A new study published in the Biochemical Journal by Ito et al. establishes that a ‘Phos-tag’™-binding assay can be exploited to measure phosphorylation of a recently identified LRRK2 substrate (Ras-related protein in brain 10 (Rab10)), and to compare and contrast relative catalytic output from disease-associated LRRK2 mutants. Powerful in vivo chemical genetic approaches are also disclosed, in which the catalytic activity of LRRK2 is unequivocally linked to the extent of Rab10 phosphorylation and the effects of chemically distinct LRRK2 inhibitors are matched with on-target inhibition mechanisms mediated through LRRK2 and its substrate Rab10. These important findings should simplify the generic analysis of Rab10 phosphorylation in model biological systems and are likely to be applicable to other substrates of LRRK2 (or indeed other kinases) for which phospho-specific antibodies are either absent or unsatisfactory.Protein kinases catalyse the addition of phosphate groups to Ser/Thr and Tyr residues in cognate substrates and are mutated or hyperactive in a variety of diseases, making them important targets for rationally designed drugs. A good example is the Parkinson's disease-associated kinase, leucine-rich repeat kinase 2 (LRRK2), which is mutated (and probably hyperactive) in a small, but significant, subset of patients. An exciting new approach for personalised therapy is the development of central nervous system (CNS)-active small-molecule kinase inhibitors, which could be employed to ‘normalise’ LRRK2 signalling in affected cell types. However, the development of such drugs requires validated assays for the analysis of target engagement and the assembly of a set of tools for interrogating LRRK2, and its substrates, both  These pportance . Howeverportance –7, in la2+:phosphate:Phos-tag acrylamide complex (2+) complex that is copolymerised in the gel prior to electrophoresis [‘A new study by Ito, Alessi and colleagues from GlaxoSmithKline (GSK) and The Michael J Fox Foundation for Parkinson’s Disease  publishe complex . This tephoresis . This apphoresis ,14 were phoresis –18.FiguBiochemical Journal study, which builds upon a recent ground-breaking phosphoproteomic study that uncovered Rab GTPase family members as new substrates  for LRRK2 [Continuing successes with kinase inhibitors in the oncology clinic have marked out protein kinases with atypical signalling profiles in other diseases, such as inflammation and neurological disorders, as potential targets for therapeutic intervention . One sucor LRRK2 . This woor LRRK2 . Getting2+-based reagents (synthesised in-house) with commercial Rab10 monoclonal antibodies [In their recent study , Ito et tibodies , the LRRin vitro using a pT73 Rab10 antibody, which also picked up the slower migrating Rab10 protein [Further work exploiting the Phos-tag system demonstrated that HA-tagged Rab10 phosphorylated by overexpressed R1441G LRRK2 also led to significant retardation of Rab10 in cells. These data were backed up  protein . Consist protein  blocked  protein .50 value shift for MLi-2 of some two orders of magnitude between wild-type LRRK2 and A2016T LRRK2-expressing MEFs represents unequivocal evidence that LRRK2 and Rab10 are a physiological kinase substrate pair in this system [in vivo evidence also comes from a mouse lung analysis, where a D2017A knockin blocked Rab10 phosphorylation, and an A2016T knockin [in vivo. Such approaches are very powerful for evaluating the effects of this and other target-validated LRRK2 drug-like compounds, in pathological models of LRRK2 signalling.An impressive validation of the Rab10 Phos-tag technique also comes from the finding that expression of a ‘drug-resistant’ A2016T) allele of LRRK2 decreased the ability of the LRRK2 inhibitor MLi-2 to block the appearance of the slower migrating Rab10 phosphorylated protein. Indeed, the ICs system . FurtherT allele in vivo and might have a profound effect on the choice of selection of Phos-tag Rab10 assays over LRRK2 phospho-specific antibodies, especially under conditions where Rab10 phospho-specific antibodies (such as Rab10 pT73) are ineffective or limiting.An important new analytical use for Phos-tag gels pertaining to Rab10 phosphorylation was also described in the new study. Initially, the authors provided evidence that MEF cells derived from knockin mouse models expressing Parkinson's-associated R1441G or G2019S LRRK2 mutants exhibit increased levels of retarded (phosphorylated) Rab10 compared with control mice when analysed using Phos-tag gels with appropriate small-molecule controls. Complimentary phospho-specific antibodies that recognise pS935 LRRK2 did not mirror this trend, probably because this phosphorylation site is under the complex control of other kinase(s); in the case of the R1441G LRRK2 mutant, this site did not show any change in phosphorylation . It is t32P labelling and Edman degradation) and 32P-based kinase assays [Cell signalling researchers are working in a golden age of chemical biology. Guided by probe and clinical compound candidates emerging from enlightened pharmaceutical companies and supported by commercial enterprises lying at a new interface between industrial and academic research, the traditional lack of access to small molecules is no longer a major impediment to research progress. Arguably, scientists are now challenged with new sets of issues, led by the twin problems of compound target validation  and target specificity (can a compound be employed to rule-in/rule-out a protein in a specific event?). Although phosphorylation site mapping has been revolutionised by proteomics and mass spectrometry workflows, most day-to-day experiments still rely on workhorse approaches for evaluating the activity of protein kinases, usually through indirect analysis of their substrates. Of course, protein phosphorylation can still be established and quantified using classical biochemistry approaches . Instead, electrophoretic retardation simply reflects phosphorylated Rab10 that is readily accessible to the Phos-tag affinity reagent. This technique might therefore also be useful in other scenarios where analysis of kinase and substrate pairings need to be either quantified, or is not well-served by phospho-specific antibodies. Given the findings of Ito et al. examining LRRK2 and Rab10, it will be very interesting to conduct a side-by-side comparison of Phos-tag derived and phospho-specific antibody data for a large panel of kinases and their substrates. This significant undertaking might even change the way we analyse or report the effects of disease-associated mutations on their substrates in the future, as could be the case for LRRK2 in both Parkinsonian models and stratified patients.The study by Ito et al.  also rep kinases . A centrCNS, central nervous system; LRRK2, leucine-rich srepeat kinase 2; MEFs, mouse embryonic fibroblasts; MLi-2,cis-2,6-dimethyl-4-(6-(5-(1-methylcyclopropoxy)-1H-indazol-3-yl)pyrimidin-4-yl)morpholine; MS, mass spectrometry; Phos-tag, Phosphate-binding tag; PINK1, PTEN-induced putative kinase 1; Rab10, Ras-related protein in brain 10.The Author declares that there are no competing interests associated with this manuscript.


Schizophrenia is one of the most devastating heterogeneous psychiatric disorders. The dopamine hypothesis is the longest standing pathoetiologic theory of schizophrenia based on neurochemical evidences of elevated brain striatal dopamine synthesis capacity and increased dopamine release in response to stress. Dopamine and cyclic AMP-regulated phosphoprotein of relative molecular mass 32,000 (DARPP-32) is a cytosolic protein highly enriched in the medium spiny neurons of the neostriatum, considered as the most important integrator between the cortical input and the basal ganglia, and associated with motor control. Accumulating evidences has indicated the involvement of DARPP-32 in the development of schizophrenia; i. DARPP-32 phosphorylation is regulated by several neurotransmitters, including dopamine and glutamate, neurotransmitters implicated in schizophrenia pathogenesis; ii. decrease of both total and phosphorylated DARPP-32 in the prefrontal cortex are observed in schizophrenic animal models; iii. postmortem brain studies indicated decreased expression of DARPP-32 protein in the superior temporal gyrus and dorsolateral prefrontal cortex in patients with schizophrenia; iv. DARPP-32 phosphorylation is increased upon therapy with antipsychotic drugs, such as haloperidol and risperidone which improve behavioral performance in experimental animal models and patients; v. Genetic analysis of the gene coding for DARPP-32 propose an association with schizophrenia. Cumulatively, these findings implicate DARPP-32 protein in schizophrenia and propose it as a potential therapeutic target. Here, we summarize the possible roles of DARPP-32 during the development of schizophrenia and make some recommendations for future research. We propose that DARPP-32 and its interacting proteins may serve as potential therapeutic targets in the treatment of schizophrenia. Schizophrenia is a severe mental disorder with a worldwide lifetime risk of approximately 1% of the population. This schizophrenic syndrome is characterized by cumulative psychiatric deficits including positive symptoms  and negative symptoms  causing impairments of working and semantic memory . The accTo date, many hypotheses have been proposed to explain the disease, including dopamine hypothesis , inflammBrain dopaminergic neuronal pathways are critical in maintaining multiple functions, including movement, rewards and stress . Changesc-fos and NGFI-A [One well-studied target for the phosphorylation activity of dopamine receptors is “Dopamine and cAMP-regulated phosphoprotein” of relative molecular mass 32,000 daltons (DARPP-32). DARPP-32, also known as phosphoprotein phosphatase-1 regulatory subunit 1B (PPP1R1B), was initially found as a substrate of protein kinase A (PKA), activated by dopamine receptors D1R in the brain neostriatum . In the d NGFI-A . The linNeuronal enzymes and their regulators and substrates play a fundamental role in brain function. On one hand, they are under the control of gene transcription, but they are also regulated by post-translational activities, such as phosphorylation and translocation to sub-cellular organelles. DARPP-32 is expressed at low levels prenatally, with the onset at day 14 of gestation in the rostral part of the primordial olfactory tubercle . On day Glutamatergic neurotransmission, including NMDA and other glutamatergic receptors have been involved in schizophrenia . There aIn the mammalian brain, PPP1R1B gene encodes two different transcripts of DARPP-32 protein, a full length DARPP-32 and a truncated DARPP-32. Truncated transcript encodes a protein that lacks the first 36 residues of N-terminus, which contains the phosphorylation site Thr 34 and the PP-1 interacting domain , and theThe activity of DARPP-32 depends on the state of phosphorylation at multiple regulatory sites, including Thr34, Thr75, Ser97 and Ser130. Phosphorylation of DARPP-32 by protein kinases and phosphatases is schematically presented in Figure 2+ and the activity of calcium-dependent protein phosphatase PP2B , leading to dephosphorylation of DARPP-32 [It was also reported that activation of D2R increased the neuronal influx of CaDARPP-32 . ActivatDARPP-32 . BlockinDARPP-32 . AdenosiDARPP-32 .2+/calmodulin-dependent protein kinase II (CaMK II). Activation of these kinases also results in DARPP32-Thr75 phosphorylation [Besides phosphorylation at Thr34 by PKA, DARPP-32 is also phosphorylated by cyclin-dependent kinase 5 (CDK5) at Thr75, converting DARPP-32 into a PKA inhibitor . Activatrylation . Thereforylation .Casein kinase 1 (CK1) and casein kinase 2 (CK2) are additional protein kinases inducing the phosphorylation of DARPP-32. CK2 phosphorylates DARPP-32 at Ser97, while CK1's phosphorylation site is Ser130 . Phospho2+-sensitive PP2A/PR72 [The phosphorylation state of DARPP-32 is determined by both protein kinases and phosphatases. As shown in Figure P2A/PR72 . The hetP2A/PR72 . ActivatP2A/PR72 . Thr34 dP2A/PR72 . ii. PhoP2A/PR72 . Both PPP2A/PR72 . DephospP2A/PR72 . iii. PhP2A/PR72 . HoweverP2A/PR72 . HoweverAs described above, it has long been suggested that brain mesocorticolimbic dopaminergic system dysfunction, expressed by increase in dopamine levels or dopamine receptor sub-types expression levels, are involved in the pathogenesis of schizophrenia. Based on this theory, current antipsychotic drugs act as the pharmacological antagonists of D2R. The schizophrenic behavior may be expressed in a specific animal phenotype and may be analyzed under controlled conditions in the laboratory. For example, amphetamine, by increasing synaptic monoamine levels, can induce psychotic-schizophrenic like symptoms including reduced social behavior , 63, defThe glutamate hypothesis of schizophrenia based on NMDA receptor hypofunction arose from the observation that administration of noncompetitive NMDAR antagonists, such as phencyclidine (PCP) or ketamine, to healthy individuals, induces hallucinations, delusions, negative symptoms and cognitive impairment that mimic schizophrenia, and exacerbates those symptoms in schizophrenic patients . AdminisConsidering findings from schizophrenic animal models, we can conclude that increased phosphorylation of DARPP-32 and its downstream effectors have a causative role in schizophrenia. These changes are associated with the behavioral responses in animals, and can be regarded as a surrogate marker of the positive schizophrenic symptoms . This coIn human patients with schizophrenia, DARPP-32 was significantly decreased in the dorsolateral prefrontal cortex circuit (DLPFC) , 71. MorUnfortunately, there are on this topic inconclusive reports as well. No change in DARPP-32 protein levels were found in schizophrenic prefrontal and cingulate cortex and T lymphocytes of elderly patients with schizophrenia , 76. MutDopamine D2R is the main target of antipsychotic drugs used for schizophrenia therapy. Chronic blockade of either D1R by SCH-23390 or D2R by raclopride does not increase the level of expression of DARPP-32 protein in specific rat brain regions, including striatum, thalamus, hippocampus and frontal cerebral cortical pole . HoweverNurr77 is an immediate-early gene of the nuclear receptor super family, which is highly expressed in the brain prefrontal cortex, dorsal striatum, and nucleus accumbens. Several investigations indicate that Nurr77 mediates biological effects of dopamine receptors blockade or stimulation. Nur77 is also involved in the antipsychotic motor lateral effects of antipsychotic agents , 88. It Neurotrophins, particularly brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF), are crucial mediators involved in the regulation of neuronal survival, proliferation, differentiation, neurodevelopment and maintenance of neurons in the central and peripheral nervous systems –91. The The most extensively single nucleotide polymorphism (SNP) studies of BDNF gene is rs6265, which produces a G/A amino acid substitution  at codon 66  . This fu−/− mice showed delayed and decreased expression of DARPP-32, while BDNF treatment in vivo increases the level of DARPP-32 [Preclinical experiments demonstrated that BDNF elevates the temporal and perhaps spatial expression of DARPP-32 , and BDNDARPP-32 . FurtherDARPP-32 . The inhDARPP-32 .Dysfunction of dopamine system triggers a shift of the balance between the phosphorylation and dephosphorylation of DARPP-32, which is highly associated with abnormal psychiatric behavior. An adjustment of this imbalance is supposed to be beneficial for the treatment of CNS disorders, including schizophrenia . StimulaCurrently, most of the medical translational studies focused on the phosphorylation state of DARPP-32, and they represent a promising direction . On the In conclusion, in the last three decades, scientific investigations have proved that DARPP-32 acts as a major integrator signaling phosphoprotein, activated by a diverse array of neurotransmitters, such as dopamine, glutamate, serotonin, adenosine, and gamma-aminobutyric acid in the CNS . Importa


Lymnaea stagnalis . We studied the effects of dopamine on voltage-dependent Ca2+ current and synaptic vesicle release from the VD4. We report that dopamine inhibits voltage-dependent Ca2+ current in the VD4 by both voltage-dependent and -independent mechanisms. Dopamine also suppresses synaptic vesicle release downstream of activity-dependent Ca2+ influx. Our study demonstrated that dopamine acts through D2 receptors to inhibit interneuron synaptic transmission through both voltage-dependent Ca2+ channel-dependent and -independent pathways. Taken together, these findings expand our understanding of dopamine function and fundamental mechanisms that shape the dynamics of neural circuit.D2 dopamine receptor-mediated suppression of synaptic transmission from interneurons plays a key role in neurobiological functions across species, ranging from respiration to memory formation. In this study, we investigated the mechanisms of D2 receptor-dependent suppression using soma-soma synapse between respiratory interneuron VD4 and LPeD1 in the mollusk  Lymnaea stagnalis, rhythmic DAergic inhibition of respiratory interneuron output is key to mediating aerial respiration behaviour i) was measured using a FURA-2 ratiometric Ca2+ imaging system, as described previously ;,61;ICa [., 1995; ,43. Hereker Cd2+ . We demoICa and direct inhibition of synaptic vesicle release all contribute to DAergic presynaptic inhibition in interneurons. It may be hypothesized that whereas the Gβγ subunit-mediated voltage-dependent inhibition allows for rapid activity-dependent regulation of synaptic transmission, the voltage-independent inhibition of ICa and direct inhibition of vesicle release machinery may allow for more persistent and nuanced inhibition of interneuron synaptic output [L. stagnalis. In the context of the respiratory CPG network, the VD4 controls the muscles that close the pneumostome [2+ channels, may be important for the well-documented hypoxia and learning-induced modulation of CPG activity and aerial respiration behaviour in L. stagnalis [In this study, we provide the first demonstration that inhibition of c output ,51,76,95umostome . Increasumostome . Therefotagnalis ,8.ICa, and voltage-gated Ca2+ channel-independent inhibition of synaptic vesicle release. In future studies, the relative sensitivity and function of each pathway and any potential interactions between them will be further investigated to expand our understanding of the fundamental mechanisms that shape interneuron function and neuronal network dynamics.In this study, we provide the first demonstration that DA suppresses synaptic output of a molluscan respiratory interneuron through both voltage-dependent and -independent inhibition of


Here, we found that Cdk5-p25 phosphorylated tau in vitro at Ser404, Ser235, Thr205 and Ser202 in this order. In contrast in cultured cells, Ser404 was preferentially phosphorylated by Cdk5-p35, whereas Thr205 was not phosphorylated. Ser202 and Ser235 were phosphorylated by endogenous kinases. Tau exhibited ~12 phosphorylation isotypes in COS-7 cells with different combinations of phosphorylation at Thr181, Ser202, Thr231, Ser235 and Ser404. These phosphorylation sites were similar to tau phosphorylated in mouse brains. FTDP-17 tau with a mutation in the C-terminal region had different banding patterns, indicating a different phosphorylation pattern. In particular, it was clear that the R406W mutation causes loss of Ser404 phosphorylation. These results demonstrate the usefulness of the Phos-tag technique in the quantitative analysis of site-specific in vivo phosphorylation of tau and provide detailed information on in situ combinatory phosphorylation of tau.Tau is hyperphosphorylated in the brains of patients with tauopathies, such as Alzheimer’s disease and frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). However, neither the mechanism of hyperphosphorylation nor its contribution to pathogenesis is known. We applied Phos-tag SDS-PAGE, a phosphoaffinity electrophoresis, to the analysis of tau phosphorylation  Tau is a microtubule-associated protein primarily expressed in the axons of neurons. Tau regulates microtubule dynamics and transport of organelles along microtubules in axons124578in vitro1718in vitro and in cells.Cyclin-dependent kinase 5 (Cdk5) is a proline-directed protein kinase, which phosphorylates Ser or Thr residues followed by Pro; i.e., (S/T)P sequences101213in vivo phosphorylation. To address this issue, Kinoshita et al. invented the method Phos-tag sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)in vivo phosphorylation of the p35 Cdk5 activatorin vivo tau phosphorylation, we have recently reported that there is abundant non-phosphorylated tau in mouse and human brainsin vivo analysis of pathologically phosphorylated tau, however, we needed to determine the physiological phosphorylation states in detail. Here, we used the technique in the analysis of tau phosphorylation by Cdk5 in vitro in cultured cells and brain tissues. We identified a number of novel and interesting aspects of the phosphorylation of tau, including FTDP-17 mutant tau, by Cdk5.Phosphorylation of proteins has been studied using radioisotope labeling, mass spectroscopy, and anti-phospho-specific antibodies. These methods are powerful to detect phosphorylation but are difficult to use to quantify in vitro primarily at four sites, Ser202, Thr205, Ser235 and Ser4041718in vitro reaction conditions.Cdk5 phosphorylates tau in vitro Cdk5-phosphorylated tau because its reaction was lost by phosphorylation at Thr205 , and the upper band might have a single phosphorylation at a particular non-Cdk5 site. About half of tau 4A was phosphorylated at the site in COS-7 cells. The minor bands slightly increased in intensity with co-expression of Cdk5-p35  or by Cdk5-p35 in cultured cells . The AlaThese experiments enabled us to assign Ser202, Ser235 and Ser404 to respective tau bands in COS-7 cells. To assign phosphorylation sites completely, however, we needed to identify the one unknown major phosphorylation site found in tau 4A , lane 4.1722From these data, we assigned most phosphorylation sites to each tau band detected in COS-7 cells. There are approximately 12 bands, including minor ones, for tau with different phosphorylation combinations in the absence of Cdk5-p35 , left. T24Cdk5 is overactivated by cleavage of p35 to p25 with calpain in neurons2627FTDP-17 tau is causative for FTDP-17 disease and is hyperphosphorylated in diseased brains6306303218Ser404 was the preferential Cdk5 phosphorylation site . Cdk5 pr34FTDP-17 mutations are distributed throughout tau, although the majority is found in the microtubule-binding (MTB) region6Because the changes in the banding pattern were more apparent during co-expression with Cdk5-p35, we thought it possible that phosphorylation at Cdk5 sites was affected by these C-terminal mutations. To identify which phosphorylation site is affected by these C-terminal mutations, we examined phosphorylation at the major Cdk5 sites in COS-7 cells by two-dimensional phospho-peptide map analysis  and immuCOS-7 cells are a non-neuronal cell line. It is important to determine whether tau in the brain has a phosphorylation profile similar to tau in cultured cells. Because mouse tau is nine amino acids shorter than human tau, and multiple isoforms of tau are expressed in the brain, we decided to analyze the phosphorylation of human tau expressed in mouse brain using the JLPL3 P301L transgenic mouse. It was shown above that P301L tau has the identical phosphorylation profile to tau WT in COS-7 cells . Becausein vitro, in cultured cells and in brain tissue using the Phos-tag SDS-PAGE technique. Cdk5 phosphorylated tau at Ser404, Ser235, Thr205 and Ser202 in this order in vitro. Among these major in vitro phosphorylation sites, Ser202 and Ser235 were phosphorylated by endogenous kinase(s) in COS-7 cells. Tau occurs as ~12 different phosphorylation isotypes in COS-7 cells, with a maximum of five phosphorylation sites, all of which are proline-directed sites. Ser404 was phosphorylated preferentially by Cdk5-p35, but Thr205 was rarely phosphorylated. Mouse brain tau showed some similarity in phosphoisotypes, but not the extent of phosphorylation, to those of tau expressed in cultured cells. The C-terminal mutations of FTDP-17 tau affected the banding pattern of tau in Phos-tag SDS-PAGE. In particular, it was clearly shown that the R406W mutation eliminated phosphorylation at Ser404. These results increase greatly our understanding of physiological and pathological phosphorylation of tau.In this report, we characterized the phosphorylation of tau by Cdk5 in vitro Cdk5 sites1718in vitro by Cdk5-p35 in the order of Ser404, Ser235, Thr205 and Ser202  in the absence of Cdk5 activity  Nevertheless, we have assigned phosphorylation sites in different phosphorylated states in cultured cells. These results demonstrate that the Phos-tag would be a powerful method to characterize Anti-tau Tau5 and AT270 P-T181) were purchased from ThermoFisher Scientific . Anti-human tau Tau-12 was described previously1 were pug for 20 min, the supernatant was used as the brain extract.All animal experiments were performed according to the guidelines for animal experimentation of Tokyo Metropolitan University. The study was approved by the Research Ethics Committee of Tokyo Metropolitan University . All efforts were made to reduce the suffering of animals used. The P301L tau transgenic mouse line (JNPL3) was housed in the animal facility of the Tokyo Metropolitan Institute of Medical Science, as described previously2, 100 mM NaCl, 0.5% (v/v) Nonidet P-40, 0.4 mM Pefabloc, 10 mM NaF, 10 mM β-glycerophosphate, 10 μg/ml leupeptin and 1 mM dithiothreitol on ice for 10 min. After centrifugation at 15,000 × g for 15 min, each supernatant was retained.COS-7 cells were maintained in Dulbecco’s modified Eagle’s medium (Sigma) supplemented with 10% (v/v) fetal bovine serum , 100 U/ml penicillin, and 0.1 mg/ml streptomycin. Cells were transfected with the indicated plasmids using HilyMax transfection regent , according to the manufacturer’s protocol. After transfection for 24 h, the cells were lysed in 20 mM HEPES, pH 7.4, 1 mM MgClEscherichia coli BL21-CodonPlus (DE3)-RP using a Mono S column with an AKTA purifier 2, 0.1% Nonidet P-40, and 1 mM ATP.Cdk5-p25 was purified from Sf9 cells infected by baculovirus encoding Cdk5 and p25, as described previously32P]orthophosphate 24 h after transfection. The cell lysate was boiled in the presence of 0.5 M NaCl, and the heat-stable supernatant was obtained by centrifugation at 100,000 × g for 30 min. Tau was immunoprecipitated using Tau5 antibody, followed by binding to protein G Sepharose , as described previouslyCOS-7 cells were labeled by incubation for 4 h with [2, as described previously20Laemmli’s SDS-PAGE was performed using 12.5% (w/v) polyacrylamide gels, and Phos-tag SDS-PAGE was performed using 7.5% (w/v) polyacrylamide gels containing 50 μM Phos-tag acrylamide  and 150 μM MnClHow to cite this article: Kimura, T. et al. Quantitative and combinatory determination of in situ phosphorylation of tau and its FTDP-17 mutants. Sci. Rep.6, 33479; doi: 10.1038/srep33479 (2016).


Comparison and correlation across the southern North Sea Basin confirms that the glaciation is the equivalent of that during the Late Saalian Drenthe Stadial in The Netherlands. The implications of this correlation are presented. Before the glaciation occurred, the Fenland Basin did not exist. It appears to have been initiated by a subglacial tunnel valley system beneath the Anglian  ice sheet. During the subsequent Hoxnian  interglacial, the sea invaded the drainage system inherited following the glacial retreat. The evolution through the subsequent ca 200 ka Early to Middle Wolstonian substages, the interval between the Hoxnian (Holsteinian) temperate Stage and the Wolstonian glaciation, represents a period during which fluvial and periglacial activity modified the landscape under cold climates, and organic sediments were laid down during a warmer event. Palaeolithic humans were also periodically present during this interval, their artefacts having been reworked by the subsequent glaciation. The deglaciation was followed by re-establishment of the rivers associated with the deposition of Late Wolstonian  gravels and sands, and later, deposits of the Ipswichian interglacial  including freshwater, then estuarine sediments. Subsequent evolution of the basin occurred during the Devensian Stage  under predominantly cold, periglacial conditions.Detailed investigation of landforms and their underlying deposits on the eastern margin of Fenland, East Anglia, demonstrated that they represent a series of glaciofluvial delta-fan and related sediments. Associated with these deposits are glacially dislocated sediments including tills, meltwater and pre-existing fluvial sediments. These ‘Skertchly Line’ deposits occur in the context of a substantial ice lobe that entered Fenland from the N to NE, dammed the streams entering the basin and caused glacial lakes to form in the valleys on the margins. Bulldozing by the ice lobe caused a series of ice-pushed ridges to form at the dynamic margin, especially at the ice maximum and during its retreat phases. Meltwater formed a series of marginal fans that coalesced into marginal accumulations in the SE of the basin. The ice lobe is named the Tottenhill glaciation. Further investigations of the Fenland margin have revealed the extent of the Tottenhill glaciation in the Fenland Basin, to the south and west, in sufficient detail to demonstrate the nature of the Tottenhill ice lobe and the landscape left on deglaciation. The ice lobe is likely to have been prone to surging. This is indicated by the low gradient of the ice lobe, the presence of underlying ductile Mesozoic clays, the evidence of ice-marginal flooding and the presence of arcuate glaciotectonic push moraines. Regional correlation, supported by independent numerical geochronology, indicates that the glaciation occurred  The Fenland receives rivers from the Chalk uplands to the east and south and from the Lower Cretaceous and Jurassic uplands to the west. The rivers discharge into the wide bay of the Wash, itself an important geological feature, and so drain to the North Sea. With extensive areas now slightly above and slightly below the present sea level, enclosing minor islands, the geology is sensitive to changes in sea level. These have affected the nature of sediments deposited in the Fenland in the last half million years, a period covering much sea-level change associated with the growth and decay of Middle and Late Pleistocene ice sheets.ca 11.7 ka, shows the accumulation of freshwater and brackish sediments in a well-marked basin in the Fenland. This complex sequence of peats and inorganic sediments has been described in detail by Godwin )et al.  for the et al. ). Surginet al. , and thiet al. .9.Examination of the bedrock topographic map  figure  shows a Examination of the bedrock surface and geological maps of the floor of the Wash indicate a continuation of the features observed beneath Fenland . The subA third deep valley aligned east–west at the mouth of the Wash  joins thThose rivers that formed following the Anglian deglaciation of the basin would have probably followed courses through the depression controlled by the incompletely filled tunnel valleys. Following the Anglian glaciation, the only available exit to the North Sea towards the northeast, was the Wash. The Wash gap certainly existed in some form at the end of the Anglian glaciation because Hoxnian-age marine deposits occur in the Nar Valley, near Kings Lynn, Norfolk, and at Peterborough, Cambridgeshire, showing that the northern Fenland area was open to the sea at this time, implying an estuary may have existed here (cf. ).Secondly, if the ‘proto-Soar/Bytham’ stream is correctly identified as having adopted a course eastwards through the South Witham Gap in the Jurassic escarpment in the post-Anglian period (cf. ), the riThirdly, because this south Lincolnshire–Wash course must have been overridden by the subsequent ‘Tottenhill ice sheet’ as it advanced into the Fenland, the gravel braidplain could have provided a second significant source of the frequent quartzose pebbles in the Feltwell Formation glacial sediments of the East Fenland margin (cf. §3.3).10.Following the period of uncertainty regarding the timing and extent of glaciation during the late Middle Pleistocene in Britain (cf. §2), recent research has demonstrated that the Fenland was glaciated during the Wolstonian  Stage, intermediate between the Hoxnian and Ipswichian stages , especially from Warren Hill (Three Hills) and Lynford, Suffolk and Tottenhill, Norfolk and the presence of an interglacial palaeosol developed on the deposits' surface ([ca MIS 8), based on OSL ages [The age of the Tottenhill glaciation advance to the Skertchly Line complex in Fenland during the Late Wolstonian Substage  has been independently demonstrated based on litho- and morphostratigraphical relationships in the region, optically stimulated luminescence (OSL) ages from individual localities  glaciation is equated with MIS 6 cf. ,152–155)–155152–1et al. ,15 and Get al.  discusseet al. ,156,157.d et al.  and Gibbd et al.  concluded et al. ,158. Thed et al. –163) becSubsequent ice recession appears to have resulted in separation of the ice sheets, as indicated in geophysical plots ,157, resca 260 ka, i.e. twice the duration of the Weichselian Stage, division of the former into early, middle/mid- and late substages, based broadly on the equivalent MIS cycle divisions; MIS 11b–10, MIS 9–8 and MIS 7–6, respectively, has been proposed by Head & Gibbard [ca 155–126 ka) of the Late (Younger) Saalian in northern Europe . Thope cf. ,167) and andca 26The dating of the Tottenhill glaciation to the Late Wolstonian also indicates that the preceding period, the Early to Middle Wolstonian substages, lasted over 200 ka. While various cold-stage fluvial and related accumulations, as well as the High Lodge interglacial/interstadial event, have been identified within this interval, they generally occur in river valleys entering the basin or on the basin margins, rather than within the basin itself. This implies that erosion was the dominant process shaping the basin throughout the Wolstonian Stage.ca MIS 5e) interglacial sequences, occurring at or close to the modern floodplain level in river valleys throughout the region, confirm that the present drainage system was in place by this time. Given the fact that it is known that Ipswichian sea level was somewhat higher than at present [Subsequent Ipswichian Stage , implieet al. , Lewis [et al.  and Lee  [et al. . The gra [et al. , based o [et al. ), while 11.2.The arrival of glacial ice in the Anglian Stage caused a profound change in the Fenland region landscape evolution. The Anglian glaciation began when an ice lobe advanced across the North Sea Basin from the NE, overriding the pre-existing landscape throughout the north and east of East Anglia. This advance deposited a complex suite of interbedded tills and associated meltwater sediments, collectively termed the North Sea Drift Formation , but it et al. [rinnen of northern Europe, they undoubtedly result from subglacial drainage discharge under high hydrostatic pressure [Throughout East Anglia a series of deep, steep-sided valleys are cut through the Chalk and associated Mesozoic bedrock. These valleys form a radiating pattern, broadly parallel to the dip of the Chalk, that is, they trend west–east in the north and north–south in the south and north–east–south–west in the west of the region. Investigation of these ‘tunnel valleys’, by Woodland , Cox 17, Cox andet al. ,177 amonpressure ,178.Around the Fenland a series of these tunnel valleys occur to the east, southeast and west, broadly parallel to the ice movement of the Lowestoft ice sheet. These valleys were potentially confluent at the northern end of the basin, radiating southwards in fan-like pattern, possibly in response to bedrock strike and/or movement of the British ice lobe. In particular, a substantial northeast-aligned tunnel valley occurs south of Spalding, Lincolnshire . This depression continues ENE beneath the Wash, where it is over 60 m deep and is partially filled by glacial deposits . Beyond,During deglaciation, meltwater rivers and glacial lakes became concentrated in the series of these large channel-like valleys, the alignment of which was determined by the subglacial ‘tunnel valley’ courses, especially where these valleys were only partially infilled by glacial sedimentation. In the Nar Valley ,140 wher11.3.Following deglaciation during the latest Anglian time, local rivers established new courses on the deglaciated terrain. Initial freshwater sedimentation gave way to estuarine/marine deposition as a glacio-eustatic marine transgression entered the valleys. According to Ventris  the mariIn southern Peterborough, on the western side of the Fenland Basin, the Woodston Beds ,181,182 The Hoxnian interglacial marine sequences in the Fenland valleys represent the lateral equivalent of the remarkably parallel Svarte Bank Formation infilling of the Silver Pit tunnel valley, on the North Sea floor ,139, furThe absence of Hoxnian-age freshwater and estuarine/marine deposits from the Fenland, south of a line from the Nar and Wissey valleys interfluve in the east to southern Peterborough, is significant. It implies that the land to the south must have exceeded the maximum marine transgression height, i.e. at least 25–26 m O.D. The occurrence of high ground, capped by gravel of the Ingham  stream and possibly also Lowestoft Formation deposits, forming a WNW–W extension of the interfluve across what is now the Fenland Basin, has already been mentioned. To have acted as an effective barrier, such high ground would have had to extend across the entire Fenland Basin, forming the Hoxnian North Sea coast. The presence of such high ground implies that the southern part of the Fenland Basin did not exist during Hoxnian times.11.4.Climatic deterioration, following the Hoxnian interglacial, returned in the earliest Wolstonian. This cooling, accompanied by glacio-eustatic sea-level fall, and the return of periglacial climates, resulted in rivers extending and adopting courses focused on the Wash area. The lack of deposits preserved from this interval, probably a result of later glacial scouring, makes it difficult to present anything other than a cursory review of events in the Fenland at this time, although detail can be increased by including evidence from the surrounding areas.ca 200 ka period was dominated by cold climates, punctuated by occasional warmer climate events, one such being that represented at High Lodge, Mildenhall, Suffolk, where the High Lodge clayey silts and underlying sands infill one of a group of doline hollows in the Chalk bedrock. This forested cool-climate, temperate, interglacial or more probably interstadial event occurred within the Wolstonian Stage (possibly equated to MIS 7), while the solution hollow in which the deposits accumulated must have developed shortly before, following the melting of permafrost at the end of the preceding cold period, i.e. potentially the Middle Wolstonian Substage  [in situ Palaeolithic flake industry has been recovered associated with lake sediments. Artefacts, including hand-axes, have also been frequently recovered from Feltwell Formation proglacial deposits in the district. Although the occurrence of the artefacts at High Lodge attests to the occupation of the doline and of immediate areas to the west of the Warren Hill ridge by Palaeolithic humans, the general occurrence and distribution of artefacts indicates that the humans were present throughout the Fenland district throughout much of the period from the end of the Hoxnian Stage interglacial to the Late Wolstonian glaciation.What is certain is that during post-Hoxnian/pre-Tottenhill glaciation times a major period of evolution must have occurred in the Fenland Basin, markedly lowering the landscape associated with streams entering Fenland. This o MIS 8) . PalaeolDeposition during this period represents an exception; however, fluvial incision operating in concert with periglacial thermal erosion of the Mesozoic clays flooring the basin being the norm for the Early to Middle Wolstonian interval. An example is the River Lark at Mildenhall. Here, erosion of the Lark Valley (presumably under a cold climate), immediately adjacent to the Warren Hill ridge to the south and west, caused associated recession of the Chalk escarpment eastwards towards the ridge containing the High Lodge doline, causing local landscape levels in the area to be reduced by at least 25 m cf. ,32,183).,183.32,1Elsewhere in the areas marginal to the Fenland, river deposits representing this interval notably occur beyond the Tottenhill glaciation maximum limit. For example, in the Nene Valley the sequences underlying Terrace 3, and abutting the Woodston Beds date from this time, but are separated by an erosional interlude. The gravels underlying the River Great Ouse Terrace 2 surface at Stoke Goldington may also belong to this period .et al. [While additional warm climate events have been claimed to be represented by fluvial deposits, for example in the Peterborough area, e.g. by Langford & Briant  and Langet al. , the equ11.5.Climatic deterioration in the Late Wolstonian followed the temperate event represented at High Lodge (cf. above). During this time  the Fenland region was glaciated by the Tottenhill advance (the deposits referred collectively to the Feltwell Formation). However, before the arrival of the ice lobe, periglacial conditions returned. Evidence for these conditions occurs at the Warren Hill Beech Clump locality where periglacial ‘rubble-chalk’ diamicton slope material, derived by freeze–thaw weathering of the underlying Chalk bedrock, is present. The cryoturbated upper surface of this local diamicton indicates subaerial exposure of the slope of the bedrock ridge .During the Late Wolstonian, a substantial ice lobe advanced down the eastern side of Britain, entered the Fenland and continued to flow and fan outwards broadly towards the east, southeast, south and west. The advancing ice appears to have preferentially followed the Mesozoic clay outcrops towards the south, southwest and southeast, the ice apparently stalling against the rising ground underlain by the more resistant Jurassic limestones and associated rocks in the west and the Chalk in the east. The initial impact of the glacial ice-lobe advance towards its Skertchly Line maximum limit is poorly represented, although it is likely to have disrupted drainage, as well as eroded pre-existing bedrock and Quaternary materials alike. Evidence of this is found in the recycling of pre-existing materials in the glacial deposits. At the Beech Clump site the horizontally bedded sands rest directly on the rubble-chalk diamicton, indicating that they accumulated in a waterbody presumably dammed by the ice as it advanced towards the local hillslope. The combination of a ductile Mesozoic clay subglacial substrate and subglacial flooding would have made the ice lobe prone to surging, a situation important in the subsequent evolution of glacial sediments and landforms in Fenland.Throughout the Fenland area the colour, texture and clast content of the glacial diamicton associated with this advance is predominently yellow-brown to red-brown in colour but it varies across the area, apparently being dependent upon the incorporation of local materials. The earliest glacial sediment recorded is yellow-brown silty clay-rich diamicton interpreted as basal-facies glacial diamicton (lodgement till) at the High Lodge site. Here it is overlain by glaciofluvial deposits. These sediments, together with the silts including the fossil assemblages and artefact industry, have been glaciotectonically dislocated and locally overridden by glacial ice post-depositionally. However, the general paucity of a regional diamicton (till) sheet from this glaciation may, in part, result from the surging behaviour of the ice lobe.Following the advance of a glacial ice lobe into the Fenland Basin, the ice reached the marginal area in the southeast and was probably halted by the rising ground of the chalk hills to the east and south. From the evidence collected, it is likely that the ice advanced locally a few kilometres further towards the east and southeast up the valleys (e.g. the Lark) than the later ridges, to account for the finds of glacial diamicton (till) beneath the overlying sand and gravel accumulation, noted at some sites. This also accounts for the push structures noted above and for the deposition at the Rampart Field and equivalent localities.Deposition of these ice-contact delta-fans (the Skertchly Line), which prograded into proglacial, ice-dammed lake or lakes in immediate contact with the ice lobe  arose frThe glacio-marginal delta-fans consistently indicate a maximum lake-water level of approximately 30–32 m O.D. in Lake Paterson  althoughExtension of the ice lobe further to the southwest and west has, until now, remained less certain, a consequence of later fluvial activity. In the Cam Valley, the 4th Terrace ridge at Stow-cum-Quy and Little Wilbraham and the Travellers' Rest Pit Gravels northwest of Cambridge appear to relate to marginal drainage of the glacier. However, tracing the ridge complexes described above, it is clear that the western lobe reached a maximum position possibly as far as Cottenham, continuing westwards towards Over, northwest to Bluntisham and Huntingdon, thence to the Folksworth interfluve, and to the Yaxley to north of Farcet ridge, reaching southeast of Peterborough. At Peterborough the complex internal structure of the ridge closely mirrors that seen in the eastern Fenland margin localities. It indicates both local ice-front oscillations and meltwater discharge producing marginal fans. Equally, here glaciolacustrine deposits, overlain by deltaic sands and gravels, indicate that the ice-dammed local streams (in this case the River Nene), causing a proglacial lake to form, which was later locally overridden. Finally, the overlying diamicton, rather than originating by subaquatic slide, almost certainly represents a glaciotectonic thrust. It is interesting to note that the substantial depression, occupied by the Whittlesey Mere and Ugg Mere basins, occurs within the semicircular, concave arc of this Yaxley to Wennington ridge complex . From PeDiversion of the streams entering the Fenland Basin at the glacial maximum is repeatedly indicated. Langford  considerFurther evidence of glacial stream diversion, drainage of the local rivers, including the Cam and the Great Ouse, in common with the rivers of the Peterborough district would have been forced to flow marginal to the ice lobe(s) cf. ,135,136),136135,1A consistent picture therefore emerges of a two-lobed ice advance, the margin of which was dynamic in its behaviour, the ice reaching a maximum position zone, yet oscillating over minor distances as it terminated within waterbodies dammed in the valleys entering the basin. The role of the water in potentially causing or amplifying these oscillations is unknown but changes of level during the ‘stillstand phase’ might be expected.Having reached its maximum extent, the ice began to retreat but throughout the period maintained its dynamic activity, the ice front oscillating to generate the series of ice-pushed ridges that are now the Fenland islands and its margin and associated erosion hollows from which the diamicton material was quarried. These hollows may be the origin of the depressions which were to become the meres in the Holocene, all of which are in the marginal area of the glaciation. Once deglaciation began, the ice appears to have continued its oscillatory behaviour, the active ice-front stillstands or minor re-advances punctuating the general ice-lobe retreat. As many as four or possibly five such stillstands are indicated by these ridge complexes, which occur as bedrock/diamicton thrust structures on the Mesozoic clay bedrock, whereas on the Chalk, in the Mildenhall–Newmarket area, the stillstands are marked by ice-contact meltwater stream spreads. Here, possibly as many as four positions are indicated, which together with the push-moraines identified suggest a minimum total of six, or potentially seven or more stillstands.As the ice lobe retreated, it is possible that it fragmented into two sublobes, separated by the north–south aligned ridge complex at March. A similar north–south orientated ridge occurs in the Littleport–Ely area and again might imply further fragmentation of the lobe structure.During this period the Downham Market to Watlington gravel spread, along the northeastern Fenland margin, probably formed as a kame-terrace-like deposit laid down by northwards flowing meltwater marginal to the Tottenhill ice lobe. The altitudinal relationship of this unit to the Tottenhill delta demonstrates that the former spread postdates the latter. This interpretation implies that the kame-terrace almost certainly accumulated during a stillstand phase in the local ice recession and that by this time drainage was aligned northwards towards the North Sea via the Wash gap. This contrasts with the eastward drainage that occurred during the phase represented by the glacial lacustrine deposits at Tottenhill itself.Assuming those rivers continued to flow throughout the period the Tottenhill ice lobe occupied the basin, deglaciation would have been expected to see the rivers reorganizing their courses on the newly deglaciated basin terrain. However, once the ice had retreated sufficiently to the north, the rivers would be expected to have migrated towards the basin centre. Such a process might explain the substantial distribution of NE-trending gravel and sands to the north.In the southeastern Fenland, the substantial meltwater deposits that occur as discrete, laterally separated accumulations appear to have been laid down during progressive ice retreat that began in the east with the ice front retreating westwards in a punctuated series of stillstands. The distribution of these spreads, generally unrelated to the modern rivers yet cutting through the Fenland island ridges, suggests that they accumulated during the deglaciation, and that they are not ‘normal’ fluvial accumulations. That they originate from ice-contact, southward-flowing meltwater, their gradients aligned broadly towards the NE implies that they were deposited at tunnel portals. The fan-like form of the spreads suggests that they represent subaerial ice-front alluvial fans locally confluent fluvial and ice-marginal drainage alignment towards the Fenland Basin centre. This distribution is confirmed by the relationship of the spreads to the underlying bedrock topography, the gravels being aligned towards the central Fenland Valley.Following deglaciation, the rivers re-established the drainage system. Downcutting caused the rivers to lower their valleys, cutting through the pre-existing glacial sediments, and in places, scouring them away completely. On the basis of the bedrock floor map, it appears that the rivers adopted a northward course towards the Wash and the southern North Sea Basin; an alignment possibly encouraged by glacial erosion and glacio-isostatic depression of the region. The rivers were apparently confluent north of the March ‘island’, the Cam system passing northwestwards from Cambridge. The Great Ouse passed to the east and southeast of Chatteris where it was linked to the River Nene. Realignment of the river Witham in the northwest of the basin may also have occurred during this interval.In the Nar Valley this period is represented by emplacement of the cold-climate Wormegay Gravels. As mentioned above, this sequence is important because it demonstrates that downcutting and deposition under cold-climate conditions persisted after the glacial retreat. Moreover, this was followed by a second phase of downcutting, and deposition occurred, during which time the immediately pre-Ipswichian-age Marham Gravel was laid down. Gravel and sand units occur in comparable positions to the Marham unit throughout Fenland at sites including Block Fen, Somersham, Mepal, Maxey, etc.Likewise in the Nene and Welland, the 2nd Terrace gravels and potentially the basal segments of the 1st Terrace accumulations almost certainly represent the same depositional system. Overall, therefore, these coarse arenaceous deposits represent cold-climate accumulations, predating the Ipswichian Stage, i.e. the deglaciation was not immediately followed by interglacial climatic amelioration. Their distribution demonstrates that the interval between the Tottenhill glaciation and the beginning of the last interglacial saw the Fenland Basin achieving its modern form. The deposition of cold-climate gravelly sediments flooring the river valleys, and immediately predating the Ipswichian interglacial accumulations, is a pattern repeated throughout lowland Britain and indeed in neighbouring countries, e.g. The Netherlands ,186.11.6.Carpinus (Ip III). The interglacial sediments vary from sands and fines to highly organic muds, with Jurassic or Cretaceous clays or Late Wolstonian gravels below and Devensian gravels above. A fluvial sequence within a terrace of the Great Ouse Valley shows a sequence of alluviation during the interglacial [In the period of climatic amelioration marking the end of the Wolstonian, the beginning of the Ipswichian saw rising sea levels and deposition of sediments in the river valleys. These are recorded in a large number of sites in the Fenland, and combine to give much detail of the afforestation of the Fenland in the interglacial, with the pollen zones characteristic of the Ipswichian, particularly the succession from mixed oak forest (Ip II) to forest with rglacial .Carpinus zone, when there is an increasing diversity of the non-tree pollen, with higher frequencies of pollen of, inter alia, Gramineae, Compositae and Plantago species. This change is seen in the pollen diagram from the site at Histon Road, Cambridge, underlying the Terrace 3 surface of the Cam Valley [A further significant point of interest in the vegetation succession of the period is a change towards the end of the m Valley  and at Cm Valley , indicatThe response of lowland English rivers to interglacial climatic conditions has been considered by Gibbard & Lewin . They deThe interglacial sediments include both freshwater and brackish/marine deposits. It is possible to demonstrate the presence of a marine transgression into Fenland during the Ipswichian, and its approximate timing. Brackish/marine sediments, identified by analyses of the molluscs, occur at March , SomershThe heights O.D. of these brackish/marine sediments at particular sites are shown in 11.7.The Devensian Stage is represented in the Fenland by gravels forming terraces in the lower reaches of the rivers which drain to the Wash. They extend below the Holocene sediments of the Fenland, and are separated from them by a thin horizon of clay with scattered pebbles in the Peterborough area, the Crowland Bed of Horton  and to tDevensian terraces are present in the valleys of the Welland, Nene, Great Ouse, Cam and Nar rivers, as described above. They are less well developed in the Wissey, Little Ouse and Lark valleys, potentially a consequence of their relation to the local bedrock Chalk aquifers. There is a wealth of information within or related to the gravel sequences, ranging from the rich palaeobotanical contents of fine and organic sediments within the gravels, as at Somersham , to indiThe study of the Devensian aggradations has led to two areas where taphonomy has complicated environmental interpretations. The first is the occurrence of gravelly beds containing fossils possibly reworked, most often occurring in the basal parts of aggradations, where the local landscapes have been swept by the onset of aggradation .There are a considerable number of radiocarbon dates from the Fenland Devensian organic sediments or bones within the gravel aggradations, including the sites at Barnwell Station , Block F12.This summary has demonstrated that the formation of the Fenland Basin was multiple-phased, resulting from the complex interaction of cold and temperate climate processes over the last 500 ka, where easily eroded bedrock provided a preferential pathway for glacial advance during two glaciations.On the basis of the results presented here, it appears that the basin began forming during the Anglian glaciation as a consequence of a subglacial meltwater tunnel valley that breached the Chalk bedrock in the area of the present Wash. A network of tunnel valleys was apparently confluent at the northern end of the basin and radiated southwards in a fan-like pattern. This pattern was possibly dictated by response to bedrock strike and/or the movement of the Anglian ice lobe. This glaciation was marked by southwards advancing ice that overrode the entire region and in the process destroyed the pre-existing NW to SE drainage system.Following deglaciation, a new drainage system began to develop on the hummocky glacial terrain, the partially infilled tunnel valleys acting as conduits guiding the initial drainage pathways. During deglaciation glacial lakes formed in the tunnel valleys, such as the Nar Valley, resulting from the glacially impeded northward-aligned drainage. Once the blockage was removed, normal rivers occupied the valleys but they were quickly overwhelmed by marine transgression into pre-existing valleys, of the Nene at Peterborough and the Nar, near Kings Lynn, the sea reaching a maximum height of approximately 24 m O.D. As the marine transgression did not enter the March and Cam tunnel valley systems, it is assumed that they were completely infilled during the preceding glaciation. South of the line linking the Nar and Wissey valleys interfluve in the east, west to southern Peterborough, no marine deposits are found, this implying that the area was land that reached over 25–26 m O.D., forming the Hoxnian interglacial North Sea coast.Following the glacio-eustatic sea-level regression at the end of Hoxnian, the rivers followed the retreating sea to establish courses across the Fenland, superimposed on marine/glacial surface. Potentially the tunnel valleys could have provided a broad control of course alignments, but this cannot be demonstrated in the main basin area today because of the substantial subsequent erosion. The Early to Middle Wolstonian in lowland Britain was characterized by predominantly cold climates, which saw a long period of periglaciation that made a major impact on the landscape. During this period of almost 200 ka the river systems evolved and matured, valleys including terrace accumulations of coarse fluvial sediments. The rivers generally incised into the pre-existing materials, the predominantly low sea levels allowing the rivers to extend beyond the present Wash into the North Sea Basin beyond.Although the period was dominated by cold climates, warmer climate interludes also occurred. Of particular significance is the High Lodge event, represented by the sediments which occur within one of a suite of doline solution hollows in the Chalk bedrock, the doline having formed at the end of the previous Middle Wolstonian cold interval (i.e. MIS 8). This Late Wolstonian forested cool-climate, interglacial or interstadial event  included the presence of Palaeolithic humans. Humans had been present throughout much of the preceding period from the Hoxnian interglacial, their artefacts being found repeatedly in many of the deposits from the period and reworked subsequently into younger accumulations.The deterioration of the climate during the Late Wolstonian saw a return to periglacial climate with ‘rubble-chalk’ diamicton slope deposits resulting from freeze–thaw processes on exposed surfaces. The arrival of a glacier from NW–N resulted in a substantial ice lobe invading the basin, fanning out towards the margin. The advancing ice appears to have preferentially followed the Mesozoic clay outcrops towards the S, as well as towards the SW and SE, the ice jamming on the rising ground underlain by the more resistant Jurassic limestones and associated rocks in the west and the Chalk in the east. Initial blockage of the drainage system entering Fenland must have occurred but no evidence remains from this time apart from the lacustrine sands overlying the rubble-chalk diamicton at Warren Hill, Suffolk. During the advance the ice excavated materials to expand the lobate form of the basin, the material being largely removed by meltwater recycling. Diamicton from the glaciation is remarkably rare, but pre-existing diamicton inherited from the earlier Anglian glaciation was bulldozed and remobilized by the Tottenhill advance. Glaciotectonism resulted in a series of low, subparallel ridges being pushed up around the margins of the lobe on the western, southern and southeastern margins, whereas in the east the Skerchley Line limit is marked by the deposition of delta-fan and alluvial fan complexes, the former deposited into marginal lakes resulting from damming of the streams entering Fenland from the surrounding unglaciated terrain. This ponding of the rivers is seen not only in the east but also in the Great Ouse Valley west of Huntingdon, in the Nene Valley near Peterborough and in the Welland Valley. Drainage during this phase was potentially either over, under or around the margins of the ice lobe, until the lake in the Lark Valley was encountered. The latter, called Lake Paterson, progressively filled with water until the lake overspilled into the Waveney system, the water then draining into the southern North Sea. Once overspill had occurred, the breach between the Lark and Waveney valleys became progressively lowered, leaving lake sediments on the valley sides above the rivers.The Fenland ice lobe associated with the Tottenhill advance was similar in form and distribution to ice lobes that occurred at the southern limits of the last Laurentide Ice Sheet. The low gradient of the Fenland ice lobe was facilitated by the underlying easily deformable Mesozoic clays at the glacier bed combined with impeded subglacial drainage associated with the ice-marginal lakes as the ice advanced into the Fenland. As with the ice lobes associated with the last Laurentide and the Valdaian Kara Sea ice sheets, the Fenland lobe would have been prone to surging behaviour.Ice-front oscillation, probably arising from periodic surging, produced not only glaciotectonic push-moraines, but also equivalent erosional depressions on their up-glacier sides (e.g. the meres), which today are occupied by Holocene Fenland deposits. As the ice retreated, the ice front remained active so that suites of subparallel pushed ridges mark the basin margins. These ridges comprise predominantly diamicton materials but on the Chalk bedrock the stillstands comprise meltwater sands and gravels, indicating glaciofluvial discharge preferentially from a series of portals in the ice margin. Drainage towards the east was redirected northwards later to form kame-terrace deposits on the northern basin margin in the Downham Market–Nar Valley area, indicating that later in the deglaciation phase drainage ceased to flow southeastwards but was realigned northwards towards the North Sea. This change may have corresponded in part to fragmentation of the glacial lobe into two or three sublobes. Continued ice retreat saw the rivers re-establish their courses towards the centre of the Fenland Basin and exit via what is today the Wash embayment, possibly encouraged by isostatic downwarping of the basin and adjacent North Sea floor.ca 160 ka). The advance is therefore broadly coeval with similar maximum ice-lobe advances in The Netherlands, western Germany and the intervening southern North Sea Basin.Despite unsubstantiated suggestions of older ages, the numerical dating available establishes beyond doubt that the Wolstonian Tottenhill ice-lobe glaciation occurred during the Drenthe Stadial of the Late Wolstonian . Further downcutting saw the emplacement of low-level gravel and sand units in all the major tributary valleys and indeed their extensions in the Fenland itself.Carpinus (hornbeam) in the woodland assemblage. Overbank flooding of the rivers resulted from the progressive infill of channels with inorganic alluviation. This resulted from increased fluvial discharge. Consequently, fluvial sedimentation replaced estuarine conditions once sea levels began to retreat during the latter half of the event, beginning in Substage Ip III. No evidence of human occupation is known from Fenland during this temperate period. Claims that the March Gravels represent littoral deposits laid down during this Ipswichian high sea-level event have been shown to be false, the occurrence of marine fossils in the gravels being a consequence of later reworking of interglacial marine sediments by fluvial processes.The valley floor gravels in many valleys are overlain by fine-grained Ipswichian-age interglacial sediments at sites including Block Fen, Somersham, Pentney, etc. The rivers continued to the southern North Sea via the Wash. This initial freshwater alluviation ceased when marine transgression during Substage Ip II resulting from rising glacio-eustatic sea level resulted in drowning of the river valleys. This transgression is represented by intertidal sediments at Somersham. Temperate forests colonized the valleys and basin interfluve surfaces. Initially this was dominated by mixed oak tree taxa but in the second half of the period increasingly diversity of the non-tree taxa is accompanied by the establishment of The Devensian Stage in Fenland is marked by a return once again to predominant cold climates and periglacial weathering of exposed surfaces. The main river valleys all preserve remnants of terrace accumulations, especially in the Welland, Nene, Great Ouse, Cam and Nar valleys. A wealth of detail is available on the local environments and colonizing biota in these valleys, in particular at Wretton, Norfolk where there is evidence of interstadial conditions. As stated above, the occurrence of reworked marine fossils in the Devensian gravel units is common, e.g. at Somersham and the March area.Throughout the Devensian Stage and in particular following the Middle Devensian during which wide spreads of fluvial gravels and sands were laid down, a series of fluvial realignments occurred, possibly reflecting local capture and degradation of low-level confining bedrock interfluves. The migration of the river Cam from its initial northerly to a progressively more easterly alignment is a good example, but the Great Ouse appears to have undergone a similar course readjustment towards the W or NW. The Devensian also saw a wide dispersal of aeolian cover sand over the landscape east of the Fenland Basin.ca 20 ka by glacial ice of the North Sea ice lobe. The arrival of the ice blocked the Fenland drainage once more and a proglacial Lake Sparks was formed in Fenland at ca 18.5 a C14 BP, with an estimated water level of approximately 5–10 m. Although this lake probably only existed for a few decades, subsequent renewed incision by the rivers followed drainage of the lake once the ice lobe disintegrated under continuing cold, periglacial conditions of the late-glacial. Holocene Fenland deposits then accumulated on the buried valley and interfluves of the basin alike.During the Late Devensian Substage the Wash was invaded at Finally, three general conclusions can be made. The first, that the Fenland Basin results from breakthrough of the Chalk at the site of the Wash, and the excavation along its strike of the Mesozoic clay bedrock. Secondly, that the periglacial thermal erosion of the Mesozoic claylands is central to the evolution of the basin landscape occurring throughout both the Wolstonian and the Devensian cold periods (over 300 ka). Thirdly, it is interesting to note that the human occupation of Fenland during the Holocene is paralleled by its occupation in Palaeolithic times.


Protein kinases are important components of signalling pathways, and kinomes have remarkably expanded in plants. Yet, our knowledge of kinase substrates in plants is scarce, partly because tools to analyse protein phosphorylation dynamically are limited. Here we describe Kinase-Associated Phosphoisoform Assay, a flexible experimental method for directed experiments to study specific kinase-substrate interactions in vivo., which is capable of detecting subtle changes in isoform distribution.The concept is based on the differential phosphoisoform distribution of candidate substrates transiently expressed with or without co-expression of activated kinases. Phosphorylation status of epitope-tagged proteins is subsequently detected by high-resolution capillary isoelectric focusing coupled with nanofluidic immunoassayThe concept is validated by showing phosphorylation of the known mitogen-activated protein kinase (MAPK) substrate, ACS6, by MPK6. Next, we demonstrate that two transcription factors, WUS and AP2, both of which are shown to be master regulators of plant development by extensive genetic studies, exist in multiple isoforms in plant cells and are phosphorylated by activated MAPKs.As plant development flexibly responds to environmental conditions, phosphorylation of developmental regulators by environmentally-activated kinases may participate in linking external cues to developmental regulation. As a counterpart of advances in unbiased screening methods to identify potential protein kinase substrates, such as phosphoproteomics and computational predictions, our results expand the candidate-based experimental toolkit for kinase research and provide an alternative in vivo approach to existing in vitro methodologies.The online version of this article (doi:10.1186/s12870-016-0894-1) contains supplementary material, which is available to authorized users. Arabidopsis and rice four percent of genes encode kinases ATP with bead-bound GST-WUS or GST-WUS-AA as substrates for 30 min at room temperature, and then stopped by the addition of Laemmli SDS buffer. Samples were fractionated by SDS-PAGE. The gel was fixed, stained with Coomassie Blue, dried and analysed by autoradiography.For kinase assays, 300 and 100 ng of in vitro-translated, affinity purified substrate and kinase were used, respectively. As an activity control 10 μg myelin basic protein (MBP) was used as a generic MAPK substrate. The assay was carried out in 20 mM HEPES, pH 7.5, 100 μM ATP, 1 mM DTT, 15 mM MgCl


This study investigated the catch per unit effort and water quality of Lake Kalgwai Jigawa state, Nigeria for the period of 10 month (July 2012‐June 2013). The man hours, gears used, and fish catches of the Lake was determined by assessing the fishermen operating on three major landing sites of the Lake, namely Marke (Site I), Dingare (Site II), and Kalgwai (Site III). Water samples from the landing sites were collected and analyzed. Concentration of fishers ranged from 41 (April) to 51 (September). The highest number of fishing hour was observed in August (64 hr), whereas the lowest was in March (49 hr). The average catch per fisher per day ranged from 107 kg/day (December) to 144 kg/day (August) during the study period. An average of 25 days was spent fishing in each month. The result also indicated similarities in the water qualities of all the three sampling sites per months. Based on the result gotten it was concluded that lake Kalgwa is not over fished and water quality are within recommended ranges for fish production. Per capita consumption in sub‐Saharan Africa is the lowest in all regions and it is the only part of the world where consumption is declining. The main reason for this decline is the leveling off in capture fish production and the ever‐growing population. World Bank  had estiThe poor and uneconomic management of reservoir and lake fisheries is another major issue of concern. Therefore, creation of dams provides an ecosystem for the proliferation of wide range of aquatic organisms including fish thus promoting socio‐economic activities of the surrounding communities Fawole, . HoweverFishes are generally poikilotherms (cold blooded) and therefore their metabolic rates are strongly influenced by the external environment conditions. The thermal tolerance of fish for instance could be lethal, controlling, and directly influencing the responses of fish Fry, . In many22 , Dingare (Site II), and Kalgwai (Site III), respectively. Fish species were randomly sampled and examined at each site fortnightly over a period of 10 months from July 2012 to June 2013 between 6:00 a.m. and 8:00 a.m. Fish identifications as well as measurement of weight and number of fishermen were taken directly from the landing sites according to identification keys provided by Thalwar and Jhingran , FAO 19, OlaosebThe body weight of each specimen was determined was measured in grams (g) using compact balance (Model MP‐600A) sensitive electronic weighing scale. Catch per unit effort was determined using the formula:® software for descriptive and summary statistics and Genstat Discovery Edition 4 for Analysis of Variance. Data were analyzed using one way analysis of variance for a completely randomized design. Some variates were analyzed using analysis of covariance where some dependent factors were found to be significantly different (p < .05). All significantly different means were separated using Fisher's least significant difference of means.Water samples from the landing sites were collected using two liter capacity sampling bottle on monthly basis. The water samples were subjected to the following analysis as soon as possible after collection as described by APHA . Analysi3The result of catch per unit effort is as presented in Tables p < .05) between month, which ranged from 41 to 51 individuals. The highest number 51 fishers were recorded in the month of September, whereas the least value 41 fishers were in April. The highest number of fishing hours of 64 hr was observed in August, whereas the lowest was obtained in March with 49 hr. The average catch per fisher per day ranged from 107 kg/day to 144 kg/day; however, the highest value of 144 kg/day was recorded in December, whereas the least value of 107 kg/day was found in August. The number of fishing days showed no significant difference (p > .05) with similar number of 25 days for each month. The total catch per month differed significantly (p < .05). The highest catch per month was recorded for February with 158,750 kg/month and the lowest value of 111,725 kg/month for April. The total fishing hours varied between 1,225 hr in March to 1,600 hr in August. The mean catch per unit effort 114.11 kg/hr was recorded for September, whereas the least value of 75.23 kg/hr was obtained for August.The summary of mean monthly catch per unit effort of the fishers on the dam is presented in Table p > .05) among the months, the highest value 0.31 ± 0.02 was recorded in August, whereas the least value 0.25 ± 0.01 in April for site 1, the least value of 0.26 ± 0.01 was obtained for site 2 in March and December, whereas the highest record was 0.32 ± 0.02 in July and site 3 had the highest value of 0.32 ± 0.01 in the month of August, whereas the lowest record of 0.25 ± 0.01 was observed in April. The mean monthly PH of all the sites showed no significant difference (p > .05). The result indicated that the highest PH values of 6.60 ± 0.10 and .65.6 ± 0.15 for site 1 and 3 in March and site 2 exhibited in the month of July and December with the value of 6.65 ± 0.15, whereas the least PH values were recorded in October 5.80 ± 0.10 for site 1 and 6.00 ± 0.10 in September for both site 2 and 3. The monthly depth of all the three sites ranged between 4.10 ± 0.10 m and 5.40 ± 0.20 m, with highest depth (5.40 ± 0.20 m) experienced July, whereas least (4.10 ± 0.10 m) in September. The result revealed that on significant difference (p > .05) for mean monthly dissolve oxygen at all the sites, the highest value of 6.35 ± 0.15 was recorded in November, whereas the least value of 5.55 ± 0.15.The mean monthly variation in water quality parameters at the three sites is presented in Tables p > .05) in means values of the surface temperature, transparency, depth, and dissolve oxygen, however, the PH differed significantly (p < .05) across all the three sites. The parameters ranged from 26.70 ± 0.36 to 26.70 ± 0.36 for temperature, transparency 0.28 ± 0.01 to 0.29 ± 0.01, PH 6.17 ± 0.06 to 6.37 ± 0.06, depth 6.37 ± 0.06 to 4.64 ± 0.09, and dissolved oxygen 6.00 ± 0.17 to 6.18 ± 0.17 across the sites.Table 4The catch per unit effort of this study revealed the highest fish catch in February and March which was during the dry months with low rains. Similar trend was observed for the monthly total weight of fish catches. This could be attributed to the high volume of water in the dam during the rainy season and most of the species are dispersed due to the increase in surface area. This agrees with results from Jebba Lake (Halstead, Diversity of gear was observed used by fishermen in this study. The monthly CPUE variation might be linked to the difference in the number of fishers, man hours, and the category of fishing gears used. This is in line with the report of McClanahan, Kaunda‐Arara, and Omukoto , on CPUEMore so, the shallow depth of this study could also be linked to the uniformity of transparency, temperature and dissolve oxygen across the landing sites. The temperature does not seem to be stratified in the Lake, thus there is free mixing of the bottom and surface layers. This vertical mixing of the two layers brings nutrients from the bottom to the surface to enhance primary productivity which accounts for high fish species richness of the Lake. Similar to the findings of this study, Famara  had repoNone declared.


Nrk-null mice suggested the importance of Nrk in proper placental development; however, the molecular mechanism remains unknown. In this study, we demonstrated that differentiated trophoblasts from murine embryonic stem cells (ESCs) endogenously expressed Nrk and that Nrk disruption led to the enhanced proliferation of differentiated trophoblasts. This phenomenon may reflect the overproliferation of trophoblasts that has been reported in enlarged placentas of Nrk-null mice. Furthermore, we demonstrated that AKT phosphorylation at Ser473 was upregulated in Nrk-null trophoblasts and that inhibition of AKT phosphorylation cancelled the enhanced proliferation observed in differentiated Nrk-null trophoblasts. These results indicated that the upregulation of AKT phosphorylation was the possible cause of enhanced proliferation observed in Nrk-null trophoblasts. The upregulation of AKT phosphorylation was also confirmed in enlarged Nrk-null placentas in vivo, suggesting that proper regulation of AKT by Nrk was important for normal placental development. In addition, our detailed analysis on phosphorylation status of AKT isoforms in newly established trophoblast stem cells (TSCs) revealed that different levels of upregulation of AKT phosphorylation were occurred in Nrk-null TSCs depending on AKT isoforms. These results further support the importance of Nrk in proper development of trophoblast lineage cells and indicate the possible application of TSCs for the analysis of differently regulated activation mechanisms of AKT isoforms.Nik-related kinase (Nrk) is a Ser/Thr kinase and was initially discovered as a molecule that was predominantly detected in skeletal muscles during development. A recent study using  The placenta is a unique and complicated organ that plays an essential role in supporting foetal development through its involvement in various functions such as gas exchange, nutrient supply and hormone production during gestation. Because placental defects are associated with many pregnancy-related disorders , 2, a beIgf2, H19, Cdkn1 and Phlda2 malfunctions  that contained the cOmpleteTM LAS 4000 mini imaging system , according to the manufacturer’s instructions. The signal density was analyzed by ImageQuantTM TL software. As needed, membranes were stripped by incubation at 50°C in SDS-based stripping buffer  for 30 min to detect other proteins.Whole-cell extracts (30 μg) or placental extracts (100 μg) were separated using SDS-PAGE with 12% gel and subsequently transferred onto a polyvinylidene fluoride membrane. The membranes were blocked with TBST buffer  that contained 5% skim milk. After washing with TBST, the blocked membranes were incubated overnight at 4°C with a primary antibody in TBST that contained 5% BSA. After washing with TBST, the membranes were incubated for 1 h at room temperature with a secondary antibody in TBST that contained 5% skim milk. After washing with TBST, the protein–antibody complex was detected using the Chemi-Lumi One L  and the ImageQuantFor immunoprecipitation, 1 mg/ml whole-cell extracts were prepared as described above. The 250 μg proteins were incubated for 3 h at 4°C with the indicated antibody under constant rotation. Then Protein A-coated magnetic beads (Bio-Rad) were added and mixed samples were incubated overnight at 4°C under constant rotation. Magnetic beads were washed 3 times with ice-cold RIPA buffer supplemented with phosphatase inhibitors and the binding proteins were eluted using SDS-PAGE loading buffer for western blotting.Nrk-null cells. Because it has been reported that ectopically expressed NRK in culture cells is cleaved by caspases [Nrk-null cells. These anti-NRK antibodies were used at a 1:4,000 dilution for western blotting. The commercially available primary and secondary antibodies used in this study are summarized in Anti-NRK rabbit polyclonal primary antibodies were generated against a synthetic peptide (CGAAGYNGGDVGGNHGAAFN), and then purified with a column immobilized with antigen peptides (Sigma-Aldrich). We confirmed that the newly established polyclonal antibodies are able to detect several proteins in wild-type cells, but not in caspases , the lar6 cells/well in an rhBMP4-supplemented ESF5 medium, and the adherent cell number was counted 1 day after seeding. The cells were treated at 0–500 nM of MK-2206 on day 2 and maintained in this culture condition until day 4 (n = 8). Four days after seeding, the cell number was counted (n = 4) and the proliferation rate was calculated relative to the cell numbers of the untreated day 1 controls. The value of day1 was indicated as 1. The remaining cells (n = 4) were collected together, and western blotting was performed to assess the AKT Ser473 phosphorylation status.The AKT inhibitor MK-2206  was dissolved in DMSO solution, stored at −80°C as a stock solution (5 mM) and then diluted with a culture medium prior to use. Undifferentiated ESCs were seeded on a laminin-coated 24-well plate at 10Wild-type C57BL/6N and ICR mice were purchased from Japan SLC  and were maintained under specific-pathogen free conditions. The mice were housed in plastic cages with wire mesh lids and water bottles , and were put on the open racks. Mice were fed Labo MR Stock diet  and maintained on wood chip bedding . All animals were observed daily for signs of illness or abnormal behavior. All animal experiments were conducted in accordance with the Hokkaido University Guide for the Regulation on Animal Experimentation. All experiments were reviewed and approved by the Animal Care and Use Committee of the Hokkaido University (Permit Number: 10–0114 and 14–0143). All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering. Prior to our experimental endpoint, no mice became severely ill or died. In our experimental endpoint, all mice were euthanized by a high-dose (>120 mg/kg) intraperitoneal administration of sodium pentobarbital.Nrk-null ESCs and 8-cell embryos, followed by transplantation into pseudopregnant ICR females as previously described [We generated chimeric mice by aggregation of escribed . Brieflyescribed . These 8escribed . Mice weE18.5 placentas were fixed in 4% paraformaldehyde in PBS and were processed for paraffin embedding. For histological analysis, the 4-μm paraffin sections were stained with periodic acid-Schiff (PAS) and then counterstained with hematoxylin. For immunohistochemical analysis, the 4-μm paraffin sections were incubated overnight at 4°C with indicated primary antibodies. After incubation with secondary antibodies conjugated with horseradish peroxidase, sections were stained with diaminobenzidine tetrahydrochloride and then counterstained with hematoxylin.t-test. We considered P values of <0.01 to be significant.Data were presented as mean plus SD. Groups were compared using the two-tailed Student’s Nrk in trophoblast proliferation and differentiation, we first attempted to establish an in vitro culture system, wherein we could observe Nrk expression during trophoblast differentiation. As shown in Nrk mRNA expression was not detected in undifferentiated ESCs. After initiating trophoblast differentiation, Nrk mRNA expression was faintly detected on the fourth day of culture and was clearly induced after 8 days of culture. These results indicated that this culture system could be applicable for examining the role of Nrk during ESC differentiation to trophoblasts.To investigate the precise roles of Nrk-mutated EGR-G101 cells by homologous recombination with the gene-targeting vector shown in Nrk gene would disrupt the endogenous Nrk expression. EGR-G101 cells with an Nrk-destructive mutation on the X chromosome were found to be incapable of producing NRK proteins because EGR-G101 cells have an XY genotype. To select Nrk-mutated cells, we assessed the homologous recombination using PCR analysis and then examined the ability of the mutated cells to express Nrk mRNA using RT-PCR analysis. In contrast to the clear induction of Nrk mRNA in wild-type EGR-G101 cells  expressr  expressntiation . Moreoveficiency . These rNrk deficiency affected cell proliferation. After having seeded ESCs, the cell numbers were counted every day until day 4 of culture, and the proliferation rate was calculated relative to the cell numbers on day 1. To avoid cell passaging during a proliferation assay, we thus decided to detect cell proliferation until day 4. Indeed, we confirmed Nrk expression on day 4 in wild-type cells . Calculation of relative densities of phosphorylated vs total AKT revealed 2- to 5-fold increment of AKT Ser473 phosphorylation in Nrk-null trophoblasts as compared with that in wild-type control. We further assessed ERK1/2 phosphorylation and found no significant differences between wild-type and Nrk-null cells during the experiment, although a similar induction of ERK1/2 phosphorylation was observed in both types of cells during trophoblast differentiation. Thus, ERK1/2 phosphorylation may also be associated with trophoblast differentiation but may not be involved in the enhanced trophoblast proliferation that was observed in Nrk-null cells. Altogether, our results strongly suggested that enhanced proliferation observed in Nrk-null trophoblasts was caused by the upregulation of AKT Ser473 phosphorylation.To explore the molecular mechanism of enhanced proliferation that was observed in tivation –17, we d embryos . Next, w embryos , 38 usinNrk-null trophoblasts, we assessed the effect of an AKT-specific allosteric inhibitor, MK-2206, on Nrk-null trophoblast proliferation. MK-2206 binds to AKT, resulting in a decrease in AKT phosphorylation in a non-ATP competitive manner [Nrk-null trophoblasts on day 2, a dose-dependent proliferation inhibition was clearly observed on day 4 , 4 (25%), 4 (25%) and 3 (19%) lines were identified as genotype X¯X, XX, X¯Y or XY, respectively. These results indicated that Nrk deficiency did not affect the establishment efficiency of TSC lines from mouse blastocysts.We then tried to establish TSC lines from the blastocysts obtained from Nrk deficiency on TSCs, samples of undifferentiated TSCs and differentiated TSCs (day 6) were prepared as described in Nrk-null cells and confirmed that there was no big difference in the results between two cell lines with the same genotype. In wild-type cells, Nrk mRNA expression was faintly detected in undifferentiated TSC, and was clearly induced after 6 days of culture for differentiation, whereas Nrk mRNA expression was not detected in Nrk-null cells. TSC marker gene Essrb expression was highly detected in undifferentiated TSC and was decreased in differentiated TSCs (day 6) regardless of the presence or absence of Nrk. Both trophoblast giant cell marker gene Pl2 and spongiotrophoblast cell marker gene Tpbpa were not detected in undifferentiated TSCs, and were induced after 6 days of culture for differentiation regardless of the presence or absence of Nrk. Moreover, cell morphology of undifferentiated TSCs and differentiated TSCs (day 6) were not affected by Nrk deficiency  as well as in differentiated trophoblasts from ESCs (as shown in Nrk-null TSCs (day 6). To evaluate the phosphorylation level of each site, we calculated the relative densities of phosphorylated vs total AKT and confirmed 2.8- and 4.6-fold increase of AKT phosphorylation at Ser473 and Thr308, respectively, in Nrk-null TSCs (Nrk deficiency resulted in upregulation of AKT phosphorylation in newly established TSCs as well as in trophoblasts derived from ESCs.To confirm whether ull TSCs  bottom. Nrk-null TSCs (day 6), immunoprecipitation followed by western blot analysis was performed. As shown in Nrk-null cells compared with those from wild-type cells. The rate of upregulation was 4.7-, 3.1- and 3.3-fold in AKT1, AKT2 and AKT3, respectively. In contrast, threonine phosphorylation was upregulated only in AKT1 (5.4-fold) and AKT3 (2.7-fold) of Nrk-null cells. AKT2 was not phosphorylated at the threonine residue in both wild-type and Nrk-null cells. Antibody-specific immunoprecipitation was properly performed, because equivalent amounts of each AKT isoform were precipitated and detected by the corresponding antibodies.AKT is known to be comprised of three highly homologous isoforms: AKT1, AKT2 and AKT3. Each of these isoforms presents a highly homologous structure and functional redundancy with each other . Though Nrk-null trophoblasts that were differentiated from ESCs. Furthermore, we indicated that AKT was actually hyperphosphorylated in enlarged placentas of Nrk-null mice. Since AKT has been shown to be important for cell growth and survival [Nrk-null mice. This result suggested the presence of unidentified factors that were responsible for abnormal trophoblast proliferation [Akt1-deficient placentas exhibited severe hypotrophy, signifying the importance of AKT1 for normal placental development [In this study, we found that increased phosphorylation of AKT was associated with enhanced proliferation observed in survival , it is rferation . Moreoveelopment . A recenelopment .NRK induces the activation of the JNK pathway , promoteNrk-null trophoblasts, our present results revealed the induction of ERK1/2 phosphorylation in both wild-type and Nrk-null trophoblasts established in this study  with almost equivalent levels between wild-type and Nrk-null genotypes. However, their phosphorylation levels were quite different. Although the mechanism of AKT2-speficic defect in threonine phosphorylation is not clear, we can at least say that Nrk deficiency strengthened complete activation of AKT1 and AKT3 and partial activation of AKT2 in differentiated TSCs (day 6). Though the precise roles of the three AKT isoforms in trophoblasts have not been elucidated, Haslinger et al. reported that all AKT isoforms were detected in human trophoblast cell lines [In addition to the analysis in trophoblasts differentiated from ESCs, in this study, we examined the phosphorylation of AKT in newly established TSC lines from both wild-type and  in vivo . Though  in vivo  and gene in vivo , 20. Bec in vivo –21, it ill lines . They alNrk-regulating AKT phosphorylation in trophoblast proliferation and its possible control of proper placenta formation. These findings provide important implications for our understanding of placental development. Moreover, because controlled trophoblast proliferation and differentiation are essential for placental and foetal development, our experimental systems established in this study could be useful for gaining further insights into disorders that may occur during pregnancy.In conclusion, this was the first study to demonstrate the involvement of S1 Fig(TIF)Click here for additional data file.S2 FigNrk-null cells.Expression of total and phosphorylated JNK showed no significant differences between wild-type and (TIF)Click here for additional data file.S3 Fig(TIF)Click here for additional data file.S4 Fig(TIF)Click here for additional data file.S5 Fig(TIF)Click here for additional data file.S6 Fig(TIF)Click here for additional data file.