Toxoplasma gondii, TgCDPK3 regulates parasite egress from the host cell in the presence of a calcium-ionophore. The targets and the pathways that the kinase controls, however, are not known. To identify pathways regulated by TgCDPK3, we measured relative phosphorylation site usage in wild type and TgCDPK3 mutant and knock-out parasites by quantitative mass-spectrometry using stable isotope-labeling with amino acids in cell culture (SILAC). This revealed known and novel phosphorylation events on proteins predicted to play a role in host-cell egress, but also a novel function of TgCDPK3 as an upstream regulator of other calcium-dependent signaling pathways, as we also identified proteins that are differentially phosphorylated prior to egress, including proteins important for ion-homeostasis and metabolism. This observation is supported by the observation that basal calcium levels are increased in parasites where TgCDPK3 has been inactivated. Most of the differential phosphorylation observed in CDPK3 mutants is rescued by complementation of the mutants with a wild type copy of TgCDPK3. Lastly, the TgCDPK3 mutants showed hyperphosphorylation of two targets of a related calcium-dependent kinase (TgCDPK1), as well as TgCDPK1 itself, indicating that this latter kinase appears to play a role downstream of TgCDPK3 function. Overexpression of TgCDPK1 partially rescues the egress phenotype of the TgCDPK3 mutants, reinforcing this conclusion. These results show that TgCDPK3 plays a pivotal role in regulating tachyzoite functions including, but not limited to, egress.Calcium-dependent protein kinases (CDPKs) are conserved in plants and apicomplexan parasites. In  Toxoplasma CDPK3, which has recently been shown to regulate rapid egress from the host cell. The specific pathways that TgCDPK3 regulates, however, have not previously been known and so we used a quantitative phosphoproteome approach to determine phosphorylation site usage in wild type and TgCDPK3 mutant parasites before, during and after egress. This revealed >150 novel phosphorylation sites that are differentially phosphorylated between WT and TgCDPK3 mutant parasites. Some of these sites are on proteins predicted to play a role in parasite egress. However, we also identified many phosphorylation sites on proteins not thought to be involved in egress, as well as many proteins of unknown function. We confirm that basal calcium levels are affected by CDPK3 inactivation and observed a link between TgCDPK3 and another calcium-dependent kinase (TgCDPK1). Known targets of TgCDPK1 were hyperphosphorylated in the TgCDPK3 mutants, and overexpression of TgCDPK1 partially rescued the observed egress phenotype of TgCDPK3 mutants.Calcium-dependent protein kinases are plant-like enzymes of apicomplexan parasites that regulate a variety of biological processes including stage-conversion, post-translational repression and egress from the host cell. In this study, we analyzed  Toxoplasma gondii and Plasmodium species contain a number of plant-like calcium-dependent protein kinases (CDPKs) T. gondii and PlasmodiumApicomplexan parasites like T. gondii, two CDPKs have been investigated in detail. TgCDPK1 (TGGT1_059880) has been shown to be essential for the secretion of micronemes In TgCDPK3 gene show resistance to IID, but the biochemical basis for this is not yet known. TgCDPK3 also appears to be necessary for producing latent stages in mice A second phenotype that is associated with TgCDPK3 malfunction is resistance to ionophore-induced death (IID) To understand the role of TgCDPK3 during both normal conditions and ionophore-induced egress, we performed a quantitative phosphoproteome and proteome study of wild type and TgCDPK3 mutants using stable isotope labeling with amino acids in cell culture (SILAC). Our study revealed clues to the role played by this enzyme in normal physiology and induced egress.13C,15N-Lys in place of the naturally-occurring (12C or 14N) amino acids in “light” media To identify the pathways controlled by TgCDPK3, we infected human foreskin fibroblasts (HFFs) with wild type (WT) or TgCDPK3-mutant tachyzoites previously grown in either “heavy” (H) or “light” (L) conditions. “Heavy” indicates the presence of −”); 2) intracellular parasites with ionophore (“IC/ION+”); and 3) extracellular parasites with ionophore (“EC/ION+”). We measured each condition with forward and reverse labeling; that is, we labeled WT parasites with “light” amino acids and the TgCDPK3-mutant parasites with “heavy” in one experiment and reversed this labeling for the replicate experiment  from two experiments.Knock-out mutants of TgCDPK3 (RH:−” and “EC/ION+”), all analyzed in two independent runs ; 2) the log2 H/L ratio was >0.75 or <−0.75, (∼1.5 times the average standard deviation of any given experiment); sites were designated as “not different” if the log2 ratios were between 0.5 and −0.5. Sites were considered dependent on TgCDPK3 if they differed between WT and mutant parasites under one or more of the three experimental conditions, “IC/ION−”, “IC/ION+” or “EC/ION+”.We identified a subset of phosphorylation sites as reliably different between WT and mutant parasites by applying two additional criteria: 1) the site was identified in at least two datasets without conflicting ratios for different experiments  indicating that these changes are a consequence of TgCDPK3 inactivation and not a consequence of the genetic modification of the parasites independent of TgCDPK3 function (For a majority (∼58%) of the 156 phosphorylation sites discussed above we did not obtain high-confidence SILAC ratios for the untreated samples  and the same mutant strain (MBE1.1). Thus, we could not exclude the possibility that some of the observed differences in phosphorylation state are due to secondary mutations carried by this strain . To address this, we made use of a complemented MBE1.1 cell line which expresses wild type TgCDPK3 under its endogenous promoter +” using the methods described above. We then retrieved all SILAC ratios of this experiment for the 156 phosphorylation sites that we identified as different between WT and CDPK3 mutant parasites  when compared to the SILAC ratios observed for “COMP/EC/ION+”  between the distributions of SILAC ratios of all 156 differing phosphorylation sites of the “EC/IONEC/ION+” . OverallEC/ION+” . Of the A primary aim of this study was to identify phosphorylation sites that differ in their usage between the WT and TgCDPK3 mutant parasites upon ionophore treatment. Within this set of 156 changing sites, two classes are most interesting: “Class A” where the phosphosite was detected in both WT and mutant parasites in the absence of ionophore-treatment but whose usage did not differ between these two strains; and “Class B” where the phosphosite was not detected in one or other or both of the strains in the absence of ionophore-treatment. This latter class likely represents phosphorylation sites that are substantially phosphorylated only during ionophore-induced egress, assuming the absence of detection in the “IC/ION-“ condition is not caused by technical reasons, as explained below. In Class A, we identified 14 phosphorylation sites on a total of 11 proteins . Seven oTo understand more about TgCDPK3's role, we looked for proteins in our dataset that were already known or predicted to play a role in egress or motility . Among tToxoplasma and Plasmodium+” condition. These data suggest that while TgCDPK3 activity is essential for processes that regulate egress-related events, it is not the main kinase regulating phosphorylation of the motor complex components.Whether TgCDPK3 inactivation leads to a motility phenotype in the presence of ionophore remains unclear. Whereas Lourido and colleagues reported significant differences in the types of motility that TgCDPK3 mutants could perform, McCoy and colleagues observed only a slight, but not significant trend toward such differences Among the proteins that were differentially phosphorylated between wild type and mutant parasites were some that contain EF-hands, proteins that are regulated directly by calcium . We idenIn addition to the putative calmodulin, we identified two calcium-dependent kinases (TgCDPK2a (TGGT1_062170) and TgCDPK3 itself) as differentially phosphorylated in the MBE1.1 mutant vs. WT. For both kinases, the sites were hyperphosphorylated in the mutant parasites and were located within the ATP-binding loop of the kinase domain, a region where phosphorylation can play a regulatory role +_FW” condition that corresponds to the activation loop threonine (T200) of TgCDPK1 Interestingly, our dataset also indicated that two of several phosphorylation sites on proteins recently identified as targets of TgCDPK1 (TGGT1_059880). PRP (TGME49_005320) and DRPB (TGGT1_064650) TgCDPK3 mutants egress in the presence of ionophore with much slower kinetics than WT parasites st. A comparison of the ranking for each given peptide incubated with either CDPK1 or CDPK3 shows that a majority of the peptides are equally well phosphorylated by the two kinases although some are predominantly phosphorylated by one, but not the other. We did not obtain significantly enriched amino acids in any position for either TgCDPK1 or TgCDPK3, but that might be related to the technical limitation of these arrays. They sometimes contain more than 1 phosphorylatable residue per peptide; i.e., an additional serine, threonine or tyrosine residue in addition to the central serine. Since we cannot distinguish whether the central serine, or another phosphorylatable residue is phosphorylated, they have limited value for a motif analysis. However, it still allowed us to directly compare TgCDPK1 and TgCDPK3 in their linear motif analysis with the result that TgCDPK1 appears likely able to phosphorylate at least a subset of TgCDPK3 targets.We have previously shown that the egress phenotype of TgCDPK3 mutants can be rescued by overexpressing an engineered form of TgCDPK3 that has mutations in its predicted myristoylation and pamitoylation sites but only when using a strong promoter (SAG1), not when using the endogenous promoter −” condition. These results suggested a putative role for TgCDPK3 in regulating calcium levels. To test this, we measured calcium levels in the absence of ionophore in WT, MBE1.1, MBE1.1 complemented with a functional copy of TgCDPK3 (MBE1.1::CDPK3) and, as a control, MBE1.1 complemented with a nonfunctional copy of TgCDPK3 (MBE1.1::CDPK3(T239I)). Both, MBE1.1 and MBE1.1::CDPK3(T239I) showed elevated calcium levels (∼150%) compared to MBE1.1 complemented with a functional copy of CDPK3 (100%), supporting the notion that TgCDPK3 plays a role in maintaining normal intracellular calcium levels , that has recently been shown to complement the egress phenotype of MBE1.1 In addition to the calcium-dependent proteins mentioned earlier, we identified a putative calcium-transporting ATPase (TGGT1_103910) that showed differential phosphorylation between mutant and WT parasites in the “IC/IONm levels . To ruleThe aim of this study was to identify phosphorylation events that are dependent on TgCDPK3. Based on previous publications, we hypothesized that the signaling pathways controlled by TgCDPK3 might be most easily detected during ionophore-induced egress. Among the proteins that fulfilled this prediction were several that are known to be secreted into the parasitophorous vacuole (PV), the PV-membrane (PVM) or into the host cell. Some of the phosphopeptides in these proteins showed a decreased abundance in the WT samples which could be due to dephosphorylation or degradation of these proteins resulting from breakdown of the PVM. We have not further investigated these events here, but they are consistent with the fact that breakdown of the PVM, normally an early event in egress, is defective in the TgCDPK3 mutant parasites −”), it appears that this phosphorylation site has a function independent of, or in addition to, egress. A recent report on a conditional knock-out of MyoA, where loss of this protein has no effect on parasite egress  either active TgCDPK1 can phosphorylate targets of TgCDPK3, or 2) that active TgCDPK1 can activate egress and microneme secretion independent of TgCDPK3, but with much slower kinetics. Both kinases appear to have overlapping substrate specificity and the dominant localization of the overexpressed TgCDPK1 at the periphery and the increased rescue of egress supports the first option where TgCDPK1 may be phosphorylating TgCDPK3 targets at the plasma membrane. However, we cannot exclude the alternative, in which egress in the TgCDPK3 mutants is facilitated via a TgCDPK3-independent pathway. In both scenarios, the elevated levels of TgCDPK1 we observed in MBE1.1 parasites that are phosphorylated in the autophosphorylation loop might explain how TgCDPK3 mutant parasites egress slowly over time when treated with ionophore.et al., have shown that activation of PKG is partially CDPK3 dependent as inhibition of CDPK3 decreases egress triggered by Zaprinast Plasmodium falciparum as a key-regulator for calcium levels thought to be important for regulation of the TgCDPK1 orthologue in P. berghei, PbCDPK3 A possible explanation for how CDPK1 might be activated is via a PKG (TGGT1_087710) controlled pathway. Lourido in vivo.Whatever the mechanism by which TgCDPK3 and PKG are connected, our data support a broader role of TgCDPK3 and the pathways it controls. This is evident from the proteins that are differentially phosphorylated, including proteins that are important for metabolism, transcription and ion homeostasis in addition to the proteins important for egress, several of which are differentially phosphorylated even in the absence of ionophore. This is in line with our observation that basal calcium levels are elevated in TgCDPK3 mutants in the absence of ionophore. This allows for a model in which altered calcium fluxes in TgCDPK3 mutants have a profound effect on the homeostasis of the cell, which could dictate how a cell behaves under conditions of stress. The fact that CDPK3 mutant parasites don't show a measurable phenotype in cell culture points towards compensatory mechanisms that allow for normal growth. The fine balance the parasites have to strike might be easily tipped as in the case of calcium ionophore treatment, when the egress phenotype becomes evident. While this phenotype was only observed in vitro, CDPK3 mutant parasites also have a phenotype Toxoplasma parasite strains lacking TgCDPK3 (as used in this study) are still highly virulent in mice whereas Type II strains lacking TgCDPK3 activity are attenuated with severely reduced latent stages (tissue cysts) being found in the brain of chronically infected animals Type I Plasmodium berghei, PbCDPK1, that showed a role for PbCDPK1 in translational repression Toxoplasma interact to regulate several key functions, extending well beyond ionophore-induced egress.Our observation that there are differences in protein abundance in the TgCDPK3 mutants relative to WT resembles a recent study of the closest orthologue of TgCDPK3 in the malaria parasite 13C6-, 15N2-L-lysine, 84 mg/l 13C6-L-arginine, 40 mg/L unlabeled L-Proline) or light media . After 4 complete lytic cycles, parasites incorporated between 96% and 98% of heavy amino acids. For the comparative analysis parasites were seeded onto confluent human foreskin fibroblast in 150 mm dishes with an MOI of 5 in either heavy or light media. 24 hours post-infection the cells were washed once with fresh media and incubated in the presence of 1 uM A23187 or DMSO for 30 seconds. After the incubation time, the parasites were immediately placed on wet ice and quickly washed once with pre-chilled, ice-cold PBS prior to lysis in ice-cold 8 M urea containing protein and phosphatase inhibitors (Roche). We performed each experiment using 5 individual 15 cm dishes in order to monitor an average of the signaling events that take place during the first 30 seconds of ionophore treatment.Parasites lines and labeling of parasites was achieved as previously described +”.Peptide and phosphopeptide samples were prepared as previously described 4 AGC: 5×105, maximum ion time: 500 ms) followed by ten data dependent MS2 scans  of the most abundant ions. Dependent scans were configured with the following settings: 2.0 m/z isolation width, dynamic exclusion width: −0.52, 2.02, exclusion duration: 60 seconds, normalized collision energy: 35, activation time: 5 ms. Charge state screening was employed to reject ions with unassigned or +1 charge states.Phosphorylated and non-phosphorylated (flow-through) peptides were resuspended in 4% formic acid, 5% acetonitrile and analyzed by LC-MS/MS in technical duplicate on a system consisting of a MicroAS autosampler (Thermo Scientific), binary HPLC pumps (Agilent 1200 series) with flow-splitting, an in-house built nanospray source, and an LTQ Orbitrap Velos (Thermo Scientific). 2 µg of sample was loaded onto a 100 µm ID fused silica capillary packed with 18 cm of 5 µm Magic C18AQ resin (Michrome Bioresources). Peptides were eluted using a gradient of water:acetonitrile with 0.1% formic acid from 7% to 25% acetonitrile over 120 min, and then 25–40% B over 30 minutes. A top 10 method was run consisting of one MS1 scan  proteins using SEQUEST 2 H/L to 1∶1, signal to noise of each isotopic partner, and a VISTA confidence metric that accounts for chromatography quality. Weighted averages were calculated using these scores for sites and proteins for which more than one identification was made. These weighted averages are intentionally conservative, in an attempt to eliminate false-positives from the tails of the distribution. Unweighted average and standard deviation calculations have been included as well. Phosphopeptides were categorized as either mono-, bis-, or tris-phosphorylated, and separate averages were calculated for sites found in peptides of each type. The resulting data was further filtered for a minimum of 2 quantifications in each respective experiment, a minimum VISTA confidence score of 88 for the best quantification and a minimum signal to noise ratio for the best peptide of 8.Spectra were searched against a concatenated database of Human  and 2 ratios were centered on “0” based on the median SILAC ratio for each dataset.SILAC logDifferentially phosphorylated sites were identified by using the following criteria:2 fold change of 0.75 (+ or −), which is ∼1.5 times the standard deviation across the experimental datasets, and a consistent change of phosphorylation site abundance in one or more of the conditions. Note, one mismatch was allowed to capture phosphorylation sites that are just at the threshold, or missing in a single sample due to a bad SILAC quantification, to be included in the dataset. Phosphorylation site quantifications were manually curated by analyzing the MS1 elution profiles and removed if they originated from low-quality quantifications.a minimum logProtein SILAC ratios were calculated by using the median SILAC ratio for all identified peptides. Pearson correlation was calculated using a two-tailed test with a 95% confidence interval (Prism6).The open reading frame of TgCDPK1 was cloned into pGRA 32P-ATP and added to the peptide arrays for 4 h at 30 degrees Celsius. After washing and drying a high-resolution phosphor-imaging screen  was exposed. Images were acquired using a Typhoon scanner using 25 µM resolution and spot intensity values were analyzed using microarray software. Median spot intensities were generated after manual verification of each spot. Contaminations were manually removed and excluded from the analysis.Random peptide library kinase arrays  with a central serine were incubated according to the manufacturer's instructions. Briefly, 100 nM recombinant CDPK1 or CDPK3 was incubated with 50 µM Ca and 10 µM cold ATP to allow for autophosphorylation and activation of the kinase. This mix (400 µL) was then supplemented with 10 µCi of st rank) to the lowest observed phosphorylation.Each median intensity was plotted as a rank from highest  and labeled with Fluo-4 AM (2.5 µM) at room temperature with shaking for 30 minutes. Parasites were then pelleted, washed and resuspended in intracellular buffer followed by incubation at room temperature with shaking for 30 minutes to allow de-esterification. The parasites were then loaded onto 96-well plates (107 per well) and fluorescence (excitation 496 nm and emission 516 nm) was quantified using a Synergy H1 plate reader (BioTek).Naturally egressed parasites were resuspended in intracellular buffer . 200 µg of purified TgCDPK1 was injected with an equal volume of Freunds incomplete adjuvant into 6–8 week old pre-screened BALB/c mice from Charles Rivers. None of the mice showed reactivity in the pre-bleeds. Mice were boosted 3 times with 100 µg of recombinant protein every 3 weeks. Test bleeds were taken at week 6 and all mice were sacrificed at week 10 for the terminal bleed. Final bleed serum was screened for selectivity by Western blot and used in this study.Statistical analysis and graphs were made using GraphPad Prism6. Data handling was performed using Excel and MySQL using Python scripts. Heat-maps were generated using Cluster 3 , and visualized by Treeview.Figure S1Experimental conditions used in this study.(TIF)Click here for additional data file.Figure S22 SILAC ratios.Heatmap of all phosphorylation sites that differ between WT and TgCDPK3 mutant parasites, but where the protein-level is unchanged. The color bar represents log(TIF)Click here for additional data file.Table S1All quantified phosphorylation sites and proteins identified in this study. All quantified mono-, bis- and tris-phosphorylated peptides that passed the filtering are shown on individual sheet. Additional information from the individual mass-spectrometry experiments are represented in individual sheets.(XLSX)Click here for additional data file.Table S2A list of all phosphorylation sites that show differential phosphorylation site usage between CDPK3 mutants and wild type parasites.(XLSX)Click here for additional data file.


We sought to test the hypothesis that differences in the ability to acclimate to short-term temperature treatment would be revealed as differences in photosystem II maximum yield (Fv/Fm). Thermal treatments were applied to samples from 12 and 11 populations during 12 or 13 days in growth chambers and comprised: (1) 10/5 °C; (2) 20/10 °C; (3) 25/15 °C; (4) 30/20 °C (12 hours day/night temperature). In Pleurozium schreberi, there were no significant site-dependent differences before or after the experiment, while site dependencies were clearly shown in Racomitrium lanuginosum throughout the study. Fv/Fm in Pleurozium schreberi decreased at the highest and lowest temperature treatments, which can be interpreted as a stress response, but no similar trends were shown by Racomitrium lanuginosum.There is limited knowledge available on the thermal acclimation processes for bryophytes, especially when considering variation between populations or sites. This study investigated whether short-term  There is limited knowledge available on the thermal acclimation processes for bryophytes, especially when considering variation among and between populations, sites and time scales ,2,3. Theex situ temperature treatment of the bryophyte Pleurozium schreberi (Willd. ex Brid.) Mitt. originating from eight sites at differing altitudes in Hokkaido, Japan, showed that the responses were dependent upon the sampling site and that treatment effects differed between sites [A four-week study involving en sites . Those rP. schreberi [Racomitrium lanuginosum (Hedw.) Brid.) in this study to make the results comparable on an inter-species level.The main aim of this study was therefore to investigate whether short-term thermal acclimation of different populations of bryophytes showed similar patterns of site dependencies as found previously in chreberi . Howeverchreberi , it was ex situ but that Fv/Fm also, at least partly, reflected pre-treatment conditions.For short-term responses, growth rate or shoot length increases are not possible to measure and instead Fv/Fm, a measurement of the maximum quantum yield of PSII performed on dark-adapted samples, was used. Fv/Fm is a sensitive indicator of plant photosynthetic performance, but lower values may also indicate stress and/or photoinhibition , or indiex situ temperature manipulation. Based on this, the starting hypothesis was that species with significant site-specific Fv/Fm have lower acclimation capacity to different temperature conditions due to lower plasticity , while the opposite occurs in species with little significant site-specific variation in Fv/Fm due to higher plasticity  and thus they have a higher acclimation potential.A second aim was to confirm whether Fv/Fm could be used as a short-term indicator of adaptation or temperature stress in bryophytes. Decreased Fv/Fm values compared with before the start of the experiment were taken to indicate stress due to decreased photosynthetic performance, while similar or higher values were taken to indicate adaptation capability to P. schreberi prior to the experiment, while significant site differences were found in R. lanuginosum  temperature conditions  or 2 cm (R. lanuginosum) and Fv/Fm measurements were made before the experiment started. Shoots were then individually marked and placed into small transparent plastic bags (9 cm × 15 cm), and assigned to one of four temperature treatments. Samples in bags were fully hydrated with de-ionized water and suspended in a natural position in growth chambers on 5 July for P. schreberi, and on 6 July for R. lanuginosum. The growth chambers simulating different temperature conditions had 12 h day/night temperature cycle and photoperiod , which is generally considered a low value. However, P. schreberi grows underneath the vegetation canopy at all sites and was therefore likely to be shade-adapted. The light responses were assumed to be similar to those of bryophyte species growing in forests. For example, several bryophyte species show a light saturation point of less than 30 µM m–2 s–1 in laurel evergreen forests [2 did not limit growth.From each site, five moss shoots from each of four samples were randomly chosen and cut to 4 cm (toperiod . Tempera forests . FurtherR. lanuginosum) and 13 (P. schreberi) days, shoots were carefully removed from plastic bags and put in bunches to increase the surface area of chlorophyll fluorescence measurements (from each treatment and site for the treated samples), and placed in leaf clips for two hours of dark adaptation. Maximum quantum of PSII (Fv/Fm) was measured with a mini-PAM . Calculations followed [R. lanuginosum, Mt Koma 550 m above sea level), the measurement on Fv/Fm was later determined to be wrong and was not included in the analyses.Before the start of the experiment and after 12 (followed . In one P. schreberi and R. lanuginosum. Significant differences for P. schreberi to treatments were subsequently determined with Fisher’s PLSD post-hoc test. We used t-test to investigate differences in Fv/Fm before and after the experiment. All statistics were computed in IBM © SPSS © Statistics version 22 (22.0.0.1.), USA.The data were log-transformed to meet normality assumptions and then investigated for differences in Fv/Fm in response to either site (before and after) or temperature treatment (after). Since there was only one value of Fv/Fm for each site and treatment after the experiment was finished, separate one-way ANOVAs were performed for This study shows that bryophytes may exhibit differences in site dependencies in their acclimation response in Fv/Fm to short-term temperature treatments and that using Fv/Fm as an indicator revealed those differences. The results may have implications for studies assuming similar acclimation potential between species, sites or populations of bryophytes.


Drosophila and rodents. Alzheimer disease’s patients exhibit high levels of circulating GSK3β and, consequently, pharmacological strategies based on GSK3β antagonists have been designed. The approach, however, has yielded inconclusive results so far. Here, we carried out a comparative study in Drosophila and rats addressing the role of GSK3β in synaptogenesis. In flies, the genetic inhibition of the shaggy-encoded GSK3β increases the number of synapses, while its upregulation leads to synapse loss. Likewise, in three weeks cultured rat hippocampal neurons, the pharmacological inhibition of GSK3β increases synapse density and Synapsin expression. However, experiments on younger cultures (12 days) yielded an opposite effect, a reduction of synapse density. This unexpected finding seems to unveil an age- and dosage-dependent differential response of mammalian neurons to the stimulation/inhibition of GSK3β, a feature that must be considered in the context of human adult neurogenesis and pharmacological treatments for Alzheimer’s disease based on GSK3β antagonists.The PI3K-dependent activation of AKT results in the inhibition of GSK3β in most signaling pathways. These kinases regulate multiple neuronal processes including the control of synapse number as shown for  Drosophila larvae, GSK3β is represented by two splice variants (Sgg10 and Sgg39) encoded in the single gene shaggy (sgg) [Glycogen synthase kinase 3 (GSK3) ,2 is a sgy (sgg) –15. In tDrosophila and Rattus, aimed to identify possible reasons for the puzzling failure of current AD pharmacological approaches.The functional requirements of GSK3 are diverse and, consequently, the repertoire of elicited pathologies is extensive. Focusing on the nervous system, in mammalian brains, GSK3 is required for proliferation, differentiation and neurotransmission in adult neurons –20. GSK3Drosophila which is conserved in vertebrates. Activation/inhibition of PI3K or downstream elements of this pathway coherently regulate the number of synapses [Drosophila and mammals [We have previously identified a phosphoinositide 3-kinase (PI3K) synaptogenic pathway in synapses –29. Whil mammals –30, the  mammals ,31–32. T mammals . Likewis mammals . Indirec mammals  whereas  mammals .The molecular mechanisms that sustain these cellular processes involve changes in the phosphorylation status of specific residues in these kinases. Thus, after PI3K activation, AKT is phosphorylated in Serine 473 by mTORC2 and in Threonine 308 by PDK1 ; which rDrosophila neurons. In rodent hippocampal neurons, however, GSK3 inhibition yields differential results according to cell culture age and dosage. Young culture neurons show a decrease of synapses while aged cultures respond with an increase of synapse number. Also, whereas spine density increases under low concentrations of GSK3 inhibitor, high concentrations reduce the number of spines.Here, we show that the downregulation of GSK3 promotes synapse formation in D42-Gal4 was kindly provided by H. L Atwood (University of Toronto) [elav-Gal4 [UAS-AKT [UAS-GSK3DN [UAS-PI3K92E (referred here as UAS-PI3K) and UAS-PI3K92ED954A (UAS-PI3KDN) stocks originate from S. Leevers . The UAS-GSK3RNAi construct was obtained from the Vienna Stock Center (http://stockcenter.vdrc.at/control/main) (reference 101538KK) and the UAS-GSK3 strain was provided by Dr. Manuel Calleja  [Line Toronto) . Line ellav-Gal4 , UAS-AKT[UAS-AKT  and UAS--GSK3DN  were obtPrimary hippocampal cultures were obtained from P0 rat pups . Animals were anesthetized by hypothermia in paper-lined towel over crushed-ice surface during 2–4 minutes and euthanized by decapitation. Animals were handled and maintained in accordance with the Council Directive guidelines 2010/63EU of the European Parliament, and approved by the Ethical Committee of the CIBIR.4 cells/cm2 and used 5 days later, usually when cultures reached a 70–80% confluence. Culture media contained DMEM F-12 Ham  supplemented with 0.5 mM glutamine , penicillin (50 mg/ml)/streptomycin (50 U/ml) from Sigma-Aldrich (USA), and 10% FBS . Cells were serum starved for 16 hours prior to treatment, in order to reduce Akt basal activity.SH-SY5Y human neuroblastoma cells were purchased from ATCC (ref: CRL-2266). Cells were seeded at 5x104 neurons/cm2 for immunocytochemistry, and a higher density of 50x104 neurons/cm2 for biochemical experiments. In both cases, cultured cells were seeded on plastic 24-wells plates.Primary cultures of hippocampus neurons were prepared as previously described –43. Glashttp://rsb.info.nih.gov/ij/, NIH, USA) was used to determine the number of synapses per neuromuscular junction (NMJ). Individual synapses (nc82 positive puncta) were quantified by using the Point Picker plug-in as published previously [We systematically used the nc82 Mab to identify the active zone component, CAST/Bruchpilot, of synapses. The matching between presynaptic nc82 and postsynaptic GluRII immunosignals have been previously documented . Late theviously . A minimSynaptic density on hippocampal cultures was analyzed as previously described . In shor6 cells and 10 μg of plasmid were mixed in BioRad electroporation buffer (BioRad). An exponential discharge protocol  was employed. Spines were visualized by transfection with a plasmid encoding the GFP protein fused to chick β-actin under the control of the platelet-derived growth factor promoter region  [Neuronal GFP-Actin expression was obtained by electroporation before plating, employing a BioRad Cell electroporator system. Approximately 4x10don, UK) . TransfeDrosophila samples, nitrocellulose membranes were incubated with the following primary antibodies: mouse anti-GSK3 (detecting both Drosophila GSK3 splice variants) , rabbit anti-AKT  and rabbit anti-phosporylated-AKT (p-S505AKT) , with 3% non-fat dry milk (Bio-Rad) in 0.05% PBST  overnight at 4°C. They were washed with double distilled water twice and incubated with secondary peroxidase-conjugated antibodies goat anti-mouse or goat anti-rabbit IgG  for 1.5 hours at room temperature. Membranes were washed with PBST (2x10 minutes) and revealed with SuperSignal (Thermo Scientific). Western blots of Drosophila and rat materials were quantified as previously described [For escribed ,44. BandThe same Western blot protocol was employed for SH-SY5Y and neuronal cultures. Briefly, cell wells were washed with PBS (4°C) and scrapped in 60 μl of lysis buffer . Once collected, cell lysates were heated, sonicated and clarified by centrifugation at 17000 g. Protein concentration was measured using Bradford protein assay and 60 μg of total protein were loaded into each SDS-PAGE lane. After blotting, membranes were incubated overnight with the following antibodies: anti-Actin , anti-GSK3 α/β , anti-phospho-S9GSK3β , anti-Synapsin I , anti-AKT (pan) (C67E7)  and the following polyclonal antibodies: anti-phospho-S473AKT1 , anti-β-Catenin  and anti-phospho-β-Catenin (Ser33/37/Thr41) . Signal was detected using the corresponding IR-fluorescence-labelled antibodies  and visualized and quantified with an Odyssey Infrared Imaging System (Li-Cor Bioscience). Unless otherwise indicated, all biochemical experiments were performed adding the peptide at day 10 or 19 in vitro, cell lysis was done 48 hours later. The p-S473AKT and p-S9GSK3β values were related to the total AKT and GSK3β, respectively. Synapsin and phospho-β-Catenin levels were related to Actin. Average changes in phosphorylation and percentage of protein represent the normalized value with respect to the control value, always included in the same Western-blot membrane. Values over 100 indicate increase in protein levels and values below 100 indicate reduction.Activation of PI3K was achieved by a transduction peptide (PTD4-PI3KAc) with the following sequence: YARAAARQARAGSDGGpYMDMS ,46–48. TDrosophila WB. If p<0.05 in the ANOVA test, a Bonferroni posthoc t-test was applied. The Kolmogorov-Smirnov test for normality was performed where appropriate, prior to the use of the Student’s two tailed t-test. GraphPad Prism software package versions 3 and 6  were used throughout. Significant differences were noted by *p<0.05, **p<0.005 and ***p<0.0001.All data are represented as mean ± s.e.m. Statistical significance was calculated using a Student’s two-tailed t-test or ANOVA in shaggy/GSK3 reduces synapse number [UAS-GSK3) or down-regulate (UAS-GSK3DN or UAS-GSK3RNAi) the gene, we first quantified the expression changes of the two protein isoforms in larvae: SGG39 and SGG10 [elav-Gal4, elav-Gal4/UAS-GSK3 and elav-Gal4/UAS-GSK3RNAi larval central nervous systems  indicate that the upregulation of GSK3 does not cause a significant increase of isoform SGG39 levels , but a strong one  in the case of SGG10 isoform  (n = 11 brains per lane in each genotype). In turn, the downregulation by GSK3RNAi decreases the protein content to 50 ± 4% in SGG39 and to 58 ± 8% in SGG10 (p<0.05 and p<0.05 respectively) . Thus, both genetic tools effectively modify the gene expression.We showed previously that the upregulation of e number ,31. Herend SGG10 . WesternD42-Gal4/UAS-GSK3: 417±18, n = 13 larvae vs. D42-Gal4: 528±35, n = 9 larvae; p = 0.0026) . Next, we analyzed the downregulation effects: whereas the dominant negative form of GSK3 proved ineffective (not shown), the GSK3RNAi caused a significant (p<0.0001) increase: 835±38 synapses (n = 7 larvae) when compared to control: 528±35 synapses (n = 9)  in brains over-expressing PI3K  and decreases 22±10% (p<0.05) in brains over-expressing a dominant negative form of PI3K  compared to control  . Thus, PI3K modulates the phosphorylation state and, hence, the activity of AKT.As it is well known in several signaling pathways, PI3K modulates AKT activity. To confirm the case in the Drosophila has only one serine/threonine protein kinase encoding gene, Akt1 (dAkt1), homologous to mammalian c-Akt [Drosophila AKT inhibits GSK3 by specific phosphorylation of Serine 9 [Fig. 2C-D). Thus, the synaptic effects of GSK3 manipulations proceed through a feed-forward mechanism that does not involve changes in AKT phosphorylation.Next, we addressed the relationship between GSK3 and AKT. an c-Akt . InteresSerine 9 . We askein vivo between PI3K, AKT and GSK3 in the context of synaptogenesis, we tested their epistatic phenotypes. First, we confirmed the synaptic effects of PI3K to allow a comparison with the subsequent double mutant combinations. As previously reported, PI3K overexpression increases synapses  and its downregulation decreases them  compared to control  .To address the functional relationship D42-Gal4/UAS-PI3K/UAS-GSK3, 411±23, n = 8, p<0.005) or the downregulation of PI3K  . These results indicate that, following the direct upregulation of GSK3 by genetic procedures, the synaptic effects of this enzyme are independent from any potential regulation from PI3K. By contrast, the increase of synapse number elicited by the downregulation of GSK3 is abolished and reversed when PI3K is down-regulated , but maintained when PI3K is over-expressed  . Since the genetic downregulation of GSK3 still leaves about 40% expression of both isoforms , these in vivo data are compatible with a regulatory role GSK3 upon PI3K activity in the context of synaptogenesis. Thus, akin to other functional contexts, GSK3 and PI3K seem to share the same pathway for synaptogenesis where GSK3 would be a negative modulator of PI3K without evidences of a feedback regulatory loop.Next, we assayed combinations of up and down expressions of PI3K and GSK3. The data indicate that the reduction in synapse number elicited by GSK3 overexpression is epistatic over, either the upregulation  compared with controls  (p<0.0001). The combined AKT overexpression/GSK3 downregulation showed a synaptic increase of the same magnitude as the stronger of the two, AKT . Finally, when GSK3 and AKT were simultaneously over-expressed we obtained a modest but significant increase in synapse number . GSK3 combinations against a homozygous AKT null background  were not viable and, thus, we limited our study to AKT over expression condition only. Taken together, the data indicate that GSK3 levels do not modify the synaptogenic effects of AKT upregulation, placing GSK3 functionally downstream of AKT in the synaptogenic pathway.Concerning the epistasia analysis between AKT and GSK3, we first confirmed that AKT upregulation increases synapses  by stimulating its PI3K-AKT-dependent phosphorylation using an engineered peptide, and 2) by its pharmacological inhibition using two high-affinity and selective organic compounds.Material and Methods) and quantified the levels of p-S473AKT and p-S9GSK3β [Fig. 4A). In parallel, p-S9GSK3β/GSK3β levels peaks after 30 minutes, reaching plateau (157±2%) for the rest of the experiment .To examine if the PI3K activator peptide can modulate GSK3β phosphorylation, we took advantage of a PI3K previously characterized activation peptide   that decreases to basal levels (around 100% after 120 min). Levels of p-S9GSK3β/GSK3β show a similar profile with a maximum of phosphorylation after 60 min (186±3%) and a later reduction to basal levels 240 min later (100%) . As a negative control, cells were treated with the transduction PTD4 peptide without the PI3K activation domain . Similar results over p-S9GSK3β were obtained with hippocampal neurons in culture: after 48 hours treatment with the PI3K activator peptide, levels of p-S9GSK3β/GSK3β raised to a statistically significant, 127±4% (p<0.0001), over control conditions . All together, these experiments validate the PTD4-PI3KAc peptide as an effective tool to modulate GSK3 phosphorylation, and point out to an important feature of the peptide: although the activation level of AKT is weaker than that of a tyrosine kinase activator (EGF), its biological activity remains stable for a longer period of time, up to 48 hours. Further, the data demonstrate that the activation of AKT by the peptide results in GSK3β phosphorylation at Ser 9.As a positive control, we employed EGF, a S2A Fig.). SB did not affect neuronal density at a concentration range of 10 to 25 μM, but the use of 50 μM resulted on a not statistically significant reduction of 13.8% on neuronal survival . The second inhibitor, AR, did not affect neuronal survival at 1 and 10 μM, while at 50 μM induced a significant neuronal death of 16.5% (p<0.05) .To pharmacologically block GSK3β, we used two specific organic inhibitors: AR-A014418 (AR) ,55 and SFig. 5A, 5C). The effects of AR were tested at 1, 2, and 10 μM . No significant changes were observed at 1μM; while 2 μM and 10 μM reduced it 24±5% and 35±8%, respectively (p<0.05 in both experiments) . The simultaneous activation of PI3K by PTD4-PI3KAc and the direct inhibition of GSK3 with AR did not result in a further inhibition of GSK3β pathway, with an average reduction of 32±5% (p<0.005), 25±9% (p<0.05), and 24±7% (p<0.05) at 1, 2 and 10 μM of AR respectively, suggesting that both drugs act over the same pathway . The efficiency of SB inhibiting GSK3β was tested at 1, 10 and 25 μM, the levels of p-β-Catenin decreased in a 26±6% (p<0.05), 28±9% (p<0.005) and 38±8% (p<0.005) respectively , 25±8% (p<0.05) and 39±10% (p<0.05) at 1, 10 and 25 μM of SB, respectively; Fig. 5E). In conclusion, PTD4-PI3KAc and the inhibitors AR and SB effectively inhibit GSK3β at the selected dose.We next assayed the effectiveness of these drugs to inhibit GSK3 in hippocampal neurons in culture. GSK3 regulate β-Catenin levels by phosphorylation at both Serine 33 and 37 and also at Threonine 41 . Since bFig. 6A). The overlap of two presynaptic markers  was used as criteria for mature synapses as previously described [Fig. 6B, 6C) to 125±4% . This observation is in agreement with our previous data showing an increase in both excitatory and inhibitory synapses following PI3K up-regulation [Fig. 6B) and SB raised synaptic density to 112±5% (ns) and 133±5% (p<0.0001) at 10 and 25 μM respectively . To investigate potential cooperative effects, we analyzed the simultaneous activation of PI3K and inhibition of GSK3. The combined treatment of PTD4-PI3KAc with AR results in an increase of 113±5%, ns, while the use of SB resulted in synapse density raises of 120±5% (p<0.05) and 137±6% (p<0.0001), 10 and 25 μM respectively. In these three cases, the increase was similar to the individual treatments ; suggesting that GSK3 should be downstream from, and in the same pathway of PI3K in the context of synaptogenesis.The density of synapses was evaluated by immunocytochemistry on hippocampal cultures . However, the inhibition of GSK3 by AR or SB reduced synapse number instead as observed in older cultures. SB at 10 and 25 μM reduced synaptic density to 74±12% (p<0.05) and 79±4%, (p<0.0001) respectively , and AR reduced it to similar values  . As it seems, the strong pro-synaptogenic effect elicited by GSK3 inhibition in 21 DIV cultures , is opposite to the effect obtained with the same drug and dosage in 12 DIV cultures . To assess the independence of PI3K and GSK3 in the process, we measured synaptic density after a combined treatment with SB and PTD4-PI3KAc. The results indicate that activation of PI3K compensates the reduction of synaptic density caused by GSK3 inhibition , yielding mean values of 105±10 and 115±4% (10 and 25 μM of SB respectively), which are not significantly different from culture controls conditions . Thus, PI3K is not involved in the age-dependent effect caused by GSK3 inhibition.In younger (12 DIV) cultures, PI3K activation resulted also in the expected synaptic increase, 126±6% (p<0.0001) . The simultaneous activation of PI3K and inhibition of GSK3 resulted in a similar degree of augmentation, with mean values of 137±1% (p<0.005) and 141±2% (p<0.05) . These changes in Synapsin levels are consistent with the variation in synapse density .To analyze the age-dependent effect of GSK3 inhibition through an alternative method, we quantified the levels of two synaptic proteins, Synapsin and PSD95, by Western blot. Synapsin is the major component of the presynaptic vesicles and its expression has been used as a reporter of synaptogenic activity . ProteinFig. 7C, 7D) as it was observed in the aged cultures . Similar to 21 DIV, the simultaneous PI3K activation by the peptide and GSK3 inhibition by SB, at all concentrations tested, did not elevate Synapsin in young cultures . These effects on Synapsin expression were reproduced with AR . In this case, neither the 2 or 10 μM AR treatments, nor their combination with PTD4-PI3KAc, modified Synapsin levels .In 12 DIV cultures, PI3K activation increased Synapsin by 126±4%, the same effect as in the aged cultures (see above). By contrast, GSK3 inhibition by SB  did not result in a significant change of Synapsin levels . In 21 DIV cultures GSK3 inhibition by SB raised expression levels to a 128±3% (p<0.005) and the joint activation of PI3K plus the inhibition of GSK3 resulted also in a significant increase of 148±1% (p<0.05). By contrast, the same treatments applied in 12 DIV cultures did not result in significant changes in PSD95 , . Therefore, we conclude that GSK3 inhibition has a differential effect repressing/enhancing synapse formation depending on neuron age and this effect is not related to PI3K.In addition, we tested another synaptic protein, PSD95, which is a component of the glutamatergic postsynaptic side and obtained similar effects . To discriminate if the protein could localize also in the presynaptic compartment we co-immunolocalized Bassoon. The images indicate that p-S9GSK3β is not associated with the presynaptic side .We first examined the localization of p-S9GSK3β in hippocampus neurons transfected with GFP-Actin . The expFig. 9C; [Fig. 9D). Interestingly, when cultures were treated with the lowest concentration (0.5 μM) spine density exhibit an opposite effect, a significant increment of 16±5% (p<0.005) . Likely, age and dosage are just two among the several factors that may account for the diversity of synaptic responses to GSK3 inhibition.To quantify the effects of GSK3 inhibition over spine density, culture neurons were treated with a broad range of SB concentrations from 0.5 to 25 μM for 48 hours . SimilaDrosophila larvae and cultured hippocampal rat neurons. The data indicate that GSK3 inhibition in both species leads to a synaptic increase through a PI3K signaling pathway, in which GSK3 represses synapse formation.We have addressed the role of GSK3 in synaptogenesis and spinogenesis in a comparative study between the neuromuscular junction of Figs. 1D and 3A). Both results could be explained taking into account the respective amounts of both proteins. Thus, while GSK3 overexpression increases three fold the amount of Sgg10 isoform , PI3K upregulation increases p-S505AKT by 35% only . This excess of GSK3 might not be effectively inhibited by a lesser amount of p-S505AKT, with the final result of a decrease in synapse number due to the GSK3-dependent antisynaptogenic effect. In the second combination, the joint downregulation of PI3K and GSK3, the PI3K phenotype prevails. This result can also be explained through the relative protein levels elicited by the genetic tools. The RNAi against GSK3 yields a reduction of 50% in the total amount of both GSK3 isoforms, Sgg10 and Sgg39 . By contrast, PI3KDN reduces pS505-AKT around 22% . In this case, the amount of p-S505AKT could not be enough to phosphorylate, hence inhibit, all the GSK3 protein, leaving a sufficiently activated level of GSK3 to finally cause a reduction in the number of synapses. Additionally, we cannot formally rule out the possible differences between the use of dominant negative and RNAi constructs to reduce expression levels of these kinases, taking into account that, whereas the first interferes with protein function, the second attenuates protein transcription. To assess this variability in the available genetic tools, we do not observe changes in synapse number when we use GSK3DN but we observe satellite boutons as previously described  in agreement with the previous analysis of PI3K and GSK3 .In flies, synapse number increases when GSK3 is down-regulated or when PI3K is up-regulated. In the epistasis analysis, same phenotype combinations are not additive which strongly suggests that both kinases share the same pathway. However, these data do not inform about their hierarchical order. Opposite phenotype combinations could have solved the functional hierarchy. One of these combinations—joint upregulation of PI3K and GSK3 -, produced the phenotype of GSK3 alone, while the other combination—joint downregulation of PI3K and GSK3-, yielded the phenotype of PI3K alone . Also, the p-S505AKT/AKT ratio does not change in larval brains that over- or down-regulate GSK3 , suggesting again that GSK3 is downstream from AKT and does not affect AKT phosphorylation. This scenario from Drosophila neurons is consistent with evidences from other cell types and functional contexts [The three kinases, PI3K, AKT and GSK3 seem to share the same pathway for synaptogenesis because all their same phenotype combinations do not show additive effects , PI3K activation or direct GSK3 kinase inhibition induced an increase of synapse number, concomitantly with an increase of Synapsin and PSD95 expression. The lack of an additive effect of the conjoint activation of PI3K and GSK3 inhibition, suggest that both kinases share the same pathway. Results in both species confirm a direct role of GSK3 regulating synapse formation and suggest that, under physiological conditions, GSK3 activity represses synapse formation. Inhibition of GSK3 up-regulates PSD95 or Synapsin indifferently, suggesting that GSK3 could operate downstream from PI3K/AKT in the pathway that leads to synapse formation.When the same experimental procedure was performed in 12 DIV hippocampal neurons, inhibition of GSK3 by PI3K-AKT activation led to an increase of PSD95 and Synapsin expression and a concomitant raise in synaptic density. In contrast, blocking GSK3 kinase activity induced a reduction of synaptic density without affecting Synapsin or PSD95 levels.Concerning spine density, it has been previously demonstrated that PI3K activation increases spinogenesis both in cultures and in brain hippocampus ,61,67. Hin vivo, an effect that was impaired by the use of a direct GSK3 kinase inhibitor (i.e. SB) [Why GSK3 inhibition can repress/enhance synaptic formation depending on the age of the cultured neurons? To answer this question we must consider that GSK3 regulation can be independent from AKT phosphorylation. Indeed, downstream of PI3K, GSK3 is inactivated via AKT-mediated phosphorylation of two serine residues . When bi.e. SB) . Furtheri.e. SB) . All togAlternatively, a possible change in Wnt signaling along hippocampal cell culture development could also be responsible for the observed differential GSK3 effects. Wnt and PI3K-AKT pathways share a common element, Axin, which binds to GSK3 preventing its phosphorylation and the subsequent inhibition by AKT . InhibitOther authors have also proposed an additional layer of GSK3 regulation based on the existence of “primed” or “not primed” substrates. The prior phosphorylation of many GSK3 targets enhances GSK3 affinity and binding to such primed substrates ,74–75. TIn summary, this study shows that GSK3β regulates synapse number in flies and rodents through a signaling pathway that includes PI3K and AKT. In view of this conservation, it seems justified to consider GSK3 as a potential target for the treatment of several CNS diseases . NeverthS1 FigA. Control  B. GSK3 downregulation  C. Simultaneous overexpression of GSK3 and PI3K  D. Simultaneous downregulation of GSK3 and PI3K  E. Simultaneous overexpression of PI3K and downregulation of GSK3  F. Overexpression of AKT  and G. Simultaneous overexpression of AKT and downregulation of GSK3 . A high magnification view from the white square in E is shown in the upper right corner. Scale bars = 10 (A-G) and 2 μm (insert in E).Representative confocal images of larval motor neurons double immunostained against α-HRP (red) and nc82 antibody (green) from (TIF)Click here for additional data file.S2 FigA. Representative confocal images of hippocampal neuronal cultures in control conditions and after treatment with the indicated concentrations of SB 415286 or AR-A014418. Neuronal density was quantified from high-resolution images comprising 40 individual pictures . The pictures only show a detailed area of 6 mm2. Neuronal staining by MAP2B is shown in green and nuclear staining by DAPI is shown in red. DAPI marks all cells in the culture, astrocytes and neurons, while MAP2B marks only neurons. Scale bar = 50 μm. B. The effects of SB 415286 over neuronal survival were tested after 48 hours at three concentrations  and compared with control conditions (white histogram) in 12 DIV hippocampal neurons. C. Similar experiments were performed employing AR-A014418  (n = 4 coverslips from 3 different cultures). Student’s two-tailed t-test, *p<0.05.(TIF)Click here for additional data file.


Microcystis aeruginosa produces microcystin and an array of diverse metabolites believed responsible for their toxicity and/or immunogenicity. Previously, chronic rhinitis patients were demonstrated to elicit a specific IgE response to nontoxic strains of M. aeruginosa by skin-prick testing, indicating that cyanobacteria allergenicity resides in a non-toxin–producing component of the organism.The cyanobacterium species M. aeruginosa peptide(s) responsible for allergic sensitization in susceptible individuals, and we investigated the functional interactions between cyanobacterial toxins and their coexpressed immunogenic peptides.We sought to identify and characterize M. aeruginosa toxic [MC(+)] and nontoxic [MC(–)] strains were used to test IgE-specific reactivity by direct and indirect ELISAs; 2D gel electrophoresis, followed by immunoblots and mass spectrometry (MS), was performed to identify the relevant sensitizing peptides. Cytotoxicity and mediator release assays were performed using the MC(+) and MC(–) lysates.Sera from patients and extracts from We found specific IgE to be increased more in response to the MC(–) strain than the MC(+) strain. This response was inhibited by preincubation of MC(–) lysate with increasing concentrations of microcystin. MS revealed that phycocyanin and the core-membrane linker peptide are the responsible allergens, and MC(–) extracts containing these proteins induced β-hexosaminidase release in rat basophil leukemia cells.M. aeruginosa have been identified as the relevant sensitizing proteins. Our finding that allergenicity is inhibited in a dose-dependent manner by microcystin toxin suggests that further investigation is warranted to understand the interplay between immunogenicity and toxicity of cyanobacteria under diverse environmental conditions.Phycobiliprotein complexes in Microcystis aeruginosa peptides responsible for allergic sensitization and characterization of functional interactions between cyanobacterial toxins and immunogenic peptides. Environ Health Perspect 123:1159–1166; http://dx.doi.org/10.1289/ehp.1409065Geh EN, Ghosh D, McKell M, de la Cruz AA, Stelma G, Bernstein JA. 2015. Identification of  Cyanobacteria  are ubiquitous photosynthetic bacteria that have the potential to produce toxins. Cyanobacteria are primarily found in freshwater systems worldwide. In nutrient-rich water, cyanobacteria cells proliferate to form a mass called a bloom. During the past decade, cyanobacteria blooms have been of increasing concern to public health and water management officials as their potential health effects are being better recognized. Global climate change, resulting in increases in water temperatures and severe droughts in combination with increases in nutrient load, has led to massive and prolonged cyanobacteria blooms in many large bodies of freshwater in the United States, further threatening human health and the environment . SpecifiSignificant variability exists in the toxicity of cyanobacteria because some species produce toxins but others do not . InteresMicrocystis aeruginosa, Arthrospira platensis, and Aphanizomenon-flos aquae), A number of clinical studies in humans found a significant correlation between exposure to cyanotoxins and allergic reactions in sensitized individuals , 2006b. Reagents and antibodies. Goat anti-human IgE, alkaline phosphatase–conjugated rabbit anti-goat IgG, and horseradish peroxidase (HRP)-conjugated rabbit anti-goat IgG were purchased from KPL . We obtained anti-phycocyanin from Bioss , microtiter ELISA plates from Corning , microcystin LR and anti-FcεRI from EMD Millipore Corporation , Eagle’s Minimum Essential Medium (EMEM) from ATCC , and phycocyanin, G418, and pNPP substrate from Sigma-Aldrich .Study population. After signing an Institutional Review Board–approved informed consent and, if applicable, an assent, participants ≥ 6 years of age who were being evaluated for allergic rhinitis with aeroallergen skin testing also underwent SPT to extracts from M. aeruginosa–nontoxic [MC(–)] strains. Due to ethical concerns, SPT to extracts from M. aeruginosa–toxic [MC(+)] strains was not performed. Participants with dermatographia or who were taking medications that prohibited skin testing were excluded from the study. A chart review was conducted for each participant to obtain information regarding potential sources of indoor cyanobacteria exposure. All allergic and control participants were recruited locally and living in the greater Cincinnati, Ohio, area.Serum. The patient sera used for this study were collected from a previous study that longitudinally enrolled and skin tested 259 patients presenting with chronic rhinitis over 2 years ( 2 years . Serum wCultivation of M. aeruginosa and preparation of crude cell extracts. Axenic cultures of M. aeruginosa (2385 and 2386) were obtained from UTEX The Culture Collection of Algae . M. aeruginosa 2385 produces the cyanobacterial toxin microcystin , whereas 2386 does not produce microcystin toxin . Cultures were cultivated in BG11 broth medium  supplemented with 1.8 mM sodium nitrate NaNO3 and 10 mM sodium bicarbonate. Cultures were incubated and maintained under fluorescent white light (irradiance incident of 20 μmol/m2/sec) at 25°C without mixing, with a 14:10 hr light and dark cycle and humidity at 55%. Cells in logarithmic phase  were harvested by centrifugation  and washed three times with sterile 0.01 M phosphate-buffered saline (PBS), pH 7.4. Washed cells were kept at –20°C until needed.M. aeruginosa cells were resuspended in 1 mL lysis buffer . The resuspended cells were then incubated on ice for 20 min. The cells were then sonicated using a cell disruptor  at setting 7 three times for 20 sec, followed by 40-sec incubation on ice. The extract was centrifuged at 14,000 × g for 30 min at 4°C. The supernatant was then collected and either lyophilized or concentrated using Amicon Ultra-15 (10 kDa capacity) centrifuge tubes (EMD Millipore Corporation) before freezing at –80°C. The total protein yield for both the MC(–) and MC(+) was approximately 1.2 mg per 50 mg dry cell weight. The concentrated lysates were used in all experiments except the toxicity studies. For the toxicity assay, we used lyophilized lysates because the Amicon tubes were inefficient in retaining low-molecular-weight proteins such as microcystin .To obtain the crude extract, 50 mg of washed IgE-specific ELISA. IgE-specific ELISA was performed using a previously described method (p-nitrophenyl phosphate (pNPP) substrate and read at 405 nm using a Multiskan Ascent 96 Plate Reader . The results are presented as optical density (OD) or absorbance compared with controls (relative OD).d method . BrieflyELISA inhibition. Antigen specificity and cross-reactivity were determined using an ELISA competitive inhibition assay. Pooled serum from patients sensitized to M. aeruginosa was preabsorbed at 4°C overnight, with increasing concentrations of MC(–) lysate (solution phase). The MC(–) lysate–serum mixture was then added to wells coated with MC(–) and MC(+) lysates (solid phase). Subsequent steps of the inhibition assay were performed as described for the IgE-specific ELISA above.SDS-PAGE, immunoprecipitation, and immunoblot analyses. SDS-PAGE  of M. aeruginosa lysates was carried out following the method of For immunoprecipitation (IP), lysates were precleared by incubating with 10 μL of Protein A beads in IP Lysis Buffer . The precleared lysate was then preincubated with anti-phycocyanin antibody  for 1 hr at RT before adding the cyanobacteria lysate. The mixture was further incubated for an additional 1 hr at RT and then overnight at 4°C. The beads were then washed four times with IP wash buffer . After the last wash, the immune complexes were then eluted using 50 μL of Elution Buffer (Pierce). SDS-PAGE and immunoblot analysis were performed on the eluate as described above.Two-dimensional (2D) gel electrophoresis. 2D gel electrophoresis was performed using 500 μg of cyanobacteria lysate. The lysate was solubilized in 125 μL DeStreak Rehydration Solution  prior to applying to pH 3–11 range strips  for isoelectric focusing (IEF). IEF of the proteins was performed on an Ettan IPGphor apparatus  using the following running conditions: 500 V for 1 hr, 2,000 V for 1 hr, and 8,000 V for 2 hr. After focusing, the strips were then incubated in equilibration buffer  for 30 min before being mounted on a 12.5% polyacrylamide gel. Electrophoresis and immunoblotting were carried out as described above. Coumassie staining was performed using Bio-Safe Coomassie Stain  per the manufacturer’s protocol.Mass spectrometry. Nano-liquid chromatography–coupled electrospray tandem mass spectrometry (nLC-ESI-MS/MS). nLC-ESI-MS/MS analyses were performed on a TripleTOF 5600+  attached to an Eksigent  nanoLC.ultra nanoflow system. The recovered peptides were loaded (via an Eksigent nanoLC.as-2 autosampler) onto an IntegraFrit Trap Column  from New Objective Inc.  at 2 μL/min in formic acid/H2O, 0.1/99.9 (vol/vol) for 15 min to desalt and concentrate the samples. For the chromatographic separation of peptides, the trap column was switched to align with the analytical column, an Acclaim PepMap100  from Dionex-Thermo Fisher Scientific . The peptides were eluted using a variable mobile phase gradient from 95% phase A  to 35% phase B  for 15 min, from 35% phase B to 80% phase B for 1 min, and then kept at the same mobile phase composition for 2 more minutes at 300 nL/min. The nLC effluent was ionized and sprayed into the mass spectrometer using NANOSpray® III Source (AB Sciex). Ion source gas 1 (GS1), ion source gas 2 (GS2) and curtain gas (CUR), respectively, were kept at 7, 0, and 25 vendor-specified arbitrary units. Interface heater temperature and ion spray voltage was kept at 150°C and 2.5 kV, respectively. The mass spectrometer was operated in positive ion mode set to go through 2,091 cycles for 25 min, with each cycle performing one time-of-flight (TOF)-MS scan type  followed by 30 information-dependent acquisition (IDA)-mode MS/MS-scans on the most intense candidate ions, having a minimum 250 counts. Each product ion scan was operated under vender-specified high-sensitivity mode with an accumulation time of 0.05 sec and a mass tolerance of 50 mDa. Former MS/MS-analyzed candidate ions were excluded for 15 sec after their first occurrence, and data were recorded using Analyst®-TF  software.http://www.matrixscience.com/) of all entries’ protein sequences. Deamidation of asparagine and glutamine and oxidation of methionine were selected for the search parameters as variable modification, and carbamidomethyl modification of cysteine was used as a fixed modification. The enzyme selected was trypsin, with a maximum missed cleavage of 2. The peptide mass tolerance and the fragment mass tolerance were selected as ± 0.1 Da and ± 0.3 Da, respectively.Data analyses. Searches from the nLC-MS/MS analyses were accomplished using Mascot Daemon software , against an NCBInr 20120123 database  cultured in complete medium  were seeded onto 96-well plates. One day after reaching confluence, the cells were sensitized with pooled serum from either controls or patients (1:5 dilution in cell culture medium) for 16 hr at 37°C. After sensitization, cells were washed twice with Tyrod’s buffer  and then exposed for 30 min to 100 μg/mL of cyanobacterial lysate and a 1:1,000 dilution of high-affinity IgE receptor (FcεR1) in Tyrod’s buffer. Aliquots of the supernatant (40 μL) were transferred onto 96-well plates and incubated with 40 μL pNAG substrate  for 1 hr at 37°C. Total release was determined by lysing the cells with 1% Triton X-100. The reaction was stopped by adding 150 μL of 0.2 M glycine, pH 10.7. The absorbance was measured with a microplate reader (Thermo Scientific) at 405 nm, and the percentage of total β-hexosaminidase was calculated as follows: Percent degranulation = OD supernatant ÷ (OD supernatant + OD Triton X-100) × 100.Measurement of microcystin. Total microcystin was measured using a QuantiPlate™ Kit for Microcystins . All incubations were performed using a shaker incubator  at 25°C. Briefly, strain MC(+) lysates were first added to allow binding to anti-microcystin antibodies on the wells. After incubation, microcystin-enzyme conjugate was added, reincubated, and washed and then the substrate added. The enzymatic reaction was stopped, incubated for 10 min and air bubbles eliminated, and then read at 450 nm (650 nm for turbidity blank control) using a Spectra-max M2 spectrophotometer . All samples were analyzed in triplicate except the kit negative control and the microcystin-LR standard in Advantage A10 Milli-Q water  in six replicates.Cytotoxicity assay. RBL SX-38 were seeded at 104 cells/well in a 96-well plate. At 90% confluence, the cells were either left untreated or treated for 48 hr with varying concentrations of M. aeruginosa toxic [MC(+)] and nontoxic [MC(–)] strain lysates. At the end of the treatment, a CytoScan-WST-1 cell toxicity kit  was used to measure the cytotoxic effect of the lysates according to the manufacturer’s protocol. Percent cytotoxicity was calculated as follows: Percent cytotoxicity = [100 × ] ÷ (cell control).Statistical analysis. All data are reported as the sample mean ± SD. Comparisons between the means of sample groups and controls were performed using an unpaired Student’s t-test. A paired Student’s t-test was used to compare IgE reactivity of individual patient serum to MC(–) and MC(+) lysates. Differences were considered statistically significant if the Student t-test provided p-value < 0.05.Immunoreactivity of M. aeruginosa extracts with patient serum. M. aeruginosa by SPT (M. aeruginosa strains and the sera previously collected from M. aeruginosa SPT-positive patients. ELISA results (p < 0.001) with the M. aeruginosa MC(–) lysate, it exhibited no reactivity with the MC(+) lysate. Patient 2 serum had no reactivity with either lysate. Although both strains had a similar immune-reactive profile, the MC(–) lysate was significantly (p < 0.01) more immunoreactive than the MC(+) lysate, indicating that the nontoxic strain was more allergenic than the toxic strain .a by SPT . An IgE- results  demonstrM. aeruginosa peptides present in lysates of both strains. Because the immunoblot was performed under denaturing conditions, the results also suggest that the MC(+) lysate may contain an endogenous inhibitor that prevents effective IgE binding to the relevant peptide(s) in its native form (An IgE-specific immunoblot was performed to further characterize the immune-reactive peptides eliciting these responses. Three bands (> 50 kDa) were identified in all patients’ sera  for bothive form .functional characterization of M. aeruginosa extracts. To further demonstrate the specificity of the M. aeruginosa peptides identified in the serum of sensitized patients, we performed an ELISA inhibition assay using plates coated with MC(+) and MC(–) lysates. Using the MC(–) lysate as the serum inhibitor, specific IgE binding was reduced in a dose-dependent manner (50 (concentration required for 50% inhibition) values were 0.6 and 3.0 μg/mL for MC(+) and MC(–) lysates, respectively. These data confirm that the same allergen is present in both lysates and suggest that the MC(+) lysate may contain an endogenous inhibitor that prevents IgE from effectively binding to the specific allergen  that expresses a high-affinity human IgE receptor (FcεR1) (M. aeruginosa lysates have on the cells. As shown in M. aeruginosa–specific IgE responses identified by SPT and specific IgE ELISA.We then assessed whether the MC(–) lysate could release mediators using an  (FcεR1) . Prior tIdentification of M. aeruginosa immunogenic peptides. To identify the allergen(s) in the cyanobacteria that is binding to IgE, we performed 2D gel electrophoresis, followed by immunoblotting, using MC(–) lysate and pooled patient sera. In the immunoblot, five spots that bind to specific IgE in patient sera were identified (M. aeruginosa.entified . Mass sp6] held together by intermediate and weak hydrogen bonds (6] rapidly disintegrate into trimers [(αβ)3] and monomers (αβ)  of the alpha and beta subunits are 18 and 20 kDa, respectively, while that of LCM is 120 kDa. LCM is responsible for anchoring the phycobilisome complex to the thylakoid membrane, which is the structural unit of the grana (stacks of thylakoid membranes) containing the chlorophyll in cyanobacteria . In its en bonds . Upon diers (αβ) , which eIgE reactivity of purified and endogenous phycocyanin. Because phycocyanin has previously been reported to cause anaphylaxis  lysate was used, we asked whether immunoprecipitated phycocyanin could elicit IgE-specific reactivity. Indeed, endogenous phycocyanin did possess IgE reactivity on the ELISA and immunoblot , indicatEffect of microcystin on the allergenicity of M. aeruginosa. The most apparent difference between both M. aeruginosa strains is their capability of producing or not producing microcystin toxin. Only the toxic strain contains the gene that codes for microcystin , was responsible for inducing an anaphylactic reaction in a teenage patient who ingested spirulina-containing food supplement tablets. The IgE-binding allergen was found to be in its native form , similar to what we have observed , was nonallergenic, whereas the species with undetectable amounts of microcystin, Aphanizomenon flos-aquae, was the most sensitizing  and virtually identical isoelectric points  PDFClick here for additional data file.


Two quartz samples from a lower cultural horizon give a weighted mean age of 0.24 ± 0.05 Ma . The site is thus younger than 340 ka at 95% confidence, which is at variance with the previous paleomagnetic results. On the other hand, our result suggests an age of older than 140 ka for the site’s lower cultural deposits, which is consistent with recent post-infrared infrared stimulated luminescence (pIR-IRSL) dating at 160–220 ka.The Xujiayao-Houjiayao site in Nihewan Basin is among the most important Paleolithic sites in China for having provided a rich collection of hominin and mammalian fossils and lithic artifacts. Based on biostratigraphical correlation and exploratory results from a variety of dating methods, the site has been widely accepted as early Upper Pleistocene in time. However, more recent paleomagnetic analyses assigned a much older age of ∼500 ka (thousand years). This paper reports the application of  The sit fossils . Almost 230Th/234U and 231Pa/235U dating of six fossil teeth. As the two U-series methods gave inconsistent age results, the samples were judged to have experienced open-system behavior, and the 230Th/234U ages (∼100 ka) were suggested to represent the site’s minimum age [230Th/234U and 231Pa/235U ages from another rhinoceros tooth, Chen et al. [14C dating was applied to sediment samples rich in organic materials, yielding minimum ages of >40 ka [14C dating of fossil teeth (>54 ka) [Based on faunal composition Jia and Wei  first brimum age . Later, n et al.  assignedf >40 ka , 9. This4U ages ∼0 ka were>54 ka) . In addi(>54 ka)  performe4 ka . In(>54 ka) .However, further paleomagnetic studies by an international team of Sino-Norwegian scientists challenged the above consensus. Su et al.  and LøvlScholars from the prehistoric community hold different viewpoints on the possibility of a much older X-H site. While Huang et al.  consider26Al/10Be burial dating to the X-H site.To address the decade-long controversy described above, and to date the cultural deposits directly, a cross-check by an independent and well-established radio-isotopic dating method is needed. Here we report the application of 2 and to a depth of ∼12 m were removed. The hominin and mammalian fossils and lithic artifacts were recovered from a yellowish-green clay layer at depths of 8–12 m below the ground surface [The X-H site  is situated on the west bank of the Liyi River, a tributary of the Sanggan River . The Liy2 in area and ∼13 m in depth. The depositional sequence was divided into 15 layers  and only a few of the fossil bones [For better illustrating the stratigraphic position of the two samples for dating, the three cross-sections given as Figs. 2, 3 and 4 in Jia and Wei  are presil bones  of the sThe sample HJY-ST was recovered during excavations carried out by one of the co-authors, F. Xie of Hebei Provincial Institute of Cultural Relics. We collected the quartzose sample, HJY-QZ, together with F. Xie and his colleagues. According to the current rules for protecting the archaeological sites, approval from the Provincial Institute of Cultural Relics is necessary, but no specific permission is required. The field work did not involve any endangered or protected species.26Al (t1/2 = 717 ± 17 ka) and 10Be (t1/2 = 1.39 ± 0.01 Ma) in the mineral quartz. These radionuclides are formed by secondary cosmic rays that penetrate into the ground surface and react with nuclei within mineral grains. The production rates of the two nuclides depend on several factors including altitude and geomagnetic latitude, but the 26Al/10Be ratio of about 6.75 remains basically constant [26Al and 10Be. If the quartz minerals are then rapidly and deeply buried in a river terrace or a cave, then the production of cosmogenic nuclides drastically slows down or even ceases, and each radionuclide decays according to its specific half-life. Because 26Al decays more rapidly than does 10Be, the 26Al/10Be ratio decreases exponentially over time with an apparent half-life of 1.48 Ma. This basic scheme offers a means of dating burial events of quartz minerals [The burial dating method is based on the buildup and decay of cosmogenic constant . As quarminerals , 33.26Al/10Be burial dating depends mainly on the precision of 26Al and 10Be measurements. With accelerator mass spectrometry (AMS) normally 10Be and 26Al can be measured at precisions of ∼1–3% and ∼4–7% , respectively, leading to an uncertainty of ∼5–8% in 26Al/10Be and an absolute uncertainty of ∼70–120 ka in age results. The recent addition of a gas-filled magnet at PRIME Lab (Purdue University) has substantially improved the precision of 26Al measurements, making their uncertainties comparable to those for 10Be, and frequently leading to uncertainties in the 26Al/10Be ratio of <5%. With this level of precision, 26Al/10Be burial dating should be able to resolve the highly controversial issue concerning the chronology of the X-H site. If the site is really as old as suggested by the paleomagnetic studies, burial dating may provide supporting evidence for its validity. Otherwise, if the site is closer to the biostratigraphical age estimate, burial dating may give ages statistically indistinguishable from zero.The effective range of 3 overnight with heating and agitation. Magnetic and gravimetric separations were performed to separate quartz from all other contaminating minerals. The quartz grains were further treated by repeated leaching overnight in 1% HF-1% HNO3 in an ultrasonic tank to ensure complete removal of meteoric 10Be.For HJY-QZ several kilograms of quartzose deposits were collected and rinsed with water to remove silt and clay. The remaining material was taken back to the laboratory for further treatment. HCl was added to dissolve the carbonate and phosphate components. For HJY-ST the gravel was crushed to submillimeter grains. For either sample type, grains of 0.2–0.9 mm were sieved out and then leached several times in 2% HF-2% HNO3, and spiked with ∼0.2 mg 9Be carrier. Aliquots were taken from the dissolved samples for Al concentration measurement by ICP-OES. After HF volatilization, Al and Be were separated and purified on ion-exchange columns in 0.4 M oxalic acid, precipitated as hydroxides, and transformed to oxides in a furnace at 1,100°C. BeO was mixed with niobium and Al2O3 with silver powder for 10Be/9Be and 26Al/27Al measurements by accelerator mass spectrometry (AMS) at PRIME Lab, Purdue University.Purified quartz samples were dissolved in 5:1 HF/HNO26Al and 10Be concentrations and calculated burial ages are presented in The 26Al/10Be burial dating depends closely on a set of assumptions. The most important one is that samples be deposited with a 26Al/10Be ratio consistent with “simple steady-state erosion”. To meet this assumption, the quartz minerals must have experienced only one exposure-burial cycle in the past ∼10 Ma, otherwise the burial ages should be considered maxima. We cannot strictly exclude the possibility that the two X-H samples have experienced complicated exposure-burial histories, but the agreement between the two ages within measurement error and their relatively young ages render it unlikely.As in the case of other radioisotopic dating methods, the reliability of The second assumption necessary for accurate burial dating is that the samples should be buried deeply enough to ignore post-burial production of cosmogenic nuclides by muons. Since the two samples of this study were taken from the same Layer 14 at a depth of 12.3–12.5 m, and their ages are quite young, the postburial production of cosmogenic nuclides can be securely neglected.Because of the possibility for the samples to have experienced complicated exposure-burial histories, and also because the samples were collected from the lower cultural unit, their burial ages should be regarded as a maxima for the X-H site. Thus at 95% confidence, this site should be younger than 340 ka. On the other hand, the results presented here may indicate an age older than 140 ka for the site’s lower cultural deposits. However, due to uncertainties in the samples’ pre-burial histories, the younger age constraint is less stringent.The weighted mean age of the two samples, 0.24 ± 0.05 Ma, is consistent with the recent post-IR IRSL dating of the site, which yielded a range of 161 ± 13 and 224 ± 22 ka for Layers 8–11 . ConsideOur radiometric age for the X-H site is significantly younger than the age of ∼500 ka suggested by paleomagnetic studies –18. ThouH. sapiens, is currently one of the hotly debated issues in paleoanthropology .–41.H. sas Tongzi , Maba [4s Tongzi , Miaohous Tongzi , Chaoxias Tongzi , and Locs Tongzi , 47, morg horses , and the26Al/10Be burial dating has played an increasingly important role in studying long-term landscape evolution and early human evolution. This method is particularly promising in dating early hominin sites in China, where the lack of volcanic tuffs precludes the application of 40Ar/39Ar dating. By assigning a maximum age of 0.24 ± 0.05 Ma to a lower cultural horizon of the X-H site and thus controverting firmly the paleomagnetic dating, the results reported in this paper exemplify its strength. Along with the establishment of isochron technique to circumvent some of the geological complexities [26Al marks an important methodological milestone. Burial dating with 26Al and 10Be is now a well-established and reasonably precise radio-isotopic dating method. Its further application will help to establish a reliable temporal framework for the mode of human evolution in East Asia.Since its first application for deriving river incision rates , 26Al/10lexities , 51, theS1 Fig(JPG)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.S3 FigU8. Modern fluvial deposits or brown sandy topsoil (ceramic fragments bearing), 0.3–0.5 m thick, with a gradual transition to the underlying layer (not given in the figures.). U7. Sandy or yellowish brown loess, with occasional sand sub-layers, with horizontal laminae, 3–5 m thick , without clear horizontal but with vertical laminae, with a few small gravels and gravelly sandy lens, bearing Coelodonta antiquitatis tooth fragments, myospalax fontanieri fossils and Ostrich eggshells, up to 5 m thick , 0.3 m thick (Layer 5 in U2. Yellowish green (grayish brown in the north) sandy clay, with a few gravels, with sandy nodules at the top, ∼6 m thick. All the stone artifacts and most of the fossils were found in this layer Click here for additional data file.S1 Table(DOCX)Click here for additional data file.


The contribution of the Greenland ice sheet to sea-level rise has accelerated in recent decades. Subglacial lake drainage events can induce an ice sheet dynamic response—a process that has been observed in Antarctica, but not yet in Greenland, where the presence of subglacial lakes has only recently been discovered. Here we investigate the water flow paths from a subglacial lake, which drained beneath the Greenland ice sheet in 2011. Our observations suggest that the lake was fed by surface meltwater flowing down a nearby moulin, and that the draining water reached the ice margin via a subglacial tunnel. Interferometric synthetic aperture radar-derived measurements of ice surface motion acquired in 1995 suggest that a similar event may have occurred 16 years earlier, and we propose that, as the climate warms, increasing volumes of surface meltwater routed to the bed will cause such events to become more common in the future. How the Greenland ice sheet responds to the drainage of water below the ice remains uncertain. Here, the authors combine digital elevation models and Landsat optical imagery to map subglacial drainage pathways and investigate the ice sheet surface response to a periodically draining subglacial lake. The Greenland ice sheet (GrIS) has lost mass to the global oceans at an increasing rate in recent decades138Meltwater is generated at the surface of the GrIS during summer and flows under gravity in supraglacial streams, collecting in local topographic lows and forming supraglacial lakes. The locations of these topographic lows reflect the underlying subglacial topographyRemote sensing observations have shown increased ice melt extent and intensity in recent years11121311151862122232425In this study, we infer for the first time the subglacial drainage pathway from a subglacial lake in Greenland, and the dynamics of this subglacial channel. It is clear that the mechanism of dynamic response of the ice sheet to meltwater input is sensitive to the nature, and evolution, of the subglacial hydrological system.2. No evidence of the feature is detected in the Ice Cloud and Land Elevation Satellite (ICESat) data acquired in 2005 or in the ice elevation data from the 2007 Greenland Ice Mapping ProjectWe used surface extraction with triangulated irregular network-based search-space minimization (SETSM)7 m3.To investigate this unusual feature, we mapped supraglacial streams and moulins in the vicinity of the collapse basin using the SETSM DEMs . The col2930The flat and horizontal floor of the collapse basin shown in the SETSM data acquired in October 2011  suggests2, an average depth of 3±1 m, and held a maximum volume of 2.0±0.9 × 106 m of water. This equates to roughly 1/24 of the water volume required to form the collapse basin and suggests that the volume of water evacuated from the bed could not be supplied solely by the nearest supraglacial lake, even over many annual cycles of supraglacial lake filling and draining  and Landsat 8 (2014) downloaded from the US Geological Survey (USGS) Earth Explorer website. For each image, the panchromatic band 8 was used to increase the image resolution from 30 to 15 m. Some of the Landsat 7 images used suffer from missing data due to the well-known scan line corrector failure.We processed two pairs of synthetic aperture radar (SAR) images acquired during August 1995 to derive ice sheet motion6To estimate where subglacial water would be routed at the base of the ice sheet, we assumed that water would flow in the direction of the steepest hydropotential gradient, and that the effective pressure is zero, that is, that the water pressure at the bed is equal to the weight of overlying iceHow to cite this article: Palmer, S. et al. Subglacial lake drainage detected beneath the Greenland ice sheet. Nat. Commun. 6:8408 doi: 10.1038/ncomms9408 (2015).