Haynesina germanica kleptoplast pigment composition over time and at different light regimes. Six common diatom lipophilic pigments were detected, two chlorophylls  and four carotenoids , the same pigment profile as the diatom species frequently isolated at the sampling site. The xanthophyll cycle (XC) was functional with kleptoplast diatoxanthin (DT) content increase with concomitant diadinoxanthin (DD) decrease after short term light exposure. DT/(DT+DD) and DT/DD ratios increased significantly in specimens exposed to low light and high light in comparison to specimens maintained in the dark. Specimens placed in very low light after the light treatments reverted to values close to the initial ones, suggesting that H. germanica XC is functional. A functional XC is an indication of H. germanica kleptoplasts capacity for short-term photo-protection from photo-oxidative damages caused by excess of light. Furthermore, the pigment survey suggests that H. germanica preserved some chloroplasts over a longer time than others and that pigment content is influenced by previous light history. Finally, the current study highlighted seasonal differences, with higher pigment contents in winter specimens (27.35 ± 1.30 ng cell-1) and lower in summer specimens (6.08 ± 1.21 ng cell-1), a quantitative and qualitative composition suggesting light acclimation to low or high light availability, according to the season.Some shallow water benthic foraminifera are able to retain functional chloroplasts (kleptoplasts) from their food source, i.e. diatoms. Here we assessed the functionality of the kleptoplast xanthophyll cycle  and we surveyed  Benthic foraminiferal species have various particular physiological adaptations such as the capacity to store nitrate and to denitrify , 2 or toHaynesina germanica, the photosynthetic machinery was found to be functional carotene . Part ofcarotene ) and alscarotene , 24.Haynesina germanica vertical distribution is characterized by a clear maximum density at the sediment surface [H. germanica behaviour does not seem to be light driven [H. germanica kleptoplast photo-regulation might be more dependent on other physiological photo-regulation mechanisms. In diatoms exposed to high light, the plastid photoprotective capacity, i.e. the xanthophyll cycle (XC), consists of a de-epoxidation reaction which convert the pigment diadinoxanthin (DD) into diatoxanthin (DT). This process is catalyzed by the diadinoxanthin de-epoxidase and regulated by the ΔpH which occurs during photosynthetic electron transport fluxes [H. germanica kleptoplast pigment content and functionality [2 production) of kleptoplastic benthic foraminifera. Thus, here we characterized kleptoplast pigment content in freshly sampled kleptoplastic foraminifera focussing on the dominant kleptoplastic species in mudflat systems: H. germanica. We focused on kleptoplasts xanthophyll cycle pigments during a short term exposition to different light regimes; and we followed kleptoplast pigment composition over time and different light intensity, during a longer time scale experiment.Kleptoplasts might avoid photo-damage either by using physiological photo-regulation mechanisms or by using their host behavioural response, e.g. sacoglossan sea slugs close their parapodia when exposed to excess light , 25, 26. surface –36, wher surface , 38. Howt driven  and thert fluxes . This ret fluxes , and is t fluxes , 41. Litionality , 21 and Haynesina germanica , H. germanica in healthy conditions  was measured using ImageJ software [H. germanica . The cytoplasmic volume (or biovolume) was then estimated by assuming that internal test volume corresponds to 75% of total foraminiferal test volume .software  after imHaynesina germanica pigments were extracted in 300 μL of methanol at 95% (MeOH:H2O v/v) with 2% of ammonium acetate and internal standard . Foraminifera were crushed and stirred with repeated strokes of a metallic row, sonicated for one minute, subsequently maintained at -20°C for 15 min, and finally filtered . The filtrate was analyzed with an Ultimate 3000 RS (Dionex) high-performance liquid chromatography (HPLC) as described in Meleder et al. [-1), was carried out at 440 nm by comparison with pigments standards . Results are shown for major diatom pigments, chlorophylls: chlorophyll a (Chl a) and chlorophyll c1 + c2 (Chl c); and carotenoids: β-carotene (β-caro), fucoxanthin (Fuco) and its by-products ); and xanthophyll cycle pigments (XCP = DD+DT): diadinoxanthin (DD) and diatoxanthin (DT). All pigment ratios , (DD+DT) /Chl a, Chl c/Chl a, Fuco/ Chl a, β-caro/ Chl a, degraded pigments /Chl a and Fuco-like 1+2 / Fuco) are expressed in g g-1. The degradation pigments are the sum of carotenoids by-products, mainly composed of Fuco-like 1 and -2.r et al. . PigmentH. germanica specimens were removed from the sediment to prevent cells from hypothetically photo-regulating by moving in the sediment matrix. In this way we avoided possible bias driven by behaviour.In the two experiments, -2 s-1). VLL was chosen as a “dark adaption” methodology instead of full darkness because this treatment has been shown to work better in reversing diatom non photo-chemical quenching (NPQ) that often accumulates in darkness [-2 s-1, 3×100 specimens) and high light . A fourth treatment of 30 min HL:30 min VLL  was added to investigate XC reversal. All specimens were sampled and frozen as described above after their respective treatments. A HL:VLL cycle was preferred to a HL:D cycle, as with diatoms de-epoxidation driven by the chlororespiratory proton gradient might occur under dark conditions [This experiment was carried out to assess xanthophyll cycle (XC) short term functionality. All experiments were carried out in a Fytoscope  maintained at 16°C but using different light regimes. All foraminifera specimens (n = 1500) were distributed in 15 polystyrene Petri dishes (100 per Petri dish) filled with 5 mL of ASW at 35 salinity and were “dark adapted” during one hour at a very low light (VLL) intensity  with Light:Dark cycle of 6:18h. Foraminifera were sampled after 5, 10 and 15 days as described before, three replicates of 100 specimens in each treatment.This experiment was carried out to assess kleptoplast long-term pigment degradation over time. All measurements were made in a 16°C temperature controlled room and using different light regimes. All foraminifera specimens (n = 2100) were spread in 21 Petri dishes (100 specimens per Petri dish) filled with 5 mL of artificial sea water at 35 salinity and placed at 16°C for a “dark” adaption of one hour at VLL. Initial pigment composition  was estimated as described above. Following dark-adaptation, 1800 specimens were placed under two different light conditions: dark  and light . However, the compensation irradiance was reached very quickly, at light level as low as 24 μmol photons m-2 s-1 [H. germanica can have an access to light only during diurnal low tide.For both experiments (1 and 2), the light intensities applied were chosen based on experimental results obtained in our previous work (19), where  m-2 s-1 . Furtherth and 25th percentiles and the whisker length is equal to 1.0 times the interquartile range. After testing for homogeneity of variance and normality (test Kolmogorov-Smirnov), statistical analyses consisted of analysis of variance (ANOVA) for experiment 1 and a multifactor ANOVA for experiment 2 followed by a Fisher's LSD test to compare pigment composition and ratios. Differences were considered significant at p<0.05. All statistical analyses were carried out with Statgraphics Centurion XV.I  software.Data in tables and text are expressed as mean ± standard deviation (SD). Data in figures are expressed using box plots; the box plots shows the median, the tops and bottoms of each box mark the 75Haynesina germanica had a mean maximal test elongation of 255 ± 32 μm . This resulted in cytoplasmic biovolumes equal to 3.26 × 106 ± 6,38 × 103 μm3 (SD).Haynesina germanica pigments was clearly from ingested diatoms, with the typical diatom pigments Chl a, Fuco, Chl c, DD, DT and β-caro observed in freshly isolated specimens from the sediment  for ~6%, Chl c for ~4% and the sum of the degradation pigments, mainly fucoxanthin by-products (fuco-like pigment 1 and 2) for ~14%. Globally, H. germanica pigment profiles were similar throughout experiment 1; however, total pigment content was lower in condition HL/VLL compared to the other ones ; whereas, it increased to 1.23 ± 0.05 and 1.35 ± 0.06 g g-1after 30 minutes of LL and HL exposition, respectively. It was also noteworthy that DT/DD decreased to 0.61 ± 0.06 g g-1 after the treatment HL:VLL  and respective cytoplasmic biovolumes equal to 6.13 × 106 ± 4.27 × 104 μm3 (SD).-1 with Chl a accounting for ~53%, Fuco for ~22%, the XCP for ~6%, Chl c for ~6% and the sum of the degradation pigments for ~12%. The β-carotene was below detection limit.Initial pigment content was 27.35 ± 1.30 ng cellH. germanica pigment contents  of and of β-caro.Between experiment 1 and experiment 2 a clear difference (P < 0.01) was found between total -1 to 6.47 ± 1.04 and 10.64 ± 2.60 ng cell-1 after 5 days of exposure to light and dark treatments, respectively. The decrease between day 0 and day 5 was observed for most pigments, with the exception of 19'Butanoyloxyfucoxanthin and Fuco-likes (1 and 2) pigments. After the first 5 days of exposure to either light treatment, i.e. between days 5 to 15, total pigment content did not significantly changed  and four carotenoids . These pigments profiles were close to those usually found in diatoms isolated from Bourgneuf bay mudflats [H. germanica pigment composition results and kleptoplast origins [-3 and experiment 2, total pigment = 4.46 ± 0.21 fg μm-3, P < 0.01), we thus suggest that the observed difference was mainly driven by different light availability at the two seasons, with higher pigment production and/or higher plastid assimilation linked to lower light intensity in the winter. Furthermore, pigment qualitative composition reflected H. germanica plastid light acclimation to low or high light availability according to the season; i.e. Fuco/Chl a ratio, which is a microphytobenthos photo-adaptation biomarker [-1 (exp 1) and higher in December 0.43 ± 0.05 g g-1 (exp 2)). Also, the photoprotective pigment β-carotene levels were below the detection limit in the winter experiment, thus supporting the hypothesis that winter specimens were acclimated to lower light levels, similarly to the diatoms on which they fed. However, other physico-chemical parameters might affect their kleptoplast photoprotective mechanisms or pigment content such as temperature [All pigments detected and quantified in mudflats –50 and c origins , 18, 21.iomarker , varied perature  or nitroperature , requiriHaynesina germanica lives mainly in the first millimeters of intertidal mudflat sediments [-2 s-1 [H. germanica is not sensitive to light [H. germanica kleptoplasts from photo-oxidative damages caused by excessive light.ediments –36, 53 a [-2 s-1 , 55) at  [-2 s-1 ) wherebyto light  then we H. germanica cytoplasm. However, DT/ (DD+DT) ratio of H. germanica exposed to the HL:VLL cycle did not completely return to its initial state. It suggests that lumen acidification and subsequent diadinoxanthin de-epoxidase activation was not easily reversed in the kleptoplasts during the very low light cycle, thus still promoting a higher DT/ (DD+DT) ratio than in total darkness. Alternatively, no statistical differences between DT/ (DD+DT) ratio were observed between specimens exposed to LL (25 μmol photon m-2 s-1) and HL (300 μmol photon m-2 s-1), suggesting that at 25 μmol photon m-2 s-1 most of the DD was already converted in DT and thus that the light levels necessary to activate the XC were very low, i.e. < 25 μmol photon m-2 s-1 (LL) and maybe even as low as 5 μmol photon m-2 s-1 (VLL). This early de-epoxidation reaction activated at low light showed that the kleptoplasts react similarly to low-light adapted photosynthetic organisms, as they quickly increase their de-epoxidation state [-2 s-1 the kleptoplasts were already close to their maximal de-epoxidation sate. Interestingly, the maximal de-epoxidation state reached by foraminiferal kleptoplasts was high (0.55–0.6) for a short-term exposition compared to the diatoms on which they fed (0.3–0.5) [The activation of the xanthophyll cycle under light exposure is therefore still functional after plastid assimilation in on state –58. Furt0.3–0.5) , 50, 55.0.3–0.5) , with hi0.3–0.5) , 60. A c0.3–0.5) , 50, 55,-2 s-1) of starved H. germanica specimens. The effect of food deprivation showed a biphasic decrease of kleptoplast total pigment content. A rapid decrease was observed during the first five starvation days, which was followed by a slow decrease over the remaining ten days. We suggest that this trend is the result of two different processes. The rapid initial decrease would correspond to chloroplasts that were ingested but not integrated as kleptoplasts and were thus in a process of cellular digestion and the latter slower phase would correspond to true kleptoplasts that were slowly being degraded with time. This would also suggest that only some diatom chloroplasts are capable kleptoplasts. Similar results were observed in other kleptoplastic organisms, e.g. in some sea slugs species a fast chloroplast degradation was observed after sampling followed by a second period were some chloroplasts remained up to 15 days [To follow how kleptoplast pigment composition changed over time, we surveyed the pigment content and composition at two light regimes (0 and 50 μmol photon m 15 days . Howevera ratios were stable and that Fuco-likes/Fuco and (DD+DT)/Chl a ratio increased over time suggests a slower degradation of carotenoids compared to chlorophylls. Similar observations were made with other kleptoplastic species such as sacoglossan sea slugs Elysia veridis and Thuridilla hopei [a ratios seem very similar between treatments. A similar increase of Zeaxanthin in the dark was also observed in the Jesus et al. [Elysia timida, thus it is also possible that heterotrophic cells in darkness exhibit lower pH inducing an activation of the XC in the dark different from algal cells.Kleptoplast pigment composition did not change much throughout the 15 days experiment with an increase of some ratios such as Fuco-likes/Fuco suggesting that kleptoplasts were slowly degrading and thus producing fucoxanthin by-products (mainly Fuco-like 1 and 2). Interestingly, the observation that Fuco/Chl la hopei , 62. Furla hopei , 62 as cla hopei ). In thela hopei . Howevers et al.  study wiH. germanica specimens [H. germanica photosystem II maximum quantum efficiency (Fv/Fm) slowly decreased from 0.65 to 0.55 within a week; whereas, a quick decrease to 0.2 was found under light exposure (70 μmol photon m-2 s-1). In the present study the difference between pigment content was significant but not so large between specimens kept in the darkness or under light; however, this difference can be explained as Fv/Fm measured using pulse amplitude fluorometry can stay high even if only a reduced number of functional kleptoplasts remain active. Interestingly, similar observations were previously made with sacoglossans, suggesting similarities between kleptoplast functionality in different organisms [Kleptoplast functionality and pigment content are necessarily linked. In a previous experiment we assessed kleptoplast functionality as function of light and time (6 days) using starved pecimens . In darkrganisms , 62, 64.H. germanica kleptoplasts photo-regulation capacities and monitored pigment composition, over time and with different light histories. The main diatom lipophilic pigments were detected: chlorophylls (Chl a and c) and different carotenoids , similarly to diatom pigment profiles isolated at the same sampling place. Furthermore, the current study showed that there were seasonal differences between pigments from specimens isolated in winter or summer, with higher pigment contents in winter specimens, indicating that H. germanica acclimatize to ambient light levels or keep the diatom plastids light acclimation state. Haynesina germanica xanthophyll cycle was found to be functional with DT/(DT+DD) ratios increasing under light exposure and reversing back in darkness or at very low light. This suggests that H. germanica kleptoplasts retain some of their physiological photo-regulation mechanisms active after being incorporated in foraminifera cytosol. To our knowledge, this is the first evidence of a functional xanthophyll cycle in kleptoplastic foraminifera and it is indicative of the presence of a short-term photo-protective mechanism protecting H. germanica kleptoplasts from photo-oxidative damages caused by excessive light. Finally, the pigment survey suggests that H. germanica preserved some chloroplasts over a longer time than others and that pigment content is influenced by light history.This study measured


Our results identified several natural compounds with α,β-unsaturated moiety. A structure-activity relationship (SAR) analysis and the results of pretreatment with reducing reagent DTT suggested the importance of α,β-unsaturated moiety. We also examined the underlying mechanisms, and our findings suggest that the actin reorganization seen in capsaicin treatment is important for the reversibility of TJ opening. Furthermore, our analyses revealed that TRPA1 is involved in the Ca2+ influx and TJ permeability increase not only by an α,β-unsaturated compound but also by capsaicin. Our results indicate that the α,β-unsaturated moiety can be a potent pharmacophore for TJ opening.The delivery of hydrophilic macromolecules runs into difficulties such as penetration of the cell membrane lipid bilayer. Our prior experiment demonstrated that capsaicin induces the reversible opening of tight junctions (TJs) and enhances the delivery of hydrophilic macromolecules through a paracellular route. Herein, we screened paracellular permeability enhancers other than capsaicin. As TJ opening by capsaicin is associated with Ca There is an increasing need for biotechnological therapeutics (biologics) such as peptides, proteins, antibodies, polynucleotides and other macromolecular drugs, as they can act with high specificity and potency. Although recent progress in biotechnology has accelerated the processes for producing biologics on a commercial scale, improvements that address the biologics’ low oral bioavailability have been lacking. Hydrophilic macromolecular drugs are usually classified into class III of Biopharmaceutics Classification System (BSC), and for their administration, injections  remain the most common routes.3. One of the most important of which is the epithelium, across which hydrophilic macromolecular drugs must move in order to exert their therapeutic effects4. Thus, the enhancement of the paracellular delivery of hydrophilic macromolecular drugs has received considerable attention5.However, injections decrease the patients’ quality of life. Non-invasive routes such as oral, nasal, pulmonary, transdermal and buccal routes are more favorable than injections, but their use also encounters several obstacles6 and are composed of a complex combination of transmembrane integral proteins including occludin, claudins and tricellulin, along with several intracellular proteins such as zonula occludens-1 (Zo-1), which connects the transmembrane proteins to the actin cytoskeleton7. Therefore, TJ proteins and actin are key regulators of the TJs8. The opening of TJs by paracellular permeability enhancers (PPEs) is a way to increase the absorption of hydrophilic drugs across the epithelium10. In particular, reversible opening of TJ could allow such drugs to be safely and controllably absorbed. This approach is attractive because it could be applied to many different hydrophilic drugs, whereas the chemical modification of biologics to improve bioavailability requires full drug development for each compound11.The paracellular movement of solutes and macromolecules across the epithelium is restricted by tight junctions (TJs). TJs are located at the apicolateral plasma membranes of adjacent cells1), is a naturally occurring alkaloid that is responsible for a hot and spicy taste12. In previous research we demonstrated that capsaicin induces reversible opening of TJ, and we investigated the underlying mechanisms15. We observed that capsaicin treatment induced a Ca2+ influx that resulted in cofilin activation, accompanied by an F-actin alteration unique to capsaicin; in addition, F-actin decreased at bicellular junctions but increased at tricellular junctions. No change in TJ protein localization was observed upon exposure to capsaicin, but the amount of occludin was significantly decreased. Both the cofilin activation and occludin decrease contributed to the TJ opening by capsaicin. Those results suggest that capsaicin is a new type of PPE with unique mechanisms16.Capsaicin  family of ion channels, which transmits noxious stimuli2+ influx and FD4 permeability assay of a Madin-Darby canine kidney (MDCK) II cell monolayer. The screening identified several natural compounds, a few of which have an α,β-unsaturated moiety: pyrenocine A 24, pyrenocine H (3), dehydrocurvularin (4)25, and avenaciolide (5)26. Further investigation of each compound’s ability to increase TJs’ permeability, including the role of α,β-unsaturated moiety and the underlying mechanisms affecting TJ integrity revealed that the TRPA1 channel is involved in the reversible TJ opening. Our present findings may lead to new PPE research and development.TRPA1 is the other member of TRP family of ion channels which transmits a variety of noxious stimuli. Different from TRPV1, TRPA1 is activated by several cysteine-targeted compounds containing electrophiles through covalent binding at cysteine residues. Here we screened for TJ openers by conducting a Cae A Fig. , 223,24,1) induces reversible TJ opening associated with a Ca2+ influx, cofilin activation and occludin decrease. We speculated that the Ca2+ influx is one of the essential events to induce TJ opening, based on the following observations: the short treatment of Ca2+ chelator EGTA can inhibit both cofilin activation and the associated decrease in transepithelial electrical resistance (TER)13, and blocking Ca2+ signaling by trifluoperazine also inhibited a capsaicin-induced cofilin activation, capsaicin-specific actin alteration, and the decrease in TER15.In our previous research, we demonstrated that capsaicin Fig. , 1 induc2+ influx assay in an MDCK II cell monolayer. As shown in Fig. 2+ influx was induced by a positive control (ionomycin) and capsaicin in the MDCK II monolayer. The Ca2+ indicator fluorescence by ionomycin decreased at approx. 20 min, but that by capsaicin was sustained until at least 60 min.In the present study we therefore screened 795 natural and synthetic compounds for TJ openers by performing a Ca2+ influx assay system, we identified several natural and synthetic compounds. Interestingly, among the hit compounds, four natural compounds, pyrenocine A (2), pyrenocine H (3), dehydrocurvularin (4), and avenaciolide (5) have a common structural feature: an α,β-unsaturated moiety  solution with each compound was applied to the apical side of the MDCK II monolayer in transwells, and basolateral solution was collected every 30 min to measure the permeability. Figure ity Fig. .28, this coefficient is not applicable for our compounds because of their reversible pattern. We thus chose to evaluate how much larger amounts they transported cumulatively until 2 h, compared to the control  analysis.As shown above, we demonstrated that all four hit compounds induced a Ca6   and 8 (clog P: 2.05). These data suggest that compounds 6, 7, and 8 lost the Ca2+ influx and the permeability enhancement ability not because of their lipophilicity change, but rather because of the loss of the α,β-unsaturated moiety. In the case of compound 5, it has the highest clog P-value: 3.00. Notably, the difference between compound 5 and its analog compound 9 (clog P: 1.80) was found to be larger. This lipophilicity change can be one of the reasons why compound 9 is unable to induce TJ opening, even though compound 9 keeps an α,β-unsaturated moiety.To investigate other structural factors that may be required for TJ-opening ability, we estimated lipophilicity differences between each hit compound and its analogs, by calculating theoretical partition coefficients (clog P) Figs , 2A. ComN-ethylmaleimide  can be added to the β-carbon of α,β-unsaturated carbonyl compounds, a mechanism referred to as the Michael reaction. DTT pretreatment of NEM completely abolished NEM’s TJ-opening ability  within 10 min, and the phosphorylation recovery was observed after approx. 60 min Fig. . Compoun4 showed a retardation of cofilin activation compared to capsaicin, but later on, there is no difference between its effect and that of capsaicin. At 30 min, compounds 2 and 4 reduced phosphorylated cofilin to 10–20% relative to that at time 0. Compound 5 retained cofilin phosphorylation compared to capsaicin at both 10 and 30 min. The maximum reduction in cofilin phosphorylation by compound 5 was observed at 30 min, and was as high as 50% at time 0.We analyzed the extent of cofilin activation by capsaicin and three compounds by measuring the phosphorylation level of cofilin at 10 and 30 min, because cofilin activation ceased after 60 min. As shown in Fig. 2, 4, and 5 also decreased occludin, although the extent of occludin decrease varied. Again, we analyzed the relative occludin content at 10 and 30 min after treatment, to observe early events of the TJ opening phase, not the recovery phase. The densitometric analysis showed that compounds 2, 4, and 5 seem to decrease occludin at 10 and 30 min, but not significantly compared to capsaicin  reduced the TER of MDCK II cell layers, offering the TRPA1 channel as a regulator of epithelial cell barriers33. It is also noteworthy that the non-pungent capsaicin analog capsiate activates TRPA134. We thus examined whether compound 2, a representative electrophilic molecule among the four natural molecules we identified in this study, uses TRPA1 activation to open TJs.Earlier studies showed that structurally unrelated electrophilic compounds activated TRPA1 through the covalent modification of cysteine residues2+, we first examined TRPA1’s contribution to the Ca2+ influx induced by compound 2 in an MDCK II monolayer. Pretreatment with the TRPA1 antagonist A-967079 before compound 2 greatly reduced the Ca2+ influx in the MDCK II monolayer  and TRPA1-KO MDCK II monolayers. Figure 2+ influx compared to compound 2 and capsaicin. In contrast, in the TRPA1-KO monolayer, AITC completely lost its ability to induce a Ca2+ influx  MDCK II cells prepared by the CRISPR/Cas9 system. First, we checked the Calux Fig. , verifyi33.We then investigated whether an FD4 permeability increase is induced by AITC, and if so, whether it is affected by the existence of TRPA1. Figure 2 to both the WT and TRPA1-KO MDCK II monolayers. As shown in Fig. 2 in the WT monolayers  compared to 3.00 of compound 5. Because cysteine residues required for TRPA1 activation are located in cytoplasm side, it is required for compounds to pass through the plasma membrane to attack these cysteine residues of TRPA1. Hydrophilic property of compound 9 might decrease the ability to pass through the plasma membrane. Although further SAR analyses are required, these results suggest that the hydrophobicity of compound 5 can be important for the TJ-opening ability. In addition to an α,β-unsaturated moiety, each structure should have optimal hydrophobicity.Our findings indicate that the α,β-unsaturated moiety could be a pharmacophore for TJ opening. However, the introduction of a hydroxyl group into the side chain of compound TJs Fig. , indicatTJs Fig. , 2A. Com2: Ca2+ influx, cofilin activation, occludin decrease, and reversible opening of TJ. This is noteworthy because we demonstrated that both the cofilin activation and occludin decrease contribute to capsaicin’s reversible TJ opening ability15. Compound 5 showed weak cofilin activation, quicker occludin decrease, and low reversibility  and mass spectroscopy (MS) data48. We determined the structures of compounds 6 and 9 by interpreting the spectroscopic data (1D/2D NMR and MS). Compound 6: 1H NMR , δ 5.47 , 3.85 , 2.68 , 2.23 , 1.66 , 0.95 ; 13C NMR , δ 201.1, 168.4, 162.7, 162.4, 114.2, 87.7, 56.3, 46.6, 18.4, 17.5, 13.7; HRESIMS m/z 233.0788 [M + Na]+ . Compound 9:1H NMR , δ 6.45 , 5.85 , 5.02 , 4.39 , 3.52 , 3.16 , 1.77 , 1.52 , 1.45 , 1.31 , 1.29 ; 13C NMR , δ 169.7, 167.4, 134.6, 126.3, 85.1, 74.2, 62.9, 44.2, 36.1, 32.7, 29.3, 29.2, 29.0, 25.7, 24.8; HRESIMS m/z 305.1359 [M + Na]+ .Natural products were extracted essentially as described2.Madin-Darby canine kidney (MDCK) II cells were cultured in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal calf serum  and 1% penicillin-streptomycin  in a humidified atmosphere containing 5% COCapsaicin, FD4, and NEM were purchased from Sigma . LatA was from Wako Pure Chemical Industries . DTT was from Nacalai Tesque, and A-967079 was from Cayman .Antibodies to occludin (#71-1500), Zo-1 (#61-7300), and tricellulin (#700191) were purchased from Invitrogen . Anti-claudin-1 (#13050-1-AP) and anti-cofilin (#3312) was purchased from Proteintech Group, Inc.  and Cell Signaling Technology , respectively. Anti-β-actin (#125K4769) and anti-phospho-cofilin  were from Sigma and Santa Cruz Biotechnology , respectively. Horseradish peroxidase (HRP)-conjugated anti-mouse and anti-rabbit IgGs were from Kirkegaard & Perry Laboratories .All other reagents were of reagent grade and purchased from Nacalai Tesque unless otherwise noted.4 cells per well. The cells were cultured for 3 days, and the medium was changed every day to establish monolayer integrity. After specific treatments, monolayers were washed with Hank’s balanced salt solution (HBSS) and treated with fluo-8 loading buffer  at 37 °C for 1 h. Cells were washed with HBSS twice, and recording buffer (2.5 mM probenecid in HBSS) and various compounds were added. The Fluo-8 fluorescence was measured every 3 min for 60 min with a PowerscanHT fluorescence microplate reader  at the excitation and emission wavelengths of 485 and 528 nm, respectively.MDCK II cells were seeded in 96-well plates at a density of 3.4 × 1015.We measured the MDCK II monolayer permeability using FD4 as described2) coated with collagen, at a density of 3.4 × 104 cells per well. The cells were cultured for 3 days to establish monolayer integrity. Transwell plates were washed three times, incubated with Hank’s balanced salt solution (HBSS) and equilibrated for 1 h at 37 °C. HBSS (0.1 ml) containing 1.0% w/v of FD4 was placed on the apical side and each transport enhancer was added to the apical side. The basolateral side was exposed to HBSS (0.6 ml), which was refreshed at predetermined intervals. Samples collected from the basolateral compartments were analyzed for FD4 using a PowerscanHT fluorescence microplate reader  at an excitation wavelength of 485 nm and an emission wavelength of 530 nm.In briefly, MDCK II cells were seeded in 6.5 mm diameter transwells  coated with collagen at a density of 1.1 × 105 cells per well. The cells were cultured for 3 days, and the medium was changed every day to establish monolayer integrity. After specific treatments, monolayers were washed once with phosphate-buffered saline (PBS) and lysed with lysis buffer. After sonication, the cell extracts were boiled at 60 °C for 10 min, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a polyvinylidene fluoride microporous membrane , blocked with 5% skimmed milk  or setsuyakukun supporter , probed with the appropriate primary antibody and HRP-conjugated anti-IgG secondary antibody, and detected by enhanced chemiluminescence . Images were visualized using X-ray film  for Figs MDCK II cells were seeded in poly-L-lysine-coated 24-well plates at a density of 2.1 × 10After specific treatments, MDCK II monolayers grown on LAB-TEK chamber Permanox slides  were washed with PBS, fixed for 10 min with 3.7% formaldehyde, and permeabilized for 5 min in PBS containing 0.2% Triton X-100. Each antibody diluted in PBS containing 0.5% BSA was applied and incubated for 1 h at 37 °C. After washing with PBS, samples were stained with secondary antibodies as above. F-actin was labeled for 1 h with CF488-phalloidin  diluted in PBS containing 0.5% BSA and washed with PBS. The coverslips were mounted with 5 µg/ml Hoechst 33258 (Sigma) in PBS containing 60% glycerol. Fluorescence images were acquired with a Leica DMI6000B microscope .14. We sequenced the PCR products of the modified clones in order to detect the indel mutations.We established a stable MDCK II cell line with the knockout of TRPA1 using the CRISPR/Cas9 system. The non-viral all-in-one plasmid containing CRISPR Cas9 nuclease, sgRNA-targeted canine TRPA1, the neo gene for G418 selection, and mCherry as a fluorescent reporter were purchased from GeneCopoeia . Transfection was performed using Lipofectamine 3000 (Invitrogen) according to the manufacturer’s instructions. The isolation of transfectants was performed as describedP-values with ChemDraw Pro ver. 13.0.2.3021 software . We used GraphPad Prizm 6 to perform Tukey’s multiple comparison test for Fig. t-test or Dunnett’s test for Fig. p-values.We calculated the theoretical clog Supplementary Information


Polygonum aviculare L. contains ingredients that inhibit the precontraction of mouse and human airway smooth muscle (ASM). High K+-induced precontraction in ASM was completely inhibited by nifedipine, a selective blocker of L-type voltage-dependent Ca2+ channels (LVDCCs). However, nifedipine only partially reduced the precontraction induced by acetylcholine chloride (ACH). Additionally, the ACH-induced precontraction was partly reduced by pyrazole-3 (Pyr3), a selective blocker of TRPC3 and stromal interaction molecule (STIM)/Orai channels. These channel-mediated currents were inhibited by the compounds present in P. aviculare extracts, suggesting that this inhibition was mediated by LVDCCs, TRPC3 and/or STIM/Orai channels. Moreover, these channel-mediated currents were inhibited by quercetin, which is present in P. aviculare extracts. Furthermore, quercetin inhibited ACH-induced precontraction in ASM. Overall, our data indicate that the ethyl acetate fraction of P. aviculare and quercetin can inhibit Ca2+-permeant LVDCCs, TRPC3 and STIM/Orai channels, which inhibits the precontraction of ASM. These findings suggest that P. aviculare could be used to develop new bronchodilators to treat obstructive lung diseases such as asthma and chronic obstructive pulmonary disease.Because of the serious side effects of the currently used bronchodilators, new compounds with similar functions must be developed. We screened several herbs and found that  These diseases are typically treated using bronchodilators3. Nevertheless, these treatment modalities can cause various side effects, including palpitation, tremor, headache, cardiovascular disease, ischemic heart disease, cardiac failure and death from life-threatening asthma attacks5, and they can also lead to desensitization6. Therefore, new bronchodilators must be developed.Worldwide, approximately 300 million individuals have asthmaPolygonum aviculare L. (P. aviculare), a member of the Polygonaceae family, is distributed across Asia, Africa, Latin America and the Middle East, where it is used as a traditional medicine. It has antimicrobial7 and anti-inflammatory activities8. However, we found that the ethyl acetate fraction of P. aviculare (EAF) can inhibit the precontraction of airway smooth muscle (ASM).13 involving the increase in intracellular Ca2+ and sensitization of Ca2+, remodeling of the cytoskeleton and interaction between filaments, and activation of signal pathways. An increase in Ca2+ results from Ca2+ release mediated by inositol 1,4,5-trisphosphate receptors (IP3Rs) and/or ryanodine receptors (RyRs) in the endoplasmic reticulum (ER) membrane and Ca2+ influx mediated by Ca2+-permeant ion channels in the plasma membrane. Ca2+ binds with calmodulin to form a Ca2+-calmodulin complex, which then activates myosin light-chain kinase (MLCK). The activated MLCK phosphorylates myosin light chain (LC20) to induce cross-bridge cycling, which results in the contraction of muscle cells.ASM contraction is triggered by a complex pathway2+ release is transient, and thus it can trigger only a transient contraction. However, release-induced depletion of stored Ca2+ activates ion channels that induce a sustained contraction, which can be stimulate by acetylcholine chloride (ACH) and high levels of K+. It is known that high levels of K+ result in membrane depolarization that activates L-type voltage-dependent Ca2+ channels (LVDCCs), which mediate Ca2+ influx to trigger a sustained contraction. These channels can also be activated following the stimulation of muscarinic (M) receptors by ACH or methacholine (Mch). In addition, ACH can induce the activation of TRPC3 and stromal interaction molecule (STIM)/Orai channels15. These channels are permeable to Ca2+ and can mediate Ca2+ influx to cause sustained contraction. Our results suggest that these Ca2+-permeant channels can be inhibited by EAF, which results in the inhibition of the sustained contraction of ASM.Ca+ levels . As shown in Fig. 50) of EAF was 0.33 ± 0.02 mg/mL , and the maximal inhibition was 59.8 ± 4.7%. These results indicate that EAF potently attenuates ASM precontraction. However, the petroleum ether, chloroform, butyl alcohol and water fractions of P. aviculare had no such inhibitory effects.We first studied the effects of EAF on precontracted ASM induced by ACH and high K50 value was 0.33 ± 0.01 mg/mL, and the peak inhibition was 61.2 ± 2.2% (n = 6/6). These results show that EAF inhibits the ACH-induced precontraction of epithelium-denuded TRs. Although the dose-inhibition curve was steeper for these samples than that in samples with intact samples, the maximal inhibition was similar. Thus, intact samples were used in subsequent experiments unless otherwise stated.This inhibitory effect of EAF was also observed in epithelium-denuded TRs Fig. . The rem+-induced precontraction of TRs , with a peak inhibition of 55.9 ± 4.7%.Additionally, EAF reduced high KTRs Fig. . The IC5Moreover, EAF induced very small transient and moderate sustained contractions in quiescent TRs Fig. . The peaWe further studied the effects of EAF on precontraction in bronchial ASM by measuring the airway lumen area (ALA) in mouse lung slices. As shown in Figure Strips of human bronchial ASM were induced to contract by the addition of ACH, and the effects of vehicle and EAF were observed Fig. . EAF ind+ levels and ACH. It is known that high K+ levels induce plasma membrane depolarization and activate LVDCCs. The activated LVDCCs mediate Ca2+ influx, which then triggers contraction. As shown in Fig. + solution, which was almost completely inhibited by 10 μM nifedipine, a selective blocker of LVDCCs. The maximal inhibition was 99.8 ± 0.63% (n = 8/8). Moreover, single tracheal ASM cells exhibited LVDCC-mediated currents, which were completely blocked by nifedipine  was larger than that caused by Pyr3 ; however, the Pyr3-induced relaxation was not different in the absence  or presence of nifedipine , suggesting that LVDCCs, TRPC3 and STIM/Orai channels partially contribute to ACH-induced precontraction. The role of TRPC3 and STIM/Orai channels in ASM precontraction was further investigated. We used nifedipine, tetraethylammonium chloride (TEA) and niflumic acid (NA) to block LVDCC-mediated and K+- and Cl−-channel-mediated currents, respectively15, because these channels are activated by ACH. The measured currents were thus ACH-activated non-selective cation channel (NSCC)-mediated currents, recorded using a ramp ; the remaining contraction force was 1.95 ± 0.55 mN  and LVDCC blockade (with nifedipine) due to Ca2+ release from the ER. When the contraction reached a plateau, 2 mM Ca2+ was added, and additional contractions occurred. These contractions were further blocked by EAF , suggesting that the blockade of TRPC3 and STIM/Orai channels can contribute to the EAF-induced relaxation of TRs.TRPC3 and STIM/Orai channels are CaP. aviculare18 and has been shown to relax ASM22. Therefore, we fractionated EAF using HPLC . The vehicle failed to inhibit the precontraction of ASM. In addition, the inhibition in epithelium-denuded TRs was 104.8 ± 2.5%, and the IC50 was 21.4 ± 0.1 μM .We next tested whether quercetin can induce relaxation in precontracted mouse TRs. The quercetin standard induced a substantial relaxation of ACH-induced precontraction in TRs compared to the vehicle Fig. . The maxion Fig. . The max+ channels and NA-sensitive Cl−-channels were blocked)  Figure . These rWe next studied the effects of quercetin on the precontraction of human bronchial ASM. As shown in Figure +- and ACH-induced precontraction in ASM. The high K+-induced precontraction was completely mediated by LVDCCs; however, the ACH-induced precontraction was at least partially mediated by LVDCCs, TRPC3 and/or STIM/Orai channels. EAF inhibited these channels and thus almost inhibited the precontraction of ASM induced by high K+, while it partially attenuated the precontraction of ASM induced by ACH. In addition, EAF was found to contain quercetin. Standard quercetin could also inhibit ASM precontraction and currents mediated by LVDCCs and ACH-activated NSCCs. These results suggest that EAF and quercetin can inhibit ASM precontraction through the inhibition of Ca2+-permeant ion channels such as LVDCCs, TRPC3 and STIM/Orai channels , ACH, pyrazole-3 (Pyr3), bovine serum albumin (BSA), papain, collagenase H, dithiothreitol (DTT), dithioerythritol (DTE), cesium acetate, cesium chloride (CsCl), Mg-ATP, and agarose were purchased from Sigma . Nifedipine and Pyr3 were dissolved in dimethyl sulfoxide (DMSO), and other compounds were dissolved in the appropriate solutions used in the experiments.P. aviculare was purchased from Beijing Tongrentang  and authenticated by Professor Ding-Rong Wan . A voucher specimen was deposited at the herbarium of College of Pharmacy, South Central University for Nationalities.P. aviculare plants were dried, powdered and then extracted thrice with 70% aqueous ethyl alcohol. The extracts were boiled for 2.5 h, and the supernatants were collected and dried under reduced pressure. The dried products were mixed with 600 mL water, and the lipids were removed using petroleum ether. The remaining components were then extracted with ethyl acetate and dried under reduced pressure. The final product, EAF, was dissolved in 3% DMSO for all experiments.18 column  as the stationary phase and methanol:0.1% aqueous phosphoric acid (65:35) as the mobile phase. The flow rate was 1.0 mL/min. The detection wavelength was set at 366 nm.HPLC analysis was performed using an Ultimate 3000 instrument . Fractionation was performed using a ReproSil 100 CSix-week-old male BALB/c mice were purchased from the Hubei Provincial Center for Disease Control and Prevention  and housed in a temperature and humidity controlled room with a 12 h light/12 h dark cycle.26. Briefly, mice were sacrificed with intraperitoneal injections of sodium pentobarbital (150 mg/kg). The tracheae were dissected and transferred to ice-cold physiological salt solution (PSS) containing (in mM) NaCl 135, KCl 5, MgCl2 1, CaCl2 2, HEPES 10, and glucose 10 . TRs (~5 mm) were cut from the bottom of the tracheae and mounted in 10-mL organ baths containing PSS bubbled with 95% O2 and 5% CO2 at 37 °C. A 0.3 g preload was added. The TRs were equilibrated for 60 min and precontracted three times with 10−4 M ACH. After 30 min, experiments were performed with high-K+ solution containing (in mM) NaCl 60, KCl 80, MgCl2 1, CaCl2 2, HEPES 10, and glucose 10 .The contraction of ASM in TRs was measured as previously described with some modifications27. Briefly, after the mice were euthanized, the tracheae and lungs were exposed. The tracheae were cannulated. Each lung was slowly inflated with warm agarose solutions  followed by ~0.2 mL of air. The lungs were then cooled at 4 °C to gel the agarose. Single lobes were removed and sectioned into ~140-μm thick slices with a vibratome . The slices were collected and transferred to Dulbecco’s modified Eagle’s medium (DMEM) (GIBCO) and maintained in an incubator (with 95% O2 and 5% CO2 at 37 °C) for 2 h before being used in experiments. The slices were placed in a chamber and held in place with a small nylon mesh. The chamber was placed under an LSM 700 laser confocal microscope  and was perfused with Hanks’ balanced salt solution (HBSS) at a rate of ∼800 μL/min at room temperature. HBSS was supplemented with 20 mM HEPES . Images were acquired at a rate of 30 frames/min under a 10 × objective. ALA was calculated using Zen 2010 software (Carl Zeiss).Additionally, ASM contraction was indirectly measured in mouse lung slicesASM contraction was also measured in human bronchial ASM as described above. ASM strips were cut from airways and transversely mounted in chambers. The preload was set at 0.3–0.8 g based on the tissue size.28. Briefly, after the mice were euthanized, the tracheae were removed and transferred to ice-cold PSS. The tracheal muscle was dissected, minced and incubated for 22 min at 35 °C in low-Ca2+ PSS  supplemented with 2 mg/mL papain, 1 mg/mL DTE, and 1 mg/mL BSA. The tissues were then transferred to LCPSS containing 1 mg/mL collagenase H, 0.15 mg/mL DTT, and 1 mg/mL BSA for 8 min at 35 °C. The tissues were washed with LCPSS and then gently triturated to yield single ASM cells. Cells were kept on ice and used for experiments within 4 h.Single ASM cells were isolated from BALB/c mouse tracheal smooth muscle as previously described with minor modifications30. For LVDCC-mediated current measurement, Ba2+ was used as the charge carrier. The pipette solution contained (in mM) CsCl 130, EGTA 10, MgCl2 4, Mg-ATP 4, HEPES 10, and TEA 10 . The bath solution contained (in mM) NaCl 105, CsCl 6, BaCl2 27.5, glucose 11, HEPES 10, TEA 10, and NA 0.1 . The holding potential was -70 mV, and the current was measured every 10 s using 1000-ms steps from -70 to 40 mV in 10 mV increments. For ACH-activated current measurements, the pipette solution contained (in mM) CsCl 18, cesium acetate 108, MgCl2 1.2, HEPES 10, EGTA 3, and CaCl2 1 . The free Ca2+ concentration was approximately 70 nM as calculated using the WEBMAXC standard program (http://www.stanford.edu/~cpatton/webmaxc/webmaxcS.htm). The bath solution was PSS without K+ containing 10 μM nifedipine, 100 μM NA, and 10 mM TEA to block Ca2+-, Cl−- and K+-channel-mediated current, respectively. Cells were patched in the classical whole-cell configuration and held at −60 mV. The current was measured using a 500-ms ramp from −80 to 60 mV, and the values at −70 mV were used to represent the current.Current from LVDCCs and ACH-activated channels was recorded using an EPC-10 patch-clamp amplifier  at room temperature as previously described with some modifications31. Briefly, after the experiments were complete, the TRs were fixed using 4% paraformaldehyde for 10 min at room temperature, sectioned into 8-μm slices and then stained with hematoxylin and eosin. The slices were examined under a microscope.The depletion of airway epithelia was confirmed by hematoxylin & eosin staining as previously described with some modificationsThe results are expressed as the mean ± standard error of mean (SEM). Student’s t-test between two groups and one-way ANOVA among multiple groups were performed, and the statistical significance was set at P < 0.05.Supplementary Information


Triticum aestivum L.) are more easily able to generate embryogenic callus than mature embryos (MEs). To understand the molecular process of embryogenic callus formation in wheat, de novo transcriptome sequencing was used to generate transcriptome sequences from calli derived from IMEs and MEs after 3d, 6d, or 15d of culture (DC).During asexual reproduction the embryogenic callus can differentiate into a new plantlet, offering great potential for fostering in vitro culture efficiency in plants. The immature embryos (IMEs) of wheat  were annotated with a significant Blastx against nr, Pfam, Swissprot, KOG, KEGG, GO and COG/KOG databases. Comparative transcriptome analysis indicated that a total of 5194 differentially expressed genes (DEGs) were identified in the comparisons of IME vs. ME at the three stages, including 3181, 2085 and 1468 DEGs at 3, 6 and 15 DC, respectively. Of them, 283 overlapped in all the three comparisons. Furthermore, 4731 DEGs were identified in the comparisons between stages in IMEs and MEs. Functional analysis revealed that 271transcription factor (TF) genes  and 346 somatic embryogenesis related genes  were differentially expressed in at least one comparison of IME vs. ME. In addition, of the 283 overlapped DEGs in the 3 comparisons of IME vs. ME, excluding the SSEGs and TFs, 39 possessed a higher rate of involvement in biological processes relating to response to stimuli, in multi-organism processes, reproductive processes and reproduction. Furthermore, 7 were simultaneously differentially expressed in the 2 comparisons between the stages in IMEs, but not MEs, suggesting that they may be related to embryogenic callus formation. The expression levels of genes, which were validated by qRT-PCR, showed a high correlation with the RNA-seq value.This study provides new insights into the role of the transcriptome in embryogenic callus formation in wheat, and will serve as a valuable resource for further studies addressing embryogenic callus formation in plants.The online version of this article (10.1186/s12870-017-1204-2) contains supplementary material, which is available to authorized users. Efficient plant regeneration from in vitro cultured cells and tissues is a prerequisite requirement for the successful application of plant genetic engineering, which has been incorporated into the improvement and breeding of many crops . HoweverCyclamen persicum [Arabidopsis LEC1 and L1 L, encode HAP3-related transcription factors [Arabidopsis LEC2, FUS3, and ABI3, as well as maize Viviparous1 (Vp1), encode the B3 domain transcription [To understand the molecular mechanisms of gene regulation in in vitro culture, the expression of the somatic embryogenesis-related genes and signaling regulation has been researched in many species, including persicum , oil palpersicum , cotton persicum , 10, Arapersicum , soybeanpersicum  and chicpersicum , using d factors –16, whilcription , 18. Somcription  encoded cription , 21, AGLcription , and WUScription , are essTriticum aestivum L.) is one of the most important human food crops. Considerable effort and progress has been made in tissue culture system optimization over the last few decades, however tissue culture efficiency is still low and lags behind that of other plants such as rice and maize [The bread wheat  technology has high sensitivity and includes both low- and high-level gene expression –30, and fouriana , camphorfouriana , Maize [fouriana , and in fouriana  for tranfouriana , and theThe bread wheat cultivar Zhoumai 18 was grown in the Zhengzhou experimental field  of the Henan Agricultural University of China during the 2011–2012 cropping season. Harvested seeds were planted in the same experimental field in October 5 2012, after which they were transplanted into a greenhouse in January 15 2013 under the following controlled environmental conditions: 75% relative humidity, 26/20 °C day/night temperature, 12−/12-h light/dark photoperiod, and a light intensity of 10,000 lx. Anthesis was recorded in February of 2013 when 50% of the plants had reached the flowering stage.v/v) ethanol, followed by 6 min immersion in 0.1% (m/v) mercuric chloride solution with agitation, and then rinsed 4 times with sterilized distilled water. The IMEs and MEs were extracted from the sterilized seeds on a clean bench and explants were placed with the scutellum upwards in sterile Petri dishes containing solid Murashige and Skoog (MS) medium . They were then grown in a growth chamber at 22–24 °C in the dark. The MEs and IMEs were cultured in 3 biological replicates, each replicate consisting of 15 plates and each plate containing 10 embryos. The embryos were harvested at 3 DC, 6 DC, and 15 DC from 5 plates in each of the 3 replicates, after which they were snap-frozen in liquid nitrogen and stored at −80 °C until RNA extraction.Immature seeds from the main spikes were harvested 14 days after pollination. The mature seeds were soaked in running tap water for 4 h. Immature and mature seeds were surface-sterilized for 30 s in 75%  according to the manufacturer’s instructions, and then characterized on a 1% agarose gel. The purity of the RNA was examined using a NanoDrop 2000 spectrophotometer , and the integrity was examined using an Agilent 2100 Bioanalyzer . The RNA samples of the 3 biological replicates were mixed in equal amounts and used for the construction of libraries by the Biomarker Biotechnology Corporation . A total of 3 μg RNA for each sample was used as input material for cDNA library construction. The mRNA was enriched and purified with oligo (dT)-rich magnetic beads and then broken into short fragments. Taking these cleaved mRNA fragments as templates, first strand cDNA was synthesized and primed by oligo-dT and the second strand cDNA was synthesized using random primers. The resulting cDNAs were then subjected to end-repair and phosphorylation using T4 DNA polymerase and Klenow DNA polymerase.Following this, an “A” base was inserted as an overhang at the 3′ ends of the repaired cDNA fragments and Illumina paired-end solexa adaptors were subsequently ligated to these cDNA fragments to distinguish the different sequencing samples. To select a size range of templates for downstream enrichment, the products of the ligation reaction were purified and visualized on a 2% agarose gel. Next, PCR amplification was performed to enrich the purified cDNA template.RNA sequencing was performed on an Illumina HiSeq (TM) 2000 . The RNA-seq read data were deposited in the NCBI Sequence Read Archive (SRP093588).http://www.ncbi.nlm.nih.gov), the Swiss-Prot protein database (http://www.expasy.ch/sprot), the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database (http://www.genome.jp/kegg), the Cluster of Orthologous Groups (COG) database (http://www.ncbi.nlm.nih.gov/COG), and the pfam database. Functional classification by gene ontologies (GO) of all unigenes was performed using Blast2GO software.Clean, high quality reads were obtained after filtering the adaptor sequences and reads with ambiguous “N” bases and with a base quality less than Q30 . The clean reads were assembled into contigs using the Trinity method  using an optimized k-mer length (25-mer). Subsequently, the contigs were assembled into transcripts according to the paired-end information, after which they were clustered based on sequence similarity. Finally, the longest transcript in each cluster was regarded as a unigene, and the singletons were combined together as total unigenes. All unigenes were mapped to the reference sequences (wheat unigenes from NCBI) and allowed no more than one nucleotide mismatch. For annotation, all unigenes were used for the blast search and annotation against a series of public databases using BLASTx , including the NCBI non-redundant (Nr) protein databases , Arabidopsis Transcription Factor (http://planttfdb.cbi.pku.edu.cn/index.php?sp=Ath) and the alignment results of orthologous in the NCBI. Subsequently, the differentially expressed TFs were picked out from the DEGs in the IME vs. ME comparison, and between the stages in IMEs and MEs.Putative transcription factors were identified by BLASTx against the Plant Transcription Factor Database (PlnTFDB) 3.0  using thA total of 32 unigenes were selected for expression profile validation by qRT-PCR. Reverse transcription (RT) reactions were performed in 3 independent biological replicates with RNA that was individually extracted from 3 independent biological samples of six types of callus, performed using a PrimeScript® RT reagent Kit with gDNA Eraser . The first genomic DNA elimination reaction was conducted in a final volume of 10 μL including 2.0 μL of 5× gDNA Eraser Buffer, 1.0 μL of gDNA Eraser, 2.0 μL of total RNA, and 5 μL of RNase Free dH2O. Reactions were incubated at 42 °C for 2 min, and then maintained at 4 °C. The RT reactions (10.0 μL) were then used for the SYBR® Green qPCR assay in a 20-μL reaction mixture that included 4.0 μL of 5 × PrimeScript® Buffer 2 , 1.0 μL of PrimeScript® RT Enzyme Mix I, 1.0 μL of RT Primer Mix, and 4.0 μL of RNase Free dH2O. The reactions were incubated at 37 °C for 15 min, followed by 85 °C for 5 s, after which they were maintained at 4 °C.-ΔΔCt method [Real-time PCR was performed on a Bio-Rad CFX96TM Real-Time System with SYBR® Premix Ex Taq II™ . Each reaction included 2 μL of product from the diluted RT reactions (cDNA solution), 1.0 μL of each primer  that were constructed and sequenced using the Illumina HiSeq™ 2000 platform. Following data cleaning and quality checking, 155,192,839 reads (31.34 G) with Q30 values ≥80.00% were obtained from the six libraries. Among the clean reads, 100% had quality scores at Q20 level , followed by Triticum urartu , Hordeum vulgare  and Triticum aestivum  were successfully annotated by at least one database in 7 databases, i.e., Nr, Pfam, Swissprot, KOG, KEGG, GO and COG Table . In addi−5 Table . With reTo further reveal their functions, gene ontology (GO) assignments were performed using Blast2GO, with the result that 34,761 (24.44%) unigenes received at least one GO term and were classified into 3 major functional categories: cellular component, molecular function, and biological process . For the biological process category, the majority of unigenes were annotated with “metabolic process” , “cellular process”  and “single-organism process”  unigenes assigned to COG classification and divided into 25 COG categories, with “general function prediction only” (3129), “replication, recombination and repair” 2004) and “transcription” (1548) being the 3 most highly represented Fig. . Metabol and “tray expressed genes (DEGs) analysis in the comparisons of IME vs. ME at the 3 corresponding stages resulted in a total of 5194 DEGs in all three comparisons, with 3181, 2085 and 1468 DEGs at 3, 6 and 15 DC, respectively /calmodulin (CAM) , 51, gerGH3 and SAURs which were down-regulated at 15d, as well as GH3 which was up-regulated at 3d and 6d. SAUR was mix regulated (up and down) at 3d and 6d, and AUX1 was down-regulated at 3d and up-regulated at 6d. In the comparisons between stages in IMEs, ARF was down-regulated in IME6 vs. IME3 and in IME15 vs. IME6, while GH3 was down-regulated in the IME15 vs. IME6 comparison, and most of the auxin related genes are down-regulated during embryogenic callus formation from 3d of callus initiation to stage 6d of visible embryogenic callus, and to the somatic embryo generation at 15d in IME , single-organism process (78 unigenes) and cellular process  and 7 unknown genes (Table 7) showed significant differences in the different stages of IMEs, but did not show any significant difference in the different stages of MEs, indicating that the 7 unknown genes might be related to embryogenic callus formation. The qRT-PCR results validated that the 7 unknown genes and one (comp126788_c0) of the 5 annotated genes showed highly consistent expression levels with RNA-Seq .The ectopic expression of BBM can lead to somatic embryogenesis in Arabidopsis and B. napus [A. thaliana EMBRYOMAKER (EMK), another member of the AP2/ERF domain family, plays a redundant role in the maintenance of embryonic cell identity. Ectopic expression of AtEMK can promote the initiation of somatic embryos from cotyledons [Hevea. In the present study, there were 20 AP2/ERF genes differentially expressed in the comparisons of IME vs. ME at 3 stages  was identified as a marker of SE in cell cultures of B. napus , 74, whiB. napus , sweet pB. napus , and in B. napus . The A. tyledons , 79 reveArabidopsis [Arabidopsis [Arabidopsis embryonic patterning and interacting with the auxin and BR signaling pathways [Members of bHLH are involved in growth, developmental, and abiotic stress responses , axillarbidopsis , and arebidopsis . BIM1, apathways , which ipathways . In thisThe WRKY TFs have been shown to be involved in the response to biotic and/or abiotic stresses , and werArabidopisis  encode regulatory proteins involved in embryogenesis and the induction of somatic embryo development [Arabidopsis [TcLEC2, a functional ortholog of AtLEC2, is involved in similar genetic regulatory networks during cacao somatic embryogenesis [B3 domain transcription factors in elopment . LEC1 (Lbidopsis . Furtherogenesis . In thisAGAMOUS-LIKE15 (AGL15) had been reported to enhance somatic embryo development in Arabidopsis and soybean when ectopically expressed [Arabidopsis MADS box genes, AGAMOUS-Like15 (GmAGL15) and GmAGL18, constitutively expressed increased embryogenic competence of explants. ABI3 and FUSCA3 were found to be directly up-regulated by GmAGL15 [The MADS-domain transcriptional regulator xpressed . Soybean GmAGL15 . WUSCHEL GmAGL15 . In the  GmAGL15 , 93, 15 Somatic embryogenesis is a complex process. TF with auxin and stress treatment were essential for the acquisition of embryogenic competence by carrot somatic cells . In thisA. thaliana, glycerol-3-phosphate acyltransferase 2 A. thaliana, gag-polypeptide of LTR copia-type/ reverse transcriptase Integrase core domain  protein 22, xyloglucan endo-transglycosylase (XET) C-terminus, root cap, cortical cell-delineating protein, U-box domain/E3 ubiquitin-protein ligase in Table . XTH, a In summary, the combination of tissue culture of wheat embryos and RNA-seq approaches by global gene expression patterns during embryogenic callus formation were performed to investigate the regulatory genes in embryogenic callus formation. Expression and function analysis indicated that the DEGs of 271 TFs , 346 SSEGs , 39 BPGs, and 7 unknown unigenes that overlapped in IME vs. ME and in comparisons between stages in IMEs, but not in any one of comparison in ME, might play an important role in embryogenic callus formation in wheat. The present comparative profiling provided new insights into the molecular mechanisms for the regulation of embryogenic callus formation processes. However, as molecular markers are required to follow specific events during embryogenic callus formation, further experiments are required to evaluate the interaction between stress and auxin signaling during embryogenic callus development.Additional file 1:Primers used in this study for qRT-PCR. (XLSX 14 kb)Additional file 2:Lengths distribution of all assembled unigenes (XLSX 11 kb)Additional file 3:Functional annotation of unigenes against Nr, Pfam, KEGG, COG, GO Swissprot databases. (XLS 20569 kb)Additional file 4:Gene ontology (GO) classification, COG classification and KEGG classification of unigenes. a. The GO terms are summarized into three main categories: biological process, cellular component and molecular function. b. COG classification of the unigenes. c. KEGG classification of 5933 KO annotated unigenes. (XLSX 111 kb)Additional file 5:Expression of genes in the 6 libraries in wheat callus based on the results of RNA-Seq. (XLSX 6574 kb)Additional file 6:Information of DEGs in the comparisons of IME vs. ME. (XLSX 397 kb)Additional file 7:Information of DEGs in the comparisons between stages in IMEs and MEs. (XLSX 319 kb)Additional file 8:Functional annotation of all DEG against Nr, Pfam, Swissprot, KOG, KEGG, GO and COG databases. (XLSX 1510 kb)Additional file 9:KEGG enrichment analysis of all DEGs. (XLSX 65 kb)Additional file 10:List of all TFs and expression of DEGs TFs in the 6 libraries. (XLSX 93 kb)Additional file 11:Expression and annotation of differentially expressed aux and SSEGs IME vs. ME. (XLSX 86 kb)Additional file 12:KEGG pathways of plant hormone signal transduction in comparisons of IME vs. ME and between stages. (PDF 122 kb)


Importantly, sustained P110 treatment significantly improved behavioral deficits, and reduced Aβ accumulation, energetic failure and oxidative stress in the brain of the AD mouse model, 5XFAD. This suggests that Drp1/Fis1 interaction and excessive mitochondrial fission greatly contribute to Aβ-mediated and AD-related neuropathology and cognitive decline. Therefore, inhibiting excessive Drp1/Fis1-mediated mitochondrial fission may benefit AD patients.Mitochondrial dynamics, involving a balance between fusion and fission, regulates mitochondrial quality and number. Increasing evidence suggests that dysfunctional mitochondria play a role in Alzheimer's disease (AD). We observed that Drp1 interaction with one of the adaptors, Fis1, is significantly increased in Aβ-treated neurons and AD patient-derived fibroblasts. P110, a seven-amino acid peptide, which specifically inhibits Drp1/Fis1 interaction without affecting the interaction of Drp1 with its other adaptors, attenuated Aβ The vast majority of AD is sporadic (SAD), but mutations in the amyloid precursor protein (APP) and in presenilin-1 (PSEN1) and -2 (PSEN2), which are components of the γ-secretase complex that processes APP to produce amyloid-β (Aβ), have been identified in familial forms of AD (FAD), which have an earlier age of onset [Alzheimer’s disease (AD) is a late onset, progressive disease, characterized by neurodegeneration and impaired cognitive functions . As the Mitochondria are essential organelles involved in oxidative phosphorylation, ATP production, metabolism of some neurotransmitters, amino acids and nucleotides, calcium homeostasis, reactive oxygen species management and programmed cell death –7. MitocRecently, the effect of Drp1 inhibition using Mdivi-1  on Aβ-me42 to induce Aβ-induced cytotoxicity, cells expressing a APP KM670/671NL double mutant, five different AD patient-derived fibroblasts and 5XFAD mouse model.In this study, we focused on inhibiting Drp1/Fis1 interaction using P110, a selective short inhibitory peptide of Drp1/Fis1 interaction that was developed in our lab . The ben42, whereas no change was observed with its interaction with other adaptors in SHSY5Y cells treated with 5 µM Aβ42 for 24 hours , which was corrected by P110 treatment  was significantly reduced by P110 treatment , a mutation that is associated with FAS, found in a Swedish family (KM670/671NL). Cell culture studies show that this mutation increased the production and secretion of Aβand Aβ42 . We obseand Aβ42 . Phosphoand Aβ42 , 46. TheP < 0.01) and interconnectivity score  as well as elongation (P < 0.05), without affecting these parameters in WT cells .  and P110 treatment resulted in a significant reduction in both  showed altered mitochondrial activity, including mitochondrial dynamics, mitochondrial morphology and mitochondrial function , 62 and reported , 24, 27. [et al.  using an [et al.  and a veoduction . RegardlFinally, our finding of abnormal mitochondrial functions in fibroblasts of patients with AD indicates that AD is not only a disease of the CNS and that treatments that addresses only the CNS may expose pathologies in other tissues. The advantage of P110 is that it inhibits excessive mitochondrial fission in all tissues where excessive fission occurs , 66, butAll chemicals were purchased from Sigma-Aldrich  unless stated otherwise. P110 and P110A along with control peptide were purchased from Ontores, Zhejiang, China.APP  and human presenilin 1 , under transcriptional control of the mouse Thy1 promoter. 5XFAD lines from the B6SJL genetic background were maintained by crossing hemizygous transgenic mice with B6SJL F1 breeders. These mice exhibit AD-related symptoms earlier than other animal models and amyloid deposition starts in the cortex and subiculum at 2 months of age. The animals used in the P110 treatment study were implanted with a 28 days osmotic pump (Alzet) containing TAT47–57 carrier control peptide or P110-TAT47–57, which delivered to the mice at a rate of 3 mg/Kg/day, as described previously [All the experiments were in accordance with protocols approved by the Institutional Animal Care and Use Committee of Stanford University and were performed based on the National Institutes of Health Guide for the Care and Use of Laboratory Animals. We used 5XFAD transgenic mice, which overexpress two transgenes bearing five mutations linked to familial AD: human Human neuroblastoma SH5YSY cells were maintained in modified Eagle’s medium (50%) and F12 medium (50%) supplemented with 10% (v/v) fetal bovine serum and 1% (v/v) penicillin/streptomycin.Mouse Neuro2a (N2a) neuroblastoma cells stably overexpressing the human Swedish mutant  APP  and human WT APP (N2aWT) were a gift of Dr. Gopal Thinakaran (University of Chicago) and maintained in N2a growth medium as described earlier .2–95% air.AD patient fibroblasts  and fibroblasts of control healthy individuals  were purchased from Coriell Institute, USA. H3 (HDFA) was purchased from Invitrogen. All fibroblast cultures were maintained in MEM supplemented with 15% (v/v) fetal bovine serum and 1% (v/v) penicillin/streptomycin at 37°C in 5% CO47–57), P110 inactive analog (Ala DALPRGT -P110), all at 1 μM final concentration, over a 24-hour incubation in the absence or presence of Aβ42 (5 µM). N2a cells were treated with P110 at a final concentration of 1µM every 24-hour in serum free media. Similarly, for patient-derived fibroblasts, TAT or P110-TAT peptides were added once daily for the duration of the experiment at 1 μM final concentration. All experiments were carried out in defined serum free media. Cells with fewer than 18 passages were used in all experiments.SH5YSY cells were treated with P110, vehicle  and prepared according to protocols described elsewhere [1–42 peptides were dissolved in 1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP) and aliquoted into polypropylene micro-centrifuge tubes. HFIP was evaporated and the resulting peptide films were stored at −80°C. Before use, these peptide films were reconstituted at a concentration of 1 mM in dimethylsulphoxide (DMSO), then subsequently diluted to 100 μM with ice-cold DMEM (phenol red-free) and incubated for 24 h at 4°C to facilitate the formation of oligomers. Resultant peptides were stored at –80°C until use.Human synthetic Aβlsewhere . Briefly™ Red mitochondrial superoxide indicator (Invitrogen) for 10 min at 37°C according to manufacturer’s protocol and fluorescence was analyzed with excitation/emission at 510/580 nm using SpectraMax M2e (Molecular devices).To determine mitochondrial ROS production, cells were treated with 5 μM MitoSOXCells were treated with 5 μM JC-1  dye (Invitrogen) in HBSS  together with DAPI  (Thermo Fisher) for 20 min at 37°C, and the fluorescence was analyzed with excitation/emission at 485/590 nm for mitochondrial polarization and at 485/525 nm for mitochondrial depolarization using SpectraMax M2e (Molecular devices). The mitochondrial depolarization was indicated by a decrease in ratio of polarization to depolarization.™ Triplex assay was used according to the manufacturer’s instructions . Briefly, cells were plated in 100 μL medium in an opaque-walled, clear-bottomed, 96-well plate . At the end of the culturing period, viability/cytotoxicity reagent was added, mixed by orbital shaking (300 r.p.m. for 30 sec) and incubated for 30 min at 37°C. Fluorescence was measured at 400/505 nm and emission to assess cell viability, and 485/520 nm to assess cytotoxicity; and represented as % Control. Then, 100 μL Caspase-Glo 3/7 reagent was added to each well, mixed by orbital shaking (300 r.p.m. for 30 sec) and incubated for 30 min at room temperature. Luminescence was then measured to assess caspase activation as a marker of apoptosis. To evaluate mitochondria toxicity, cellular ATP levels were determined using Mitochondrial ToxGlo™ assay . Briefly, after the cytoxocity measurement, ATP Detection Reagent to the 96-well plate containing cell samples and treatments. Luminescence was measured after 5 minutes to assess ATP levels and represented as % untreated control. All measurements were done using SpectraMax M2e (Molecular devices).To evaluate cell viability, apoptosis and cytotoxicity, the ApoTox-Glo™ kit  following manufacturers instructions using SpectraMax M2e (Molecular devices).The ratio between reduced and oxidized glutathione is a good indicator of oxidative stress in cells. Cells were plated in 96-well plates and allowed to attach overnight. Post treatments, growth media was removed cells were washed with PBS, and total glutathione and GSSG were each assayed in triplicate via GSH/GSSG-Glo50 µg of lysate was added to each well of a 96-well plate containing 160 µl assay buffer ). The lysate was incubated for 5 min, and then 20 µl of 2 mM oxaloacetate was added to each well. OD was measured at 412 nm for 30 min using SpectraMax M2e (Molecular devices). The rate of citrate synthase activity was quantified relative to control cells.OCR was measured in adherent fibroblasts with an XFe24 Extracellular Flux Analyzer (Seahorse Bioscience). Each control and mutant fibroblast cell line was seeded in 5 wells of an XF 24-well cell culture microplate (Seahorse Bioscience) at a density of 30,000 cells/well in 250 μl of DMEM and incubated for 24 h at 37°C under a 5% CO2 atmosphere. The growth medium was replaced with 575 μl of prewarmed bicarbonate-free DMEM, pH 7.3, and cells were incubated at 37°C for 1 h before starting the assay. After baseline measurements of OCR and ECAR, OCR was measured after sequentially adding to each well 75 μl of oligomycin, 75 μl of FCCP, 75 μl of rotenone and 75 μl of antimycin A to reach working concentrations of 1 μM. OCR and ECAR values were normalized to protein content measured by Sulforhodamine (SRB) assay, following a standard protocol. For data analysis, the following parameters were evaluated: basal respiration, measurement before oligomycin injection subtracted by non-mitochondrial respiration (measurement after antimycin A injection); proton leak–linked respiration, measurement after oligomycin injection subtracted by non-mitochondrial respiration; ATP-linked respiration, basal respiration subtracted by proton leak–linked respiration; maximal respiration, measurement after FCCP injection subtracted by non-mitochondrial respiration; spare capacity, maximal respiration subtracted by basal respiration; complex I–linked respiration, basal respiration subtracted by the measurement after rotenone injection; and maximal complex I–linked respiration, maximal respiration subtracted by the measurement after rotenone injection.Cells cultured on 8-well chamber slides were washed with cold PBS, fixed in 4% formaldehyde, and permeabilized with 0.1% Triton X-100. After incubation with 2% normal goat serum (to block nonspecific staining), fixed cells were incubated overnight at 4°C with anti-TOM20 (1:500) . Cells were washed with PBS and incubated for 60 minutes with FITC-conjugated goat anti-rabbit IgG (1:500 dilution). The cells were then washed gently with PBS and counterstained with Hoechst 33342  to visualize nuclei. The coverslips were mounted with Slowfade-antifade reagent (Invitrogen), and images were acquired using an All-in-One Fluorescence Microscope BZ-X700 (Keyence) , 58.Parameters of mitochondrial morphology were further quantified with a custom plugin for ImageJ as described before , 70. In Cells were washed with cold phosphate-buffered saline (PBS) at pH 7.4 and scraped off using mannitol–sucrose (MS) buffer containing 210 mM mannitol, 70 mM sucrose, 5 mM MOPS (3-(N-morpholino)propanesulfonic acid), 1 mM EDTA, and protease inhibitor cocktail, pH 7.4. The collected cells were passed through a 27-gauge ½-inch needle for lysis, followed by centrifugation at 800 × g to pellet nuclei. The post-nuclear supernatant was further centrifuged at 10,000 × g for 20 min to collect a mitochondria-enriched fraction as previously described before , 58.® A/G  overnight at 4°C and washed in PBS containing 1% Triton X-100 followed by PBS. The immunoprecipitates were then washed, resuspended in the sample buffer, and boiled at 95°C to reverse cross-linking [Co-immunoprecipitation experiments in patient-derived fibroblasts were carried out as described before . In brie-linking .Protein concentrations were determined using the Bradford assay (Pierce/Thermo Scientific). Proteins were resuspended in Laemmli buffer containing 2-mercaptoethanol, loaded on SDS/PAGE gel and transferred on to nitrocellulose membrane, 0.45 µm (Bio-Rad) as before , 66. MemThe rate of hydrogen peroxide production by fresh isolated mitochondria was determined by the AmplexRed Hydrogen Peroxide/Peroxidase Assay kit (Invitrogen) following the manufacturer’s instructions.Lipid peroxides in brain mitochondria were measured using the leucomethylene blue assay, using tert-butyl hydroperoxide as a standard, by monitoring the 650 nm absorbance after 1 h incubation at RT. The aldehyde product or termination production of lipid peroxidation in brain mitochondria was determined by measuring thiobarbituric acid reactive substances (TBARS). Samples were mixed with 0.15 M phosphoric acid. After the addition of thiobarbituric acid, the reaction mixture was heated to 100°C for 1 h. After cooling and centrifugation, the formation of TBARS was determined by the absorbance of the chromophore (pink dye) at 531 nm using 600 nm as the reference wavelength.40 and Aβ42 ELISA kits (Invitrogen) following the manufacturer’s instructions.Aβ levels in mitochondrial fractions and brain cortex were measured by using human AβAliquots of mitochondria were analyzed by using the ATP Luminescent assay kit (Abcam). Mitochondria were energized with 5 mM glutamate/malate and ATP synthesis was induced with the injection of 500 μM ADP. ATP determination was measured following the manufacturer’s instructions.The Activity Chamber was used to determine general activity levels, gross locomotor activity, and exploration habits in rodents . AssessmNest-building behavior of mice was tested as described previously . Mice weMice were subjected to the wire hang test once at 6-month old . They weClasping behavior was tested in 6-month old mice: mice were suspended by the tail for 60 s and the latency for the hind limbs or all four paws to clasp for at least 3 s was recorded .t-test, and one- or two-way analysis of variance (ANOVA), as appropriate, with Tukey post hoc tests or Bonferroni post hoc tests. A p-value equals to or less than 0.05 was considered significant.All data are expressed as means ± S.D. Results were collected from three or more independent experiments in at least duplicates. Statistical analysis was performed using Student’s


The inhibition of pyrimidine biosynthesis by blocking the dihydroorotate dehydrogenase (DHODH) activity, the prime target of leflunomide (LEF), has been proven to be an effective strategy for rheumatoid arthritis (RA) treatment. However, a considerable proportion of RA patients are refractory to LEF. Here, we investigated lapachol (LAP), a natural naphthoquinone, as a potential DHODH inhibitor and addressed its immunosuppressive properties.Molecular flexible docking studies and bioactivity assays were performed to determine the ability of LAP to interact and inhibit DHODH. In vitro studies were conducted to assess the antiproliferative effect of LAP using isolated lymphocytes. Finally, collagen-induced arthritis (CIA) and antigen-induced arthritis (AIA) models were employed to address the anti-arthritic effects of LAP.We found that LAP is a potent DHODH inhibitor which had a remarkable ability to inhibit both human and murine lymphocyte proliferation in vitro. Importantly, uridine supplementation abrogated the antiproliferative effect of LAP, supporting that the pyrimidine metabolic pathway is the target of LAP. In vivo, LAP treatment markedly reduced CIA and AIA progression as evidenced by the reduction in clinical score, articular tissue damage, and inflammation.Our findings propose a binding model of interaction and support the ability of LAP to inhibit DHODH, decreasing lymphocyte proliferation and attenuating the severity of experimental autoimmune arthritis. Therefore, LAP could be considered as a potential immunosuppressive lead candidate with potential therapeutic implications for RA.The online version of this article (doi:10.1186/s13075-017-1236-x) contains supplementary material, which is available to authorized users. Rheumatoid arthritis (RA) is an autoimmune disease that is characterized by chronic articular inflammation with progressive joint destruction . The curLEF is an isoxazole derivative with a potent immunosuppressive activity that was approved for the treatment of RA in 1998 . LEF is Lapachol -1,4-naphthoquinone) is a nonpolar naturally occurring naphthoquinone found in some Brazilian medicinal plants . LAP and1H NMR  δ7.66 , 7.64 , 7.53 , 7.41 , 5.15 , , 1.72 , 1.63 ; 13C NMR  δ 187.2, 178.4, 169.6, 136.0, 133.2, 131.3, 129.7, 127.5, 125.76 124.81, 124.4, 117.9, 25.59, 20.8, 14.1; HRMS-ESI m/z cald for: [M + Na]+ = 265.0835; found = 265.0834).To a solution of LAP  in ethanol (20 ml) was added NaOH  and the reaction mixture was stirred for 24 h. After consumption of LAP, the reaction mixture was concentrated under reduced pressure and the solid residue was washed with dichloromethane (4×) and petroleum ether (4×) to afford a purple solid of 518 mg, 95% yield ; DSM338 (PDB id: 4OQV); O57 (PDB id: 4JS3); a brequinar analogue (PDB id: 4JTU); 221290 (PDB ID: 2WV8); amino-benzoic acid inhibitor 715 (PDB id: 3KVL); LEF derivative inhibitor 1 (PDB id: 3F1Q); another brequinar analogue (PDB id: 2B0M); and antiproliferative agent A771726 (PDB id: 1D3H). The crystal structure of hDHODH in complex with antiproliferative agent A771726 (PDB id: 1D3H) has been considered for flexible docking with A771726 and LAP, and the calculations were carried out using the GOLD (Genetic Optimisation for Ligand Docking) 5.2 software . GOLD wax = 49.65, y = 42.13, z = –1.54), keeping the Leu46 side chain flexible (using a rotamers library). The number of docking simulations to be performed with each inhibitor was specified under 10 GA  runs, once each docking run can evolve to different ligand poses (pose = conformation + orientation). Thus, ten poses of highest score (top-ranked GOLD solutions) obtained for each compound were selected by using the CHEMPLP score function. In this case, a Piecewise Linear Potential (fPLP) is used to model the steric complementarity between protein and ligand, and for CHEMPLP the distance- and angle-dependent hydrogen and metal bonding terms from other fitness function also implemented in GOLD, so called ChemScore, are considered. CHEMPLP has been found to give the highest success rates for docking pose prediction as well as virtual screening experiments against diverse validation test sets and it was here chosen as the fitness function. Based on this CHEMPLP function, GOLD classifies the orientations of the molecules by a decreasing order of affinity (scores) with the binding site of the receptor  × 100.A total of 1 × 10Male DBA/1 J mice were injected i.d. at the base of the tail with 200 μg bovine type II collagen  emulsified in Freund’s complete adjuvant (CFA) on day 0. Mice were boosted i.d. with CII (200 μg emulsified in Freund’s incomplete adjuvant (IFA)) on day 21. Mice were monitored daily for signs of arthritis. Scores were assigned based on erythema, swelling, or ankylosis present in each paw on a scale of 0 to 3, giving a maximum score of 12 per mouse. After arthritis induction, mice were treated orally with LAP (3 mg/kg and 10 mg/kg) or LEF (3 mg/kg) or saline daily. The clinical score was addressed every day after collagen boost. All mice were euthanized for histologic assessment of the hind limbs 4 weeks after the boost.Femur-tibial joints were collected 4 weeks after CII boost, fixed in 4% (vol/vol) buffered formalin and decalcified in 10% EDTA for 2–3 weeks. The tissues were then trimmed, dehydrated in ethanol, and embedded in paraffin for the preparation of the slides. Histological assessment was carried out following routine staining. Joint sections were stained with hematoxylin and eosin (H&E) to analyze synovitis  or Safranin-O to visualize proteoglycan depletion and cartilage destruction. The severity of the joint damage was scored according to the criteria described by Wang et al. : 0 = no Interleukin (IL)-17 and interferon (IFN)-γ cytokines were measured by enzyme-linked immunosorbent assay (ELISA) from hind paw homogenate of an individual mouse using antibodies according to the manufacturer’s instructions . The results were expressed as pg of cytokine/mg of tissue.Myeloperoxidase (MPO) activity in tissue homogenates was used as an index of neutrophil infiltration into paws from CIA mice as previously described .Serum concentrations of aspartate aminotransferase (AST) or alanine aminotransferase (ALT) were measured using commercial kits according to the manufacturer’s instructions .Mycobacterium tuberculosis; Sigma-Aldrich, St. Louis, MO, USA). Booster injections of mBSA in IFA were given at 7 and 14 days after the first immunization. On day 21 after the first immunization, arthritis was induced by an intra-articular injection of mBSA (30 μg). During the AIA protocol, LAP (10 mg/kg) or saline (vehicle) was given orally every day from 12 to 21 days after first immunization.Mice were immunized with methylated bovine serum albumin  as described previously . Briefly4 (mean ± SEM)/joint.Leucocyte infiltration into the joints was assessed 6 h after intra-articular challenge with mBSA as previously described . BrieflyThe titers of serum anti-mBSA antibody were measured by ELISA as previously described .5 cells) of draining lymph nodes  and spleen from naive and mBSA-immunized (treated or not with LAP) mice were stimulated in a 96-well round-bottom plate with mBSA (100 μg/ml) for 96 h. Next, the supernatant was collected to measure the levels of IL-17A, IFN-γ, and IL-4 by ELISA .Cell suspension (1 × 10t test (for three or more groups) comparing all pairs of columns, or two-tailed Student’s t test (for two groups). P < 0.05 was considered statistically significant. Statistical analysis was performed with GraphPad Prism .Statistical analyses were performed using one-way nonparametric analysis of variance (ANOVA) followed by Bonferroni’s 1H and 13C NMR and high-resolution mass spectrometry. Analysis of the LAP sodium salt stability on plasma and acid media (similar to the stomached conditions) was carried out using ultra-performance liquid-chromatography tandem mass spectrometry (UPLC-MS/MS) and it confirmed the instantaneous conversion of LAP sodium salt into a neutral molecule of LAP. The individual plasma profiles of LAP and LAP sodium salt after intravenous administration were identical and were best described by a two-compartmental open model, with a volume of distribution of 0.19 ± 0.03 l/kg, a total clearance of 0.04 ± 0.01 l/h/kg, and a half-life of 4.1 ± 1.1 h , using a flexible docking approach implemented in the GOLD software . As a co μM Fig. , indicatWe next assessed the antiproliferative effect of LAP. To this end, freshly isolated mouse CD4 T cells were labelled with Dye Efluor 670 and stimulated with anti-CD3/CD28 in the presence of LAP or LEF  for 4 days. As shown in Fig. The antiproliferative effect of LEF is completely reversed by supplementation of uridine, supporting that DHODH is the target for LEF . We thenWe next examined the therapeutic potential of LAP in two experimental models of arthritis. The first model was collagen-induced arthritis (CIA), a well-established T cell-dependent preclinical model for RA . TreatmeFinally, we investigated the immunomodulatory effects of LAP in a second model of experimental arthritis. To this end, we employed the antigen-induced arthritis (AIA) model in C57BL/6 mice, which also requires a T-cell response for the generation of the acute articular inflammation . BrieflyIn the present study, we conducted a series of in silico, in vitro and in vivo studies describing the biological activity and pharmacokinetic properties of LAP, which is a novel immunosuppressive drug that attenuates experimental autoimmune arthritis through inhibition of DHODH activity. Firstly, we synthetized LAP and performed chemical modifications to improve its solubility in water. In accordance with a previous report , we founLAP is a naturally occurring naphthoquinone that has been reported to exhibit antitumor, anti-inflammatory, and antimicrobial activities, but the molecular mechanism underlining these effects is poorly understood –15. It wIn the pathogenesis of RA, it is well accepted that the influx and proliferation of T cells in the synovial space play a critical role in the articular inflammation and joint destruction , 27, 30.Synovial tissue infiltrating inflammatory cells from RA patients are more resistant to apoptotic events, contributing to their accumulation and, consequently, the persistence of inflammation . The exaIn summary, this study demonstrated that LAP is a novel immunosuppressive drug that attenuates experimental autoimmune arthritis through inhibition of DHODH activity. Therefore, LAP could be considered as a potential immunosuppressive lead candidate with potential therapeutic implications for RA.


Mycobacterium tuberculosis clinical isolates previously identified in central Los Angeles, CA, in the 1990s using a PacBio platform. Isolate TB282 represents a large-cluster strain that caused 27% of the tuberculosis cases, while TB284 represents a strain that caused disease in only one patient.We report here the genome sequences of two  Mycobacterium tuberculosis strains cause disease in many persons, whereas others only cause disease in a single or a limited number of patients  and the Hierarchical Genome Assembly Process (HGAP) (Genomic DNA was isolated using the cetyltrimethylammonium bromide (CTAB) method . Each ges (HGAP) , with des (HGAP) .N50, 11,370 bp) and an average coverage of 82.95× for TB282, and a total 38,740 reads with a mean read length of 7,239 bp  and an average coverage of 52.73× for TB284. Using filtered reads, a genome assembly with a single contig of 4,424,438 bp was obtained for TB282, and for TB284, an assembly with three contigs of 4,394,243 bp, 4,245 bp, and 1,737 bp, respectively, was obtained.The single SMRT cell sequencing resulted in a total of 61,505 reads, with a mean read length of 7,292 bp (Both genome sequences have a mean G+C content of 65.6% and contain 45 tRNA genes and three rRNA genes. TB282 has 4,285 protein-coding regions, while TB284 has 4,230 protein-coding regions. The two genomes were compared by performing whole-genome alignment using Mauve  to identThe genome sequences of this pair of isolates, one representing a widespread strain and the other a unique strain, will provide an additional resource for comparative genomic analyses in studying the bacterial factors important for tuberculosis transmission.M. tuberculosis strains TB282 and TB284 have been deposited at DDBJ/ENA/GenBank under GenBank accession numbers CP017920 and MNBY00000000, respectively.The genome sequences of the


Correction to:Molecular Psychiatry21, 749–757; doi:10.1038/mp.2016.49P-values for the nine reported loci have changed slightly  and the P-value for the index SNP rs62353264 on chromosome four is now 5.5 × 10−8 (previously 3.7 × 10−8).The GWAS of neuroticism conducted within the Queensland Institute of Medical Research (QIMR) Berghofer Medical Research Institute cohort did not include covariates of age, sex, genotyping batch and 10 principal components. Adding these covariates does not substantially change the pattern of results within the meta-analysis, but