P. schrenkiana needles' water extract, organic extract fractions, and key allelochemical DHAP was systemically investigated at the temperature rising 2 and 4°C based on the data‐monitored soil temperature during the last decade in the stage of Schrenk spruce regeneration (seed germination and seedling growth). The results showed that the criterion day and night temperatures were 12°C and 4°C for seed germination, and 14°C and 6°C for seedling growth, respectively. In the presence of water extract, the temperature rise of 2°C significantly inhibited the germination vigor and rate of P. Schrenkiana seed, and a temperature rise of 4°C significantly increased the inhibition to the seedling growth (P < 0.05). Among the three organic fractions, the low‐polar fraction showed to be more phytotoxic than the other two fractions, causing significant inhibitory effects on the seed germination and growth even at low concentration of 0.1 mg/mL, and the inhibition effect was enhanced as temperature increased. The temperature rise significantly enhanced the promotion effect of DHAP, while the inhibition effect of temperature rise became less important with increasing concentration of DHAP. This investigation revealed that autotoxicity of P. schrenkiana was affected by the climate warming. As expected, it provided an insight into the mechanism and effectiveness of allelopathy in bridging the causal relationship between forest evolution and climate warming.In order to explore how plant autotoxicity changes with climate warming, the autotoxicity of  The phenomenon of one plant's growth inhibited by chemicals released from another plant into environment is generally defined as allelopathy  in Dinghu Mountain, Peng et al.  is an endemic species in Middle Asia and the mountains of Asia. In China, Schrenk spruce is mainly distributed on the northern and southern slopes of Tianshan Mountains, and the northern slope of Kunlun Mountain West. As widely documented, however, the natural regeneration of Schrenk spruce has been in jeopardy  were recorded hourly at each of survey sites during the sampling period, and the temperature data collected from 8:00 am to 8:00 pm were automatically averaged as a daily mean temperature, while the data collected from 8:00 pm to 8:00 am were automatically averaged as night mean temperature.Five field sites having different climatic conditions in the middle elevations on the northern slops of the Tianshan Mountains, including Zhaosu (ZS) forest center  and Gongliu (GL) forest center  in the western Tianshan, Xinjiang Agricultural University (XAU) forest education center  and Xingjiang Tianshan forest ecosystem research station of State forestry bureau (SFB)  in the central Tianshan, and Hami (HM) forest center  in the eastern Tianshan Fig. . Real‐tiP. Schrenkiana were collected from the parent trees located at the XAU forest education center  on 28–30 September 2012. All selected P. Schrenkiana plants were 30–35 m tall, 80–100 years old, and healthy and had no infection disease. After the collection, the cones were dried in paper bags at room temperature for 7 days and then threshed by hand to get seeds.  at room temperature for 48 h. Sequentially, the solution was extracted three times with the same volume of diethyl ether, ethyl acetate, and n‐butanol. The residues were concentrated under the reduced pressure to yield the fractions of diethyl ether (1.96 mg/g dry mass), ethyl acetate (4.67 mg/g dry mass), and n‐butanol (14.18 mg/g dry mass), respectively, and the residual water (9.19 mg/g dry mass) after the organic solvent extraction. Bioassay‐guided fractionation of the diethyl ether fraction (3.74 g) with the strongest autotoxicity was conducted in silica gel column chromatography  to give a yellow crystal (986 mg) which was identified as 3,4‐dihydroxy‐acetophenone (DHAP). All the treated solutions were kept in a refrigerator at 4°C for use.A total of 200 g of dry Stock DHAP solution of 100 mmol/L was prepared by dissolving pure DHAP into the distilled water. Considering the measurable levels of DHAP in the soil, the stock solution was diluted to concentrations of 5, 2.5, 1.0, 0.5, 0.25, and 0.1 mmol/L as treatment solutions and water as control for bioassays. The experiments of seed germination and seedling growth were conducted according to ISTA .P. Schrenkiana were placed in each sterile Petri dish (15 cm diameters) lined with Whatman #3 filter paper. Each Petri dish was added with 10 mL treatment solution which might be one of the original water extracts, water residue after the organic solvent extraction, the fractions of diethyl ether, ethyl acetate, and n‐butanol at 0.1, 0.5, 1.0, and 2.5 mg/mL, and the solutions of DHAP at 5, 2.5, 1.0, 0.5, 0.25, and 0.1 mmol/L. Then, the Petri dishes were placed in programmable illuminated incubator for a light/dark (L/D) cycle of 12 h/12 h accompanied with a temperature cycle of 12/4°C, 14/6°C, and 16/8°C, respectively. Treatments were conducted by a complete randomized design, and each treatment was replicated three times. Germination (radicle emergence) was measured within 7 or 21 days after treatment.In triplicate, 100 seeds of the surface‐sterilized P. Schrenkiana seeds were preceded in plastic boxes (20 × 15 × 10 cm) lined with Whatman #3 filter paper for 5–6 days until radicles emerged. A total of 100 grain of successful germination seeds were placed in Petri dishes in three replicates, and 10 mL of various treatment solutions (same as germination) was added to each Petri dish. Seedlings were incubated in an programmable illuminated incubator for a L/D cycle of 12 h/12 h accompanied with a temperature cycle of 14/6°C, 16/8°C, and 18/10°C, respectively. Five seeds were randomly sampled from each Petri dish, and the length of shoot and root was measured with a vernier caliper . Fresh weight of seedlings was also recorded (Mettler Toledo Instrument Ltd). The measurements were taken on the third day after incubation and continued once every 3 days for a total of 30 days.Pregermination of P. Schrenkiana were presented as the mean ± standard error of three replications. The significant differences among treatment solutions and control on seed germination and seedling growth of P. Schrenkiana were first examined by ANOVA (P < 0.05) and then analyzed using Fisher's LSD test for multiple comparisons among the different treatments using SPSS software.The average day and night temperature as well as average month temperature was calculated in Excel software and statistically analyzed linear regression using SPSS software, (SPSS Chicago). All results of seed germination and seedling growth of The changes of soil temperature in the five survey sites showed that mean soil temperatures of day/night in the periods of seed germination from May 15 to June 14 and seedling growth from June 15 to July 15 increased 3.1–3.9 °C/1.1–1.9°C and 1.7–3.3 °C/1.9–2.4 °C, respectively (Table S1). In the germination periods, the minimum soil temperatures of day/night (9.2°C/3.7°C) appeared at the sampling points of ZS and the maximum temperatures (15.6°C/6.5°C) at GL site, respectively. In the seedling growth periods, the maximum soil temperatures during the day (18.3°C) and night (8.8°C) appeared at GL site, and the minimum soil temperatures of 12.3°C during the day appeared at ZS site, while the minimum soil temperatures of 4.9°C during the night appeared at GL site with altitude 2600 m and SFB site with altitude 2700 m.The data of monthly soil temperature change at the five survey sites have been collected from 2003 to 2012 (Table S1). During the 10 years, average day and night temperatures have increased 0.5–0.6°C and 0.2–0.3°C in the germination period and increased 0.5–0.7°C and 0.3–0.4°C in the seedling growth periods, respectively. Monthly average soil temperature due to anomalous climate change in 2010 was much higher than those in other years. Based on the above analysis, the benchmark day and night temperatures were determined to be 12°C and 4°C for seed germination, and 14°C and 6°C for seeding growth, respectively.P. Schrenkiana seeds was measured to determine the effects of water extract or organic fractions as temperature increased (Table P. schrenkiana seeds enhanced significantly as temperature increased. In the presence of water extract (1.25 mg/mL), the temperature rise of 2 to 4°C significantly inhibited the germination vigor and rate of the seeds (P < 0.05). For the low‐polar fraction (diethyl ether extraction) at the concentrations of 0.1, 0.5, and 2.5 mg/mL, the initial temperature rise of 2 °C significantly reduced the seed germination vigor and rate, the further temperature rise of 2 °C also enhanced the inhibition but with lesser effect, and at the concentration of 1.0 mg/mL, however, temperature rise of 4 °C showed more significant effect of inhibition to the seed germination (Table n‐butanol extraction) and medium‐polar fraction (ethyl acetate extraction), a low concentration of 0.1 mg/mL significantly promoted the germination vigor and rate. With increasing temperature by 4 °C, the vigor was significantly enhanced, but not the rate . The effect of water residue (1.25 mg/mL) on seed germination and seedling growth did not differ significantly from the effect of water (control).The effects of water extract and organic fractions on the seedling growth of ngs Figs  and 3. IFor the low‐polar fraction, temperature rise of either 2°C or 4°C significantly increased the inhibition to plumules and radicle length of seedling Fig. . For theIn general, the key allelochemical DHAP at low concentration of 0.1 mmol/L promoted both the germination vigor and rate, but inhibited at concentrations from 0.25 to 5.0 mmol/L. By comparison, the effect of concentration was dominant, while the effect of temperature rise became less important with increasing concentration. For example, increasing temperature by 4°C remarkably enhanced the promotion effect of 0.1 mmol/L DHAP and significantly increased the inhibition effect of 0.25 or 0.50 mmol/L DHAP, but only slightly increased the inhibition effect of 2.5 or 5.0 mmol/L DHPA on the vigor and rate of seed germination Table .The temperature rise of 2 or 4°C significantly increased the promotion of 0.1 mmol/L DHAP on the plumules length of seedlings, but not affected the inhibition effect of 0.25, 1.0, and 2.5 mmol/L DHAP. The temperature rise of 2°C significantly increased the inhibition of 0.5 and 5 mmol/L DHAP on the growth of seedlings. Also, temperature rising significantly increased the promotion of 0.1 and 0.25 mmol/L DHAP to radicle length of seedlings, did not change the effect of 0.5 and 5.0 mmol/L DHAP, and significantly increased the inhibition of 1.0 and 2.5 mmol/L DHAP. Furthermore, the temperature rise of 4°C significantly increased the promotion of 0.1 mmol/L DHAP and the inhibition of 2.5 mmol/L on the fresh weight of seedlings, did not change the effect of 0.25 and 5.0 mmol/L DHAP, and only slightly increased the inhibition of 0.5 and 1.0 mmol/L DHAP  changed from 3.41 to 12.62°C, 2.89 to 11.06°C, 3.37 to 12.59°C, 3.68 to 11.75°C, and 3.68 to 12.50°C, respectively, and the temperatures of the five sites during the seedling growth period (middle of June to middle of July) changed from 4.30 to 16.45°C, 3.66 to 13.75°C, 4.23 to 16.56°C, 4.59 to 16.49°C, and 4.96 to 16.50°C, respectively. Also, the average soil temperature at the regeneration stage of P. Schrenkiana has increased 0.5–0.7°C at day and 0.2–0.4 °C at night during the last decade. The survey data were very valuable for the study of global climate change and regional responses.Based on the simulation of a regional climate model with 2 × COPinus laricio have very strong inhibition to its seed germination  Hook] in China  showed to be more phytotoxic than other two fractions, causing significant inhibitory effects on the seed germination and growth even at low concentration of 0.1 mg/mL, and the inhibition effect was enhanced with the increasing temperature or concentration. On the other hand, low concentration (0.1 mg/mL) of medium‐polar (ethyl acetate) and high‐polar (n‐butanol) fractions could promote the seed germination and seedling growth, and the promotion effect was generally enhanced with increasing temperature; at concentration of 0.25–1.0 mg/mL, however, the two fractions could inhibit the seed germination and seedling growth, and the effects of temperature rise on the inhibition strength were variable; at high concentration of 2.5 mg/mL, the two fractions could result in more significant inhibition to seed germination and seedling growth, and the inhibition strength was significantly enhanced with rising temperature.In this preliminary investigation, it was found that there was variable and synergistic effect of temperature, chemical polarity, and concentration on Centaurea maculosa stimulated roots growth in Gaillardia aristata and Lobelia erinusat 10 μg/mL, but had a significant inhibitory effect at 400 μg/mL. Needle‐leached DHAP had a similar effect on some of the plants, which was verified by tests. In this investigation, 0.1 mmol/L DHAP could act as promoter and 0.5 mmol/L DHAP as a inhibitor, and 0.25 mmol/L DHAP might be the inflection point of changing action direction. More interestingly, the concentration of DHAP corresponding to the inflection point decreased as the temperature increased .Click here for additional data file.


Here, we demonstrate that DYRK1B also undergoes trans-autophosphorylation on serine-421 (S421) in vitro and in cells and that this site contributes to DYRK1B kinase activity. Whilst a DYRK1BS421A mutant was completely defective for p-S421 in cells, DYRK1B inhibitors caused only a partial loss of p-S421 suggesting the existence of an additional kinase that could also phosphorylate DYRK1B S421. Indeed, a catalytically inactive DYRK1BD239A mutant exhibited very low levels of p-S421 in cells but this was increased by KRASG12V. In addition, selective activation of the RAF-MEK1/2-ERK1/2 signalling pathway rapidly increased p-S421 in cells whereas activation of the stress kinases JNK or p38 could not. S421 resides within a Ser-Pro phosphoacceptor motif that is typical for ERK1/2 and recombinant ERK2 phosphorylated DYRK1B at S421 in vitro. Our results show that DYRK1B is a novel ERK2 substrate, uncovering new links between two kinases involved in cell fate decisions. Finally, we show that DYRK1B mutants that have recently been described in cancer and metabolic syndrome exhibit normal or reduced intrinsic kinase activity.The dual-specificity tyrosine-phosphorylation-regulated kinase, DYRK1B, is expressed de novo during myogenesis, amplified or mutated in certain cancers and mutated in familial cases of metabolic syndrome. DYRK1B is activated by The online version of this article (doi:10.1007/s00018-015-2032-x) contains supplementary material, which is available to authorized users. CDK, MAPK, GSK and CLK families) of the eukaryote kinome ATP in a total volume of 50 µl for 20 min at 30 °C as described previously  were immunoprecipitated from whole cell lysates with anti-FLAG antibodies and then assayed for kinase activity by incubating with 50 μM Woodtide with two additional lysines attached to the 39A or S432P]ATP (2.5 µCi per reaction) in a buffer containing 7 mM MOPS (pH 7.2), 3.5 mM glycerol 2-phosphate, 7 mM MgCl2, 1.4 mM EGTA, 0.56 mM EDTA and 0.07 mM DTT. Reactions were incubated at 30 °C for 1 h and terminated by the addition of 17 µl 4× Laemmli Buffer and heating at 95 °C for 5 min. Reactions were separated by SDS-PAGE, transferred onto PVDF membrane and 32P incorporation was detected by autorad followed by probing with the indicated antibodies.FLAG-DYRK1B (WT or S421A) or empty vector was expressed in HEK293 cells for 24 h in the presence of 10 µM AZ191. FLAG-DYRK1B was isolated from whole cell extracts by anti-flag immunoprecipitation and used as a substrate in an ERK2 in vitro kinase assay. Each 50 µl reaction contained 0.4 µg GST-ERK2 , 20 µl substrate beads and 50 µM [γ-DYRK1B siRNA  and GFP siRNA (Qiagen 1027310) were dissolved in nuclease-free water to generate stock solutions of 20 µM and used at a final concentration of 30 nM. For each well of a six well plate, 5 µl of Lipofectamine 2000 was diluted in 250 µl OptiMEM and incubated at room temperature for 5 min. siRNA were diluted in 250 µl OptiMEM prior to the addition of 250 µl of the Lipofectamine-OptiMEM solution. The transfection mixes were incubated at room temperature for 20 min to allow the formation of siRNA complexes. Cells were seeded the previous day in Pen/Strep-free medium to achieve a confluency of 50 % on the day of transfection. 500 µl of the transfection solution was added to each well of the tissue culture plate and the cells were incubated 37 °C in a humidified incubator with 5 % (v/v) CO2 for 16 h. After this 16 h, the medium was changed to fresh Pen/Strep-free media containing DMSO or 1 µM Selumetinib. The cells were incubated for a further 48 h at 37 °C in a humidified incubator with 5 % (v/v) CO2 prior to harvesting.1, S, G2/M) and the fraction of dead cells (those with sub-G1 DNA content) was assessed by staining fixed cells with propidium iodide followed by flow cytometry as described previously [Cell cycle distribution  within the activation loop. However, once translation is complete mature DYRK1B phosphorylates substrates on Ser/Thr residues in trans. We wished to develop an assay to monitor the kinase activity of mature DYRK1B in cells, hoping that this might serve as a biomarker to support the development of DYRK1B inhibitors. To determine if DYRK1B could autophosphorylate on other residues, we analysed potential auto-phosphorylation sites on recombinant DYRK1B by proteolytic digestion and mass spectrometry (MS). Using a combination of endoproteases we achieved 85 % coverage of DYRK1B and identified twelve phosphorylation sites including p-Y63 and p-Y273 that have been previously reported (Supplementary Table 1). A targeted MS method was developed to quantify these phosphorylation sites in Cos1 cells expressing Myc-DYRK1B using multiple reaction monitoring (MRM). This analysis identified four phosphorylation sites in cells but only one, S421 in the peptide IpSPLGALQHGFFR, was inhibited when cells were incubated in the presence of AZ’294, an early stage precursor of the DYRK1B inhibitor AZ191 [Y271F/Y273F or the catalytic lysine mutant DYRK1BK140R (kinase-dead or KD); these results demonstrated that p-S421 in cells was strongly dependent on DYRK1B kinase activity  was completely defective. However, since only ~50 % of DYRK1B was phosphorylated at S421 when over-expressed  so that we could distinguish S421 phosphorylation in cis or in trans  expressed alone was not phosphorylated on S421, nor was wild-type EGFP-DYRK1B able to phosphorylate FLAG-DYRK1BS421A (SA). However, both EGFP- and FLAG-tagged wild-type DYRK1B auto-phosphorylated on S421, and this was blocked by AZ191 or harmine. Finally, FLAG-DYRK1BD239A (KD) was phosphorylated on S421 when co-expressed with wild-type EGFP-DYRK1B and this was blocked by AZ191 or harmine, suggesting this was an intermolecular phosphorylation  KRAS drives activation of multiple protein kinase cascades and (ii) expression of activated KRAS has previously been proposed to activate DYRK1B by up to 20-fold [D239A (KD) was transiently expressed with KRASG12V for 24 h in HEK293 cells. DYRK1B proteins were then isolated from whole cell extracts by immunoprecipitation and assayed in an in vitro DYRK1B kinase assay; activity was again normalised to the amount of DYRK1B that was recovered in the immunoprecipitation  exhibited negligible kinase activity that was unaffected by KRASG12V expression. Control blots confirmed that KRASG12V was expressed and active since it activated endogenous ERK1/2 , which cannot auto-phosphorylate on S421  was expressed in HR1 cells [D239A (KD) became phosphorylated on S421 and this was blocked by the highly selective MEK1/2 inhibitor, selumetinib (AZD6244/ARRY-142886) [KRAS activates multiple effector pathways including the RAF-MEK1/2-ERK1/2 pathway and various PI3K-dependent pathways. However, we noted that S421 is immediately followed by a proline residue (P422) in a Ser-Pro motif, the minimum required for phosphorylation by proline-directed kinases such as ERK1/2. To address if activation of the ERK1/2 pathway alone could increase p-S421, DYRK1B-142886)   . When DYD239A in HM3 cells; these are HEK293 cells which harbour an inducible MEKK3 fusion protein (∆MEKK3:ER*) that activates the ERK1/2, JNK and p38 pathways  in HR1 cells and stimulated them with 4HT for various times to activate ΔCRAF:ER*. Activation of ERK1/2 was observed as early as 15 min after 4HT addition and persisted for the full 120 min timecourse  following activation of ΔCRAF:ER*.If DYRK1B is a direct substrate of ERK1/2 then the kinetics of S421 phosphorylation on DYRK1B should closely follow the kinetics of ERK1/2 activation in cells and should also match the phosphorylation of bona fide ERK1/2 substrates such as BIMEL . To addr32P incorporation and pS421-DYRK1B western blot. However, 32P was still incorporated into DYRK1BS421A in the absence of AZ191 (Lane 6), albeit to a lesser extent than wild-type DYRK1B, suggesting that there are other sites of DYRK1B auto-phosphorylation in vitro in addition to S421, consistent with our MS analysis (Table 32P incorporation and p-S421 western blot and this was blocked by the ERK1/2 inhibitor SCH772984 [S421A (Lane 8) although to a lesser degree, indicating that ERK2 may also be able to phosphorylate other sites on DYRK1B in vitro. Taken together these results demonstrated that in addition to auto-phosphorylation, DYRK1B is also phosphorylated on S421 by ERK1/2 in cells and in vitro.To determine if ERK2 could directly phosphorylate DYRK1B at S421, recombinant, active GST-ERK2 was incubated with FLAG-DYRK1B in an in vitro kinase assay Fig. b. In thiCH772984  (Lane 5)G13D/NRASQ61L) with 1 µM selumetinib this strongly and immediately inhibited ERK1/2 signalling and increased the expression of DYRK1B after a delay of 8 h. This was accompanied by an increase in DYRK1B mRNA which was apparent after 5 h of selumetinib treatment . Treatment with actinomycin D, an inhibitor of transcription, prevented the increase in DYRK1B expression observed in response to selumetinib treatment  suggesting that the increase in DYRK1B expression observed upon MEK inhibition reflects de novo gene transcription. Finally, similar results were obtained in the A375 melanoma cell line (BRAFV600E) , where selumetinib caused a dose-dependent inhibition of ERK1/2 signalling and an increase in expression of DYRK1B as well as BIMEL, which is known to be repressed by the ERK1/2 pathway [It has previously been show that first generation, pan-MEK1/2/5 inhibitors PD98059 and U0126 can increase DYRK1B mRNA and protein , 45. Not pathway . Since i pathway   exhibited identical kinase activity to wild-type DYRK1B; DYRK1BR102H and DYRK1BS234G exhibited 40–50 % reduction in kinase activity and DYRK1BQ275R exhibited a 90 % reduction in kinase activity  severely.Systematic sequencing of cancer genomes has revealed rare mutations in DYRK1B including L28P, R102H, S234G and Q275R . Howevertrans-phosphorylation mechanism whereas Y273 is an intramolecular cis-phosphorylation event. To our knowledge, this is the first description of a serine or threonine auto-phosphorylation site on DYRK1B. S520 in DYRK1A was previously identified as a site of auto-phosphorylation that was required for full DYRK1A kinase activity [cis, in contrast to the intermolecular phosphorylation of S421 in DYRK1B described here. Indeed, given the position of S421, situated away from the kinase active site, it is difficult to imagine it could be phosphorylated in cis through an intramolecular mechanism. Regardless, it seems that S421 phosphorylation also contributes to DYRK1B kinase activity, either directly or by an indirect mechanism involving accessory proteins, since the S421A mutant exhibited a consistent 50 % reduction in kinase activity; indeed, this is likely to be an underestimate of the impact of the S421A mutant since only half of over-expressed DYRK1B was phosphorylated at S421. Whilst a twofold modulation of kinase activity may seem modest, it is important to remember that even a 50 % increase in expression and activity of the closely related DYRK1A has profound physiological effects in mice and phenocopies some Down’s syndrome defects [Drosophila, mice and human patients including microcephaly, growth retardation and behavioural defects [Our results identified S421 as a site of DYRK1B autophosphorylation since it was present in purified recombinant wild-type DYRK1B incubated in vitro with ATP, absent in kinase-dead DYRK1B and strongly (though not completely) inhibited by DYRK1B inhibitors in vitro or in cells. These drugs do not prevent autophosphorylation on tyrosine  in cells  except aactivity ; in this defects , 15 whil defects , 50, 51.RIS421PL) shortly after alpha helix H/kinase sub-domain XI [th in terms of frequency out of the 2500 identified mutation positions across kinase domains, suggesting a potential wider significance for phosphorylation at this site. Of the 61 human CMGC kinases listed at http://kinase.com/human/kinome/groups/cmgc.aln only 7 lack a Ser or Thr at the position corresponding to Ser421 of DYRK1B. More significantly, S421 in DYRK1B is a Pro-directed site (Ser-Pro) and within the closely related human DYRK family  the corresponding site is a Ser-Pro or Thr-Pro in all cases except DYRK1A (Gln-Pro) raising the possibility that these kinases may also be regulated by phosphorylation at this site. This site is also Ser-Pro or Thr-Pro in DYRKs from lower eukaryotes including S. pombe Pom1p, S. cerevisiae Yak1p, C. elegans MBK-1 and MBK-2 and D. melanogaster dDYRK2 and dDYRK3, indicating evolutionary conservation of the site. In terms of functional relevance, intermolecular phosphorylation at S421 suggests that DYRK1B may be able to form dimers. Indeed, the structure of the closely related DYRK1A [ΔCLV exhibits reduced kinase activity against exogenous substrates compared to the full length DYRK4 [S421 occurs close to the highly conserved arginine residue  and activates multiple protein kinase signalling pathways. In contrast to previous reports of a 9- to 20-fold activation [G12V caused only a very modest (10–15 %) increase in activity that was not statistically significant. This was not due to a deficit in our KRAS construct as KRASG12V strongly activated endogenous ERK1/2 in the same transfections. These results suggest that any effect of activated KRAS on DYRK1B effector pathways such as Hedgehog [G12V could actually increase DYRK1B phosphorylation at S421. Through a combination of conditional kinases and selective kinase inhibitors we were able to show that ERK1/2 activation alone could increase p-S421, commensurate with established ERK1/2 substrates, and ERK2 could phosphorylate S421 in vitro. This is the first report to clearly define DYRK1B as a substrate of ERK2. A previous study suggested that ERK2, JNK or p38 might phosphorylate DYRK1B but stopped short of specifying the kinase, identifying the phosphorylation site or the effect on DYRK1B function [We noted that inhibitors of DYRK1B Ser/Thr kinase activity left a residual p-S421 signal in cells, prompting a search for other kinases that might target this site. For this purpose we co-expressed DYRK1B with KRAStivation  we consiHedgehog  may be ifunction .CIP1 and/or p27KIP1 and thereby promote cell cycle arrest and differentiation [Together with previous studies, our results reveal a complex interplay between ERK1/2 signalling and DYRK1B. Since phosphorylation of S421 may enhance DYRK1B kinase activity, ERK1/2 inhibition could decrease DYRK1B activity. In contrast, previous studies have indicated that inhibition of ERK1/2 signalling may increase DYRK1B expression , 45 and ntiation , 45, 55.ntiation . ERK1/2-There is growing interest in identifying synthetic lethal drug combinations in the treatment of cancer . For exaDYRK1B is over-expressed due to gene amplification in certain tumours, including pancreatic cancer , 28, andWe found that these mutations were either neutral with respect to DYRK1B activity (L28P) or caused a consistent reduction in kinase activity ; in the case of the Q275R mutation this effect was severe, with an approximate 90 % reduction in kinase activity. Q275 lies within the activation loop of DYRK1B; whilst activating, oncogenic mutations are found in this region at significant frequency in some protein kinases, mutations in this loop, including the introduction of bulky and highly positive side chain such as arginine, are also known to inhibit activity by impacting on activation loop conformation and flexibility. At this stage we cannot rule out the possibility that these mutations might confer kinase-independent properties upon DYRK1B; for example, by driving DYRK1B to a specific cellular location or engaging with particular partner proteins. Indeed, some of the effects of DYRK1B in metabolic syndrome were apparently independent of DYRK1B kinase activity . RegardlG12V does not increase DYRK1B kinase activity. This may be because DYRK1B expressed in cells is already active so that any effects of KRAS are modest and incremental; this may require single cell analysis to resolve. Alternatively, different KRAS effectors may exert opposing effects on DYRK1B; so whilst activation of RAF-MEK-ERK may promote S421 phosphorylation, that would enhance activity, other KRAS effector pathways may exert opposing effects on DYRK1B. Indeed, our mass spectrometry analysis has identified at least two other phospho-Ser sites on DYRK1B expressed in cells that are not autophosphorylation sites; the kinases for these sites and the functional consequences remain unknown. Finally, mutants of DYRK1B found in cancer or metabolic syndrome are either neutral with respect to kinase activity or exhibit significant decreases in kinase activity. At least one of these, R102C in metabolic syndrome, is defined as a gain-of-function mutation [In summary, we demonstrate that phosphorylation of S421 contributes to DYRK1B kinase activity and that S421 is a site for DYRK1B autophosphorylation and phosphorylation by ERK1/2, defining DYRK1B as a new substrate of ERK1/2. Our results reveal opposing effects of ERK1/2 signalling on DYRK1B which may serve to fine tune DYRK1B activity. However, despite the ability of ERK1/2 to phosphorylate S421, KRASmutation  but clea(A) MelJuso cells were incubated with 1 µM Selumetinib for the time points indicated. Cells were lysed in TRIzol, RNA was extracted and cDNA was generated by reverse transcription. The cDNA was used as the template in a QPCR reaction using SyBr green PCR master mix with DYRK1B or B2 M primers. The level of DYRK1B expression was normalised to that of the housekeeping gene B2 M. (B) MelJuso cells were treated with 0 or 1 µM Selumetinib in the absence or presence of 10 µM Actinomycin D for 16 h. Whole cell extracts were separated by SDS-PAGE, transferred onto PVDF membrane and immunoblotted with the specified antibodies. Data are taken from a single experiment representative of three separate experiments. (C) A375 cells were treated for 24 h with the indicated doses of selumetinib. Whole cell extracts were fractionated by SDS-PAGE, transferred onto PVDF membrane and immuno-blotted with the indicated antibodies. Data are taken from a single experiment representative of two separate experimentsSupplementary material 1 Specific inhibition of MEK-1/2-ERK1/2 signalling increases DYRK1B mRNA levels and require new gene transcription. Supplementary material 2 Sites of DYRK1B autophosphorylation identified in vitro. Recombinant DYRK1B was allowed to autophosphorylate in the presence of ATP. Coomassie-stained bands of (0.5 µg or 1 µg) were reduced, cysteines blocked, and digested with: trypsin only; AspN and trypsin; chymotrypsin only; chymotrypsin and trypsin. The resultant peptides were analysed using the LCMSMS workflow on the QSTAR Elite mass spectrometer. Peptides and phosphorylation sites were identified using Mascot. *Precise location of single phosphorylation site not known


H. sapiens, M. musculus, R. norvegicus, D. melanogaster, C. elegans, S. pombe and S. cerevisiae. From the scientific literature and public databases, we totally collected and integrated 54,148 phosphoproteins with 483,001 phosphorylation sites. Multiple options were provided for accessing the data, while original references and other annotations were also present for each phosphoprotein. Based on the new data set, we computationally detected significantly over-represented sequence motifs around phosphorylation sites, predicted potential kinases that are responsible for the modification of collected phospho-sites, and evolutionarily analyzed phosphorylation conservation states across different species. Besides to be largely consistent with previous reports, our results also proposed new features of phospho-regulation. Taken together, our database can be useful for further analyses of protein phosphorylation in human and other model organisms. The dbPAF database was implemented in PHP + MySQL and freely available at http://dbpaf.biocuckoo.org.Protein phosphorylation is one of the most important post-translational modifications (PTMs) and regulates a broad spectrum of biological processes. Recent progresses in phosphoproteomic identifications have generated a flood of phosphorylation sites, while the integration of these sites is an urgent need. In this work, we developed a curated database of dbPAF, containing known phosphorylation sites in  As one of the most well-studied and ubiquitous post-translational modifications (PTMs) in proteins, phosphorylation is a conserved mechanism in both eukaryotes and prokaryotes, participates in almost all of biological processes, and reversibly determines the cellular dynamics and plasticity1234236et al. identified and quantified 3,457 proteins with 5,249 phosphorylation siteset al. totally quantified 31,480 phosphorylation sites in 7,280 proteins across 14 rat organs and tissueset al. developed a new platform of EasyPhos, which can rapidly quantify over 10,000 phosphorylation sites from cell or tissue samples in a single run14151619Recently, rapid progresses in high-throughput liquid chromatography/mass spectrometry (LC-MS) techniques have greatly advanced the identification of phosphorylation sites1112H. sapiens, D. melanogaster, C. elegans and S. cerevisiae30S. cerevisiae322534Due to the data accumulation, the collection and integration of numerous phosphorylated substrates with their sites from different studies have emerged to be a great challenge. The first phosphorylation database, PhosphoBase, was developed in 1998 and only contained 398 experimentally identified phosphorylation sites at that time222628H. sapiens, M. musculus, R. norvegicus, D. melanogaster, C. elegans, S. pombe and S. cerevisiae. The detailed annotations together with original references were provided for each protein entry. Using the new data set, we detected significantly over-represented sequence motifs around phospho-serine (pS), phospho-threonine (pT) and phospho-tyrosine (pY) sites for each species, separately. From the results, we observed that the most significant pT motifs are highly conserved across different organisms, while pS sites prefer to occur at intrinsically disordered regions with low-complexity. We also predicted potential PKs for phospho-sites and demonstrated that different PK groups or families play different roles in the regulation of phosphorylation. In addition, we systematically analyzed the conservation states of phosphorylation sites, and observed that the phosphorylation is similarly conserved across different species. Taken together, the dbPAF database can serve as a useful resource for further studies of protein phosphorylation in human, animals and fungi.During the past decade, we also collected and curated phosphorylation sites beyond directly using known data sets from public databases. Previously, we developed two phosphorylation databases of dbPPTH. sapiens, M. musculus, R. norvegicus, D. melanogaster, C. elegans, S. pombe and S. cerevisiae. Totally, we collected 294,370 non-redundant phosphorylation sites of 40,432 proteins from 115 published papers , while only 22.49% and 11.67% of phosphorylation events occur in pT and pY sites, respectively  to determine the conservation status of PTM sitesH. sapiens, M. musculus, R. norvegicus, D. melanogaster, C. elegans and S. cerevisiae, respectively. Based on the data set, the average rRCS values were calculated as about 55~85%, exhibiting a strong difference across eukaryotesIn a previous study, Minguez S. pombe) to 84.34% (in R. norvegicus), and there was not a significant difference across different organisms , a widely-used online tool for detecting phosphorylation motifs from the phosphoproteomic datap-value <0.000001. The phosphorylation motifs were calculated for pS, pT and pY sites, respectively. The most significant motifs of the three types of residues for each species were diagrammed in sequence logos  for pairwisely detecting orthologs among the seven species. All orthologous proteins among different species were multi-aligned by Clustal Omega RCSpWe analyzed the conservation distributions of phosphorylation sites in Np/N was defined as the Residue Conservation Ratio (RCR)N is the number of sequences with the maximum branch length (MBL), which is the maximum branch distance between any two species that contain a conserved phospho-site. The Np is the number of phospho-sites observed in the column. Then the calculated RCSp values of phosphorylated sites were mapped into the reference distribution to calculate the relative RCS or rRCShttp://itol.embl.de/)The How to cite this article: Ullah, S. et al. dbPAF: an integrative database of protein phosphorylation in animals and fungi. Sci. Rep. 6, 23534; doi: 10.1038/srep23534 (2016).


Widespread use of this approach has been slow among molecular biologists, in part because the methods require significant computational expertise. Here we present PhyloBot, a web-based software tool that makes ancestral sequence reconstruction easy. Designed for non-experts, it integrates all the necessary software into a single user interface. Additionally, PhyloBot provides interactive tools to explore evolutionary trajectories between ancestors, enabling the rapid generation of hypotheses that can be tested using genetic or biochemical approaches. Early versions of this software were used in previous studies to discover genetic mechanisms underlying the functions of diverse protein families, including V-ATPase ion pumps, DNA-binding transcription regulators, and serine/threonine protein kinases. PhyloBot runs in a web browser, and is available at the following URL: http://www.phylobot.com. The software is implemented in Python using the Django web framework, and runs on elastic cloud computing resources from Amazon Web Services. Users can create and submit jobs on our free server (at the URL listed above), or use our open-source code to launch their own PhyloBot server.The method of phylogenetic ancestral sequence reconstruction is a powerful approach for studying evolutionary relationships among protein sequence, structure, and function. In particular, this approach allows investigators to (1) reconstruct and “resurrect” (that is, synthesize  PLOS Computational Biology Software paper.”“This is a in vitro. Comparisons with the modern counterparts led to the discovery of key amino acid residues responsible for the biochemical diversity among related members of a gene family . The meMany questions in molecular and cell biology could be addressed using ancestral protein analysis. One obstacle is that the typical protocol for ancestral reconstruction involves multiple steps that require significant expertise with computational phylogenetics. In brief, the protocol begins with a collection of orthologous protein sequences sampled from diverse species. Next, the sequences are aligned to each other, their phylogenetic relationships are inferred, probabilities of ancestral sequences are computed at internal phylogenetic nodes, and then mutations  are identified on every phylogenetic branch. The rigorous application of this protocol can be challenging because it is not implemented as a single software package. Rather, ancestral reconstruction currently requires dozens of software tools, the computational skills to combine them, knowledge about phylogenetic models, and the programming abilities to deal with multiple file formats (many of them esoteric).PhyloBot, described here, is new software that automates ancestral sequence reconstruction. It provides a user interface that greatly simplifies the reconstruction process, and also includes visual tools to analyze ancestors. Specifically designed for bench scientists unfamiliar with bioinformatics, the software runs in web browsers and it requires no installation on users’ computers. Rather, PhyloBot uses elastic computing resources in the Amazon cloud. Moreover, results from PhyloBot analyses are portable: every ancestral reconstruction receives a permanent URL that can be shared with colleagues and used in publications. We believe PhyloBot is a significant methodological advance for computational molecular biology, one that will hopefully inspire widespread use of ancestral protein analysis.PhyloBot is a web portal that automates the reconstruction of ancestral amino acid sequences. The portal provides interactive web tools to compose and launch analysis jobs on remote supercomputers. The tools are easy-to-use and conceal a great deal of underlying automation. To start, users upload a FASTA-formatted text file containing a collection of related protein sequences . There ia priori to be true. If this tree is provided, PhyloBot will use it to restrict the phylogenetic analysis to evolutionary hypotheses that match the constraints.The front page of the PhyloBot portal provides a control panel to compose new analysis jobs , and to http://www.phylobot.com, or launch their own instance of PhyloBot using its open-source code.PhyloBot is engineered using Python Django, and it currently runs on cloud computing resources from Amazon Web Services. When a job is launched, PhyloBot acquires elastic compute nodes from Amazon. This means that all jobs are launched instantly, and there is no queue to wait. Users are welcome to use an instance of PhyloBot available at The inference of homology between sites in related protein sequences  is a necessary first step for phylogenetic analysis. Many alignment methods have been proposed , 21, andPhyloBot evaluates the consistency of sequence alignments by mapping the aligned position of every residue to its corresponding position in other alignments . This coPhyloBot infers phylogenies using a maximum likelihood (ML) method implemented in RAxML . BrieflyAs a consequence of the model-fitting step, PhyloBot finds ML trees for all combinations of sequence alignments and evolutionary models in its collection. This means that phylogenetic conclusion drawn from one method-model pair can be assessed for robustness across alternate methods and models . DiffereThe accuracy of every tree branch is estimated using approximate likelihood ratio tests (aLRT), implemented in PhyML v3.0 ,17. The The aLR for a particular branch can be interpreted as an estimated likelihood ratio between two different evolutionary hypotheses. In the first hypothesis, the true tree is the ML tree containing the branch in question. In the second hypothesis, the true tree is an alternate tree in which the branch does not exist. Using this framework, it can be said that the existence of specific phylogenetic split is estimated to be “X times more likely” than the next-best hypothesis in which that branch doesn’t exist.Lazarus [PhyloBot reconstructs ancestral protein sequences at every internal node of every ML tree, for all combinations of sequence alignment method and evolutionary model. Ancestors are reconstructed using the empirical Bayes approach , as implLazarus  to contrLazarus . PreviouLazarus . FollowiPhyloBot provides web tools to compare ancestral protein sequences at different points in evolutionary history. Ancestral sequence comparison is a direct means to generate testable hypotheses about which residues in a protein determine its unique biochemistry. In many protein families, all members perform an analogous function, such as binding a class of substrates, but individual members exhibit biochemical variation in this function. Sequence comparisons between present-day proteins often suggest a large number of possible amino acid changes to explain observed biochemical differences. In contrast, comparisons between ancestral sequences on relevant phylogenetic branches may reveal a smaller set of candidate residues with fewer false-positives .http://www.phylobot.com/cmgc, http://www.phylobot.com/mcm1, and http://www.phylobot.com/VATPase. The methods of ancestral reconstruction can be applied to nearly any protein family, regardless of its age or diversity. The accuracy of a reconstruction is correlated with conservation; this means that functionally important interaction domains are generally reconstructed with higher accuracy than poorly conserved regions, such as polypeptide linkers.PhyloBot has been used to discover genetic mechanisms underlying biochemical diversity in several protein families, including protein kinases , DNA-binPhyloBot provides an ancestral library viewer to interact with results from completed analyses . In pracThe methods of ancestral reconstruction are ideal for examination of protein families with one or more diverse biochemical functions that can be assayed in molecular experiments. In these cases, PhyloBot is well-suited to guide experimentalists toward identification of the residues that determine functional variation across a protein family.http://www.phylobot.com, and its source code is available at https://github.com/vhsvhs/phylobot-django. Future versions of PhyloBot will include an expanded suite of alignment methods and phylogenetic models.PhyloBot is available to use at


Schistosoma mansoni infection has been correlated with IgE responses to the adult worm. Molecular targets of this response are gaining interest as markers of immunity and as indicators of allergenic properties. Few protein families contain IgE antigens  and one of the most highly represented are the tropomyosins. Alternative splicing generates numerous tropomyosin isoforms, which in parasites is likely to induce a range of anti-tropomyosin responses in the host. Here we examine human IgE and the counteracting IgG4 responses to splice variants of S. mansoni tropomyosin (SmTpm). It was possible to show life-cycle transcription profiles for 12 of 20 predicted splice variants from the four SmTpm genes. We expressed recombinant protein of four variants of TpmII  with considerable differences in sequence. TpmII.4 and 8 were muscle, and TpmII.3 and 7 non-muscle, types. IgE and IgG4 responses to all four proteins were measured in a population of 228 infected boys and men (7–76 years) from a region of Uganda endemic for S. mansoni. Levels of these antibodies were not dependent on age and did not change following anthelminthic treatment. IgE to TpmII.3 was common in the cohort (>60%) and IgG4 to TpmII.3 less so (33%). IgE to TpmII.7 was rare (6.5%), but IgG4 to TpmII.7 was more common (49%). In regression analysis, a detectable IgE response to TpmII.3 was associated with reduced re-infection 2 years after treatment and an IgG4 response to TpmII.7 with increased re-infection. Different isoforms generated by alternative splicing are targeted by different components of the anti-Tpm IgE/IgG4 response. Only some of these are associated with immunity.Resistance to  For people living where the parasite is endemic, repeated exposure to infectious cercariae can result in life-long infection. Partial immunity does gradually develop and epidemiological studies show that this is correlated with an IgE response to the worm  protein family are dominant IgE antigens. IgE responses to a number of these proteins  have been associated with resistance to infection  is another likely target for the host IgE response. Tpm has been described as an invertebrate “panallergen”  with ove simplex . The can simplex . Within  simplex , at diff simplex . In vert simplex  and ther simplex . IgE bin simplex , but the simplex .S. mansoni Tpm (SmTpm) is predicted from genomic studies , select four splice variants with markedly different domain structures, express these as recombinant proteins and assess IgE and IgG4 responses to the four molecules in a cohort of infected males from a region endemic for S. mansoni.Alternative splicing of  studies  but it i studies . In this22.1S. mansoni eggs, cercariae, cercarial heads, cercarial tails, 24 h schistosomulae and 7 week adults were prepared as previously described . Smp_022170, Smp_031770, Smp_044010, Smp_085290 and Smp_032490 were analysed for conserved domains by BLAST searches of NCBI/GenBank databases (https://www.ncbi.nlm.nih.gov/genbank/) and sets of primers were designed to uniquely identify each putative alternatively spliced isoform of every gene by PCR . The reaction products were then visualised by ethidium bromide (EtBr)/agarose gel electrophoresis. In the case of a negative result, the reaction was repeated at annealing temperatures 2–4 °C lower than the optimum, until non-specific binding of the primers became apparent.All reactions were performed according to the following cycling protocol: an initial denaturation at 98 °C for 30 s, 35 three-step cycles of 98 °C for 10 s, The primers for Smp_031770.3 (TpmII.3) were later found to not be specific to this variant alone; they also amplify the novel isoform Smp_031770.16 (TpmII.16) identified in this study.2.3KC904503.1, KC904504.1KC904505.1 and KC904506.1). While cloning the CDS of Smp_031770.3 (TpmII.3), a novel 699 bp isoform that was not predicted by EVM2 in the S. mansoni genome was found. This sequence was uploaded to GenBank (KC904507.1) as Smp_031770.16 (TpmII.16).The coding DNA sequences (CDS) of four SmTpm II (Smp_031770) variants, TpmII.3, 4, 7 and 8, were amplified from cercarial cDNA using the following primers: 5′-ATG AAG CTT CAG ATA GAC CAGC-3′ and 5′-TTA GAT ATT TTC TAC TTC AGT AAA CAT GG-3′ for TpmII.3 (699 bp), 5′-ATG GAA CAT ATT AAA AAG AAA-3′ and 5′-TTA GTT TCC AGT AAG TTC TG-3′ for TpmII.4 (855 bp), 5′-ATG GAA GAA GCT TTA TCA G-3′ and 5′-TTA GTT TCC AGT AAG TTC TG-3′ for TpmII.7 (720 bp) and 5′-ATG GAA CAT ATT AAA AAG AAA-3′ and 5′-TTA GAA ATG CAC CTC ATC-3′ for TpmII.8 (504 bp). Amplicons were gel-purified and cloned directly into the pJET1.2/blunt vector using the blunt-ended CloneJET PCR cloning kit . Recombinant plasmids were sequenced . The mRNA coding sequence of SmTpmII.3, 4, 7 and 8 were uploaded to GenBank (Accession Nos. 2.4S. mansoni RNA Polymerase II (RpII) were assayed by qPCR using the DyNAmo Flash SYBR Green qPCR Kit (Fermentas). One microlitre of diluted cDNA  was used as the maximum template amount per 20 μl reaction. Primers 5′-GCT GAA GTC GCC TCA CTA CA-3′ with 5′-TTT CAT CAG CAG CTT TAC TTG C-3′ for TpmII exon E (121 bp) and 5′-CGAAAATGATGTCAGCCTCA-3′ with 5′-CCGTTCTTGTGATTCCTCGT-3′ for RPII, were used at a final concentration of 0.521 μM. Reactions were performed according to the recommended cycling protocol for the kit, on an Eco Real-Time PCR System  and high-resolution melt curves were performed over a 55–98 °C range. qPCR amplicons were cloned into pJET1.2/blunt and sequenced as described in Section Transcription levels of the common TpmII exon E and of control gene ΔCt method  or between XbaI and XhoI sites (TpmII.7). Constructs were sequenced to verify inserted sequences. GST-TpmII fusion proteins (5′ GST) were expressed in Escherichia coli, isolated by affinity chromatography on glutathione–agarose , reapplied to glutathione–agarose in PBS containing 1% Triton X-100 , washed with PBS and then cleaved at 22 °C for 16 h with 50 units (U) of thrombin per mg of fusion protein to release free TpmII.3, 7 or 8 and 4 h with 30 U of thrombin per mg of fusion protein to release TpmII.4. In each case the absence of contaminating GST was confirmed by ELISA using rabbit-anti-GST antisera (Sigma) and horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (Sigma).Full CDS for TpmII variants 3, 4, 7 and 8 were amplified by PCR from the recombinant plasmids . Antigens were prepared at a concentration of 100–200 μg/ml in a 10 mM phosphate buffer, pH 7.5, containing 50 mM NaF. Thermal denaturation of the antigens was followed at 222 nm from 25 to 95 °C in 2 °C steps with an equilibration time of 30 s and an averaging time of 1 s at a 1 nm bandwidth. Renaturation was followed from 51 °C for TpmII.4, and 8, from 81 °C for TpmII.3, and from 75 °C for TpmII.7. Wavelength scans were performed before and after thermal denaturation at 25 °C between 190 and 260 nm in 0.5 nm intervals with an averaging time of 1 s at a 1 nm bandwidth. Baseline values were obtained by concurrent CD spectrometry of buffer alone. Dynode voltages did not fall outside of the linear range in any scans. Melting temperatures were calculated by taking the maxima of the derivatives of thermal denaturation curves, and percent helicity calculations were performed using the CONTINLL analysis program  and the 2.7S. mansoni-infected male inhabitants aged 7–76 years were selected by simple random sampling (S. mansoni eggs per gram (epg) of faeces . Blood samples were collected from participants before and 9 weeks after treatment with 40 mg/kg of Praziquantel (PZQ). Of the 228 selected males, 216 donated blood samples before treatment and 202 donated samples after treatment. To determine the efficacy of treatment and intensity of re-infection, quantitative parasitology was also carried out at 9 weeks, 8 months and 2 years post-treatment. In line with Ugandan national health policy, infected participants were treated at 2 years (but not at 9 weeks or 8 months).IgE and IgG4 responses to TpmII variants 3, 4, 7 and 8 were measured in a sample of adult males resident in the fishing village Musoli, located on Lake Victoria, Uganda. Full details of the study area, population and sample selection have been described elsewhere . Brieflysampling . WrittenEthical clearance for the study was obtained from the Uganda National Council of Science and Technology .2.8o-phenylenediamine substrate solution (Sigma). Plasma samples collected from 26 uninfected Europeans (after written informed consent was obtained) were tested concurrently with each assay, and all measurements were performed in duplicate. O.D. values were read using a Powerwave HT microplate spectrophotometer  at 490 nm. Standard curves were generated for every plate with the O.D. values obtained from the wells coated with serial dilutions of human myeloma IgE  or IgG4 (Sigma).Plasma IgE and IgG4 levels were measured by isotype-specific ELISA. A coating inhibition assay was employed to estimate the saturating coating concentrations for each antigen  as described  and 15 μ2.9P < 0.05 was considered significant.The influence of post-treatment anti-TpmII antibody (IgG4 and IgE) levels on intensity of re-infection 2 years post-treatment was examined using linear regression analysis, with adjustment for age. Analysis was restricted to individuals for whom treatment was efficacious, defined as at least a 90% reduction in epg and <100 epg 9 weeks post-treatment. Due to positive skew, both egg counts (epg) and anti-TpmII antibody levels were log transformed after the assay cut-offs were added to remove zero values. Age was included as a categorical variable, comprising five groups of approximately equal size. Log-likelihood ratio tests were used to determine significance. 33.1S. mansoni genome: Smp_044010 (TpmI), Smp_031770 (TpmII), Smp_022170, Smp_085290 and Smp_032490. However, as a result of alternative splicing it is predicted that there may be as many as 20 Tpm isoforms in the organism  and a detailed exon chart of 3.3It was not possible to quantify individual splice variant transcripts by qPCR. Whilst non-quantitative differentiation was achieved using nested conventional PCR , primers3.4Onchocerca volvulus  were chosen in the hope of subsequently capturing a representative immunological profile of the entire repertoire of splice variants of this gene. Amino acid sequences of the four variants are aligned in volvulus  that coivolvulus . This mo3.5The four selected splice variants were expressed in bacteria and http://swissmodel.expasy.org/). The same coiled-coil alpha-helical structure was obtained for each of the four proteins, typical of chain C crystal structure for Tpm . Then, to ensure that the secondary structure of the recombinant antigens was as predicted by the modelling, we performed CD spectroscopy on all four purified Tpm proteins. This indicated that the recombinant TpmII.3, 4 and 7 were indeed entirely alpha-helical, but that TpmII.8 contained a small, more disordered helical component at room temperature . With the exception of a post-treatment drop in the proportion of individuals with a detectable TpmII.7-IgG4 response (Tukey’s Honestly Significant Difference (HSD) test; P = 0.03), prevalence of responders was also not affected by treatment (P > 0.1). For each of the four Tpm isoforms, IgE and IgG4 responses were often present in the same individuals. That is, the two responses were not mutually exclusive and the less prevalent response was detected in a subset of those with the more prevalent response.The purified recombinant antigens were used to measure TpmII isoform-specific antibody responses in a cohort of 228 n = 146) of the whole cohort had detectable IgE to TpmII.3, significantly more than to the other three isoforms . In contrast, IgE to TpmII.7 was very rare  and significantly less prevalent than IgE to the other three isoforms (P < 0.05). The most prevalent IgG4 response was that to TpmII.7 (n = 112), significantly more than IgG4 to TpmII.3, 4 or 8 . TpmII.7 C, detectS. mansoni TpmII isoforms and immunity to re-infection, allowing for age. Anti-TpmII.3 IgE levels were significantly negatively correlated with re-infection intensity 2 years post-treatment . The swapping of a small number of exons may make a functional difference to the protein but not greatly influence antigenicity. For immunological study however, we chose four isoforms, TpmII.3, 4, 7 and 8, with marked regional differences, especially in their N and C-terminal domains. TpmII.4 is a highly representative 284 residue, muscle-type isoform. It is homologous to known Tpm allergens from shrimp (Pen a1) and other invertebrates , includiDespite all the published data on continuous epitopes, it seems that, in the major allergens at least, most IgE epitopes are of the conformational (discontinuous) type . CorrectOnly a small number of protein families contain allergens/IgE-antigens . We beliThere were significant differences between the antibody responses to the four different isoforms. Most striking was the difference between the two non-muscle molecules, TpmII.3 and TpmII.7. The response to TpmII.3 was predominantly IgE, with few people producing TpmII.3–IgG4. The response to TpmII.7 was predominantly IgG4 with few individuals producing IgE. Given previous work linking the IgE:IgG4 balance to immunity, it is notable that those with the IgE-dominated response to TmpII.3 were more resistant to re-infection and those with the IgG4-dominated response to TpmII.7 were more susceptible. The antibody responses to the muscle-type TpmII.4 and its truncated form, TpmII.8, were very similar. They share many of the same epitopes, have the same developmental expression pattern and if they are both in the muscle, are likely to have a similar exposure pattern to the host immune system. We have not established why the two non-muscle forms evoke such different responses, although they do have entirely different N-terminal domains and the rest of the molecule is also relatively dissimilar. Nothing is known about their tissue distribution or their accessibility to the host immune system but these may be important factors in their antigenicity.S. mansoni produced IgG against parasite Tpm (Loverde and colleagues showed that individuals infected with site Tpm  but ours


Estrogen can modulate neuronal development and signalling by both genomic and non-genomic pathways. Many of its rapid, non-genomic effects on nervous tissue have been suggested to be mediated via the activation of the estrogen sensitive G-protein coupled receptor (GPER1 or GPR30). There has been much controversy over the cellular location, signalling properties and endogenous activators of GPER1. Here we describe the pharmacology and signalling properties of GPER1 in an immortalized embryonic hippocampal cell line, mHippoE-18. This cell line does not suffer from the inherent problems associated with the study of this receptor in native tissue or the problems associated with heterologously expression in clonal cell lines. In mHippoE-18 cells, 17β-Estradiol can mediate a dose-dependent rapid potentiation of forskolin-stimulated cyclic AMP levels but does not appear to activate the ERK1/2 pathway. The effect of 17β-Estradiol can be mimicked by the GPER1 agonist, G1, and also by tamoxifen and ICI 182,780 which activate GPER1 in a variety of other preparations. The response is not mimicked by the application of the classical estrogen receptor agonists, PPT, (an ERα agonist) or DPN, (an ERβ agonist), further suggesting that this effect of 17β-Estradiol is mediated through the activation of GPER1. However, after exposure of the cells to the GPER1 specific antagonists, G15 and G36, the stimulatory effects of the above agonists are replaced by dose-dependent inhibitions of forskolin-stimulated cyclic AMP levels. This inhibitory effect is mimicked by aldosterone in a dose-dependent way even in the absence of the GPER1 antagonists. The results are discussed in terms of possible “Biased Antagonism” whereby the antagonists change the conformation of the receptor resulting in changes in the agonist induced coupling of the receptor to different second messenger pathways. B receptors in proopiomelanocortin expressing neurons in the hypothalamus (see -cyclic AMP  protein kinase A radiometric binding assay nonyl]estra-1,3,5(10)-triene-3,17-diol  and (2)-2-[4-phenoxy]-N,N-dimethylethanamine citrate (Tamoxifen citrate) were purchased from Tocris Bioscience . Forskolin was obtained from Abcam Biochemicals . We thank Professor Jeffrey Arterburn, New Mexico State University, Las Cruces, New Mexico, USA for initial samples of G36.17β-Estradiol, aldosterone and IBMX were purchased from Sigma-Aldrich ; G1, G15, G36, 2,3-50’s of 5.2 x 10−10 M and 1.1 x 10−9 M respectively. The responses to both compounds declined at all concentrations tested above 3 x 10−9 M.GPER1 is able to activate a wide range of second messenger pathways in different cell types (see ). Howeve−6 M, did not produce any significant increases in cyclic AMP levels at any of the concentration tested. However, it did produce a significant decrease in forskolin-stimulated cyclic AMP levels in these cells at concentrations between 10−10 M and 10−9 M.The mineralocorticoid, aldosterone, has also been suggested to be able to activate GPER1 in a number of different preparations from the cardiovascular system and other cell types , 29–31. α and ERβ [−10 M and 10−9 M) than ICI 182,780 (between 10−9 M and 3 x 10−9 M). Tamoxifen was also slightly more potent than ICI 182,780 with EC50’s of 6.4 x 10−10 M and 3.6 x 10−9 M respectively.In many preparations GPER1 responses have been shown to be induced by drugs which have traditionally been shown to be antagonists of the classical estrogen receptors, ER and ERβ . Thus, w−6 M  and G36 (10−6 M)  G1 still showed a significant increase in cyclic AMP levels even in the presence of G15 (10−6 M). In contrast, the inhibitory effect of G1 in the presence of G36 showed a threshold effect only between 3 x 10−9 M and 10−8 M. The inhibitory effect of G1 appeared to be almost an order of magnitude more potent in the presence of G15 than in the presence of G36.Similarly, the dose-dependent effects of G1 on forskolin-stimulated cyclic AMP levels in mHippoE-18 cells were also blocked by both G15 (10(10−6 M) . However−6 M) to see if they were also likely to be mediated via GPER1. It can be seen that the dose-dependent stimulatory effects of both Tamoxifen and ICI 182,780 on cyclic AMP levels were both blocked by G15 .Since GPER1 activation has also been shown to be capable of coupling to the activation of the MAPK pathway in a number of different tissues  in the immortalized hippocampal cell line, mHippoE-18. Application of 17β-Estradiol to the mHippoE-18 cells induces a rapid, and dose-dependent, increase in forskolin-stimulated cyclic AMP levels in these cells. The response is not mimicked by the application of the classical estrogen receptor agonists, PPT, (an ERα agonist) or DPN, (an ERβ agonist), suggesting that this effect of 17β-Estradiol may be mediated through the activation of GPER1. This suggestion is supported by the fact that the response is mimicked, in a dose dependent way, by the GPER1 agonist, G1 , and by  in the iGPER1 has been shown to increase cyclic AMP levels by the Gs-mediated activation of adenylyl cyclase in a range of other tissues and when exogenously expressed in a range of clonal cell lines –6, 35. T−6 M G36 both 17β-Estradiol and G1 produce a dose-dependent inhibition of forskolin-stimulated cyclic AMP levels in these cells. Similarly, in the presence of G15, G1, Tamoxifen and ICI 182,780 also produce a dose-dependent inhibition of forskolin-stimulated cyclic AMP levels in these cells. It is not clear why 17β-Estradiol did not produce a similar inhibition of this response in the presence of G15 but it did in the presence of G36. This may reflect differences in the properties of the GPER1 conformations induced by exposure to G15 and to G36.The results of the present study using the proposed specific GPER1 antagonists G15 and G36, indicate that 17β-Estradiol and G1 can modulate cyclic AMP levels in mHippoE-18 cells by multiple pathways. In the presence of 10At the present time the molecular mechanisms underlying this novel inhibition of forskolin-stimulated cyclic AMP levels in mHippoE-18 cells are not known. There would seem to be at least two possible general mechanisms. First, the inhibition of the forskolin-stimulated cyclic AMP levels by G15 and G36 could reveal the presence of a second receptor activated by 17β-Estradiol, G1, Tamoxifen and ICI 182,780, which is possibly coupled to a Gi-mediated inhibition of forskolin-stimulated cyclic AMP levels, the effects of which are normally masked by the stimulation of cyclic AMP levels by the activity of GPER1. The presence of such a second Gi-coupled receptor might also explain why, at high concentrations in the absence of the antagonists, G1 has a larger apparent efficacy in increasing cyclic AMP levels than 17β-Estradiol. If 17β-Estradiol had a larger efficacy for this second receptor than G1, its effects could be inhibited more if both receptors are activated at the same time. However, this could simply be because G1 has a higher efficacy than 17β-Estradiol at GPER1 in this preparation. Previous studies have reported the coupling of a range of GPCRs, such as the β2-adrenergic receptor  and the A second possibility would be that GPER1 adopts different conformations in the presence of so called biased ligands , 52. ThuThe fact that aldosterone alone can specifically mimick the actions of 17β-Estradiol and G1 in the presence of G36 on the inhibition of forskolin-stimulated cyclic AMP levels in mHippoE-18 cells, but does not mimick their actions on the potentiation of forskolin-stimulated cyclic AMP levels in mHippoE-18 cells, is interesting in view of the current controversy surrounding the possible actions of aldosterone on GPER1 (see –31 and hThus, mHippoE-18 cells  can prov


H1 and cytotoxic T cell activity against tumors. While canonical signal transduction through effector Smads has been well-defined as a requirement for TGFb-mediated inhibition of T cells, essential non-canonical pathways have not, to date, been defined. This abstract describes the identification of Platelet Endothelial Cell Adhesion Molecule-1 (PECAM-1), CD31, as a novel facilitator of non-canonical TGFβ signal transduction in T cells. Subcutaneously injected tumor cells known to require TGFβ-mediated suppression of immunity for clearance grew more slowly in PECAM-1-/- mice relative to wild type counterparts, and T cells isolated from PECAM-1-/- mice demonstrated relative insensitivity to TGFβ-induced inhibition of IFNγ production and proliferation. Similarly, human T cells lacking PECAM-1 expression demonstrated decreased sensitivity to TGFβ in a manner that could be partially restored by re-expression of PECAM-1. Phosphorylation of PECAM-1 on an Immunoreceptor Tyrosine-based Inhibitory Motif (ITIM) and resultant binding of the inhibitory Src homology 2 domain-containing tyrosine phosphatase-2 (SHP-2) was observed after co-incubation of T cells with TGFβ and anti-CD3, and inducible co-localization of PECAM-1 with the TGFβ receptor complex was identified using co-immunoprecipitation, confocal microscopy and proximity ligation assays. These studies indicate an unexpected role for PECAM-1 in enhancing crucial inhibitory functions of TGFβ in T cells and suggest that targeting of the PECAM-1/TGFβ inhibitory axis represents a novel means to overcome TGFβ-dependent immunosuppression within the tumor microenvironment.Transforming Growth Factor β (TGFb) is an immunosuppressive cytokine that inhibits pro-inflammatory functions of T cells, and is a major contributor to abrogating T


Live-cell imaging and genetic tools reveal a new way in which pigment cells communicate in zebrafish Related research article Eom DS, Bain EJ, Patterson LB, Grout ME, Parichy DM. 2015. Long-distance communication by specialized cellular projections during pigment pattern development and evolution. eLife 4:e12401. doi: 10.7554/eLife.12401Image Three types of pigment cells in the skin of zebrafish arrange themselves into a striped patternHow the zebra got its stripes is a frequent topic in popular literature about the natural world. For developmental biologists, however, it is much easier to study the biology of pattern formation in zebrafish, where the tools of experimental embryology and genetics have proved to be very useful. Now, in eLife, David Parichy from the University of Washington and co-workers – including Dae Seok Eom as first author – report an important step forward in our understanding of pattern formation . Making The elegant stripe patterns seen in adult zebrafish start to emerge in the juvenile fish, when the cells responsible for the different colors organize themselves into intricate patterns. Pigment cells called melanophores form the dark stripes, while xanthophores and iridophores form the regions between stripes. How does this come about? Work from several groups over the last decade has highlighted three general principles. First, although all the pigment cells originate in the same area of the zebrafish embryo, they have unique and separate developmental histories. For example, the melanophores and iridophores in adult fish belong to lineages that are separate from the pigment cells found in larvae .Second, genetic mutations that act primarily on one type of pigment cell can alter the distribution of other types of pigment cells in the adult fish . This deEom et al.’s findings add an intriguing new wrinkle to the story. They used time-lapse imaging to follow genetically marked melanophores, xanthophores and iridophores in the skin of young zebrafish as the adult stripes begin to form. The experiments used a new type of instrument – a spinning disk confocal laser microscope – that allows images to be acquired more rapidly than with the conventional scanning approach, and is especially powerful when coupled with the genetic tools available in zebrafish.When Eom et al. looked at zebrafish that had been genetically engineered to express a fluorescent marker specifically in xanthophore cell membranes, they noticed thin filaments projecting from the xanthophores. These filaments sometimes extended for hundreds of microns, seemingly to deliver small packets of cargo (vesicles) to melanophores. Eom et al. found that these filaments, which contain cytoplasm, are distinct from previously known projections and dubbed them 'airinemes'. A key aspect of pattern development in zebrafish is that the cells that will become the melanophores and xanthophores – which are initially disorganized in the skin – move in a carefully directed manner to form the adult stripes. Eom et al. realized that the majority of airinemes were produced by immature xanthophores, which then contacted immature melanophores located in between stripes. By using a genetic trick to prevent the production of airinemes, Eom et al. demonstrated that these structures play critical roles in the formation of clearly defined stripes. When the airnemes are missing, the immature melanophores persist in the regions between the stripes, and the edges of the stripes are ragged and ill-defined . A seconWhile the genetic tools employed by Eom et al. are incredibly sophisticated, we are most excited about how they have been applied in the context of live-cell imaging to reveal a new form of cellular communication. This underscores one of the most important principles in biology—to look carefully—and raises the obvious question as to where should one look next?An unsolved mystery of color pattern biology is how the appearance of stripes or spots can be remarkably stable within a species, yet evolutionarily flexible between species. For example, all leopards have rosette markings, but the same underlying mechanism can give rise to stripes in tigers or spots in cheetahs. One possible explanation stems from Alan Turing's work on chemical reaction-diffusion systems in the early 1950s: Turing showed how stable patterns can develop spontaneously in chemical systems, and that small changes in the components of the system can lead to large changes in the patterns produced.A role for this type of system in zebrafish is somewhat controversial, in part because it doesn’t account for the different types of pigment cells involved. However, the same may not hold true for zebras and other mammals in which a single type of pigment cell is uniformly distributed in the skin.Zebras will never enjoy the advantages of zebrafish when it comes to experimental genetics and embryology. However, advances in DNA sequencing are furthering the reach of observational genetics, perhaps even to exotic mammals with diverse color patterns like civets and servals. The question "How the zebra got its stripes" may not have the same answer as the question "How the zebrafish got its stripes". Vive la différence.


Finally, we confirm that ephrinB1/Fc treatment induces tyrosine phosphorylation (activation) of EphB2, while deletion of the tyrosine kinase domain (VM) of EphB2 eliminates the receptor stimulation-induced GSK-3β inhibition and tau dephosphorylation. We conclude that activation of EphB2 receptor kinase arrests tau hyperphosphorylation through PI3K-/Akt-mediated GSK-3β inhibition. Our data provide a novel membranous target to antagonize AD-like tau pathology.Abnormal tau hyperphosphorylation is an early pathological marker of Alzheimer’s disease (AD), however, the upstream factors that regulate tau phosphorylation are not illustrated and there is no efficient strategy to arrest tau hyperphosphorylation. Here, we find that activation of endogenous EphB2 receptor by ligand stimulation (ephrinB1/Fc) or by ectopic expression of EphB2 plus the ligand stimulation induces a remarkable tau dephosphorylation at multiple AD-associated sites in SK-N-SH cells and human embryonic kidney cells that stably express human tau (HEK293-tau). In cultured hippocampal neurons and the hippocampus of human tau transgenic mice, dephosphorylation of tau proteins was also detected by stimulation of EphB2 receptor. EphB2 activation inhibits glycogen synthase kinase-3β (GSK-3β), a crucial tau kinase, and activates phosphatidylinositol-3-kinase (PI3K)/Akt both  Hyperphosphorylation of microtubule associated protein tau is an early pathological marker of Alzheimer’s disease (AD)2457in vitro and in vivo induces tau hyperphosphorylation with impairments of the cognitive functions, whereas inhibition of GSK-3β improves tau pathologies and memory deficits131415171813Several protein kinases have been reported to play a role in AD-like tau hyperphosphorylation, among them, glycogen synthase kinase-3β (GSK-3β) is the most implicatedEph receptor is a member of receptor tyrosine kinases (RTKs) that play a critical role in the development of the central nervous system222325222324In the present study, we activate the endogenous EphB2 receptor in SK-N-SH cells, mouse hippocampal neuron culture and human tau transgenic mice by using ephrinB1/Fc (the chimeric agonist of EphB2), or by ectopically expressing EphB2 with application of ephrinB1/Fc in HEK293-tau cells that do not express endogenous EphB2. Then we measured the phosphorylation level of tau and the GSK-3β-related signaling pathway. We demonstrate that activation of EphB2 induces tau dephosphorylation at multiple AD-related sites with mechanisms involving the EphB2 kinase-coupled PI3K/Akt activation and GSK-3β inhibition.in vivo effects of EphB2 on tau phosphorylation, we injected ephrinB1/Fc into the hippocampal CA3 region of 10 m-old human tau transgenic mice for 45 min and then measured tau phosphorylation level. The results show that in vivo stimulation of EphB2 also reduces tau phosphorylation in hippocampal CA1, CA3 and dentate gyrus (DG) shown by immunohistochemistry  do not express EphB2 . By treahemistry  and immuhemistry . These dTo explore the mechanisms underlying the EphB2-induced tau dephosphorylation, we measured the alterations of GSK-3β, the most implicated kinase in regulating tau phosphorylationThe activity of GSK-3β is regulated by PI3K/AktTo further verify the role of PI3K/Akt-mediated GSK-3β inhibition in EphB2 stimulation-induced tau dephosphorylation, we first used wortmannin, a PI3K inhibitor, in SK-N-SH cells. We found that simultaneous inhibition of PI3K almost abolished the ephrinB1/Fc-induced GSK-3β phosphorylation at Ser9 and tau dephosphorylation , suggestin vivo effects of EphB2 stimulation in tau dephosphorylation and the involvement of PI3K/Akt/GSK-3β pathway, we infused ephrinB1/Fc or Fc as control into the hippocampal CA3 region of the human tau transgenic mice and then measured the alterations of tau phosphorylation and PI3K/Akt/GSK-3β signaling pathway. We observed that stimulation of EphB2 by ephrinB1/Fc increased phosphorylation (activation) of PI3K at P85 and Akt at Ser473 with a simultaneous inactivation of GSK-3β and dephosphorylation of tau at Ser396 and Thr231  and reverse (ephrin) signaling. Like other Eph receptors, the monomer form of EphB2 receptor has no activity, and association of EphB2 with its ephrin ligands activates the receptor. The ephrinB ligands can be divided into three subgroups, namely B1, B2 and B3. We choose to use ephrinB1 because it is the most abundantly expressed form in the brainThe kinase domain and the PDZ-binding domain are two major structural domains of EphB2. Upon binding, the dimerized or aggregated Eph-ephrin receptors can transduce signals via phosphorylation of the conserved tyrosine residues234526Taken together, we find in the present study that stimulation of EphB2/ephrinB1 attenuates tau phosphorylation through PI3K/Akt/GSK-3β-mediated pathway. Therefore, EphB2/ephrinB1 receptor could be an upstream factor in attenuating AD-like tau pathology.EphB2 wt-flat, delta VM (VM domain knockout), and delta PDZ (PDZ domain knockout) plasmids were kind gifts of Prof. Matthew B. Dalva in Department of Neuroscience, University of Pennsylvania School of Medicine. The ephrinB1 and Fc were purchased from R&D Systems . The ephrinB1/Fc chimera or Fc control was preclustered by incubating with anti-human Fc IgG at 37 °C for 30 min following the manufacturer’s instructions, and the final concentration of ephrinB1 or Fc is 2 μg/ml. The information for antibodies employed was listed in in vivo studying the effects of EphB2/ephrinB1/Fc on tau dephosphorylation. Mice were housed in the Animal Resource Center at Tongji Medical College, Huazhong University of Science and Technology. All experiments conducted on animals were performed according to protocols approved by the institutional Animal Care and Use Committee.Ten month-old human tau transgenic mice Klt Tg(MAPT)8cPdav/J) were used for in vivo effects of EphB2 stimulation on tau dephosphorylation, ten ~10 months old human tau transgenic mice, which express six isoforms of tau and show an age-dependent development tau pathology  were used. For the brain infusion, the mice were anesthetized with 6% chloral hydrate (6 ml/kg) and placed in a Jiangwan-II stereotaxic instrument . After the scalp was incised, the skull was cleaned and a hole (diameter 0.5 mm) was made for the injection. The co-ordinates of AP-2.18, L-2.6, V-2.3 (in mm from bregma) for CA3 area of hippocampus were selected according to the stereotaxic atlas of Franklin and Paxinos. A sterilized needle connected to a 5 μl syringe (Hamilton) was used to deliver ephrinB1/Fc chimera or Fc control in 1 μl to the left hippocampus of the mice, after 45 min, the mice were sacrificed for further studies.To study the Human neuroblastoma SK-N-SH cells  that express endogenous EphB2 receptor and tau proteins were cultured in Dulbecco’s modified eagle’s medium (DMEM) supplemented with 10% fetal bovine serum  and grown at 37 °C in a humid atmosphere containing 5% carbon dioxide, and ephrinB1/Fc was incubated with the cells for 45 min to activate EphB2.HEK293 cells that do not express endogenous tau were transfected with the longest human tau (tau441) or the pcDNA 3.0 vector, and the cell line with stable expression of tau (HEK293-tau) or the vector (HEK293/vector) were established from our group2 containing atmosphere at 37 °C. After cultured for 7 days, more than 90% of the cells were neurons in vitro. Then the cells were harvested for Western blotting after ephrinB1/Fc treatment for 45 min.The primary neurons from E18 rat hippocampus that express endogenous tau proteins and EphB2 receptor were seeded at 30,000–40,000 cells per well in neurobasal medium supplemented with 2% B27/0.5 mM glutamine/25 mM glutamate. Half of the culture medium was changed every 3 days with neurobasal medium. All cultures were kept in a humidified 5% COFor immunohistochemistry staining of the brain sections, mice were perfused with 4% paraformaldehyde (PFA) in 10 mM phosphate-buffered saline (PBS). The brains were removed and fixed overnight in 4% PFA at 4 °C. Coronal sections (25 μm) were collected on a vibratome  and placed in PBS with 0.1% sodium azide. Immunohistochemistry was performed on brain sections by incubating free-floating sections with primary antibodies overnight at 4 °C. The sections were further incubated with biotin-labeled secondary antibodies for 1 h at 37 °C and visualized with the DAB tetrachloride system. The images were taken using a microscope  at identical settings for each of the conditions. Quantitative analyses were carried out by an experimenter blind to treatment groups using an image analysis system .For cell line studies, SK-N-SH cells were plated on diametric glass coverslips coated with poly-D-lysine (10 μg/ml) in 24-well culture plates. After treatment with ephrinB1/Fc or Fc control, cell culture medium was carefully removed. After two rinses with PBS, the cells were fixed in a freshly prepared solution of 4% paraformaldehyde for 15 min. After two more rinses in PBS, the cells were permeabilized in 1% Triton X-100 in PBS for 15 min. Then the cells were incubated in 3% BSA in PBS for 1 h and then incubated with primary antibody at 4 °C overnight. The immunoreactivity was probed using Oregon Green 488-conjugated secondary antibodies  for 2 h at room temperature. The immunofluorescence images from mouse brains or cells were taken by using a confocal laser scanning microscope .3VO4, 1 mM edetic acid, 1 mM benzamidine, 1 mM PMSF, and a mixture of aprotinin, leupeptin, and pepstatin A (10 μg/ml each). After measurement of protein concentration in the extracts using BCA kit , a final concentration of 10% β-mercaptoethanol and 0.05% bromophenol blue were added, and the samples were boiled for 10 min in a water bath for Western blottingversus IOD of DM1A protein).The cells or the hippocampal tissues were rinsed twice in ice-cold PBS (pH 7.5) and lysed with buffer containing 10 mM Tris-Cl, pH 7.6, 50 mM NaF, 1 mM NaActivity of GSK-3β was measured using GSK-3β Activity Assay Kit  by following the manufacturer’s manufacture’s instructions. The principle of the colorimetric method is based on the activity of GSK-3β to phosphorylate the target sequence GPHRSTPESRAAV in the presence of ATP and then through reactions that involve pyruvate kinase and lactate dehydrogenase to oxidize NADH (reduced nicotinamide adenine dinucleotide) into NAD (nicotinamide adenine dinucleotide), and the absorbance was read at 340 nm. In this assay, the GSK-3α activity was inhibited by Aloisine A (a GSK-3α inhibitor provided in the kit).P < 0.05.Data were evaluated as the mean and standard deviation (mean ± SD). Statistical analysis of the quantitative multiple group comparisons was performed using the one-way analysis of variance (ANOVA) followed by Duncan’s test; whereas pairwise comparisons were performed using the t test by SPSS software . Results were considered to be statistically significant with How to cite this article: Jiang, J. et al. Stimulation of EphB2 attenuates tau phosphorylation through PI3K/Akt-mediated inactivation of glycogen synthase kinase-3β. Sci. Rep.5, 11765; doi: 10.1038/srep11765 (2015).


Ciconia ciconia) populations have been object to several conservation measures such as reintroduction programs, habitat improvement or supplementary feeding in the last decades. Although recent white stork censuses revealed an upward trend of most of the western populations, evaluations of the relative importance of certain conservation measures are still scarce or even lacking. In our study we analyzed the effect of supplementary feeding on the reproductive success of white storks in conjunction with other factors such as weather or nest site characteristics. We present data of 569 breeding events at 80 different nest sites located in variable distances to an artificial feeding site at Affenberg Salem (south-western Germany) collected from 1990–2012. A multilevel Poisson regression revealed that in our study population (1) reproductive success was negatively affected by monthly precipitation in April, May and June, (2) pairs breeding on power poles had a lower reproductive success than pairs breeding on platforms or trees and (3) reproductive success was significantly higher in pairs breeding in close distance to the feeding site. The number of fledglings per nest decreased by 8% per kilometer distance to the feeding site. Our data suggest that supplementary feeding increases fledgling populations which may be a tool to attenuate population losses caused by factors such as habitat deterioration or unfavorable conditions in wintering habitats.European white stork ( Ciconia ciconia) populations in Western Europe decreased tremendously after 1945 leading to a severe threat of extinction or even extinction in most West European countries White stork (Procambarus clarkii) are discussed to influence population recovery of white storks White stork censuses in 1995 and 2004 revealed an upward trend of most western populations In most of the west European white stork populations reproductive success is low or highly variable Supplementary feeding is a common element of wildlife management that can aid in the establishment of a viable, self-sustaining population by increasing body condition, growth rates, survival, social interactions or reproductive success e.g. . HoweverWhite storks, while breeding, forage on average in an area of not more than 1500 m from the nest site The study was conducted at Affenberg Salem (N 47°45′43″ E 09°14′44″) Baden-Württemberg, Germany . Affenbe2. The number of storks visiting the feeding site was highly variable depending on season  and most likely also on food availability in the surroundings. During the breeding season number of storks visiting the feeding site was highest but at times dropped to 0. Outside the breeding season number of storks at the feeding site was in general considerably lower. The majority of the population is migrating and therefore not present during winter feeding. We tried to adjust the quantity of food so that 30 min after the food had been distributed no more food was left at the feeding site. As an estimate for adjustment, we used the time storks needed to empty the feeding site the respective day before. As consequence feeding time per day and the amount of food provisioned were limited. The amount ranged between . Storks were able to ingest up to 12 chickens per feeding into their crops and transport them to their nests (pers. obs. D. Zinner).At a defined feeding site at Affenberg Salem, supplementary food  was provided twice a day (11.00 h and 16.45 h) during the breeding season (including the nestling period) and once a day (14.00 h) outside the breeding season. The food was distributed randomly over an area of approximately 1000 mad libitum and were only available for a few pairs. As a measure for reproductive success we used number of fledged chicks per breeding pair (nest) per year. As potential predictor variables we included (1) distance between nest site and feeding site , assuming that storks nesting closer to the feeding site will obtain supplementary food more regularly and in larger quantities, (2) weather data for the breeding and nestling periods, i.e., monthly rainfall and average temperature for April, May and June 1990–2012, obtained from the closest meteorological station of the Deutscher Wetterdienst in Konstanz, which is about 10 km from the feeding site and in addition (3) type of nest base: (a) on artificial platforms, (b) wild nests on houses and in trees (not particularly supported nests) or (c) on power poles. Data are available from Breeding pairs were determined following standardized census methodology We used a multilevel Poisson regression to examine potential associations between the number of fledglings and the distance to the feeding site while controlling for other potential predictors variables. As covariates we assessed monthly rainfall and average temperature in April, May and June of respective years and nest base type. We included nest ID as a random factor to model the dependency of breeding success due to the same nest .In a first step, we included all independent variables in the model and subsequently removed variables with a p>0.1. We then compared this final model with (1) a model with replacing weather data with ‘year’ as a categorical independent variable (‘good stork years’ vs. ‘bad stork years’) and (2) a model with distance replaced by a categorical variable with either two (<300 m or >300 m) or three levels  . The thrA Poisson model assumes that the variance is equal to the mean. If there is more variation in the number of fledglings than predicted by the model  standard errors are often underestimated and therefore p-values  become overoptimistic (type I error inflation). To account for possible overdispersion we used an observation–level random effect to model possible overdispersion assuming that overdispersion arises from errors taking on a log-normal mixing distribution Besides handling of white stork chicks for ring application, no other handling of storks was done. For all nests included in this study ring application was done by one of the authors (RH) and Walter Angst who hold a ringing permit issued by Regierungspräsidium Tübingen and Landratsamt Bodenseekreis (Germany). All handling of the storks was done in compliance with the laws of Baden-Württemberg (Germany). Number of fledglings was determined from a distance, if necessary with the help of binoculars. For these observational data collection no specific permission was required.Over a period of 23 consecutive years we collected data from 569 breeding events at 80 nest sites. The minimum number of nests per year was 10 in 1991. This number increased to 64 in 2012 . The number of fledglings per brood ranged from 0 to 5 . ‘Bad years’ for stork reproduction were 1991  and 2007 . In contrast, ‘good years’ were 2003  and 2011 . On average 2.46  young storks fledged if breeding occurred in close proximity to the feeding site (<300 m); 1.99  chicks fledged in nests which were between 300 m and 3 km away form a feeding site and only 1.70  chicks fledged from nests if the distance to the feeding site was more than 3 km.2 = 0.045).Our overall final model explained 28.7% of the variance. The fixed effects ‘nest base’, ‘distance to feeding site’ and ‘monthly precipitation in April, May and June’ explained 15.5% and the random factor ‘nest ID’ 13.2% . Monthly2 = 0.075). The type of nest-base tends to have an influence with nests on power poles having 31% less fledglings than storks breeding on platforms and 29% less fledglings than storks breeding in wild nests, whereas nests on platforms and wild nests on trees and buildings yielded similar numbers of fledglings  than storks breeding in a distance of less than 300 m from the feeding site. Using distance as a variable with three levels suggests that storks breeding in a distance of more than 3 km from the feeding site have 36% less fledglings  than those breeding close to the feeding site (<300 m) while storks breeding in an intermediate distance (300 m–3000 m) have 20% less fledglings. But this difference is not significant compared to those living less than 300 m to the feeding site . There is also no significant difference in the number of fledglings between those breeding in an intermediate and a long distance to the feeding site . The distance factor accounted for 4.6% of the explained variance.Using distance as a categorical variable with two (<300 m or >300 m) and three levels  resulted in a model with a slightly smaller AIC  than the model with distance as a continuous variable. The 2-levels model suggests that storks breeding in a distance larger than 300 m from the feeding site have 29% less fledglings Click here for additional data file.