The effective treatment of bipolar disorder (BD) represents a significant unmet medical need. Although lithium remains a mainstay of treatment for BD, limited knowledge regarding how it modulates affective behavior has proven an obstacle to discovering more effective mood stabilizers with fewer adverse side effects. One potential mechanism of action of lithium is through inhibition of the serine/threonine protein kinase GSK3β, however, relevant substrates whose change in phosphorylation may mediate downstream changes in neuroplasticity remain poorly understood. Here, we used human induced pluripotent stem cell (hiPSC)-derived neuronal cells and stable isotope labeling by amino acids in cell culture (SILAC) along with quantitative mass spectrometry to identify global changes in the phosphoproteome upon inhibition of GSK3α/β with the highly selective, ATP-competitive inhibitor CHIR-99021. Comparison of phosphorylation changes to those induced by therapeutically relevant doses of lithium treatment led to the identification of collapsin response mediator protein 2 (CRMP2) as being highly sensitive to both treatments as well as an extended panel of structurally distinct GSK3α/β inhibitors. On this basis, a high-content image-based assay in hiPSC-derived neurons was developed to screen diverse compounds, including FDA-approved drugs, for their ability to mimic lithium’s suppression of CRMP2 phosphorylation without directly inhibiting GSK3β kinase activity. Systemic administration of a subset of these CRMP2-phosphorylation suppressors were found to mimic lithium’s attenuation of amphetamine-induced hyperlocomotion in mice. Taken together, these studies not only provide insights into the neural substrates regulated by lithium, but also provide novel human neuronal assays for supporting the development of mechanism-based therapeutics for BD and related neuropsychiatric disorders. Despite serious, dose-limiting, adverse effects and lack of efficacy in roughly 60% of patients, the mood stabilizer lithium remains at the forefront of bipolar disorder (BD) treatment due to the absence of better alternatives3. Multiple targets of lithium have been discovered, but the capacity of this agent to limit the activity of glycogen synthase kinase-3ß (GSK3β) at therapeutically relevant doses has attracted significant attention8. This interest is supported by the multiple connections that have been established between dysfunction of GSK3 and the pathophysiology of different neuropsychiatric disorders. Recent human genetic studies, in combination with accumulating evidence from molecular, cellular, pharmacological, and behavioral studies, have consistently recognized the importance of a balance in the activity of GSK3 for mental health10. In light of these observations, pharmacological control of GSK3 signaling networks is being actively pursued as a potential therapeutic target to treat these severe illnesses.Treatment of mental illness represents one of the greatest unmet medical needs of our time14. Which GSK3 substrate(s) may be most relevant to the mood and cognitive enhancing properties of GSK3 inhibition by lithium remains unclear and none have been unambiguously associated with the mood and cognitive enhancing properties of GSK3β inhibition. In a recent study, our group revealed an unsuspected link between lithium-dependent GSK3β inhibition and the critical cytoskeleton regulator collapsin response mediator protein 2  using patient-derived cellular models of BD15. The activity of CRMP2, which is predominantly expressed in the nervous system and exists as either homo- or heterotetramers, can be greatly influenced by multiple post-translational modifications, including phosphorylation, glycosylation, oxidation, and proteolysis18. Phosphorylation of CRMP2 inactivates it and prompts it to become unbound from the cytoskeletal elements it regulates. As a phosphorylation target of GSK3β, CRMP2 is potentially relevant given its roles in neural development, neuritogenesis, and synaptic function26. Furthermore, defects in CRMP2 expression and phosphorylation have been reported in relation with many brain disorders, including Alzheimer’s disease27, multiple sclerosis29, Rett syndrome31, in addition to BD and schizophrenia33. Hence, it is possible that the regulation of CRMP2 activity by lithium represents a key component of this drug’s efficacy in BD treatment.GSK3 contributes to the regulation of many proteins implicated in a wide range of cellular functions36) or genetically more complex . In addition to human disease modeling, an exciting opportunity presented by hiPSC technology is the capacity to probe the cellular mechanism of action of drugs used to treat neuropsychiatric disorders and to develop high-throughput phenotypic assays for novel therapeutic discovery43. For these reasons, we and others have developed neural models through the derivation of homogenous, self-renewing, cultures of neural progenitor cells (NPCs) from hiPSCs46. These hiPSC-derived NPCs can be expanded to provide the sufficient number of cells needed for large-scale high-throughput screening (HTS), an approach successfully implemented for the discovery of WNT signaling pathway modulators47. Here, we report the successful use of hiPSC-derived neural cells not only to dissect the effects of lithium on neuronal targets, but also to use that knowledge to develop high-throughput cell-based assays that could, in an unbiased fashion, identify compounds from a library (including FDA-approved drugs) that mimic lithium’s behavioral effects in the amphetamine-induced hyperlocomotion (AIH) test.Human induced pluripotent stem cell (hiPSC) technology is being used increasingly to create genetically accurate cellular models of diseases, including neuropsychiatric disorders, whether monogenic , 30% Ham’s F-12 , supplemented with B-27 (Invitrogen), 20 ng ml−1 each epidermal growth factor , and basic fibroblast growth factor  as previously described in ref. 47. Unless mentioned otherwise, the NPC line 8330-834 was used throughout this study. To obtain postmitotic neurons, NPC differentiation was initiated by removal of growth mitogens  from the expansion media and cultures were maintained with medium replacement twice per week. To dissociate and re-plate neuronal cultures differentiated en masse, cultures in T75 flasks were washed once with phosphate-buffered saline (PBS), treated with 3 ml of Accutase (Sigma) for 10 min at 37 °C, and then gently triturated several times. Finally, after dilution with fresh media, cells in suspension were passed through a 40 μm cell strainer to remove clumps, counted and spun down. To prepare lysates for western blotting, 1 × 106 cells were seeded per well in 6-well plates. For high-content p-CRMP2 imaging assays, 40,000 cells were seeded per well in 96-well plates (Corning 3904). Re-plated differentiated neuronal cultures were allowed to recover for 1 week before treatment with compounds.hiPSC-NPCs derived from a healthy control subject was generated as reported in ref. 15. For the purpose of this manuscript, mass spectrometry data from this previous publication was reprocessed using an updated human reference proteome UniProt database (release 2014_10) subtracting a set of 150 common laboratory contaminant proteins with Spectrum Mill software (Agilent Technologies). Similar MS/MS spectra acquired on the same precursor m/z within ±60 s were merged. MS/MS spectra were excluded from the analysis if they were not within the precursor MH+ range of 600–4000 Da, or if they failed the quality filter by not having a sequence tag length >0. Spectra were allowed ±20 p.p.m. mass tolerance, 30% minimum matched peak intensity, and ‘trypsin allow P’ enzyme specificity with up to four missed cleavages. The fixed modifications were carbamidomethylation at cysteine and SILAC: Arg (6–10 Da), Lys (4–8 Da) mix. Allowed variable modifications were phosphorylation of serine, threonine, and tyrosine residues, oxidized methionine, pyroglutamic acid on N-termini glutamines, and acetylation of protein N-termini with a precursor MH+ shift range of −18 to 258 Da. Individual spectra were automatically assigned a confidence score using Spectrum Mill autovalidation module. A target–decoy FDR of 1.2% was used. A Spectrum Mill Protein-Var Mod Site Comparison was generated to identify only phosphopeptides. For data analysis, phosphopeptides not observed in both SILAC experiments and decoy peptides were removed. In addition, any phosphopeptides that did not have at least 1 fully localized phosphorylation site were removed, and any phosphopeptides not quantified in both replicates for at least 1 condition of CHIR-99021 treatment were excluded. Finally, ratios were median normalized and transformed into log2 space. Protein–protein interaction network modeling of differentially regulated phosphoproteins was performed using VisANT 5.0, a freely available (http://visant.bu.edu) network modeling software program48.NPCs treated with 0, 1, or 10 μM CHIR-99021 were differentially labeled with ‘light’, ‘medium’, or ‘heavy’ SILAC media. In total, two replicates were performed. For the first replicate the 0 μm CHIR-99021 treatment was labeled with heavy media, the 1 μM treatment with light media, and the 10 μM treatment with medium media, while in the second replicate the 0 μM treatment was labeled with light media, the 1 μM treatment with medium media, and the 10 μM treatment with heavy media. Complete description of SILAC labeling and subsequent quantitative analysis of protein phosphorylation is available in the supplemental information of our previous publicationT514 , and total CRMP2  were purchased from Cell Signaling Technologies .Protein lysates for western blot analyses were prepared from NPCs or 4-week differentiated human neuronal cultures in 6-well plates treated with GSK3 inhibitors at the indicated concentrations for 6 or 24 h. Western blotting was performed using 4–12% Bis-Tris pre-cast mini gels (Invitrogen) for electrophoresis, PVDF membrane for protein transfer, and overnight incubation at 4 °C for primary antibodies. The antibodies recognizing p-CRMP247. The following primary antibodies were used to assess neuronal differentiation: TuJ1 , MAP2 , SMI312 , and GFAP . To evaluate CRMP2 expression and phosphorylation levels with ICC, the same p-CRMP2T514 and CRMP2 antibodies from Cell Signaling Technologies as for western blot analysis were used. For the high-throughput high-content screening assay, neuronal cultures were co-immunostained for p-CRMP2T514, the dendritic marker MAP2, and the nuclear protein HDAC2 .Fluorescence ICC was performed according to procedures previously describedT514, MAP2 , and HDAC2 (nuclear marker). Immunostained neurons were either scanned on a laser-scanning cytometer , with a 488 nm laser setting of 100% power and 550-volt detection setting, or imaged with an IN Cell Analyzer 6000 automated confocal microscope  for high-content analysis. Data are expressed as mean ± SEM of at least three replicates. Unpaired t-tests were used to determine treatment significance for all compounds tested. Stars of significance indicate a significant effect for a treatment dose compared with control .With the exception of lithium, which was added manually, compounds were applied to 4-week differentiated human neuronal cultures prepared in 96-well plates using a CyBio Well vario automated liquid handling system equipped with a 96-pin head . More precisely, 100 nl of each compound solubilized in DMSO and arrayed in compound plates was transferred to 96-well assay plates with 100 μl culture media per well. After a period of 24 h, unless specified otherwise, cells were fixed for 30 min at room temperature with 4% paraformaldehyde (PFA) in PBS, washed with PBS to remove PFA, and then immunostained for p-CRMP2T514 intensity and MAP2 intensity measured from identified neurites. From population of MAP2-positive neurons, measured p-CRMP2T514 and MAP2 intensities, reported as averages for each well, were used to calculate percentage of p-CRMP2T514 to MAP2. Finally, the number of MAP2-positive neurons was used to evaluate toxicity of compounds.High-content images were acquired using an IN Cell Analyzer 6000 automated confocal microscope  with a ×20 objective. A total of 4–6 fields of images from each well of 96-well plates were collected. Image analysis was performed with the IN Cell Investigator Software v1.6.2. Precisely, after identification of all nuclear objects, a collar ring was drawn around each nucleus and filters were then applied to identify MAP2-positive neurons in the mixture of cells according to the following analysis criteria: (1) size of nuclear objects: too small (<50 μm)—cell debris or too large (>160 μm)—cell clumps, (2) intensity of nuclear staining: too dim (<2197 pixels)—falsely identified objects, (3) intensity of MAP2 staining in the collar ring: low MAP2—not neurons, and (4) coefficient variation (CV) of MAP2 staining in the collar ring: CV > 1—neurons. Neurites were traced out from all nuclei on the basis of the MAP2 signal, and both p-CRMP249. The following compounds were administered to mice either intraperitoneally (IP), subcutaneously (SC), or by gavage  in a volume of 0.01 ml/g body weight as following: methamphetamine 3.5 mg/kg, IP in saline (Sigma-Aldrich); LiCl 85 mg/kg, IP in saline (Sigma-Aldrich); ropinirole 10 mg/kg, IP in dH2O (Sigma-Aldrich); oxiracetam 100 mg/kg, IP in dH2O ; Huperzine A 1 mg/kg, PO in dH2O with 40% volume 0.1 M HCl (Sigma-Aldrich); sulpiride 10 mg/kg, IP in saline with three drops of Tween-80 ; ganaxolone 10 mg/kg, IP in 20% 2-hydroxypropyl-beta-cyclodextrin (Tocris Bioscience); forskolin 1 mg/kg, SC suspended in 10% cremaphor (Tocris Bioscience); NNC-711 5 mg/kg, IP in saline and sonicated (Tocris Bioscience). A cohort of 32 C57BL/6J mice and another one of 32 DBA/2J mice were used to test huperzine A, oxiracetam, and ropinirole. A third cohort of 32 C57BL/6J mice was used to test forskolin, ganaloxone, NNC-711, and sulpiride. Each cohort was tested once per week. For statistical analyses, the effect of compounds was initially analyzed by two-way ANOVA’s between-subjects factors lithium (vehicle or lithium) and compound (vehicle or compound). In addition, between-group analysis of separate cohorts of six mice per group was used in each separate experiment to test the effect of sulpiride pretreatment on AIH also in comparison with vehicle, lithium, and lithium plus sulpiride. For group allocation, there was no blinding and randomization.AIH assays were performed as described in Gould et al.8. Importantly, although comparative analysis of the two key regulatory GSK3 phosphorylation sites (using GSK3β numbering), tyrosine 216 (Tyr216) and serine 9 (Ser9)52  that is dominant to effects of Tyr216 phosphorylation54. Therefore, in addition to lithium directly inhibiting GSK3 kinase activity through competitive inhibition of Mg2+ at the ATP-binding site, but not to ATP itself or substrates55, its targeting of molecular events contribute to an indirect inhibition of kinase activity through augmentation of GSK3 Ser9 phosphorylation in an AKT-dependent manner that leaves Tyr216 largely unaffected56. In contrast, CHIR-99021 limits phosphorylation of residue Tyr216 via directly altering the intrinsic kinase activity of GSK3 by blocking the kinase’s ATP-binding site, although an effect of increasing dephosphorylation through an indirect phosphatase-mediated mechanism that lithium does not invoke cannot be ruled out. In either case, the result is a comparable diminution in the levels of GSK3 substrate phosphorylation.To address which phosphorylation sites in the human neural proteome are under the control of GSK3, we first set up to identify candidate targets that are directly changed by pharmacological inhibition of GSK3 in hiPSC-derived NPCs using quantitative SILAC-based mass spectrometry (MS). To inhibit GSK3 with high selectivity and efficacy for this MS-based quantitative proteomics screen, we chose to use the cell-permeable, ATP-competitive, aminopyrimidine derivative CHIR-99021, which is recognized as one of the most selective and potent inhibitors of GSK3 identified to date15 by re-analysis of the peptide level data through an updated computational pipeline and the latest version of the UniProt human proteome database, including removal of a set of 150 common laboratory contaminant proteins, a complete list of all high confidence, phosphopeptides identified by SILAC-MS upon treatment of hiPSC-derived NPCs with two concentrations of CHIR-99021 (1 or 10 μM) or vehicle control (DMSO) was generated  upon CHIR-99021 (10 μM) treatment were used to confirm the sequence of the enriched phospho-motif  upon CHIR-99021 (10 μM) treatment mapped to 398 unique proteins with 37 proteins (grouping GSK3α/β into one protein) represented by at least 3 or more regulated phosphopeptides as shown in Fig. 59, which is also known to be a GSK3 substrate.Having validated the global change in the phosphoproteome maps to the known GSK3 phospho-motif, we next performed a global, systems-level analysis of the change in the neural phosphoproteome upon CHIR-99021 treatment. The total of 575 peptides were identified as differentially regulated (p < 0.05) upon CHIR-99021 (10 μM) treatment and constructed an interaction network to visualize their known protein–protein interactions using VisANT (5.0)48. The resulting network graphs shown in Fig. Having identified which individual proteins were most significantly altered by CHIR-99021 treatment, as well as verified that multiple of these were known substrates of GSK3, we next sought to create an integrative, systems-level understanding of the signaling networks affected by this treatment. For this computational analysis we considered a total of 90 unique proteins with at least 2 or more phosphopeptides among the set of 575 differentially regulated phosphopeptides  at serine 522 (Ser522), a necessary priming step, and subsequent phosphorylation by GSK3 at Ser518, Thr514 and Thr50962, resulting in inactivation of CRMP2’s tubulin-binding properties that regulates microtubule assembly . Robust reduction was detected from 6-h treatment samples at the lowest dose tested for both CHIR-99021 (0.1 μM) and lithium (1 mM) with a complete reduction at higher concentrations, whereas the total amount of CRMP2 protein did not change . Together, these results validated the original MS-based proteomic findings using an orthogonal, antibody-based detection method and indicate that p-CRMP2T514 is highly sensitive to GSK3 inhibitor treatment in hiPSC-derived NPCs. Moreover, the observation of the effect of lithium at therapeutically relevant doses suggests CRMP2 phosphorylation may be relevant to the mood stabilizing properties of GSK3 inhibitors as well as lithium.To confirm and extend these MS-based proteomic findings with CHIR-99021, and to determine if they could be also observed with the much less potent, but clinically relevant mood stabilizer and GSK3 inhibitor lithium, NPCs were treated with different doses of CHIR-99021 or lithium for 6 or 24 h and western blotting performed on total cell lysates with a phosphospecific antibody that recognizes CRMP2 when phosphorylated at Thr514  and the band was nearly not visible at 20 mM treatment.We further examined the effect of CHIR-99021 and lithium treatments on p-CRMP2ing Fig. . Four-weT514 in each lysate was evaluated by western blotting .To extend the observations with CHIR-99021 and lithium, we next examined several existing GSK3 inhibitors that have distinct chemical structures and cover a range of GSK3 inhibitory activities . Unexpectedly, treatment with roscovitine had no evident effect on p-CRMP2T514 or p-CRMP2S522 levels , and a similar concentration range (1–10 μM) was demonstrated to reduce p-CRMP2S522 levels in rat NPCs65, since our experiments lacked a positive control for intracellular CDK5 inhibition other than p-CRMP2S522 we cannot rule out insufficient CDK5 target engagement as an additional explanation for the lack of observed effect.Many but not all GSK3 substrates require priming phosphorylation of three amino acids adjacent to the GSK3’s phosphorylation sites. In the case of CRMP2, CDK5 is known to phosphorylate CRMP2 at Ser522, priming it for subsequent phosphorylation by GSK3β that to preferentially phosphorylates Ser/ThrXXX(pSer/Thr) motifs, where X is any residueT514 levels in human neurons, we also used our recently discovered novel series of pyrazolo-tetrahydroquinolinone GSK3 inhibitors /BRD3937 conveys selective and potent inhibition of GSK367. Having established cellular assays in hiPSC-derived NPCs that can report on GSK3 inhibition47, and given that this compound series had not previously been tested in non-transformed human cell lines, we first tested the activities of the racemic compound JB1121(rac) and its two separated enantiomers for their ability to stimulate canonical WNT signaling pathways and promote NPC proliferation. Notably, JB1121(ent-2) increased WNT signaling reporter activity at 12.5 and 25 μM  failed to show activity. In both assays, JB1121(ent-2) produced higher activity than the racemic mixture at each concentration. As a more target-focused, neural assay we next tested whether these novel GSK3 inhibitors could modulate p-CRMP2T514 levels. Three concentrations  of JB1121(rac) and separated enantiomers were used to treat hiPSC-derived NPCs  and JB1121(ent-2) effectively reduced p-CRMP2T514 in human NPCs and neurons in a dose-dependent manner whereas JB1121(ent-1) was inactive even at the highest concentration (10 μM) tested. In summary, cellular activities obtained in reducing p-CRMP2T514 in the hiPSC-derived NPCs or neurons for GSK3 inhibitors across multiple structurally distinct series correlate well with their in vitro GSK3 inhibitory activities.In an effort to expand the panel of GSK3 inhibitors tested for their ability to regulate p-CRMP2T514 levels. Based on an extended set of assay development and refinements, we used human neurons differentiated for 4 weeks with growth factor withdrawal that showed a robust degree of morphological and marker-specific differentiation , we sought to develop a human neuron-based functional assay for GSK3β inhibitors through quantification of p-CRMP2T514 level with the potency of selected GSK3 inhibitors and compound concentrations by western blotting   as well as racemic JB1121(S/R) confirming the capability of the assay to report on highly selective GSK3 inhibitors in addition to CHIR-99021.Given the strong correlation of p-CRMP2514 Fig.  with a rμM) Fig. 66,67. A T514 immunofluorescence detection to a high-content, single-cell level assay by collecting high-resolution images on an automated confocal microscope . For this screen, all compounds were initially tested at 10 μM with a 24-h treatment duration, with CHIR-99021 (10 μM) as a positive control. As shown in Fig. T514 level to <90% of the averaged signal from all wells were a diverse set of GSK3 inhibitors  to create a graphical visualization of the underlying protein-chemical interaction network. The resulting interaction network connected the p-CRMP2T514 modulators identified in our screen within three main submodules: GSK3 signaling, lysine deacetylation, and neurotransmission /BRD3937 shown to reduced p-CRMP2T514 levels in our human neuronal assays72. In the AIH assay, d-amphetamine inhibits dopamine and other monoamine transporters, resulting in increased extracellular striatal dopamine and behavioral hyperactivity in the animals7. Methamphetamine may also modulate GSK3β signaling in the nucleus accumbens73, and our recent evidence suggests that methamphetamine increases CRMP2 phosphorylation in both rodent and human neurons15. To select p-CRMP2 inhibitory compounds for testing in the AIH assay, we focused on compounds without measurable direct GSK3β inhibitor activity or reported KDAC (HDAC) inhibitor activity since, as shown in our previous studies15, we have already demonstrated that selective Class I HDAC inhibition is capable of attenuating locomotion in the AIH test and to have antidepressant-like activity in both the forced swim test and social defeat paradigm75. On the basis of these criteria and compound availability we selected ropinirole, oxiracetam, huperzine A, sulpiride, NNC-711, forskolin, ganaxolone for in vivo behavioral testing after acute, systemic dosing either alone or in combination with lithium.Several behavioral paradigms have been used as clinically-predictive of treatment response even though they cannot truly replicate a complex, polygenic human psychiatric condition. The amphetamine-induced hyperlocomotion (AIH) assay has historically been used to assess the anti-manic-like activity of pharmacological agents, including the series of compounds represented by JB1121, but was normalized by lithium when it decreased p-CRMP2T514 levels15. Normalization of CRMP2 activity was also accompanied by an improvement in dendritic morphometrics and neuronal activity15. With the identification here of multiple GSK3 inhibitors also having this effect, many of which have anti-manic-like and/or antidepressant-like activity in rodent models , as well as a very narrow therapeutic index. A more specific and faster-onset pharmacologic treatment for BD would be desirable. Here, we significantly extend our previous finding that lithium regulates p-CRMP2T514 suppressing compounds using STITCH suggested a common property of modulating lysine deacetylases (KDACs) among many of the active compounds. While selective Class I KDAC inhibitors are capable of attenuating AIH and having antidepressant-like activity in the forced swim test, as well as in social defeat paradigms75, the relevant neural substrates mediating these behavioral effects have not been elucidated. While the acetylation of GSK3β protein (Lys205)76 and three acetylation sites on CRMP2  have been reported, other factors could also mediate the effect of these KDAC inhibitors on p-CRMP2T514. Since it is known that protein phosphatases can form complexes with HDACs78, we speculate that PP2A phosphatase is actively counteracting CRMP2 phosphorylation by GSK3β and this regulation is enhanced upon KDAC inhibition. Consequently, it would be interesting in the future to test whether KDAC inhibitors augment lithium’s effect in vivo in animal behavioral tests in a manner dependent upon PP2A phosphatase activity towards GSK3β and its related interactor AKT that governs the Ser9 inhibitory phosphorylation56.Analysis of the targets of the p-CRMP2A major consideration in the utilization of high-content screening using stem cell derivatives for drug discovery in human disease is developing a biologically-relevant secondary assay toward validating the relevance of candidate compounds. Toward this end, we subjected candidate compounds identified in our ex vivo cellular assays to in vivo behavioral testing. Overall, the in vivo test results did reveal potential candidates with relevant functional outcomes in terms of attenuating AIH, a behavioral test that has been widely used as a surrogate system to probe the pharmacology and neurobiology of dopamine-mediated behavior and lithium-responsiveness. However, the behavioral effects were not completely predictable based solely on the biochemical activity toward CRMP2 in hiPSC-derived neurons Fig. . MultiplOf note, different degrees of effects of lithium on ambulation prior to amphetamine administration were observed in separate experiments even within the same inbred mouse strain background. The observation seems most likely due to variation in penetrance of the lithium response between individual mice despite otherwise genetic homogeneity. For each experiment, the pre-amphetamine baseline recordings served internal controls for post-amphetamine ambulation for testing effects of lithium and other compounds. Therefore, our conclusions are independent to the variance of lithium effects observed for separate experiments.T514 suppressors identified that were profiled for in vivo activity, NNC-711, a reported blocker of GABA uptake via inhibition of SLC6A1 )79, attenuated AIH when tested independently, but showed no additive effect when tested in combination with lithium  receptors81. In addition, consistent with our findings of decreased p-CRMP2T514, huperzine A has been shown to increase levels of inhibitory GSK3αS21/βS9 phosphorylation and cause accumulation of β-catenin in brains of APPswe/PS1dE9 double transgenic mice and in cultured human neuroblastoma cells82. Correlated with these changes, huperzine A treatment increased APP processing leading to decreased levels of pathological forms of Aβ82. Similar results in terms of increased GSK3βS9 phosphorylation have also been reported with in vivo administration of huperzine A and analogs to 3xTg-AD mice containing transgenes expressing three mutations associated with familial Alzheimer’s disease 83. In the AIH test, huperzine A decreased AIH alone and showed additive or synergistic effect with lithium -expressing medium spiny neurons (MSNs) that play a critical role in mediating neurotransmission within the indirect pathway of the basal ganglia circuit, but not dopamine D1 receptor (D1R)-expressing MSNs within the direct pathways85. In these same studies, a double knockout of GSK3β and CRMP2 in D2R-expressing MSNs was shown to prevent the knockout of GSK3β from attenuating AIH. Correlated with these behavioral changes in the CRMP2 knockout was an observed decrease in dendritic complexity and spine density of MSNs in the striatum, similar to our previous report15. Taken together, these observations further support the conclusion that CRMP2 plays a critical role in regulating the neurocircuitry that may be involved in affective behavioral control.While this manuscript was in preparation, replicating earlier studies from the Caron laboratory using conditional Cre-recombinase expressing miceT514 levels and demonstrating efficacy in a mouse behavioral test of dopaminergic signaling—provide new cellular and molecular tools to dissect the role of neuronal GSK3 and CRMP2 signaling and investigate potentially therapeutically relevant mechanisms of lithium action. The extension of these assays to include NPCs and neurons from BD patient hiPSCs will enable ex vivo studies of lithium-sensitive signaling pathways that may help identify novel targets and leads for the development of more specific and safer novel therapeutics for BD and other neuropsychiatric disorders.In summary, the global phosphoproteomic data and novel methodology implemented in our chemogenomic screen along with the compounds discovered here as functional mimetics of lithium—reducing p-CRMP2Zhao_CRMP2 manuscript_Supplemental MaterialZhao_CRMP2 manuscript_Supplemental Table


The insertion of all screws was standardized to 400 N‐cm insertion torque. Peak reverse torque was measured immediately after screw placement (control), or after the completion of the respective loading cycles. The difference between treatment groups was compared using univariate analysis of variance. The analysis revealed a significant difference in peak reverse torque of the screws among the treatment groups (p = .000). The mean reverse torque values equaled 343.5 ± 18.3 N‐cm for non‐cycled controls, 303.3 ± 25.9 and 296.0 ± 42.9 N‐cm after 5000 and 10,000 cycles, respectively. Among all treatment groups, screws placed in the distal bone segment tended to have lesser peak reverse torque reduction than those placed in the proximal segment and the difference was proportional to the number of cycles . Cyclic loading may have a negative effect on the primary stability of screws placed in locking plate constructs used to bridge segmental bone defects and could contribute to the risk of screw loosening.The objective of this study was to evaluate the ex vivo effect of cyclic loading on the stability of screws placed in locking plates used to bridge segmental bone defects. The primary interface stability was assessed using peak reverse torque. Eighteen, 8‐hole stainless‐steel 4.5 mm locking plates and 4.0‐mm self‐tapping locking‐head screws were used to stabilize 40‐mm segmental defects in goat tibiae. Treatment groups included control constructs without cyclic loading ( In order to mimic the clinical scenario in which the bone segments would have little to no influence on stability, a complete segmental mid‐diaphysis tibia defect was created and stabilized with a locking plate and screw construct and then subjected to up to 10,000 cycles of compressive axial loading. The stability of locking‐head screws was evaluated measuring the PRT required to remove the screws. This study hypothesized that there will be an effect of the cyclic loading on the stability of the locking‐head screws placed in locking plate constructs.22.1n = 6), (2) constructs subjected to 5000 loading cycles , and (3) constructs subjected to 10,000 loading cycles . The bones were removed at the level of femorotibial and tarsocrural joint and dissected from surrounding soft tissues, including ligaments and tendons. The proximal and distal end of the bones were potted in polymethyl methacrylate (Jorgensen Laboratories) within polyvinyl chloride (PVC) (Lowe's Home Improvement) molds to achieve an external diameter of 50.8 mm  having been used in a previous orthopedic study. These 18 tibiae were randomly and equally assigned to one of three treatment groups, including (1) control constructs with no cyclic loading  with a 60 mm long solid central portion between the screw holes were used to bridge a 40 mm long, segmental tibial defect. The plates were applied to the cranial, medial surface of the tibiae and fixed with eight 4.0 mm self‐tapping locking‐head screws , four proximal to the segmental defect (position from 1 to 4) and four distal to it (position from 5 to 8)  and #8  to fix the plate against the bone surface. For each screw position, screw holes were drilled using a 3.2 mm drill guide (Veterinary Orthopedic Implants) and an orthopedic power drill (model number: ND‐1001) with a 3.2 mm drill bit (Veterinary Orthopedic Implants). Drilling was continued until the tip of the drill bit reached beyond the far cortex. A depth gage (Veterinary Orthopedic Implants) was used to measure the depth of drilled screw holes to estimate the required length for the screws (range of length: 28–34 mm). The screws were placed manually with a hand‐held screwdriver , but not tightened. In order to mimic a non‐load sharing fracture, a 40 mm full‐thickness osteotomy was created in the center of the tibiae with a diamond crusted bone saw . During an osteotomy, care was taken to leave equal 10 mm distances between the screw holes adjacent to osteotomy and the margins of the osteotomy  in chronological order. All screws were placed within the plates according to Arbeitsgemeinschaft für Osteosynthesefragen/Association for the Study of Internal Fixation guidelines. During screw placement, care was taken that the cutting flute of each self‐tapping screw extended beyond the far cortex to allow for sufficient bone purchase within the far cortex. All screws extended two to three threads beyond the far cortex to ensure full engagement of the screws. After all screws had been placed, the screws were tightened using a digital hand‐held torque measuring device  with the insertion torque limited to 400 N‐cm as recommended for 4.0 mm locking screws by the manufacturer and previous reports.2.3n = 6) did not undergo biomechanical testing and PRT was measured immediately after all screws had been tightened. The results of the control PRT measurement served further as a reference point for the PRT measurements following cyclic loading.The constructs assigned to the control treatment group  or 10,000 cycles  of compressive axial loading.In order to mimic short‐term postoperative loading conditions, constructs assigned to remaining treatment groups were subjected to either 5000  to the tibia during walking.2.4The measurements of PIT and PRT as well as results of biomechanical testing were recorded and organized using Excel spreadsheets . The statistical analysis was conducted using SPSS statistics software  and the power of the study was calculated using PS power and sample size analysis software . Descriptive statistics, including mean values and standard deviations, were calculated for each variable. Data in each variable was assessed for the normality of distribution using the Kolmogorov–Smirnov test and for the equality of variance using Levene's test.t test.Mean PIT and PRT data from all screw positions and mean PIT and PRT data from the individual screw positions (from 1 to 8) were compared between the treatment groups. Mean PIT and PRT values were further compared between the individual screw positions within the treatment groups. Results of PIT and PRT measurements were subsequently combined for the proximal (from 1 to 4) and distal (from 5 to 8) screw positions and compared within the treatment groups. The comparison was conducted using a univariate analysis of variance test and Tukey's post hoc with PIT and PRT as the dependent variables and treatment group as well as screw position as fixed effects. Further comparison of the combined screw positions was conducted using independent samples two‐tailed student t test. Statistical significance was established at p < .05. Accounting for the difference in PRT results between the treatment groups found in this study and methods of statistical analysis, the power of the study to detect the true difference was calculated to be β = 0.85.The results of biomechanical testing, including actuator displacement and stiffness of the constructs, were compared between the treatment groups subjected to cyclic loading. The comparison was conducted using independent samples two‐tailed student 3p = .000). The average measured PRT equaled 343.47 ± 18.3, 303.26 ± 25.9, and 296.04 ± 42.9 N‐cm in C, G5K, and G10K treatment group, respectively (Table p = .010) as well as between C and G10K treatment group (p = .000) .The difference in PRT between G5K and G10K was not statistically significant . The hysteresis load‐creep curve did not significantly change its shape during testing which confirmed the cyclic stability of tested constructs .The detailed analysis of the PRT measurements from the individual screw positions revealed that screws placed in positions 2, 3, and 4 had the greatest PRT reduction after 5000 and 10,000 cycles  experienced a lesser reduction in PRT than the screws placed more adjacent to it. This phenomenon was less pronounced for the distal screws possibly due to the greater bone mineral density (BMD) and thicker cortex of the distal tibia.Significant PRT reduction was observed after 5000 cycles and an additional, 5000 cycles did not significantly decrease the PRT. This observation implies that PRT reduction occurred within 5000 cycles and plateaued during the subsequent 5000 cycles. Future research could explore the effect of cyclic loading in the range from 0 to 5000 cycles and that of greater than 10,000 cycles. However, the focus of this study was to evaluate the effect of cyclic loading on the stability of the screws in the postoperative period and the number of cycles was chosen accordingly to the previous study to mimic the postoperative loading conditions in the most suitable way.Torque reduction measured immediately after placement of the screws documented the expected “time 0” difference between the insertion and reverse torque. This difference emphasizes the importance of careful screw insertion to optimize the stability of the screw and plate interfaces. The small standard deviation among PRT results within the control group confirmed the consistency of screws placement in this model and justified the use of a torque‐limiting screwdriver. The standard deviation became proportionally greater relative to the number of cycles applied to the constructs under consistent, controlled cyclic loading which confirms the significant effect of cyclic loading on screw stability. The absolute lengths of the screws varied between the implants, however, the screw and bone contact length (working length of the screw) was similar among the implants since the cutting‐flute of self‐tapping screws extended beyond the far cortex warranting the good screw purchase. The screw working length and not the screw absolute length has been associated with the mechanical stability of the implant.The model of the non‐load‐sharing fracture using a full‐thickness segmental tibial defect was designed based on the experience in our lab. The rationale behind the size of the defect gap was to create the possible shortest working length of the plate to reduce the local yield stress experienced by the plate and screws.Primary implant stability has been strongly correlated with implant osseointegration. Osseointegration results from peri‐implant osteogenesis and is influenced by several factors including implant material, the status of adjacent bone, mechanical stability, and loading conditions applied to the implant.Limitations of this study include the ex vivo nature of the experiment. All factors which could have potentially influenced the results of the study were controlled throughout the experiment and no confounding effects were found. However, extrapolation of these results to in vivo conditions and clinical patients should be performed with caution until future research can be done. The micromotion of the screw implants was not directly evaluated in a quantitative manner. This can be done using linear or rotational variable differential transformers.5This study documented a negative effect of cyclic loading on the early post‐implantation stability of screws placed within a locking plate construct used to stabilize non‐load‐sharing fractures ex vivo. Reduced primary stability could further negatively affect the osseointegration of the entire construct and impair the healing process. The results of this study will serve as a reference point for further studies on the primary stability of locking screws used in locking plate constructs.The authors declare that there are no conflict of interests.Remigiusz M. Grzeskowiak conducted internal fixation procedures, biomechanical testing, organized and analyzed data as well as wrote the manuscript. Laura R. Freeman assisted with the preparation of the specimens, internal fixation procedures, and biomechanical testing. David P. Harper supervised data collection and analysis as well as manuscript writing. David E. Anderson supervised data collection, analysis as well as manuscript writing. Pierre‐Yves Mulon mentored the project, performed internal fixation procedures, biomechanical testing as well as supervised data collection, analysis, and manuscript writing. All authors have read and approved the final submitted manuscript.


Shame and embarrassment related to Parkinson's disease (PD) are rarely addressed in clinical practice nor studied in neuroscience research, partly because no specific tool exists to detect them in PD.Objective: To develop a self-applied assessment tool of shame and embarrassment specifically related to PD or its treatment, to promptly identify the presence and severity of these two emotions in PD.Methods: Identification and selection of relevant items were obtained from the collection of PD patients' opinions during support groups and interviews. Several further items were added following a literature review. Subsequently, a two-phase pilot study was performed for identification of ambiguous items and omissions, and to obtain preliminary data on acceptability, reliability, validity and relevance of the new scale (SPARK).Results: A total of 105 PD patients were enrolled in the study. Embarrassment was reported in 85% of patients, while shame was present in 26%. Fifteen percent of patients did not describe any shame or embarrassment. On average, the intensity of these two emotions was low with a marked floor effect in SPARK items and subscales. However, SPARK total score inter-individual variability was important (range 1–84 out of 99). Acceptability and quality of data were satisfactory with no floor or ceiling effects (2.9% each) or missing data. Internal consistency  was 0.94 for total score and 0.73–0.87 for subscales. The scale correlated ≥0.60 with instruments measuring related constructs. Content validity was satisfactory. SPARK total score strongly correlated with impaired health-related quality of life (rS = 0.81), the propensity to feel embarrassed or ashamed , and anxiety (rS = 0.72) and depression (rS = 0.63) levels. Moderate to high correlations were observed between SPARK total score and apathy (rS = 0.46) and a more pronounced personality trait directed toward harm avoidance (rS = 0.46). No significant differences in SPARK scores were found by sex, education level, PD duration, Hoehn and Yahr stages or PD phenotype.Conclusion: Preliminary analysis of psychometric properties suggests that SPARK could be an acceptable and reliable instrument for assessing shame and embarrassment in PD. SPARK could help healthcare professionals to identify and characterize PD-induced shame and embarrassment. Patients affected with Parkinson's disease (PD) perceive non-motor symptoms as serious challenges and barriers to a satisfying quality of life . PD-relaShame and embarrassment are two negative self-conscious emotions associated with painful states, where the self  is focal in attention. The individual believes that she/he has failed to meet appropriate standards of conduct, and thinks that she/he has done so in the eyes of others. No consensus has been reached on how shame and embarrassment differ . IntuitiShame and embarrassment in PD may emerge from different sources: (1) PD symptoms, especially visible motor symptoms but also non-motor symptoms; 2) increasing physical dependence and need for help induced by PD; and (3) deteriorated body image  Shame and embarrassment arising from PD symptoms ; (2) Shame and embarrassment arising from the increasing physical dependence and need for help induced by PD ; (3) Shame and embarrassment arising from the deteriorated body image ; (4) Consequence of related shame and embarrassment on patient's self-esteem ; (5) Stigmatization ; (6) Type of emotion . A summary score was calculated by adding up all individual item scores, for a maximum of 99 points. The self-assessment SPARK questionnaire takes ~5 min to perform.Thirty-five PD patients with no dementia were enrolled. A neurological assessment was performed including a brief medical history aimed to determine PD duration, stage of the disease established by Hoehn and Yahr scale , levodopPatients' comments provided during the debriefing questionnaire were discussed between the authors. Comments judged relevant were used to create the final version of the SPARK scale  that wilDescriptive statistics of the sample characteristics and the applied rating scales were carried out. SPARK psychometric properties were studied only in the 35 PD patients who took part in phase 3 of the study. The following psychometric properties were analyzed, following the Classical Test Theory (CTT) :- Data quality and acceptability , 26: mis- Reliability in terms of internal consistency , 28: CroS) was calculated between SPARK  and scores obtained with the two previously validated scales measuring shame and embarrassment as well as scores obtained with scales measuring depression, anxiety, apathy, type of personality and quality of life impact, constructs that theoretically should be related with the shame and embarrassment. A high correlation was established if the coefficient value rS was ≥0.60 , Hoehn and Yahr (S = 0.30–0.70). For content validity, in addition to the input from experts opinion and literature review during the construction process, the qualitative evidence from the pre-testing debriefing questionnaire with patients was analyzed, to ensure that items in the scale were representative of the construct being measured . Th. ThS) wameasured . For theA total of 105 patients were enrolled in our study: 44 patients for phase 1, 26 for phase 2 and 35 for phase 3 of the pilot study. Demographic and clinical characteristics are reported in The phase 3 of our study analyzed SPARK's psychometric properties. The SPARK total mean score was 23.97  . SPARK tRegarding internal consistency , CronbacS = 0.81), PFQ-2 total and Shame subscale , Embarrassment scale (rS=0.68), STAI State and Trait  and BDI-II (rS = 0.63) (S = 0.46) and TPQ Harm avoidance (rS = 0.46). A negative weak to moderate correlation was observed between SPARK total score and age (rS = −0.37). PD duration, LEDD, motor score and type of PD phenotype showed weak to moderate correlations with SPARK. All SPARK subscales significantly correlated with PDQ-8 (rS ≥ 0.60). SPARK subscales PD Symptoms and Body image strongly correlated  with PFQ-2 Shame subscale; and PD Symptoms and Stigma strongly correlated  with the Embarrassment scale. PD duration, LEDD and MDS-UPDRS part 3 showed weak correlations with SPARK.SPARK total score strongly correlated with PDQ-8 (r = 0.63) . ModeratRegarding known-groups validity , SPARK tRegarding internal validity, SPARK subscales correlated from 0.31 to 0.74 between them . In termSPARK is a new self-administered questionnaire assessing shame and embarrassment induced by PD. The aim of our study was to show how SPARK was conceived and designed. In addition, some psychometric properties have been tested to orient the developers toward potential problems with the current structure. Preliminary analyses of the psychometric properties suggest that SPARK could be an acceptable and reliable instrument for assessing shame and embarrassment in PD. Higher scores of shame and embarrassment were related to impaired health-related quality of life and higher levels of depression and anxiety. Consequently, PD-related embarrassment and shame probably deserve our attention. SPARK could be a useful tool for healthcare professionals and researchers to identify and rate these two negative emotions, as well as to better understand what clinically promotes these two painful and disruptive emotions.Regarding the psychometric analysis, SPARK had a satisfactory acceptability and data quality with no missing data, due to good procedures during data collection. Internal consistency and internal validity were acceptable, suggesting that the scores of our instrument were an adequate reflection of the dimensionality of the construct (embarrassment and shame) that we thought to measure. Content validity was very satisfactory, with the vast majority of patients thinking that the questionnaire was relevant to their current situation and could be helpful for their healthcare professional for their follow-up. SPARK was judged by patients as easily understandable and of adequate length, taking about 5 min to complete. The content validity was excellent, probably due to the SPARK construction and testing process which involved a collaborative effort with multiple exchanges between PD patients and healthcare professionals specialized in PD.In terms of the frequency of shame and embarrassment induced by PD, most of our patients (85%) were experiencing embarrassment whereas shame concerned far fewer patients (26%). When shame was present, it was associated with embarrassment in 100% of cases. Our results argue for the fact that embarrassment and shame are two closely related self-conscious emotions belonging to the same continuum of emotion, varying on a range of factors such as intensity, public exposure and physical reaction . To the In terms of the intensity of shame and embarrassment, SPARK total score had a mean of 24 out of 99 with a wide range of scores (1–84) showing that the severity of the shame and embarrassment varied greatly among patients. Floor effect in SPARK items and subscales indicated that most patients showed low levels of shame and embarrassment. However, SPARK scores were associated with a lower level of health-related quality of life, as well as with higher levels of depression and anxiety. Shame and embarrassment may contribute to psychological difficulties such as personal distress, self-identity alteration, social isolation, depression, and social anxiety –36. The The wide range of SPARK scores among patients probably reflects the inter-individual variability of the experience of shame and embarrassment. These two emotions vary depending on self-awareness, personality traits, level of self-esteem and self-blame, and culture –41. Our The role of PD neuropathology itself in the experience of shame and embarrassment is unknown. According to our study, an indirect and a direct role of PD are probable. An indirect role is probable through symptoms caused by PD as well as the increasing physical dependence and the deteriorated body image. However, a direct role of PD on the emotional experience might also be associated, but remains to be demonstrated. PD is secondary to neurodegeneration involving predominantly dopaminergic neurons . Higher No relationship was found between the intensity of PD-induced shame and embarrassment and PD duration and the severity of motor symptoms. These results might suggest that other factors might be involved in the intensity of the shame and embarrassment. A longitudinal study investigating the evolution of SPARK scores depending on the phase of PD would be interesting. Our hypothesis is that the intensity of shame and embarrassment might be higher before the dopaminergic replacement therapy is introduced and during the early post-diagnosis phase when the patient is learning to adapt to her/his disease. Another hypothesis would be that shame and embarrassment are little related with motor symptoms as compared with neuropsychiatric symptoms.Nevertheless, our scale contains several limitations. Three items presented low item-total corrected correlations with their respective subscales. Two pairs of items showed high inter-item correlation which might suggest redundancy. This aspect will be checked during the validation study in a larger sample. As pointed out by the debriefing questionnaire, some aspects contributing to shame and embarrassment were missing. Some new items were consequently added  in order to capture the topic as comprehensively as possible. The final version of SPARK will be utilized during the future validation study. As mentioned by several patients and because of the type of rating scale that we chose, SPARK measures the intensity of shame and embarrassment but not the frequency of the occurrence of these two emotions. SPARK also does not differentiate clearly if shame and embarrassment are internal or external. Internal shame or embarrassment describes the negative evaluation a person applied to her/himself whereas external shame or embarrassment relates to the evaluation of what the person believes others think about her/him i.e. the distressing awareness that “I think others view me negatively” . The amoIn conclusion, the SPARK scale could be a reliable questionnaire which promptly measures the severity of shame and embarrassment specifically induced in patients in the context of PD. A validation study would be useful to confirm this assumption. The availability of this rating scale could raise awareness on these two emotions in PD. The SPARK scale could help healthcare professionals to identify the problem of shame and embarrassment affecting PD patients and to better understand what clinically promotes these two emotions. Our study demonstrated that PD-associated embarrassment is extremely frequent. Shame and embarrassment were associated with a lower level of health-related quality of life, as well as with higher levels of depression and anxiety. As such, PD-related shame and embarrassment deserve our attention. Further studies are needed to deepen the understanding of the subject, such as studies exploring clinical, cultural or socioeconomic factors influencing these two painful emotions. The clinical implication of this score system could be important especially in patients who score high on SPARK total score and shame sub-item as shame probably contributes to psychological difficulties such as personal distress, depression, suicidal ideation, and social encounter avoidance. A high SPARK score should alert healthcare professionals to the potential presence of psychological difficulties. The SPARK questionnaire could therefore help healthcare professionals to implement psychological support to patients' management in a timely fashion in order to help patients cope with their disease. Cognitive-behavioral intervention strategies such as systematic desensitization, role playing, thought stopping, disputing the inner critic, identification of irrational thinking and dysfunctional cognitive schemas could be clinically beneficial for these 2 emotions , 50. TheAll datasets presented in this study are included in the article/The studies involving human participants were reviewed and approved by Geneva Ethics Committee (SwissEthics). The patients/participants provided their written informed consent to participate in this study.VF, SC, MF, MM, ID, G-MH, PM-M, CR-B, and PK: conception and design of the study. VF, SC, MF, MM, JA, PB, and CR-B: acquisition and analysis of data. VF, SC, MF, MD, MM, ID, G-MH, JA, PB, PM-M, CR-B, and PK: writing and review of the text and preparing tables and figures. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Olea europea L. subsp. europaea var. sylvestris (Mill) Lehr (Oleaster) is a wild endemic olive tree indigenous to the Mediterranean region. Olea europea leaves represent a natural reservoir of bioactive molecules that can be used for therapeutic purposes. Aim of the study: This work was conducted to study antidiabetic and antihyperglycemic activities of flavonoids from oleaster leaves using alloxan-induced diabetic mice. The mode of action of flavonoids against eight receptors that have a high impact on diabetes management and complication was also investigated using molecular docking. Results: During 28 days of mice treatment with doses 25 and 50 mg/kg b.w, the studied flavonoids managed a severe diabetic state (<450 mg/dL), exhibiting a spectacular antidiabetic and antihyperglycemic activity, and improved mice health status compared to diabetic control. The in-silico mode of action of oleaster flavonoids revealed the inhibition of protein tyrosine phosphatase 1B (PTP1B), Dipeptidyl-peptidase 4 (DPP4), α-Amylase (AAM), α-Glucosidase inhibition, Aldose reductase (AldR), Glycogen phosphorylase (GP), and the activation of free fatty acid receptor 1 (FFAR1). Conclusion: The findings obtained in the present work indicate that the flavonoids from the oleaster may constitute a safe multi-target remedy to treat diabetes.Background:  Diabetes is the most common metabolic disease worldwide with several underlying causes leading to hyperglycemia . With twThe number of new cases is rising. The International Diabetes Federation expects over 700 Million cases by 2045 against 425 million recorded in 2017 . The sciOlea europaea subsp. europaea L. including two varieties: cultivated and wild species, respectively called europaea (cultivars) and sylvestris (oleaster), which are naturally distributed all around the Mediterranean region +), while the high-CE scan generates fragment ion information. The source parameters were set as follows: nebulizing gas flow 2.9 L/min, heating gas flow 10 L/min, interface temperature 300 °C, DL temperature 250 °C, heat block temperature 400 °C, drying gas flow 10 L/min.The analysis was performed using a Shimadzu Ultra-High-Performance Liquid Chromatography , coupled to an MS/MS detector . The MS/MS was operated with electrospray ionization (ESI) and controlled by Lab Solution software, which simultaneously provided quick switching from low energy scan at 4 V (full scan MS) to high energy scan (10–60 V ramping) during a single LC run. The low-CE experiments provided information about the intact molecular ion .Qualitative analysis was performed in flow injection with a mobile phase consisting of acetonitrile:water + 0.01% formic acid  Such files could not be directly used to dock simulation, thus utilizing AutoDockTools v1.5.6 , and theThe X-ray crystal structures of the eight receptors were retrieved from the Protein Data Bank (PDB))  . The 6th molecule was Oleuropein which belongs to secoiridoid as a characteristic molecule of the Olearaceae family . Hyperglycemia was suppressed afterward, and the FBG was significantly maintained (p < 0.001) with a reduction of 79, 79 and 44% (second week), 81, 81 and 63% (third week), 82, 82 and 73% (fourth week) respectively, compared to the negative diabetic control. The antidiabetic activity of the extract was better even than that obtained with Glibenclamide and reached normal glycemic values in the last week .Within the first week, reduction reached 76% due to oleaster leaves flavonoids at a dose of 25 and 73% for a dose of 50 vs. 36% for Glibenclamide compared to the negative control group (p < 0.001). Hyperglycemia leads to body weight loss due to the intense usage of fats and structural proteins as a replacement for carbohydrates [The bodyweight development and biochemical parameter of the different experimental mice groups are represented in hydrates , which ihydrates . Oral adp < 0.001). However, the plasma level of transaminases was remarkably reduced in the blood of diabetic animals after treatment with oleaster flavonoids (p < 0.001). Our study was in agreement with earlier data which showed that oleaster flavonoids exhibiting a remarkable hepatoprotective effect against possible damage that may be caused by the injection of Alloxan monohydrate [p < 0.001). However, the treatment of diabetic mice with oleaster flavonoids significantly decreased their urea and creatinine levels in comparison with the untreated diabetic animals (p < 0.01). Elevated levels of urea and creatinine are considered markers for renal damages [The results obtained showed that significant increase in the activities of ALAT and ASAT (liver biomarkers) was observed in the untreated diabetic animals compared to normal animals . After 12 h of treatment, glucose values in plasma of positive control mice significantly increased compared to groups treated with oleaster flavonoids  (p < 0.001). These results may reflect a long-term effect and the powerful management of the hyperglycemic status of diabetic mice.The antihyperglycemic activity of oleaster flavonoids is represented in The selected receptors for this study are highly involved in diabetes manifestations or management. Only the binding poses of ligands with the highest affinities and bonds with known active sites were selected and analyzed. Affinity results are summarized in O-glucoside and quercetin-3-O-hexose-deoxyhexose with quercetin as the attached sugar moieties were removed after ingestion before absorption can take place [It’s important to note that in the molecular docking simulations, we replaced both quercetin-3-ke place .Protein tyrosine phosphatase 1B (PTP1B) is an important enzyme in insulin receptor dephosphorylation . GeneticO-glucoside has the highest affinity to PTP1B. This finding indicates that oleaster flavonoids exhibit their antidiabetic activity via inhibition of PTP1B. Previous research indicates that the eradication or reduction of PTP1B improves glucose tolerance and the regulation of insulin. PTP1B inhibition also decreased the concentration of triglycerides in the adipose tissue under over-nutrition conditions and was not associated with any apparent toxicity [According to the docking results, Luteolin-7-toxicity .Dipeptidyl-peptidase 4 (DPP4) is a glycoprotein found on several cell surfaces. It selectively cleaves N-terminal dipeptides from various substrates like growth factors, cytokines neuropeptides, and the incretin hormones  .The active site of DPP4 in its entirety is considered to contain residues 39–51 and 501–766 and is known as the α/β-hydrolase domain ,27. The Human G-protein coupled receptor 40 (hGPR40), also known as free fatty acid receptor 1 (FFAR1), is a seven helical transmembrane domain receptor that recognizes long-chain free fatty acids and induces insulin secretion . FFAR1 iO-glucoside and Oleuropein) have affinities for the active site. Even though only two ligands have an affinity to the FFAR1 active site, activation of this protein remains one of the possible modes of action of the oleaster flavonoids. FFAR1 is of particular interest because the triggering of insulin secretion is glucose-dependent, which makes it a major focus for the treatment of type 2 diabetes, as agonists will improve glycemic regulation and minimize the risk of hypoglycemia [Free fatty acids bind to FFAR1 by coordinating their free carboxyl group to three amino acids, Arg183, Tyr2240, and Arg258, which are located close to the extracellular domain . hGPR40 glycemia .α-Amylase (AAM) is an enzyme that works as a catalyst for the hydrolysis of α-linked polysaccharides into α-anomeric products . α-AmylaThe AAM active site contains three catalytic residues: Asp197, Glu233, and Asp300. Many residues are also important for the enzyme activity: Arg337, Arg195, Asn298, Phe265, Phe295, His201, Ala307, Gly306, Trp203, Trp284, Trp59, Tyr62, Trp58, His299, His101 ,36,37,38All five tested ligands have an affinity to the active site. The highest affinity was noted for Amentoflavone, and Oleuropein has the lowest among the studied series. According to our findings, α-amylase inhibition is also one of the major modes of action of the oleaster flavonoids as all the ligand exhibit activity toward it. α-amylase is a part of the digestion associated with carbohydrate breakdown.Peroxisome proliferator-activated receptor gamma (PPAR-γ or PPARG), also known as the glitazone receptor, plays his role by enhancing the storage of fatty acids in fat cells, which increase insulin sensitivity and reduce lipotoxicity .The PPARγ ligand-binding domain is folded into a helical sandwich to provide a binding site for ligands. It is located at the C-terminal end of PPARγ. It is composed of about 250 amino acids, activation by full agonists occurs through hydrogen bond interactions between the Ser289, His323, Tyr473, and His449 residues of the PPARγ ligand-binding domain .Detailed analysis of the active site of PPARγ reveals that none of the ligands could bind with it. Reference molecule Rosiglitazone (an antidiabetic drug of the thiazolidinedione class) has shown that this molecule binds perfectly with the active site. These findings indicate that PPARγ is not involved in the global mode of action of oleaster flavonoids.Alpha-glucosidase enzymes catalyze the hydrolysis of starch and dietary carbohydrates to simple sugars (glucose) for intestinal absorption, which, in turn, results in increased blood glucose levels .The residues involved into the activity of the enzyme are Asp404, Asp518, Arg600, Asp616, and His674 Trp376, Ile441, Tp516, Met519, Trp613, and Phe649 Leu405, Trp481, Asp645, and Arg672 ,43.Good affinities with the active site were noted for all ligands. The highest affinity was observed for Amentoflavone. It corroborates that α-glucosidase inhibition can be one of the major modes of action of the oleaster flavonoids. Inhibiting the function of this enzyme may help reduce postprandial hyperglycemia.Aldose reductase (AldR), a key enzyme in the polyol pathway or sorbitol-aldose reductase pathway, catalyzes NAD(P)H-dependent reduction of glucose to sorbitol, leading to excessive accumulation of intracellular reactive oxygen species (ROS) in various tissues , that maGlycogen phosphorylase (GP) catalyzes the hydrolysis of glycogen to generate glucose-1-phosphate . The 280Only four ligands have shown affinities for the active site. The highest affinity was again noted for Amentoflavone. Glycogen phosphorylase is considered as one of the causes of hyperglycemia as it is responsible for generating glucose. Inhibiting this enzyme by different ligands present in oleaster flavonoids may lead to considerable improvement of the glycemic status of diabetic people.O-glucoside, Rutin, and Quercetin. Oleuropein had the lowest binding affinities among the studied series.Among all the ligand tested, it is obvious that Amentoflavone is the most polyvalent molecule with the best affinities to almost all selected receptors, followed by Luteolin-7-Olea europea exhibited very interesting results in diabetic control as well presented in this work, and therefore we could confirm that oleaster flavonoids could be a game changer in the treatment of diabetes.Besides its nutritional and cosmetic values, the wild olive tree (oleaster) presents a promising source of bioactive molecules that can be exploited to fight some diseases. This work provides big data on the antidiabetic activity, the safety as well as the potential mode of action of oleaster flavonoids using molecular docking.


Anomalies in plutonium concentration and isotopic ratio suggest forensic insight into specific activities on site, such as clean-up or structural change. Local and global 239Pu contributions on-site are estimated at (34 ± 1)% and (66 ± 3)%, respectively, with mass concentrations of (183 ± 6) fg g−1 and (362 ± 13) fg g−1. The latter is consistent with levels at undisturbed and distant sites, (384 ± 44) fg g−1, where no local contribution is expected. The 240Pu/239Pu ratio for site-derived material is estimated at 0.05 ± 0.04. Our study demonstrates the multi-faceted potential of trace plutonium assay to inform clean-up strategies of fast breeder legacies.Trace-level plutonium in the environment often comprises local and global contributions, and is usually anthropogenic in origin. Here, we report estimates of local and global contributions to trace-level plutonium in soil from a former, fast-breeder reactor site. The measured  This study proposes a method to differentiate between local plutonium-based contamination in soils versus trace plutonium stemming from global dispersion in the past, such as fallout from detonation and atmospheric testing of nuclear weapons. Anthropogenic contributions occur local to their source of production, for example, from nuclear plant effluents, reactor accidents, accidents involving nuclear weapons and plutonium-powered space probes etc., and also globally from fallout. Following the measurements Seaborg et al.2 of Canadian pitchblende (yielding a 239Pu mass concentration of 550 fg g−1), Krey et al.3 estimated the 240/239Pu ratio due to fallout, at 0.176 ± 0.02: this has been used widely for comparison by subsequent studies. Harley4 suggested a distribution of the oxide from weapons testing of 2 mCi km−2 (~485 fg g−1 assuming deposition in the first 5 cm and a bulk soil density of 1.33 g cm−3) in the north temperate zone. The depth-resolved assessment of plutonium residues in Lake Ontario sediments followed, yielding a 240Pu/239Pu ratio6 consistent with Krey et al. Subsequently, Buesseler et al.7 reported measurements of a dated coral record from the North Pacific of 239Pu mass (~55 fg g−1) and 240/239Pu ratio (>0.2), the latter reflecting the predominance of fallout in this environment, in agreement with the prior art3. Plutonium-239 abundance of the contrasting residues from the Cigar Lake natural ore body (Canada) were reported8, resolved with depth, in the range 17–6200 fg g−1. Kelley et al.9 reported on the global distribution, including a sample from Wick (Scotland), ~50 km from the site at Dounreay studied in this research, with a 240Pu/239Pu ratio of 0.182 ± 0.001. Muramatsu et al. published an analysis of samples from the Marshall islands10 with a 240Pu/239Pu range of 0.065–0.306 and 239Pu mass 75–500 fg g−1. In 2013, Steier et al.11 reported accelerator mass spectrometry (AMS) measurements of samples from Sellafield (UK) with a 240Pu/239Pu range of 0.183–0.228 (±0.001) and, in 2015, Armstrong et al.12 reported a 10-year assessment of samples from Savannah River (USA) with 240Pu/239Pu range of 0.0757–0.3035.Minute quantities of plutonium occur naturally in the terrestrial environment4 stated, it remains ‘…desirable…to distinguish…local sources from global fallout’ for exactly this reason. Given this context, the motivation for this study is to determine whether the local contribution to trace plutonium abundance on a fast breeder site might be discerned from the global contribution, to inform the extent to which clean-up of the local component might be necessary.Terrestrial plutonium abundance, albeit deposited over decades, varies slowly with time due to the long half-lives of most plutonium isotopes and relatively slow natural transport mechanisms, in the absence of human intervention. The ability to discern local sources of plutonium from global fallout is important to inform decisions concerning nuclear legacies, particularly the clean-up of contaminated land. As Harley239Pu, has also been the focus of nuclear reprocessing, mixed-oxide and metal fuel manufacture, and nuclear weapons fabrication operations for ~70 years worldwide, albeit on a localised basis. Of particular relevance to this research is 239Pu produced by breeding in the mid-to-late 20th century and the legacy of these activities.In addition to the variation in trace elemental plutonium, the isotopic distribution varies too. Plutonium-239 is the more probable product of activation in reactors relative to the higher-mass plutonium isotopes. Industrially, fissile Pu, in particular 239Pu, the higher-mass plutonium isotopes  have few specific applications and have not been manufactured on a comparable scale. Since their formation is dependent on the neutron fluence and energy spectrum, estimating the extent to which they are produced can require knowledge of the associated neutron environment, in which they are made. Elemental plutonium concentration in soils is dependent on many factors, in addition to the way in which it was formed, such as geographic location, precipitation and soil composition etc. By contrast, the link between a specific isotope, considered via the isotopic ratio, and its origin often yields a more tangible association13, for example: a pre-detonation weapon safety test might cause 239Pu to dominate; fallout from atmospheric detonations can comprise higher-mass isotopes through to 244Pu (refs. 15), whereas a relatively low 240Pu/239Pu ratio might reflect the low proportions of 240Pu desirable in some applications, such as that obtained by breeding. Material dispersed in fallout will often have a higher proportion of 244Pu (ref. 16), whereas higher-burnup manufacture in thermal spectrum nuclear reactors, might afford an inventory effectively limited to 240Pu and 241Pu, generally with a higher proportion of 240Pu; the decay of 241Pu affords for the activation of americium products by neutrons and the formation of curium isotopes17, also less prominent in contributions from weapons fallout. While the isotopic association, particularly to identify plutonium in fallout, is well understood we are not aware of its use to discern the local contribution to breeder-borne material in the environment: hence the focus of this study.In comparison with 241Pu levels in environmental samples18, but the relevant lines for the other isotopes can be too weak. Alternatively, detection limits achieved with sector-field ICP-MS have advanced significantly of late providing, for example, trace isotopic plutonium evidence associated with Fukushima20. To discern isotopics at trace levels AMS has been used widely22. Fifield et al.25 suggest a limit of detection for 239Pu two orders of magnitude lower for AMS than for α spectrometry.Trace plutonium can be estimated by α-assay of environmental samples, e.g., calcined shellfish or leached terrestrial minerals, etc. and γ-ray spectrometry can be used to infer trace 239Pu) relative to the amount of fissile material consumed. A fast neutron spectrum is required to achieve the neutron multiplicities necessary to offset neutron losses due to absorption and leakage. Originally, plutonium breeding26 constituted a closed fuel cycle unhindered by what was forecast to be the relatively limited abundance of 235U. Several major projects followed, for example: the Prototype Fast Reactor at Dounreay (UK)27, Phénix (France)28, KNK II (Germany)29, FBTR (India)30, Jōyō (Japan)31, BN-600 (Russia)32 and the EBR-2 (USA)33. Although interest in breeding declined, when global uranium abundance proved higher and consumption proved lower than forecast, a number of the next-generation, Generation IV, reactor designs are breeders34.Plutonium breeding aims to achieve a positive yield  permitted under the various authorisations and permits that the site has operated under. There are also expected to be some localised spots of contamination arising from historical movement of materials around site. In contrast with dispersed contamination on-site, individual particles arose at Dounreay as a result of an accidental discharge at sea. These have a very different isotopic composition consistent with irradiation in the materials test reactorTwelve soil samples were collected on-site (nine samples), near to  and far away  from Dounreay, near Thurso, on the north coast of Scotland. The nine samples from Dounreay were collected from a 220-m long invasive trench excavation of 1 m depth (assuming 0.5 m average depth after random sampling). Off-site samples include: an off-site-near sample taken near to the Dounreay site but not on it, to cater for local variations due to dispersion of material from activities on-site beyond site boundaries, fallout in the local area and uranium-rich geological deposits in the area. Two off-site-far sources of samples were taken off-site 700 km to the south: Malham in Yorkshire and Biggin in Derbyshire, both UK. These were taken from locations exhibiting no evidence of having been disturbed and that are isolated from industrial activity. Approximately 5 kg of soil was collected per sample; each was oven dried and ground to ensure a uniform and homogenous grain size distribution prior to preparation for 5 g per sample being used for measurement by AMS. A map indicating the locations from which the samples were taken is given in Fig. 240Pu/239Pu ratio on-site is observed to be anomalously low, relative to the global average, but qualitatively consistent with the reduced yield of 240Pu expected in plutonium bred with a fast neutron spectrum. We observe anomalies in plutonium concentration and 240Pu/239Pu ratio associated with two on-site samples, which suggest the potential to provide forensic insight into actions on-site at some point in the past, such as localised sites of contamination or the removal of buildings. Local and global contributions to 239Pu on-site are estimated at (34 ± 1)% and (66 ± 3)%, respectively, with mass concentrations of (183 ± 6) fg g−1 and (362 ± 13) fg g−1. The latter is consistent with our measurements of the average at undisturbed and distant sites (384 ± 44 fg g−1), where no local contribution is expected. We compare our measurement of the site average 240Pu/239Pu with that of one of the local anomalies to determine the ratio for site-derived material, which we estimate to be 0.05 ± 0.04. The novelty in our study is that we apply AMS to soil samples from a fast breeder site, we separate local and global contributions, where the isotopic inventory of the local component is consistent with that derived from a fast breeder, we identify the potential for forensic analysis of two-specific, inventory-based anomalies on-site and we estimate the 240Pu/239Pu ratio for local material, self consistently, which is observed to be consistent with material of breeder origin.Here, we report estimates of the local and global contributions to trace-level plutonium in soil samples from the former fast-breeder reactor site at Dounreay, UK, made with AMS. The average 239Pu, 240Pu, 242Pu and 244Pu isotopes in the samples are given in Fig. AMS results for the concentration of the 240Pu/239Pu, 242Pu/239Pu and 244Pu/239Pu atomic ratios are shown in Fig. The 239Pu concentrations and 240Pu/239Pu ratios, respectively. A complete dataset for concentrations and ratios for each sample is provided in the For the different isotopic concentrations presented in Fig. 239Pu concentration data suggests that the DSRL samples are statistically consistent with one another, except for DSRL3 and DSRL8: DSRL3 has a level greater than neighbouring sample locations from the same site (1001 ± 30 fg g−1) and DSRL8 a level that is lower (289 ± 9 fg g−1). Including DSRL3 and DSRL8, the weighted average 239Pu mass abundance for these samples is (481 ± 48) fg g−1. Excluding DSRL3 and DSRL8 from the weighted average yields a −1. A similar trend is also exhibited for samples DSRL3 and DSRL8 in the 240Pu and 242Pu mass concentration data, relative to the concentrations of these isotopes for the other samples, confirming DSRL3 and DSRL8 as outliers.The variance across the 239Pu concentration is consistent with the on-site samples (except DSRL3 and DSRL8), whereas the data for the off-site-far  samples are lower than the on-site average, at (341 ± 10) fg g−1 and (428 ± 13) fg g−1, respectively. This implies the ambient plutonium level in the Dounreay area may be slightly elevated, relative to Malham and Biggin. Aside from the scenario that this excess derives from legacy activities on-site, it could also be due to (i) increased levels of global fallout in the Dounreay area relative to Malham and Biggin, (ii) additional plutonium consistent with the relatively high indigenous uranium content in the area, 3.00–1470 μg g−1 (ref. 36), (iii) stronger binding of elemental plutonium to soil media in the Dounreay area due to physicochemical differences between the ground composition of these two, disparate areas, (iv) differences in elevation and rainfall, i.e., given annual precipitation (https://en.climate-data.org/europe/) being higher at Malham  and Biggin  relative to that of Caithness , of the order ~43% and ~23%, respectively, given some downward migration is expected4. The elevation in 239Pu is not replicated for 240Pu and is at the limit of the uncertainty in the 242Pu data.The off-site-near sample datum for 244Pu concentrations  and off-site-near (DSRL B1A) are consistent with each other and, interestingly, they are consistent with the mass assessment of the sample from Biggin in Derbyshire. The sample from Malham in Yorkshire is high, at (30 ± 8) × 10−3 fg g−1 relative to the average on-site (3.9 ± 0.8) × 10−3 fg g−1; given the consistent levels for 239Pu, this excess of Malham over Biggin in 244Pu can be understood when the isotopic ratios are discussed, which follows below. Malham and Biggin are located at similar elevations, as specified earlier.The ons Fig.  merit pa239Pu, given the effect of latitude, rainfall and soil type cannot be accounted for entirely, the comparison index for 239Pu concentration , various sites in the Arctic13 , of Canadian pitchblende2, Cigar Lake8 (again recognising the range involved) and Savannah River12 . Comparison with measurements at two villages off-site from Semipalatinsk-21 in Russia37 is also included (Kainar and Azkhar), having contributions from the site in that case and global fallout. However, the scatter in this comparison is significant and the range reported in some of the prior art is large. Of particular relevance is that while Malham and Biggin exhibit 239Pu levels consistent with global fallout, Fig. 239Pu in trace plutonium in the on- and near-to-site samples.Returning to ion Fig.  has beenAll of the plutonium in on-site samples derives from global fallout without any contribution from Dounreay Fast Reactor (DFR) activities. The isotopic composition of this global contribution on-site and near-to-site would therefore be expected to be uniform and consistent with the global average.Dispersed plutonium on-site derives from both global fallout and authorised discharges from stacks on-site . The isotopic composition of these two contributions is likely to be different to one another and that of the combination different to the global average (this scenario is perhaps most likely given the trace levels with which we are concerned).The majority of plutonium in the samples derives from local activities with an isotopic composition contrasting to that of scenarios (1) and (2), given an open scenario immune from global deposition is inconceivable.As plutonium concentrations in soils can be influenced by soil properties that can contrast on a very localised basis, the ratios of the plutonium isotopes for each of the samples can yield information complementary to that derived from isotopic mass. Three hypothetical scenarios merit consideration in this regard:M and comprises the global and local contributions MG and ML, respectively, then,For scenario (2), where the total plutonium mass is denoted ML = 0 and MG = 0, respectively. Where MG and ML ≠ 0, each component will have a specific isotopic composition,For scenarios (1) and (3), 244Pu is more significant in global fallout than in reactors, especially breeders, this component of ML  is expe(MG (Eq. ). Note: 239Pu, 240Pu and 242Pu  for scenario (2) becomes,With respect to the correlation across samples observed between the mass of 2Pu Fig. , global 240Pu, Fig. 240Pu mass  and 3), the isotopic ratio is hypothesised to be uniform across samples in each case, and therefore the ratio data  because 240Pu production is lower.Recapitulating, with reference to DSRL3 and DSRL8, the former is high and the latter is low in terms of 239Pu isotopic excess coupled with a relatively low R240/239 implies a greater association with material from on-site activities rather than fallout, since it is isotopically different to the material adjacent to it. Given the trace levels at which these measurements have been made, it could be associated with, for example, a previously unknown feature or the remnants of a localised case cleared up in the past; suggesting an interesting, localised forensic indication of material likely to be of breeder origin.Concerning DSRL3, the relative DSRL8, by contrast, is low relative to the on-site average for elemental plutonium and, therefore, might be consistent with an area that has been sheltered at some point  or due to the soil having been removed locally at some point. Since it does not exhibit an isotopic distinction correlating to site activity (in contrast to DSRL3), these hypotheses appear likely to relate to a restriction of the dispersion of elemental plutonium comprising a combination of material of local and global origin.R244/239  × 10−5. Since 244Pu is the only plutonium isotope not affected by reactor contamination, its lower abundance on-site may indicate the effects of decontamination activities or lower retention in soils on-site etc. A comparison of the 244Pu/239Pu ratios and the 244Pu and 239Pu concentrations for on- and near-to-site with far-from-site samples suggests an additional contribution on-site of 239Pu and the loss of some 244Pu. It is known that the UK falls in a zone where worldwide fallout of radioactivity from nuclear weapons tests exhibits a relatively steep gradient, with increasing northerly latitude39.The data for 239 Fig.  indicateR240/239 is consistently low for all of the on-site data, in comparison with both the global average3 and the off-site-far data can be appreciated from Fig. R240/239 for the prior art to that for this research). The data from this research fall consistently below the majority of the examples in the prior art associated with fallout, including non-industrial sites nearby (Wick9), an industrial site with a relatively long history of discharges from a variety of activities, and thus elevated R240/239 (Sellafield11) and one with a history of weapons-grade plutonium production (Savannah River12). In particular, the difference in ratio to Wick (a location near to Dounreay) suggests the difference is a characteristic local to the Dounreay site. Sites synonymous with contamination by material with a very low R240/239, such as Nagasaki40, Semipalatinsk-21 and Mayak37 are much lower, as expected.The observation that 240Pu and 239U (and thus 239Pu), respectively: in each, ‘A’ denotes the cross section at thermal energies and ‘B’ that at fast (1 MeV) energies. While the need for a fast neutron spectrum in 239Pu breeding to yield the neutron economy necessary relative to the thermal case is well known, it is the context with respect to the yield of 240Pu that is relevant here: at fast neutron energies , the microscopic cross sections are significantly different at ~1 b and >100 b, respectively. This illustrates the absence of a thermal neutron contribution in a fast breeder resulting in a reduced yield of 240Pu relative to that of a thermal spectrum reactor. It is worthy of note that, while fallout from a detonation would also derive from a neutron spectrum harder than a thermal spectrum fission reactor, it would also have a much higher associated neutron flux, resulting in R240/239 of a similar order to a thermal spectrum yield41, and consistent with what is observed in the average of the off-site-far data, i.e., 0.167 ± 0.005. Hence, the lower R240/239 observed for the on-site samples in this research is consistent with the material comprising a combination of anthropogenic plutonium produced locally by a fast breeder and global fallout, on the basis that less 240Pu is produced in a fast breeder relative to that of a thermal spectrum reactor42 or fallout, because of the greater thermal neutron component in the former and the higher neutron flux in the latter. It is noted that, while DSRL3 is relatively high in terms of elemental plutonium mass concentration, the R240/239 for this sample is low, consistent with more of it being derived locally from breeder-borne material, as per this argument. The DSRL3 ratio being low relative to the other on-site samples is consistent with there being a relative excess in breeder material as per the spot contamination scenario suggested above.Further corroboration can be drawn with reference to Fig. 239Pu mass contribution that is local, 240Pu/239Pu isotopic ratios where the total derives from local and global contributions, viz.,R240/239 is the 240Pu/239Pu ratio measured in this research on-site at Dounreay, 3 (0.176 ± 0.02), and the ratio for the local contribution, 43. Substitution in Eq. (239Pu abundance on-site of (34 ± 1)% which, using the weighted average for the mass concentration and R240/239 on-site  of (545 ± 9) fg g−1 and (0.126 ± 0.002), respectively, suggests local and global contributions of −1 and −1, respectively. The latter estimate is consistent with the average of off-site-far measurements made in this research, (384 ± 44) fg g−1, as representing an independent measurement of Equation  can be r239Pu contribution with the same −1 (40% of the on-site weighted average) and 43 above. However, the uncertainties are greater via this route.Alternatively, Eq.  can be a4, at various sites in the Arctic13, of Canadian pitchblende2, Cigar Lake8  and Savannah River12, notwithstanding the effects of soil type and rainfall unaccounted for. An excess is apparent relative to distant, non-industrial sites in the UK.Plutonium-239 concentration at Dounreay agrees with prior estimates of global fallout in the north temperature zone by HarleySpecific characteristics have been observed that might correlate with structural activities on-site, i.e., the influence of sheltering by structures on-site or localised operations that have removed soil at some point.240Pu/239Pu ratio for on-site and near-to-site samples suggests a forensic correlation consistent with the material expected from breeder activities produced with a fast spectrum to maximise neutron economy, which yields a reduced proportion of 240Pu relative to that of 239Pu.The consistently low 240Pu/239Pu ratio relative to neighbouring samples from nearby locations is observed consistent with a spot contamination scenario.An elemental excess of Pu combined with a low 239Pu contribution to site activities, and of the associated R240/239, have been made. The former is consistent with an off-site-far estimate for the global contribution in the UK and the latter is consistent with that expected for breeder material.Estimates of the local AMS has long been known to be capable of class-leading sensitivity with respect to the important requirement of trace actinide assay in the natural environment, and particularly the challenge associated with plutonium contamination. In this research, it has been applied to a comparison of soil samples from a legacy fast breeder site, a sample near to site and those far from site. The research confirms:R240/239. Such an incident might involve material dispersed by accident or where trace quantities of material with a ratio distinct from the global average for fallout arises, for example, as part of a forensic investigation.While all fast breeder sites might be subject to local contamination by plutonium at trace levels, the extent to which our findings might be extrapolated will be dependent on the history of activities on a specific site, historical discharge authorisations and local practice. This research is also relevant to contamination scenarios aside from breeding, where the local material might be discerned by an anomalously low 44, and Vila-Alfageme et al.45. Briefly, the weighed soil sample was placed in a TeflonTM beaker, moistened and spiked with ~30 pg of 242Pu. The 242Pu spike was kindly provided by the Centro Nacional de Aceleradores, Seville, Spain, and is traceable to the reference material R15-20 supplied by the National Physical Laboratory, United Kingdom. The Pu isotopes were leached from the soil by addition of concentrated Suprapur® nitric acid and hydrogen peroxide in a hotplate following the guidelines in Sakaguchi et al.46. The acidic solution was centrifuged and filtered three consecutive times, evaporated to dryness and diluted in 8 M Suprapur® nitric acid. The valences of Pu were adjusted using 3 N sodium nitrite prior to co-precipitation with clean Fe(OH)3 to ensure complete extraction of Pu isotopes. The iron precipitates were re-dissolved in 8 M Suprapur® nitric acid and subject to sequential extraction of Pu and other isotopes  using TEVA® and UTEVA® columns (Triskem). The eluate containing the Pu fraction was coprecipitated using 1.25 mg of clean Fe3+ solution and evaporated to dryness. All Pu isotopes were converted to the oxide form by heating to 650 °C and mixed with 1.3 mg of niobium prior to pressing in AMS cathodes. Procedural blanks (n = 7) prepared with deionized water were processed in the same way as the samples and were included in every analytical batch to assess the background contribution to Pu isotopes. Each of the samples was assessed by AMS at the ETH-TANDY AMS facility, Zürich, Switzerland47. For the 244Pu analysis, two samples were prepared and assessed for the DSRL1 source of samples, yielding data DSRL1 and DSRL1S2.Quantities of 4–5 g of each sample were subject to a simple acid leaching followed by iron co-precipitation and extraction chromatography prior to AMS. Details about the chemical procedures are reported in Chamizo et al.Supplementary information