05) among the forms LCL (141 ± 60.1 cells/mm2), BDCL (76 ± 14.9 cells/mm2), and ADCL (79 ± 45.8 cells/mm2). In terms of the spectrum of ACL caused by L. (V.) braziliensis, the only observation to be added here refers to the fact that the subpolar form BDCL (292 ± 125.8 cells/mm2) showed a higher expression (p< 0.05) than both the polar ADCL (79 ± 45.8 cells/mm2) and the subpolar BDCL (76 ± 14.9 cells/mm2) forms, both caused by L. (L.) amazonensis. Additionally, there were no differences (p> 0.05) in expression between the LCL/L.b (399 ± 140 cells/mm2) and BDCL/L.b (292 ± 125.8 cells/mm2) forms, although both were greater (p< 0.05) than the polar ML/L.b form (56 ± 66.1 cells/mm2)  .L. (L.) amazonensis, the polar ADCL form (277 ± 70.4 cells/mm2) demonstrated greater TLR9+ expression (p< 0.05) than the central LCL form (150 ± 48.7 cells/mm2), although no differences were observed (p> 0.05) between the polar ADCL and the subpolar BDCLforms (228 ± 60.1 cells/mm2), which likewise did not demonstrate any significant differences (p> 0.05) when compared to the central LCL form. Additionally, when comparing the spectrum of ACL caused by L. (V.) braziliensis, the subpolar BDCL form (221 ± 47.5 cells/mm2) demonstrated greater TLR9+ expression (p< 0.05) than either the LCL (90 ± 67.9 cells/mm2) or ML (48 ± 38.6 cells/mm2) forms; no significant differences were observed (p> 0.05) between the latter two forms (LCLandML)  .TLR2+ and 4+ with clinical infection by L. (V.) braziliensis, with greater expressions of those receptors in the LCL and BDCL forms, but weaker interactions with clinical infection by L. (L.) amazonensis, with only modest expression in the LCL and BDCL forms. In terms of TLR9+ expression, we identified stronger interactions with clinical infection by L. (L.) amazonensis as opposed to the expressions of TLR2+ and 4+, indicated by greater expression in the ADCL/L.a and BDCL/L.a forms; significant expression of TLR9+ in the BDCL form caused by L. (V.) braziliensis was also noted  braziliensis: 1) TLR9+versus CD4+T-cells: a significant negative correlation was seen only in the LCLform (r = -0.608 p = 0.03) (TLR2+versus TGF-β+: a significant negative correlation was only seen in the LCL form (r = -0.691 p = 0.01) (TLR4+versus TNF-α+: a significant negative correlation was only seen in the ML form (r = -0.862 p = 0.02) (TLR4+versus IL-10+: a significant positive correlation was only seen in the ML form (r = 0.952 p = 0.003) (TLR9+versus IL-10+: a significant positive correlation was only seen in the MLform (r = 0.828 p = 0.04) , the following results will be restricted to only five cases that showed significant correlations (p< 0.05)–all of them related to clinical forms caused only by  = 0.03) ; 2) TLR2 = 0.01) ; 3) TLR4 = 0.02) ; 4) TLR4= 0.003) ; and, 5) = 0.04) .TLR2, 4, and 9 within the context of the entire clinical-immunopathological spectrum of ACL caused by the two Leishmania species with the greatest pathogenic potential for humans [L. (V.) braziliensis and L. (L.) amazonensis] and their relationship with CD4+ and CD8+ T-cells, and TNF-α+, IL-10+ and TGF-β+ cytokines, aiming to better understand the immunopathogenesis and/or the immunopathology of that disease.This is the first study undertaken in Brazil  that analyzed the involvement of TLR2, 4, and 9 were the major interest of the present work, we could not fail to also consider the importance of other immunological factors, such as CD4+ and CD8+ T-cells, and the TNF-α+, IL-10+ and TGF-β+ cytokines that take part in the T-cell immune responses against ACL. In this way, despite there have been some earlier research concerning the participation of CD4+ and CD8+ T-cells in the immunopathogenesis of ACL, no agreement has existed about what cell types are most well-represented in the cell infiltrates found in cutaneous lesions of that disease  to modulate T-cell immune responses in ACL to opposite immunopathogenic poles (such as CD4+/Th1 [L. (V.)braziliensis] or CD4+/Th2 [L. (L.) amazonensis]) ) . Within mination , 19. Thi+, must be considered with a certain caution as it is generally regarded as a pro-inflammatory cytokine with a pivotal role in macrophage activation and parasite control . Similarly, the two regulatory cytokines (TGF-β+andIL-10+) demonstrated strong expression in terms of the infection spectrum caused by L. (L.) amazonensis but weak expression in terms of the infection spectrum caused by L. (V.)braziliensis. This indicates, once again, that the antigenic environment exercises a crucial influence on the functional activities of those cytokines. Corroborating those results, recent work showing a lesser IL-10+expression in the central LCL form caused by L. (V.)braziliensis than in the same LCL form caused by Leishmania (V.) spp. should be mentioned–for it indicates that the functional activity of that cytokine can be differentially influenced in the same clinical form (LCL) caused by different Leishmania species of the same subgenus , however, have not yet been described.In considering the roles of  tropica –46. The TLR2, 4, and 9 are present in all forms of the clinical-immunopathological spectrum of ACL, although with a stronger interaction of TLR2 and 4 with L. (V.) braziliensis, as indicated by the greater expression of those receptors in the LCL and BDCL forms of the disease associated with that species. On the other hand, there appears to be a strong interaction of TLR9 with L. (L.) amazonensis, in light of its greater expression in the ADCL and BDCL forms; there is also significant expression of that receptor in the BDCL form in response to L. (V.) braziliensis. Based on those analyses, and their similarity to observations concerning the expressions of the TNF-α+, TGF-β+, and IL-10+ cytokines, there are also almost dichotomous expressions of TLR2, 4, and 9 in the clinical-immunopathological spectrum of ACL caused by L. (V.) braziliensis and by L. (L.) amazonensis, suggesting the possibility that specific antigens of those Leishmania species may be influencing the modulation of the innate immune response against infection.We demonstrated here that in vitro studies have indicated that the interactions of TLRs with different Leishmania species may lead to different immune responses  indicated support for the results of the present analysis with clinical samples of ACL caused by L. (V.) braziliensis and L. (L.) amazonensis—analyses that indicated greater expression of TLR2 and 4 within the clinical forms caused by L. (V.) braziliensis. In that way, our results also revealed a high expression of TFN-α+ which corroborates previous evidence indicating that the immune response on that side of the disease spectrum is predominantly of the CD4+/Th1-type  have been established, it becomes necessary try to explain those differences. A number of factors can interfere with TLR expression on cell surfaces [TLR2 and 4 and responsible for more than 95% of the surface composition of Leishmania)] have crucial roles in the initial events of Leishmania-macrophage interactions . That may explain the higher interaction of TLR9 with the side of the spectrum associated with L. (L.) amazonensis, principally in those BDCL and ADCL clinical forms.Little has been observed in terms of the role of iliensis . Accordiiliensis , TLR9 hailiensis , TLR9 is DTH+/-) , 19. It tivation . This saL. (V.) braziliensis demonstrated the lowest expressions of TLR2, 4, and 9. This seems to reflect the characteristics of the predominantly lymphoplasmacytic cellular infiltrate present in that form of the disease, with wide numerical supremacy over the macrophages that serve as target cells to be double-marked by the immunohistochemistry of TLR2, 4, and 9 . It is therefore possible that this immunohistochemical strategy may have been influenced the weak expression of those receptors in ML form of the disease. While that clinical form shows low TLR expression, a positive correlation between TLR4 and 9 with IL-10+ was noted, which corroborates the weak IL-10+ expression in the ML/L.b form–which, as was described above, is characterized by strong CD4+/Th1-type immune response and significant IFN-γ and TNF-α expressions [While the current analysis yielded quite satisfactory results, an interesting and somewhat surprising finding must be noted that is related to the fact that the greatest cellular reactivity with the ML form in the clinical-immunopathological spectrum of ACL caused by ressions , 5, 6.S1 Table(PDF)Click here for additional data file.S1 FigL.a) and mucosal leishmaniasis (ML/L.b), compared with the other clinical forms.(*): p< 0.05; (⊢⊣): significant differences found between anergic diffuse cutaneous leishmaniasis (ADCL/(TIF)Click here for additional data file.S2 FigL.a) compared with the other clinical forms.(*):p< 0.05; (⊢⊣): significant differences found between anergic diffuse cutaneous leishmaniasis (ADCL/(TIF)Click here for additional data file.S3 FigL.b) compared with the other clinical forms.(*) = p< 0.05. (⊢⊣) = significant differences found between anergic mucosal leishmaniasis (ML/(TIF)Click here for additional data file.S4 FigL.a) compared with the other clinical forms.(*) = p< 0.05. (⊢⊣) = significant differences found between anergic diffuse cutaneous leishmaniasis (ADCL/(TIF)Click here for additional data file.S5 FigL.a) and mucosal leishmaniasis (ML/L.b), with the other clinical forms.(*) = p <0.05. (⊢⊣) = significant differences between anergic diffuse cutaneous leishmaniasis (ADCL/(TIF)Click here for additional data file.


Cadmium translocation from roots to shoots is a complex biological process that is controlled by gene regulatory networks. Pak choi exhibits wide cultivar variations in Cd accumulation. However, the molecular mechanism involved in cadmium translocation and accumulation is still unclear. To isolate differentially expressed genes (DEGs) involved in transporter-mediated regulatory mechanisms of Cd translocation in two contrasting pak choi cultivars, Baiyewuyueman  and Kuishan’aijiaoheiye , eight cDNA libraries from the roots of two cultivars were constructed and sequenced by RNA-sequencing.10 vs. BCd0 (690), KCd10 vs. KCd0 (2733), KCd0 vs. BCd0 (2919), and KCd10 vs. BCd10 (3455), were identified. Regulatory roles of these DEGs were annotated and clarified through GO and KEEG enrichment analysis. Interestingly, 135 DEGs encoding ion transport  related proteins were identified. The expression patterns of ten critical genes were validated using RT-qPCR analysis. Furthermore, a putative model of cadmium translocation regulatory network in pak choi was proposed.A total of 244,190 unigenes were obtained. Of them, 6827 DEGs, including BCdZIP2, ZIP3, IRT1, HMA2 and HMA4) and tonoplast-localized transport genes  than low Cd cultivar (Kuishan’aijiaoheiye). These genes, therefore, might be involved in root-to-shoot Cd translocation in pak choi.High Cd cultivar (Baiyewuyueman) showed higher expression levels in plasma membrane-localized transport genes  contains supplementary material, which is available to authorized users. Cd is readily transferred from t organs –3. The at organs . To prevt organs , 5; and OsHMA2 function in insertion mutant results in decreased leaf and grain Cd concentrations. OsHMA3 plays a role in the sequestration of Cd into vacuoles in root cells [OsHMA3 expression in the Cd-sensitive Δycf1 mutant can cause a wild-type tolerance [OsNramp1 [OsNramp5 [Several processes are involved in the distribution and translocation of Cd in plant roots –8, incluot cells , 11, andolerance . FurtherOsNramp1  and OsNrOsNramp5 –15 were Arabidopsis [Viola yedoensis [High-throughput cDNA sequencing (RNA-seq) is a recently developed approach to transcriptome profiling that has been used to discover and characterize genes, analyze functional gene variation, and identify and quantify rare transcripts , 17. Usibidopsis , ramie [bidopsis , radish edoensis . In addiedoensis –23. ThesBrassica rapa L. ssp. chinensis) is an important leafy vegetable crop. Variability among pak choi cultivars in Cd concentration has been reported [Pak choi  to obtain assembled unigenes from pak choi root transcriptome; (ii) to investigate the gene expression patterns in the roots of two distinct pak choi genotypes under long-term Cd exposure; and (iii) to identify DEGs involved in Cd-transport regulatory networks.6, 114.5 × 106, 105.8 × 106 and 118.3 × 106 raw reads were generated from two replicate libraries for BCd0, BCd10, KCd0 and KCd10, respectively  were categorized into 56 functional groups consisting of 25 biological process, 21 cellular components and 10 molecular function subcategories Fig. .Fig. 2GeThe sequence similarity search was performed against the KOG databases to obtain the functional annotations of assembled unigenes. 61,894 unigene sequences (25.34%) with significant homology were assigned to 26 KOG categories Fig. . Among tTo further characterize molecular-level gene functions in terms of biological system networks, the assembled unigenes were mapped against the KEGG protein database. 53,311 unigenes with significant matches were assigned to 132 KEGG pathways, of which the top five were ‘ribosome’, ‘carbon metabolism’, ‘biosynthesis of amino acids’, ‘protein processing in endoplasmic reticulum’ and ‘RNA transport’ , KCd10 vs. KCd0 (2733 unigenes), KCd0 vs. BCd0 (2919 unigenes) and KCd10 vs. BCd10 (3455 unigenes) . There were 2239 DEGs that showed similar expression patterns in the two cultivars, while 2076 DEGs showed opposite expression patterns , ‘legionellosis’ (10 unigenes), ‘antigen processing and presentation’ (7 unigenes) and ‘carbon fixation in photosynthetic organisms’ (6 unigenes) etc. , P1B-ATPase , ABC transporters, vacuolar cation/proton exchanger 4 (CAX4), oligopeptide transporter (OPT) families and metal tolerance proteins (MTPs)  and HMA3 were tended to be up-regulated in both cultivars compared to their respective controls. All unigenes encoding CAX4 and cyclic nucleotide gated channels (CNGCs) were up-regulated in Baiyewuyueman and down-regulated in Kuishan’aijiaoheiye compared to their respective controls. Based on pairwise comparisons, members within the some families such as OPTs, ATP synthases, V-type proton ATPase (V-ATPase), P-type ATPase superfamily (P-ATPase), inorganic pyrophosphatase (PPase), cadmium/zinc/copper-transporting ATPase (HMAs) and metal tolerance proteins (MTPs), had considerably variable in expression pattern in the four groups. Moreover, the same member within the same pairwise comparison also had a different expression pattern; for example, two unigenes (c127139_g1 and c107331_g1) encoding a multidrug resistance protein (MRP) were not consistent with the pattern of expression in KCd10 vs. KCd0. In addition, for KCd10 vs. BCd10, all unigenes encoding ZIPs, IRT1, HA6t, NRTs, P-type ATPase superfamily (P-ATPase) and copper transporters (COPTs) were down-regulated. The majority of unigenes belonging to ABC transporters (68.4%) and calcium-binding proteins  were identified as down-regulated between Baiyewuyueman and Kuishan’aijiaoheiye in Cd treatments. Surprisingly, several critical Cd-related transporters such as natural resistance-associated macrophage proteins (Nramps), zinc induced facilitators (ZIFs) and ABC transporter G family member 35 (PDR8) were similar in the four groups.In the four comparisons, 135 DEGs were identified as having high similarity with diverse transporters such as zinc transporters , FeZIP2, ZIP3, IRT1, MRP7, MTP3, COPT5, CAX4, HMA2, HMA3 and HMA4 were identified to relevant to the regulation of Cd translocation. The expression of these genes and their biological functions are listed in Table 10 than in KCd10, and were dramatically up-regulated in BCd10 vs. BCd0. Two strongly up-regulated genes (MTP3 and HMA3) and one significantly down-regulated gene (CAX4) were observed in KCd10 vs. KCd0. Meanwhile, the relative expression of ZIP2, ZIP3, IRT1, COPT5, MRP7, HMA2 and HMA4 were similar between the control and Cd-treatment in Kuishan’aijiaoheiye. The expression patterns from RT-qPCR were similar to those of the RNA-Seq-based gene expression patterns , HMA3 and HMA4 (KCd10 vs. BCd10) did not show consistent expression levels between RT-qPCR and Illumina sequencing data  in KCd10 vs. KCd0. These results suggest that Baiyewuyueman could more effectively activate gene expression under Cd treatment. In addition, 2265 DEGs were specifically regulated by Cd in Kuishan’aijiaoheiye, while only 137 DEGs in Baiyewuyueman  [2+ transport to root cells have been reported [ZIP3, ZIP10, IRT1, OPT3, MRP7, PDR9, CAX4, HMA2 and COPT5, showed higher expression in Cd-treated Baiyewuyueman than in Kuishan’aijiaoheiye. However, other heavy metal transporters including HMA , PDR , MRP2, HKT1 and OPT1, displayed lower expression levels in Baiyewuyueman roots than in Kuishan’aijiaoheiye  , 11, 34,reported , 28. Stureported , 21, 23.reported  identifi, PDR 1, , MRP2, He medium . However2+, Mn2+, Cu2+ and Fe2+ [Arabidopsis [2+ can enter root cells through ZIP transporters, such as ZIP2, ZIP3 and IRT1 [IRT1 has also been described as an Fe transporter protein that is highly expressed in the cortex cells of Arabidopsis roots [IRT1 accumulated higher levels of Cd and Zn than wild-type plants in Arabidopsis [IRT1 also mediates Cd2+ influx into root cells through the plasma membrane of the root epidermis and cortex cells. We found that ZIP2, ZIP3 and IRT1 are expressed at higher levels in Baiyewuyueman than in Kuishan’aijiaoheiye  [Cd is often sequestered in the plant vacuole as Cd-chelate complexes , 49, 50.CAX4 , HMA3 [3X4 [HMA3 , MTP3 [5A3 [MTP3 , 53 and P3 [MRP7 , are invoot apex . Moreove [AtHMA3 , AtMRP7  [AtMRP7 , and AtC [AtCAX4  have beeiye Fig. , implyinonoplast . In Arabn et al.  showed tytosolic . Our res-ATPase) .Taken together, we proposed a schematic model to depict a regulatory network of root-to-shoot Cd translocation in pak choi Fig. . The low10 vs. BCd0), 2733 (KCd10 vs. KCd0), 2919 (KCd0 vs. BCd0) and 3455 (KCd10 vs. BCd10) play significant roles in response to Cd stress. Furthermore, 135 DEGs encoding ion transport related proteins  were identified. Five plasma membrane-localized transport genes  and five tonoplast-localized transport genes , showing higher expression levels in Baiyewuyueman than in Kuishan’aijiaoheiye, might be responsible for cultivar differences in root-to-shoot Cd translocation in pak choi.The current study identified 6827 unigenes as DEGs in pairwise comparisons. Among them, 690 (BCd0) and 10 mg/kg Cd (Cd10) as CdCl2•2.5H2O. The sand (1.2 kg) was filled in each pot (13 cm × 12 cm) and kept moist for 2 weeks prior to the experiment. Seeds were sown directly into pots. The treatments were arranged in a completely randomized design with six replicates (pots).Pot trials were conducted in a growth chamber with 16 h light at 25 °C and 8 h darkness at 16 °C. Based on the previous work [One week after sowing, the seedlings were thinned to six plants per pot. The plants were fertilized daily with 50 ml of nutrient solution as previously described . Root sa0, B2_Cd0, B1_Cd10, B2_Cd10, K1_Cd0, K2_Cd0, K1_Cd10 and K2_Cd10 were constructed and sequenced as previously described [Total RNA of root samples was isolated using Trizol® Reagent (Invitrogen) and purified using the RNeasy Plant Mini kit (Qiagen) according to the manufacturer’s instructions. A total of 3 μg RNA per sample was used for library preparation and sequencing. Eight cDNA libraries named B1_Cdescribed . The libThe raw reads in fastq format were initially processed with in-house Perl scripts. After filtering the adapter sequences, reads containing ploy-N and low-quality bases, clean reads were obtained. These clean reads were then used for further data analysis.The left files (read 1 files) from all libraries/samples were pooled into one large left.fq file, and right files (read 2 files) into one large right.fq file. Transcriptome assembly was performed based on the left.fq and right.fq using Trinity  with minE-value cutoff of 10−5.The assembled unigenes were used in BLAST searches against public databases including NCBI non-redundant protein (Nr) and non-redundant nucleotide sequences (Nt), Protein family (Pfam), euKaryotic Ortholog Groups (KOG), Swiss-Prot, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and Gene Ontology (GO) with an The clean reads were mapped back onto the assembled transcriptome reference sequences by RSEM  (no mism0 (B1_Cd0 and B2_Cd0), BCd10 (B1_Cd10 and B2_Cd10), KCd0 (K1_Cd0 and K2_Cd0) and KCd10 (K1_Cd10 and K2_Cd10). The DESeq R package (1.10.1) was used to identify DEGs between two groups according to the method described by Anders & Huber [P-values were adjusted using the Benjamini and Hochberg’s approach for controlling the false discovery rate. A unigene found by DESeq with an adjusted P-value <0.05 was considered significantly differentially expressed.The DEGs were screened as previously described , 59. Pri & Huber . DESeq pP-value <0.05) [P-value <0.05) [Functional enrichment analysis was performed by the GOseq R packages based Wallenius non-central hyper-geometric distribution (corrected e <0.05)  and KOBAe <0.05) . ClusterActin gene as the internal control gene was calculated using ratio = 2Cτ−ΔΔ. The specific primers for RT-qPCR were designed using Beacon Designer 7.0 software  and are listed in Additional file Total RNA was isolated as described above. First strand cDNA fragments were synthesized using the PrimeScript® RT reagent kit . RT-qPCR was performed on an ABI 7300  using a SYBR Premix EX Taq kit (Takara) in a 20 μl reaction mixtures, containing 2 μl of diluted cDNA, 0.2 μM forward and reverse primer, and 10 μl 2 × SYBR Green PCR Master Mix. The PCR reaction protocol was 95 °C for 30 s, 40 cycles of 95 °C for 5 s and 60 °C for 30 s. The fluorescence was measured via a 65–95 °C melting curve. All reactions were performed in triplicate with each replicate measured three times; the relative expression level of the selected unigenes using the Additional file 1: Table S1. KEGG pathways of the assembled unigenes in pak choi. (XLSX 239 kb)Additional file 2: Table S2. Complete lists of differentially expressed genes in four groups based on pairwise comparisons. (XLSX 6819 kb)Additional file 3: Table S3. The enriched GO terms of up and down-regulated DEGs in four comparisons. (XLSX 153 kb)Additional file 4: Table S4. The significantly enriched pathways of up and down-regulated DEGs in four groups based on pairwise comparison. (XLSX 16 kb)Additional file 5: Table S5. The DEGs involved in transport activities in two pak choi cultivars under Cd stress. (XLSX 84 kb)Additional file 6: Table S6. The specific primer sequences of ten DEGs validated by RT-qPCR analysis. (XLSX 11 kb)


Pseudomonas jessenii C2 and Pseudomonas jessenii H16 were isolated from low-Cu and high-Cu industrially contaminated soil, respectively. P. jessenii H16 displayed significant resistance to copper when compared to P. jessenii C2. Here we describe genome sequences and interesting features of these two strains. The genome of P. jessenii C2 comprised 6,420,113 bp, with 5814 protein-coding genes and 67 RNA genes. P. jessenii H16 comprised 6,807,788 bp, with 5995 protein-coding genes and 70 RNA genes. Of special interest was a specific adaptation to this harsh copper-contaminated environment as P. jessenii H16 contained a novel putative copper resistance genomic island (GI) of around 50,000 bp.The online version of this article (doi:10.1186/s40793-016-0200-8) contains supplementary material, which is available to authorized users. Copper is an essential micronutrient in most organisms and required as a co-factor in biological processes such as redox reactions  , 2. HowePseudomonas spp. are ubiquitous inhabitants of soil, water and plant surfaces belonging to the Gammaproteobacteria. Pseudomonas spp. has an exceptional capacity to produce a wide variety of secondary metabolites, including antibiotics that are toxic to plant pathogens [Pseudomonas jessenii was also found to be an important rhizobacterium conferring protection against a number of soilborne plant pathogens [P. jessenii C2 and P. jessenii H16 were isolated from low-Cu soil and high-Cu soil from an abandoned wood impregnation site in Hygum, Denmark, respectively [P. jessenii H16 was able to grow in medium containing high concentrations of copper, whereas P. jessenii C2 was sensitive to high copper concentrations. Here, we present the genome sequences, a brief characterization and annotation of P. jessenii C2 and P. jessenii H16.athogens , 7. Pseuathogens . P. jessectively . The Hygectively , 10. P. Denmark. The high-Cu site was contaminated almost exclusively with CuSO4 more than 90 years ago [-1, respectively [Bacteria were isolated from replicated soil subsamples (n = 3) and diluted, spread-plated on Pseudomonas-selective Gould’s S1 agar [P. jessenii H16 was able to grow at high concentration of Cu (2 mM) on one-tenth strength LB agar, whereas P. jessenii C2 only grew with up to 0.125 mM Cu.A highly copper contaminated high-Cu soil and a corresponding low-Cu soil were collected (0–20 cm depth) from a well-described Cu gradient field site in Hygum, ectively . Bacteri S1 agar . For eac S1 agar . Two of Strain C2 and H16 were both Gram-reaction negative. Cells of strain C2 and H16 were rod shaped with rounded ends and motile. The cells of C2 were 2.12–2.45 μm  in length compared to 0.49–0.62 μm  in width Fig. . The celBraunschweig, Germany [16: 1ω7c and/or iso-C15: 0 2-OH (36.4 % in P. jessenii C2 and 40.1 % in P. jessenii H16); C18 : 1ω7c (15.3 % in P. jessenii C2 and 10.8 % in P. jessenii H16) and C16 : 0 (28.8 % in P. jessenii C2 and 34.6 % P. jessenii H16).Fatty acid analyses were performed by the Identification Service of the DSMZ,  Germany . The fatBiochemical properties were tested using API 20NE (BioMérieux) for Strains C2 and H16. In the API 20NE system, positive reactions for both strains were observed for nitrate reduction and production of arginine dihydrolase; negative reactions were observed for indole production, urease activity, Lysine and ornithine decarboxylase and gelatin hydrolysis  with sequence similarities of 99.87 and 99.14 %, respectively. Phylogenetic analysis was performed using the 16S rRNA gene sequences of strains C2, H16 and related species. Sequences were aligned and phylogenic trees were constructed using Maximum Likelihood method implemented in MEGA version 6 [Comparative 16S rRNA gene sequence analysis using the EzTaxon database  indicateP. jessenii C2 and P. jessenii H16 has been deposited at DDBJ/EMBL/GenBank under the accession numbers JSAK00000000 and JSAL00000000. The version described in this paper is the first version. A summary of the project and the Minimum Information about a Genome Sequence [Next-generation shotgun-sequencing was performed at the Beijing Genomics Institute . The whole genome shotgun project of Sequence  are showP. jessenii C2 and P. jessenii H16 were aerobically cultivated on Pseudomonas-selective Gould’s S1 agar at 28 °C [CA, USA) with Qubit dsDNA BR Assay kit  and amounted to 55 ng/μL of DNA for P. jessenii C2 and 48.2 ng/μL of DNA for P. jessenii H16.P. jessenii H16 and P. jessenii C2 was determined by BGI using the Illumina Hiseq2000 with a 500 bp library constructed [de novo as described previously [Denmark). P. jessenii H16 generated 78 contigs with an n50 value of 279,014 bp. P. jessenii C2 generated 64 contigs with an n50 value of 224,893 bp.The genome sequence of structed , generateviously  using CLThe genes in the assembled genome were predicted based on the RAST database . The preP. jessenii C2 contained 6,420,113 bp with a G+C content of 59.83 %, 5881 predicted genes, 5814 were protein-coding genes, 63 tRNA genes and 4 rRNA genes. In total, 5179 genes were assigned to biological functions and 635 were annotated as hypothetical proteins. P. jessenii H16 contained 6,807,788 bp, with a GC content of 59.02 %, 6065 predicted genes, and 5995 were protein-coding genes, 65 tRNA and 5 rRNA genes. Among the protein coding genes 5344 were assigned to biological functions, while 651 were annotated as hypothetical proteins. The properties and statistics of those two genomes are summarized in Table 1B-type ATPase family, which is highly conserved from bacteria to humans [P. jessenii C2 and P. jessenii H16 contained genes encoding a copper-transporting P1B-type ATPase (CopA) with conserved CPCALG motif [+-ATPase is present on the genome of P. jessenii H16 as part of the GI discussed later. P. jessenii H16 also contained ccoI encoding a Cu+-ATPase catalyzing a slower rate of efflux for copper insertion into cytochrome c oxidase [cop operon, comprising copABCDRS had been reported in related P.fluorescens SBW25 and P.putida KT2440 [P. jessenii strains contained copCDRS probably encoding proteins responsible for copper uptake, however, only P. jessenii H16 also contained copAB as part of the GI. Both P. jessenii C2 and P. jessenii H16 contain an arsenic resistance determinant (arsRBCH) [chrA) [Genes conferring resistances to heavy metals in the two studied strains are listed in Table o humans . Both P.LG motif , a coppe oxidase . The prea KT2440 , 28. BotarsRBCH)  a gene i) [chrA)  (Table 6) [chrA) , 31.TablP. jessenii H16 contained an additional putative metal fitness/pathogenicity island when compared with P. jessenii C2. It encompasses about 50,000 bp beginning at a gene encoding a sulfur carrier protein (KII37703) and ending with genes encoding Tn7 transposition proteins (KII37740-KII37743). This potential pathogenicity/fitness island harbored several copper resistance determinants including the cus determinant encoding CusABCRS  involved in periplasmic copper detoxification [KII37893, KII37715, KII37716, KII37709, KII37717) could be identified  were only identified in P. jessenii H16. Similar copper fitness islands could also be detected in P.extremaustralis 14-3b (AHIP00000000), isolated from a temporary pond in Antarctica; Pseudomonassp.Ag1 (AKVH00000000) isolated from midguts of mosquitoes and P. fluorescens FH4 (AOHN00000000) [KII37721) and the transcriptional repressor NcrB (KII37723) [merTRCAB (KII37733-37737) encoding a mercury-resistance determinant are present on this island [P. jessenii H16, these putative GI have conferred this strain with additional heavy metal resistance capability, which may be transferred to other bacteria via Tn7 transposons and are highly relevant for adaption to this specific copper contaminated niche.fication , 33. In ndViewer . Based o0000000) –37. ThisII37723) . Moreoves island . Many ofP. jessenii C2 isolated from low-Cu soil and P. jessenii H16 isolated from high-Cu soil were determined and described here. H16 provided an insight into the genomic basis of the observed higher copper resistance when compared with C2. Based on analysis and characterization of the genome, P. jessenii H16 is predicted to be resistant to a number of heavy metal(loid)s, such as Hg2+, Ni2+ Cr2+ and As3+. Comparative genomic analysis of those two strains suggested acquisition of a fitness island encoding numerous genes involved in conferring resistance to Cu and other metals as an important adaptive mechanism enabling survival of P. jessenii H16 in its Cu contaminated habitat. Possibly, P. jessenii H16 may have potential for bioremediation of copper contamination environments.The draft genome sequences of