The elevated stability of the mutant receptor contributes towards oncogenic potential, preventing termination of signaling by receptor degradation. Here, we show that inhibition of phosphatases leads to a marked increase in phosphorylation of wild-type EGFR and EGFRvIII, indicating that both undergo cyclic rounds of phosphorylation and dephosphorylation on all investigated tyrosine residues, including Tyr1045. Still, we observe elevated stability of the mutant receptor, suggesting phosphorylation as insufficient to cause degradation. Hyperphosphorylation of EGFRvIII was hindered only by EGFR tyrosine kinase inhibitors. Co-immunoprecipitation as well as semi-native Western blotting structural analyses together with functional investigation of EGFRvIII’s phosphorylation following depletion of wild-type EGFR by shRNA or EGF-mediated degradation indicated homodimerization as the predominant quaternary structure of the mutant receptor. Dimers were observed only under non-reducing conditions, suggesting that homodimerization is mediated by covalent bonds. Previous reports indicated cysteine at position 16 to mediate covalent homodimerization. Upon its substitution to serine, we have observed impaired formation of dimers and lower phosphorylation levels of the mutated oncogene. Based on the obtained results we propose that EGFRvIII is predominantly regulated dynamically by phosphatases that counteract the process of trans-phosphorylation occurring within the homodimers.Despite intensive research no therapies targeted against the oncogenic EGFRvIII are present in the clinic. One of the reasons is the elusive nature of the molecular structure and activity of the truncated receptor. The recent publications indicate the EGF-bound wild-type EGFR to  Glioblastoma (GB) is the most common primary brain tumor, characterized by a very high mortality rate. The current first line treatment, a combination of neurosurgery with adjuvant radio- and chemotherapy, extends patients life on average from 4 to 14 months . BesidesDespite intensified research on the structure-function relationship in the context of dimerization and ligand binding by wild-type EGFR (EGFRwt), our understanding of the receptor activation mechanism remains incomplete. This is also true for EGFRvIII protein complex formation and dynamics. Several reports focused on the state in which EGFRvIII is present in the plasma membrane. Chu and colleagues indicated that EGFRvIII is present only as monomers . In contIn this study, we show that EGFRvIII is constitutively active as determined by elevated phosphorylation status compared to the wild-type receptor in the absence of ligand stimulation. This phosphorylation pattern is likely to be a convergence of the kinase activity and regulation by phosphatases, as determined by elevated phosphorylation caused by treatment with pan-phosphatase inhibitors. As EGFR monomers undergo trans-phosphorylation, we exploit phosphorylation status as a readout to study structural and functional aspects of the EGFRvIII dimerization. Using EGFR kinase inhibitors as well as manipulation of the protein structure and expression levels, we determine that EGFRvIII undergoes phosphorylation in a homodimer with no interactions with the wild-type receptor observed. Furthermore, our data indicate that EGFRvIII is prone to generating stable covalent dimers via Cys16, however, a population of transient complexes is likely to exist as well.high cells, which endogenously express full-length and truncated form of EGFR , 3231, 3high cells. Firstly, we have transiently transduced cells with lentivirus containing shRNA targeting the EGFRwt mRNA or a scrambled control  variant of EGFRvIII, which carries a single point mutation  in the ATP-binding pocket that renders the kinase inactive Figure  5, 40]., 40.5, 4As previous reports have indicated the cysteine at position 16 of the mutant receptor to be free, we have investigated whether it might be involved in formation of covalent dimers , 15. To low line, characterized by much smaller fraction of EGFRvIII-positive cells, exogenously expressing either naïve EGFRvIII or the C16S variant [This observation has been confirmed using reducing Western blotting on the DK-MG variant . The latThe aspect of EGFRvIII dimerization was controversial ever since first characterization of the mutant receptor , 13, 14.Secondly, previous reports on the structure-function relationship of EGFRvIII have indicated that the deletion of the protein fragment from the extracellular domain has generated a free cysteine at position 16, that is highly reactive , 45. Thehigh line, with no degradation observed even following 24 h incubation with cycloheximide  was present Figure . This reIn summary, we have shown that EGFRvIII trans-phosphorylates itself in homodimers in a continuous manner. This process is actively counteracted by a single or multiple tyrosine phosphatases, but not by protein degradation, which does not occur despite phosphorylation of key residues, suggesting steric hindrance. Results of a number of experiments investigating direct interaction as well as functional aspect of dimerization have indicated the wild-type receptor not to play a role in the process of EGFRvIII phosphorylation. Combined with the fact that this process is actively suppressed by EGFR TKIs, it can be concluded that EGFRvIII trans-phosphorylates itself in a continuous manner. The free cysteine at position 16 mediates formation of stable, covalent dimers, with small fraction of the EGFRvIII likely to dimerize transiently.high and DK-MGlow lines was described previously [DK-MG cells were purchased from Leibniz-Institut DSMZ  and cultured in RPMI 1640 . Obtaining of U87-MGvIII clone 4.12, DK-MGeviously .AD293 cells were purchased from Agilent Technologies  and cultured in complete DMEM High Glucose . Parental cell line was either transfected or transduced with lentiviral vectors carrying appropriate transgenes (prepared as described below) and positively selected using puromycin .2, 16% O2, 37°C).The media used for culturing of cell lines were supplemented with 10% FBS , 1% penicillin/streptomycin , 0.2% Gentamycin . Cells were cultured under standard cell-culture conditions , according to manufacturer guidelines. Primers used for generation of C16S - 5′CCTGTGGGGCCGAC AGCTATGA3′ and 5′CTCGGACGCTCGAGCCGTGA T3′; KD - 5′CGTCGCTATCGCCGAATTAAGAGAAG C3′ and 5′GGAATTTTAACTTTCTCACCTTCTGGG3′; to insert FLAG at the C-terminal - 5′GATGACGACAAG ACCGGTACCCAGCTTTCTTGTACAAAG3′ and 5′GTC TTTGTAGTCCTCGAGTGCTCCAATAAATTCACTGC 3′. Resulting linear plasmids were phosphorylated with T4PNK , ligated by T4 DNA Ligase  and purified by DpnI digest . Gateway LR Clonase II Enzyme mix  was used to subclone the mutated EGFRvIII cassette to pLV1-puro-DEST or pIRESneo3-DEST vectors.C16S were established by transduction with lentivirus obtained using the LENTI Smart kit  as described previously [Stable cell lines expressing EGFRvIII, EGFRvIIIKD, EGFRvIII-FLAG or EGFRvIIIeviously . HT-1080eviously .5 cells per well, incubated overnight to allow for adhesion and serum starved for subsequent 24 h. Serum free medium containing appropriate inhibitors was used to pre-treat cells for 30 min, unless indicated otherwise, prior to stimulation with 20 ng/mL EGF , 1 mM sodium orthovanadate – NaOVa , phenylarsine oxide – PAO  or pervanadate – pV  for 1 hour and lysed. Following concentrations of inhibitors were used: DMSO , 10 μM erlotinib , 0.5 μM afatinib ; 5 μM gefitinib , 5 μM lapatinib , 300 nM flavopiridol , 5 μM arry380 , 10 μM Nu7441  and 10 μM ruxolitinib .To analyse the effects of pharmacological inhibitors, cells were seeded in 6-well plates at 2.5x10eviously ; using CThe constructs carrying shRNA targeting EGFRwt or scrambled (SCR) control, were generated using the Block-iT U6 RNAi Entry vector kit  and the following primers: WT-shRNA-F 5′CACCGTGAACCCCGAGGG CAAATACACGAATGTATTTGCCCTCGGGGTTCA3′; WT-shRNA-R 5′AAAATGAACCCCGAGGGCAAATA CATTCGTGTATTTGCCCTCGGGGTTCAC3′; Scr-shRNA-F 5′CACCGGGAGGTAAACGTAAGAGATAACGAATTA TCTCTTACGTTTACCTCC3′; Scr-shRNA-R 5′AAAA GGAGGTAAACGTAAGAGATAATTCGTTATCTCTTA CGTTTACCTCCC3′. Generated constructs were cloned into pLV1-puro-DEST vector using Gateway LR reaction, as described above. Plasmids were used to prepare lentiviral vectors and transiently transduce DK-MG cells. Knock down efficiency was determined on mRNA level by quantitative real-time RT-PCR, as described previously .5 cells per well, incubated overnight to allow for adhesion and serum starved for subsequent 24 h. Cells were pre-treated with cycloheximide  for 30 min and stimulated with EGF for 3 h. Cells were then incubated for another 30 min with DMSO or gefitinib prior to 1 h treatment with NaOVa, fixed for immunocytochemistry or lysed for Western blotting.As an alternative approach EGFRwt was depleted as a consequence of ligand binding. DK-MG cells were seeded in 6-well plates at 2.5 × 10Denaturing blots were performed as described previously  using inLysates prepared as for Western blotting were incubated for 4 h at 4°C with anti-FLAG M2 antibody  at 1:50 (v/v) dilution, with agitation. Subsequently, 1:50 of magnetic Protein G coated Dynabeads  was added and incubated over-night with agitation at 4°C. Beads were washed 6x with lysis buffer, boiled in reducing Laemmli buffer and supernatant was loaded onto gel for SDS-PAGE.


Linderane inhibited gluconeogenesis, reduced phosphoenolpyruvate carboxykinase (Pck1) and glucose-6-phosphatase (G6pc) gene expression, and decreased intracellular cAMP concentration and CREB phosphorylation in rat primary hepatocytes under both basal and forskolin-stimulated conditions. In rat primary hepatocytes, it also increased total PDE and PDE3 activity but not PDE4 activity. The suppressive effect of linderane on the cAMP pathway and gluconeogenesis was abolished by the non-specific PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX) and the specific PDE3 inhibitor cilostazol. Linderane indirectly activated PDE3 through extracellular regulated protein kinase 1/2 (ERK1/2) and signal transducer and activator of transcription 3 (STAT3) activation. Linderane improved glucose and lipid metabolism after chronic oral administration in ob/ob mice. Our findings revealed linderane as an indirect PDE3 activator that suppresses gluconeogenesis through cAMP pathway inhibition and has beneficial effects on metabolic syndromes in ob/ob mice. This investigation highlighted the potential for PDE3 activation in the treatment of type 2 diabetes.The role of phosphodiesterase 3 (PDE3), a cyclic AMP (cAMP)-degrading enzyme, in modulating gluconeogenesis remains unknown. Here, linderane, a natural compound, was found to inhibit gluconeogenesis by activating hepatic PDE3 in rat primary hepatocytes. The underlying molecular mechanism and its effects on whole-body glucose and lipid metabolism were investigated. The effect of linderane on gluconeogenesis, cAMP content, phosphorylation of cAMP-response element-binding protein (CREB) and PDE activity were examined in cultured primary hepatocytes and C57BL/6J mice. The precise mechanism by which linderane activates PDE3 and inhibits the cAMP pathway was explored using pharmacological inhibitors. The amelioration of metabolic disorders was observed in  Persistent hyperglycemia, the most notable characteristic of T2DM, contributes to a group of chronic complications and comorbidities . AbnormaPck1  and G6pc , then enhances hepatic gluconeogenesis and finally increases the whole-body blood glucose levels  that share a range of catalytic activities in multiple tissues. In the liver, two types of PDEs, PDE3 and PDE4, are mainly responsible for degrading cAMP . However2O2-induced oxidative damage  mice  were bred at the Shanghai Institute of Materia Medica (SIMM), Chinese Academy of Sciences (CAS), and fed with a high fat diet . All animals were maintained in a 12 h light/12 h darkness cycle with free access to chow and water. Animal experiments were conducted in accordance with guides by Institutional Animal Care and Utilization Committee (IACUC), Shanghai Institute of Materia Medica (SIMM), Chinese Academy of Sciences (CAS). The protocol was approved by IACUC, SIMM, CAS.Male Sprague-Dawley (SD) rats and male C57BL/6J mice were purchased from SLAC Laboratory Animals , and fed with a normal diet . B6.V--1), the liver was perfused with collagenase digestion medium, and the hepatocytes were collected after 100 μm mesh filtration and Percoll  centrifugation. Isolated hepatocytes were plated in collagen-coated 6- or 48-well plates (1 or 0.125 × 106 cells per well) in minimum essential medium (MEM) supplemented with fetal bovine serum , insulin , and dexamethasone  and cultured at 37°C and 5% CO2 in a humidified incubator.Primary hepatocytes were isolated from a male SD rat by Seglen’s two-step technique using a previously described protocol  with modRat primary hepatocytes were plated in collagen-coated plates for 4 h before experiments. Gluconeogenic precursors were added to evaluate the effect of test compounds on hepatic gluconeogenesis. Linderane was bought from Pufei De Biotech . The purity is 98%. It was solved in DMSO and stored at -20°C. The final concentration of DMSO in culture media was 0.1%. For the experiments conducted without pharmacological inhibitors, rat primary hepatocytes were incubated in serum-free, glucose-free Dulbecco’s modified Eagle’s medium (DMEM) supplemented with metformin  or different doses of linderane for 1.5 h, followed by 4 h of incubation in the presence or absence of gluconeogenic substrates  with or without the stimulation of forskolin . For the experiments conducted with pharmacological inhibitors, hepatocytes were pretreated with a specific inhibitor for 0.5 h and then co-treated with metformin (500 μM) or different doses of linderane for 1.5 h. Subsequently, hepatocytes were incubated in the presence of both inhibitors and metformin or linderane with or without gluconeogenic substrates for 4 h. Medium was collected to determine glucose production, which was measured by a glucose assay kit  and normalized to cellular protein concentration.TM RT reagent kit with gDNA eraser  . Target gene mRNA was quantified using SYBR Premix Ex Taq (TaKaRa). All oligonucleotide primers were purchased from Invitrogen, and the sequences are shown in Table 1. Gene expression was normalized against Rn18s (for samples from hepatocytes) or Actb (for samples from liver tissues) control RNA.Total RNA was extracted with TRIzol reagent  according to the manufacturer’s instructions. cDNA was generated by a PrimerScriptThe cAMP level in rat primary hepatocytes or liver was measured using an ELISA kit purchased from Enzo Life Sciences .Protein preparation and western blot analysis were performed as described previously . BrieflyPhosphodiesterase activity was detected by a PDE activity assay kit according to the manufacturer’s instructions  based on the modified Malachite Green method. The cAMP hydrolyzing activity measured in this method represents the activity of both PDEs and nucleotide cleavage enzymes like 5′-nucleotidase . Purifie-1) or vehicle  was administered orally to overnight fasted C57BL/6J mice . After anesthesia , livers were dissected 2 h later for PDE activity, cAMP measurement, and western blot analysis.Linderane (50 mg kg-1) or vehicle (0.25% CMC-Na) was administered orally twice daily to male ob/ob mice (6- to 7-weeks-old) for 20 days. The effect of linderane on metabolic abnormalities was investigated. Briefly, male ob/ob mice were assigned to two groups based on blood glucose level and body weight (n = 8). Linderane (50 mg kg-1) or vehicle  was administered twice daily by oral gavage for 20 days. The random-fed and fast blood glucose levels were measured at day 8, 12, 16, and 20 by an ACCU-CHEK Advantage II glucose monitor. Body weight was detected regularly throughout the whole treatment. On the last day, the mice were anesthetized by an intraperitoneal injection of sodium pentobarbital (50 mg kg-1) after 6 h of fasting, and blood samples were collected. The liver was dissected, weighed, and stored at -80°C. Serum triacylglycerol (TG) was determined by commercial kits purchased from Zhejiang Dongou Diagnostics Co., Ltd. . HbA1c was measured using kits from Roche Diagnostics GmbH . Hepatic triglycerides were extracted by a heptane-isopropanol-Tween mixture (3:2:0.01 by volume) and determined with the commercial kits mentioned above.Linderane , with 10 and 20 μM linderane resulting in a decrease of 39.8 and 65.6%, respectively. Linderane at 20 μM showed a comparable effect with 500 μM metformin under basal conditions. Forskolin, an AC activator, significantly stimulated gluconeogenesis in primary hepatocytes. Linderane exerted an inhibitory effect on forskolin-stimulated gluconeogenesis, with 10 and 20 μM linderane causing a reduction by 44.1 and 71.7%, respectively, which were similar to the effect of linderane under basal conditions . Moreover, 20 μM linderane significantly decreased the mRNA expression levels of Pck1 and G6pc, two key gluconeogenic genes, under both basal and forskolin-stimulated state .The structure of linderane, a natural product, is shown in Figure 2A). The inhibitory effect of 20 μM linderane on cAMP content appeared within 30 min and continued up to 240 min . Reduction in forskolin-induced cAMP accumulation was also observed after linderane treatment. Linderane at 10 and 20 μM decreased cAMP levels by 35.0 and 44.1%, respectively . This effect was similar to that under basal conditions. Correspondingly, the phosphorylation of CREB was suppressed by linderane under both basal and forskolin-stimulated conditions .Intracellular cAMP content and CREB phosphorylation were measured in rat primary hepatocytes after incubation with linderane. Linderane dose- and time-dependently decreased the cAMP concentration in hepatocytes. Treatment with 10 and 20 μM linderane for 2 h reduced cAMP concentration by 38.5 and 47.2%, respectively. Furthermore, 20 μM linderane presented a comparable potency with 500 μM metformin , whereas PDE4 activity was not altered . The data indicated that linderane activated PDE3 in hepatocytes. We further assessed the direct effect of linderane on PDE activity using purified PDE enzymes. Linderane did not alter the activity of total PDEs , PDE3 , and PDE4 , suggesting that linderane activated PDE3 in an indirect way.We detected the effect of linderane on PDE activity in primary rat hepatocytes. As shown in Figures 4A,B). Moreover, the gluconeogenesis inhibition caused by 10 μM linderane was fully blocked, and the reduction in gluconeogenesis caused by 20 μM linderane was also significantly diminished . A specific PDE3 inhibitor (cilostazol) and a specific PDE4 inhibitor (roflumilast) were applied in the following experiments. As shown in Figures 4D,E, the reduction in cAMP accumulation and CREB phosphorylation induced by 10 or 20 μM linderane was completely blocked by cilostazol but not roflumilast. Moreover, the inhibitory effect of linderane on gluconeogenesis was also significantly attenuated in the presence of cilostazol but not roflumilast . These data suggested that linderane suppressed hepatic gluconeogenesis mainly through PDE3 activation.To identify whether linderane inhibited gluconeogenesis through PDE activation and cAMP suppression, IBMX was used as a pan-PDE inhibitor. In the presence of 50 μM IBMX, the reduction in cAMP accumulation and CREB phosphorylation induced by 10 or 20 μM linderane was abolished . Both the ERK1/2 inhibitor SCH772984 (2 μM) and MEK inhibitor U0126 (10 μM) abrogated the effect of linderane on PDE3 activity and cAMP accumulation . Meanwhile, the reduction in gluconeogenesis induced by linderane was significantly restored . The ERK1/2 inhibitor and a STAT3 inhibitor Stattic (10 μM) attenuated the STAT3 phosphorylation by linderane . Furthermore, in the presence of Stattic, the majority effect of linderane on PDE3 activity  and gluconeogenesis  was abolished.Linderane increased the ERK1/2 and STAT3 phosphorylation in rat primary hepatocytes (in vivo effect of linderane (50 mg kg-1) on hepatic PDE activity and the cAMP pathway was evaluated in C57BL/6J mice. The total PDE activity in the liver was elevated by 33.7% after administration of linderane . Correspondingly, linderane treatment reduced the cAMP concentration  and CREB phosphorylation level in the liver . In addition, linderane treatment increased the phosphorylation of ERK1/2 and STAT3 . These data showed that linderane could activate hepatic PDEs through ERK/STAT3 and thus suppress cAMP signaling pathway in vivo.The -1, twice a day) was evaluated in male ob/ob mice. Treatment with linderane significantly decreased random-fed and fast blood glucose levels with a reduction rate of 49.4 and 30.2%, respectively, after 20 days of treatment . The long-term glycemic indicator HbA1c tended to be reduced by linderane treatment  . Furthermore, serum triglyceride levels and hepatic triglyceride contents were significantly decreased by 26.9 and 28.5%, respectively . The body weight did not change during the treatment . Linderane also caused a 42.2 and 42.6% decrease in the mRNA expression levels of hepatic Pck1 and G6pc, respectively . Long-term treatment with linderane increased the total PDE activity in the liver by 28.3% (p = 0.081)  and decreased cAMP concentration by 22.6% . In addition, linderane increased ERK1/2 and STAT3 phosphorylation and decreased CREB phosphorylation  in the liver.The chronic glucose-lowering potential of linderane  in mice caused excessive cAMP accumulation in liver, which resulted in abnormal gluconeogenic gene expression and systemic glucose dysregulation . Howeverin vivo study. A single oral administration of linderane in C57 mice increased the phosphorylation of ERK1/2 and STAT3 in the liver, which led to the elevation of PDE activity and suppression of cAMP/CREB pathway. Taken together, our results indicated that linderane activated PDE3 through the ERK/STAT3 pathway, which provided more detailed evidence for research on hepatic PDE3 regulation.Given that linderane did not increase PDE3 activity directly, the upstream regulators were investigated. To our knowledge, few studies so far elucidated the upstream molecular regulation of PDE3 in the liver, especially PDE3-dependent gluconeogenesis inhibition. An inorganic compound, orthovanadate, was reported to stimulate PDE3 through p44 MAPK in rat primary hepatocytes . Further1c level, which acts as an important glycemic indicator in blood glucose control. Apart from glucose metabolism, linderane could reduce serum and hepatic triglyceride levels, which might occur secondarily to hyperglycemia correction during long-term treatment. Overall, we demonstrated that hepatic PDE activation by linderane could significantly suppress gluconeogenesis and improve blood glucose and lipid metabolism in the diabetic ob/ob mouse model.In diabetic patients, aberrant hepatic gluconeogenesis during fasting disrupts the whole-body glucose balance and contributes to persistent hyperglycemia. This effect is at least partly triggered by abnormal enhancement in the cAMP/PKA/CREB pathway . Chronicob/ob mice. These findings highlighted the prospect of hepatic PDE3 for maintaining whole-body energy homeostasis and provided further information for the development of hepatic PDE3 as a potential target for T2DM therapeutics.We presented the beneficial effect of linderane on diabetic disorders and its underlying molecular mechanism. Linderane indirectly activated PDE3 through ERK/STAT3 activation and subsequently suppressed the cAMP/PKA/CREB pathway, leading to gluconeogenesis inhibition in primary hepatocytes. Chronic administration of linderane decreased blood glucose levels in YL, JS, and SH designed the research. WX, YY, YF, TX, and SH performed the research. WX, SH, JS, and YL analyzed and interpreted the data. WX, SH, and YL wrote the paper. All authors gave their final approval of the version to be published.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The overall crystal structure adopting the Ca14AlSb11-type can be described as a pack of four different types of the spiral-shaped one-dimensional octahedra chains with various turning radii, each of which is formed by the distorted ((Yb/Ca)Sb6) octahedra. Four symmetrically-independent cationic sites contain mixed occupations of Yb2+ and Ca2+ with different mixing ratios and display a particular site preference by two cationic elements. Two hypothetical structural models of Yb4Ca10AlSb11 with different cationic arrangements were designed and exploited to study the details of site and bond energies. QVAL values provided the rationale for the observed site preference based on the electronegativity of each atom. Density of states (DOS) curves indicated a semiconducting property of the title compounds, and crystal orbital Hamilton population (COHP) plots explained individual chemical bonding between components. Thermal conductivity measurement was performed for Yb8.42(4)Ca5.58AlSb11, and the result was compared to compounds without mixed cations.Four quaternary Zintl phases with mixed-cations in the Yb TB-LMTO calculations using two hypothetical structural models of Yb4Ca10AlSb11 with different cationic arrangements proved that the overall stability of a crystal structure should be attributed to the site energies of four Sb sites surrounding the M2 sites. QVAL values for each atomic site in a model structure proved that the more electronegative Yb atom should prefer the M2 site with the largest QVAL value, whereas the more electropositive Ca atom should prefer the M3 site. In addition, our theoretical calculations proved that the electronic factor based on the QVAL value criterion can be the critical factor for the site preference when there was a small size difference between the substituting and the host elements. The overall DOS curves indicated a semiconducting property of the title phase with a small band-gap, and the Sb-Al and M-Sb COHP curves explained the overall stability of bonding interactions in the given structural type. The Ca substitution for Yb was insignificant to lower the overall thermal conductivity in Yb8.42(2)Ca5.58AlSb11, but the measured value was still comparable to those of previously reported compounds in the Ybx14-CaxMT11  system.Four quaternary Zintl phases in the Yb


Biochemical Journal, Alessi and colleagues teamed up with clinical colleagues and collaborators at the Michael J. Fox Foundation (MJFF) for Parkinson's research to develop, and validate, a panel of exquisitely sensitive phospho-specific Rab antibodies. Of particular interest, the monoclonal antibody-designated MJFF-pRAB10 detects phosphorylated Rab 10 on Thr73 in a variety of cells, brain extracts, PD-derived samples and human neutrophils, the latter representing a previously unrecognised biological resource for LRRK2 signalling analysis. In the future, these antibodies could become universal resources in the fight to understand and quantify connections between LRRK2 and Rab proteins, including those associated with clinical PD.The addition of phosphate groups to substrates allows protein kinases to regulate a myriad of biological processes, and contextual analysis of protein-bound phosphate is important for understanding how kinases contribute to physiology and disease. Leucine-rich repeat kinase 2 (LRRK2) is a Ser/Thr kinase linked to familial and sporadic cases of Parkinson's disease (PD). Recent work established that multiple Rab GTPases are physiological substrates of LRRK2, with Rab10 in particular emerging as a human substrate whose site-specific phosphorylation mirrors hyperactive LRRK2 lesions associated with PD. However, current assays to quantify Rab10 phosphorylation are expensive, time-consuming and technically challenging. In back-to-back studies reported in the  Parkinson's disease (PD) affects ∼1 in 100 people over the age of 60, with the number of sufferers predicted to increase to >10 million in the world's most populated nations by 2030 .In the genetic context, ∼20 genes have been directly linked to PD , althougTo establish a clear route to the clinic, phosphorylated LRRK2 substrates have been sought as potential biomarkers to monitor ‘on-target’ LRRK2 drug engagement and clinical responses. However, until recently, a lack of validated phosphorylated substrates represented a general roadblock in the field. Works from several groups have established that CNS-active, target-validated small molecule LRRK2 kinase inhibitors have the potential to interfere with LRRK2-based signalling in PD ,17. ThisRecent mass spectrometry-based phosphoproteomics uncovered multiple Rab GTPases as direct physiological substrates of LRRK2 ,13,19. TThe important discovery — human LRRK2 directly phosphorylates a wide variety of Rab proteins, coupled with the recent finding that Rab29 is itself a Golgi LRRK2 activator  — opens Biochemical Journal [Now, in a major follow-up to these studies reported in back-to-back papers published in the  Journal ,24, Ales Journal . Importa Journal ,24. Sinc Journal ,23. As sclone-1, which is superior to all previously described Rab10 antibodies in terms of specificity and sensitivity [Initially, the authors focused on raising peptide-based antibodies in a variety of immunogenic hosts, with the common goal of recognising specific phosphorylated Rab proteins. Readers are referred to Tables 1 and 2 of the Lis paper  for detasitivity . Indeed,ex vivo, using Rab 10 phosphorylation as an LRRK2 biomarker in neutrophils isolated from a variety of human subjects [A biological role for LRRK2 in the immune system has previously been described , includisubjects . It is lsubjects . As a prmust be prepared in the presence of an irreversible serine protease inhibitor such as diisopropylfluorophosphate (DIFP). Under these conditions, endogenous Rab 10 Thr35 phosphorylation can also be readily detected in neutrophils by immunoblotting [Finally, in an experiment to delight biochemical aficionados, Fan et al. demonstrate that to isolate, and quantify, LRRK2 and Rab10 from the protease-rich neutrophil environment, an extract blotting , with sublotting . MoreoveSeveral predicted impacts of these new studies are emphasised. The first is the creation of a unique, and very wide-ranging, consolidated set of pan and specific Rab antibodies that can be exploited, and perhaps refined, by pertinent researchers worldwide. They include polyclonal and monoclonal antibody reagents, the latter available for a prolonged period of time in large amounts, an important consideration for future translational appraisal. Of equal importance, many of these reagents are, or will soon be, available either commercially or collaboratively. This published panel ,23,24,35These studies represent the latest outputs from a long-term collaboration between basic and clinical LRKK2 scientists, and the MJFF charity created and inspired by Michael J. Fox, whose memorable time-travelling experiences alongside inventor Christopher Lloyd in the ‘Back to the Future’ films influenced generations of (somewhat less ambitious) research scientists. Looking forward to the future, we can safely predict that this collection of validated antibodies, many of which bear his name, are likely to have important roles in basic and clinical settings, including the specific analysis of Rab phosphorylation in PD patients.


Moreover, this research demonstrates the value of palaeoclimate records in pristine environments for supporting and extending instrumental records. Our results reinforce and extend observations from other sites that the high Arctic has already undergone rapid ecological changes in response to on-going climate change, and will continue to do so in the future.The high Arctic is the fastest warming region on Earth, evidenced by extreme near-surface temperature increase in non-summer seasons, recent rapid sea ice decline and permafrost melting since the early 1990’s. Understanding the impact of climate change on the sensitive Arctic ecosystem to climate change has so far been hampered by the lack of time-constrained, high-resolution records and by implicit climate data analyses. Here, we show evidence of sharp growth in freshwater green algae as well as distinct diatom assemblage changes since ~1995, retrieved from a high-Arctic (80 °N) lake sediment record on Barentsøya . The proxy record approaches an annual to biennial resolution. Combining remote sensing and  Atmospheric warming leads to, and is enhanced by, concurrent changes in other Arctic climate components3, most importantly sea ice loss. Less sea ice leads to a lower surface albedo, but also promotes heat trapping clouds and enhances atmospheric moisture4. Exceeding climate model predictions5, minimum (September) sea ice extent in the Arctic has decreased at a rate of 13.4% per decade6, with progressively thinner, perennial sea ice8.Over the past three decades, atmospheric warming has accelerated at a fast rate, disproportionally affecting the high Arctic. In the period 2000–2016, high Arctic (70–90°N) near-surface temperatures exceeded those of 1961–1990 by 1.8 °C10. Additional controls on lake ice and snow cover are precipitation, which determines the thickness of the lake snow cover, and surface winds, which drive vertical mixing of the lake water column.A similar melt-albedo feedback exists on the scale of Arctic lakes: higher temperatures enhance spring snowmelt, melting snow cover and exposing the lake ice cover underneath earlier in summer. The darker ice absorbs a larger fraction of the solar radiation, which further promotes melting11, they provide archives of biotic change in response to climate change without being obscured by other stressors12. The recent, rapid climate change in the Arctic has modified ecosystem functioning and caused ecosystem shifts in high-latitude lakes15. A longer ice-free season, extending the growing season for algae and other organisms, potentially leads to increased primary production and taxonomic shifts as new habitats become available15. As a result, ecological regimes may shift16 as climate related thresholds are exceeded14.As Arctic lakes are still among the least impacted by human pollution17, as well as an uneven distribution of studied locations14, e.g. resulting from inaccessibility of many high Arctic areas. In addition, ecological and climate change are generally only implicitly linked.Yet, it is challenging to accurately determine the timing and geographical extent of ecological change in the high Arctic because of the generally low sedimentation rates and resulting low temporal resolution18, and sea ice coverage in Storfjorden and Freemansundet is much more extensive year-round.Situated in the high Arctic, the Svalbard archipelago crosses several climate zones. West Svalbard is characterised by a remarkably mild climate for its near-polar location. The proximity of the relatively warm West Spitsbergen current prevents sea ice formation for most of the year. Towards the East and North, the Atlantic waters lose influence, and East Svalbard climate is increasingly controlled by the Barents Sea. Annual mean temperatures on East Spitsbergen, Edgeøya, and Barentsøya Fig.  are abou20, whereas precipitation only shows small upward trends with substantial observational uncertainties21. In the longest available temperature record on Svalbard (Longyearbyen since 192121), all years since 1989 were warmer than the 20th century average, with 2010–2015 annual mean temperatures ~3.5 °C higher. Warming is even more pronounced in non-summer seasons18, due to the earlier open water exposure in spring and delayed sea ice cover in autumn22. As a result of this ongoing warming, most Svalbard glaciers and ice caps show signs of substantial thinning23. In the array of past anomalously warm winter seasons, Svalbard has experienced rapid permafrost thawing24 and several extreme weather events, such as winter rainfall, landslides, and wet snow avalanches. Although no long-term observations are available in the east and north, atmospheric reanalyses suggest additional warming in these regions18.By far the highest density of climate records is found on the more accessible west coasts of Spitsbergen. All of these show a strong temperature increase since the 1990’s26 in line with lakes in other Arctic regions14. Results from Edgeøya and Barentsøya in East Svalbard are scarce, not only for accessibility reasons, but also because the geology, consisting of mainly easily erodible sedimentary rocks, led to the absence of suitable lake basins.The majority of ecological studies on Svalbard, which has focussed on lakes in the western region, suggests that western Svalbard has undergone ecological regime shifts under changing climatePediastrum  and pollen concentrations, and from Pediastrum and diatom percent relative abundance data. While Pediastrum and pollen data approach an annual to biennial resolution, the resolution of the diatom data is lower.Here we present a rare, ~100 year long East Svalbard sediment record from a small coastal lake , ~150 m wide (west-east)). Ecological change throughout the record was derived from Pediastrum enables the reconstruction of past water quality, habitat, catchment processes and lacustrine ecosystems11, providing complementary insight in ecological response to high Arctic climate change. Our freshwater ecological record is the first on remote East Svalbard, where direct anthropogenic impact is negligible. East Svalbard climate is colder than that of West Svalbard, and therefore more typical for the high Arctic as a whole. Furthermore, we link our ecological records to temperature time series reconstructed from nearby meteorological stations and to sea ice cover data retrieved through remote sensing to resolve any link between climate and ecological change.The combined use of diatom valves and Pediastrum and pollen data were obtained from the past ~100 years. Pediastrum and pollen concentrations as well as percent relative abundance data of diatoms, Pediastrum and pollen have been used. The proxy record approaches an annual to biennial resolution (see Methods). Chronology is based on radiocarbon dating (14C) and on 210Pb, 226Ra and 137Cs isotopes (Bq/kg) .In August 2015, a 47 cm long sediment core was recovered from a small, shallow coastal lake  dominates the assemblage, with ~20–40%. As a generalist, opportunistic species, S. pinnata can compete well under harsh conditions, such as prolonged ice cover, and is one of the most commonly observed species in the high Arctic in shallow lakes and ponds27. Two markedly significant biotic compositional changes are observed throughout the record, leading to a more complex assemblage. Diatoma tenuis, Navicula pupula, Cymbella reichardtii and Planothidium oestrupii show higher percentages from ~1950 onward, where Diatoma tenuis and Planothidium oestrupii profoundly increase from ~1995 onward. On the other hand, Hippodonta hungarica and Fragilaria capucina show a declining trend throughout the record  is observed from ~1900 to ~1995. From 1995 until 2015, Pediastrum concentrations increase even more rapidly, showing an additional tenfold increase, towards ~200,000 coenobia per gram. Moreover, Pediastrum concentrations display a more erratic pattern from ~1995 onwards, with several outliers characterising the record  are low Fig. , resultiLoss on ignition (LOI) values show a gradual increase in organic matter percentages of the sediments over the past century. Until ~1950, LOI is ~25 to 30%, between ~1950 and ~1995 LOI is ~35%. Until 2015, LOI values increase to ~40%  to show that sea ice cover around Svalbard shows signs of strong decline during the period 1979 to 2015. In the first ten years with full data coverage (1988–1997), while the ocean west of Svalbard was sea ice free for the entire year, sea ice cover over East Svalbard exceeded ~60% in most of the winter and spring (until Day of Year (DOY) 150, Supplementary Fig. ion Fig.  and S5, 2 = 0.57, p < 0.001). Ignoring local climate processes, such as the positive feedback loop between reduced ice coverage and temperature, as well as the stronger atmospheric warming in this region compared to the locations of the stations used in the reconstruction, this number should be considered a lower bound.Situated nearby the ocean, we expect that local temperature at the site increases in concert with the lengthening sea ice free season. In absence of long-term local temperature observations, we opted to reconstruct local temperature using a stacked Svalbard daily temperature record from 1960–2015, considering local climate conditions at the core site (see Methods). As a result of increasing air temperatures .P. oestrupii, C. reichardtii and N. pupula) are not particularly associated with a specific pH29, eliminating changed lake pH as the principal driver of the observed shifts. In addition, the absence of planktonic diatom taxa in the record suggests that the lake remained shallow throughout its deposition, indicating that lake level fluctuations are unlikely to have caused major changes in the diatom assemblage.Not all of these drivers are equally likely to have caused the observed changes. Overall, the diatom taxa that increase throughout the record . This would consequently result in a more ecologically complex and diverse diatom assemblage, like the assemblage observed in this study. A similar response of diatom assemblages to longer growing seasons was suggested by other studies in the Arctic32. Longer growing seasons would likely also affect ecology outside the lake by extending flowering periods, resulting in increased pollen concentrations in the lake sediments. We therefore conclude that the observed ecological patterns in this study are most likely driven by a combination of higher temperatures, reduced ice cover periods and longer growing seasons, affecting ecology through the mechanisms described above.The more recent increase of et al.25 showed that atmospheric anthropogenic nitrogen deposition notably increased recently in four lakes in West Svalbard, consequently affecting the ecosystem. This increased nitrogen availability25, possibly aggravated by growing populations of birds in the catchment, like it is the case in West Svalbard33, could overshadow the true climate impact on pristine Arctic lakes such as Andsjøen. Yet, in contrast to studies on lakes in western Svalbard25, we do not observe signs for increased nitrogen availability in the lake. Unlike Holmgren et al.25, we do not observe a pronounced increase in diatom valves over recent years. Moreover, the diatom assemblage reflects oligotrophic to mesotrophic conditions35, and the taxa that increase throughout the record  are not associated with higher nutrient levels and thus eutrophication35. Finally, although P. boryanum var. boryanum dominates the record and is associated with eutrophic waters28, this species becomes less dominant in recent years. A gradual shift towards P. orientale, a species often associated with clear waters28, is even observed throughout the record. We therefore have no reason to assume that increased eutrophication or nitrogen availability in the lake played an important role in the observed recent ecological changes in Andsjøen the way it may have played a role in West Svalbard.Besides climate change and climate change related drivers for ecological change, other mechanisms have been suggested to explain recent sudden ecological changes in other Arctic lakes. For instance, Holmgren 36. In addition, the sheltered location of our site protects it from strong winds, further limiting sea ice drift.Similarly, sea ice variability and trends are not only governed by melt caused by global warming, but also partly controlled by large-scale sea ice drift which is driven by synoptic-scale atmospheric variability and oceanic currents. However, our hypothesis that melt dominates the sea ice variability near our site is supported by observations showing that recent sea ice trends around Svalbard are largely determined by local thinning (i.e. melt) instead of driftPediastrum is rare. Moreover, the existing Pediastrum work focuses mostly on shifts in Pediastrum assemblages11 rather than Pediastrum concentrations. Our study shows a sharp increase in Pediastrum concentrations that coincide with declining sea ice cover and a longer melting season. Although based on a single location, and although the observed increase in Pediastrum could partially be caused by diagenesis37 down-core, amplifying the trend of increasing Pediastrum toward the surface, this suggests that Pediastrum in high Arctic lakes directly responds to recent climate change. The increased Pediastrum productivity may even imply increased primary productivity in high Arctic lakes resulting from longer growing seasons caused by climate change, which was already suggested for sub-Arctic Canadian lakes38. Pediastrum may be a potential productivity indicator and an indicator for productivity and a longer growing season, but has so far not been used or recognised as such. Therefore, we recommend an increased focus on Pediastrum to support conclusions of recent studies39 that Arctic lake productivity increased over the past decades under influence of climate change.It should furthermore be noted that in comparison to diatoms, which have been widely used for reconstructing recent ecological change in the Arctic, existing work using 38. This suggests climate-driven Arctic-wide ecological change occurred in recent years, regardless of the exact locality or remoteness of records or depth of the lakes. It furthermore confirms earlier realisations14 that even the most remote and northern ecosystems in the world are continuously being affected by human activities.Finally, although the temporal resolution of our study is much higher than that of most other high Arctic diatom studies, preventing a direct comparison of these records to our study and to recent climate changes, recent shifts in diatom assemblages can also be observed in other Arctic lakesFieldwork was conducted as part of the Netherlands Scientific Expedition Edgeøya Spitsbergen (SEES.NL) that took place from August 19 to 28, 2015. For the purpose of this study, a sediment core was collected on August 25, 2015 from the small lake Andsjøen, situated on Sundneset close to Freemansundet in the south east of Barentsøya Fig. . The sed14C) and on 210Pb, 137Cs and 226Ra isotopes.Chronology is based on radiocarbon dating , using the 46.5 keV line for 210Pb, the 295.2, 351.9 and 609.3 keV lines for 226Ra and the 661.7 keV line for 137Cs. The detector, connected to a computer via a Digital Spectrum Analyser (DSA-1000), counted the radionuclide activities with Genie 2000 gamma spectroscopy software. Samples were counted for 2–5 days to obtain good counting statistics or relative standard deviation (RSD). A blank (empty petri dish) was counted during 23 days. The detector was externally calibrated with a Geological Certified Reference Material IAEA/RGU-1, with reference date of 01-01-1988. A monitor standard IAEA-300 provided quality control. Excess 210Pb activities were calculated by subtracting 226Ra activity averaged for the 295.2, 351.9 and 609.3 keV lines from the measured total 210Pb activity.For 210Pb Supply) model was used to reconstruct ages from the measured 210Pb values, following recommendations by Sanchez-Carbeza and Fernández40 assuming that sedimentation rates potentially vary throughout the core. In addition, the maximum 137Cs concentration is assumed to correspond to 1962–1963 AD  when testing of nuclear weapons reached its peak. This maximum was found between 24.5 and 35.5 cm depth, at around 29.5 cm depth. All isotope data can be found in Supplementary Dataset The CF or CRS  in Groningen, the Netherlands (GrA-67206). The radiocarbon activity correction (%) is 144.62 ± 4.4 and δ13C is −33.73, which corresponds to a Bomb-Peak calibrated age of 1962–1963 or 1971–1975 AD. Given the 137Cs peak at 29.5 cm depth and the 210Pb age reconstructions, we assume that the AMS radiocarbon date at 25.5 cm depth corresponds to 1971–1975 AD.For AMS radiocarbon analysis, a A second order polynomial was fitted through the reconstructed ages to obtain an age model for the whole core. The deepest part of the core has an approximate age of 1900 AD (47 cm depth). The age of the top of the core at the sediment-water interface is 2015. See Supplementary Fig. Sedimentation rates in the core increased from circa 0.03 cm/y around 1900 AD to about 0.2 to 0.3 cm/yr in the last decade , which is high for a small Arctic lake with no inlets and outlets. The high sedimentation rate can be explained by the high moisture content in the sediments (65 to 95% near the top), making the sediments bulky.41 with the use of sodium polytungstate heavy liquid separation to remove clastic material. A known quantity of exotic spores (Lycopodium clavatum) was added to ~0.5 gram of sediment from each sample  using a suspension based on Lycopodium tablets . The pollen residues were mounted in glycerine. Samples were analysed for pollen and Pediastrum under a light microscope with a magnification of 400x. Pollen types were identified following descriptions by Yao et al.42 and Moore et al.43.Samples were prepared for pollen analysis following the standard acetolysis procedure of Fægri and IversenSalix (Willow), Dryas, Saxifraga (Saxifrages or rockfoils), Poaceae (grasses) and Caryophyllaceae (Carnation family), but we also found high (up to 25%) percentages of allochtonous pollen of mostly Pinus (Pine tree). For these reasons, we decided to omit the pollen analysis in this study.Pollen concentrations were found to be low . Pollen percentages are calculated on the basis of the total pollen sum, excluding pollen of aquatic plants. The record shows pollen representative of high-Arctic environments, such as Pediastrum species are identified following Komárek & Jankovská28 and Turner et al.44, and are only counted if on estimation >50% of the coenobium is complete. Percentages of Pediastrum are based on the total sum of identified coenobia of Pediastrum species. Pediastrum and pollen was counted until a sum of ~300 Pediastrum coenobia was reached, or until ~100 Lycopodium spores were counted, or until the whole slide was counted. All pollen and Pediastrum data can be found in Supplementary Dataset et al.45. Samples were taken from the core every cm of the 47 cm long core. The samples were dried (at 105 °C overnight to remove water from the sample) and ashed (4 h at 550 °C) to burn the organic matter from the sample. The weight difference between the weight of the ashed sample and the weight of the dried sample is the LOI (Supplementary Dataset LOI data provide information on the nature of the lithology of the Adsjøen sediments. The organic content (in weight percentage) of the samples taken from the core from the Andsjøen was determined by loss on ignition (LOI) following Heiri 46 using 30% hydrogen peroxide (H2O2) and 50% hydrochloric acid (HCl) to remove organic material and carbonates respectively. Permanent slides were prepared using NaphraxTM as mounting medium and ≥300 valves were identified per slide under oil-immersion phase-contrast light microscope at 1000x magnification. A known quantity of microspheres was added for quantification of the concentration. All diatom data can be found in Supplementary Dataset 47 using the optimal splitting by information content method and the broken stick model. Zone boundaries are placed at approximately 1949 AD and 1994 AD. Diatom taxonomy is primarily based on Krammer and Lange-Bertalot51, Foged52 and Antoniades et al.29.A total of 24 freeze dried samples with an average weight of 0.01 grams were processed with the water bath methodIn this study we use remote sensing data of sea ice cover around Svalbard from the Oceans and Sea Ice Satellite Application Facility (OSI SAF). Covering the period from January 1979 to May 2015, this dataset uses passive microwave data (PMW) from the SMMR, SSM/I and SSMIS sensors. Ice concentration is computed from atmospherically corrected PMW brightness temperatures, using a combination of state-of-the-art algorithms and dynamic tie-points. Time resolution is daily, with partial coverage prior to 1987, and full coverage available after that time. The total uncertainty of the derived sea ice extent  in the area around Barentsøya is ~13%, which varies only marginally over the time period and throughout the year. The data are presented on a Lambert Azimuthal Equal Area polar projection, with a grid spacing of 25 km.et al.18 (VP16 hereafter)  temperature series of five stations on or near Svalbard (Supplementary Table As an additional evaluation of our approach, we compared the reconstructed temperature at the core site with that of the Kapp Heuglin weather station for the two overlapping years. The difference in temperature is only 0.3 °C, which confirms the validity of our method. The associated uncertainty is defined as the mean difference between the observed temperature at the five stations and the VP16 temperature at these locations (0.5 °C, Supplementary Table https://data.giss.nasa.gov/gistemp/. Sea ice data are available on http://osisaf.met.no/. Temperature data are available through the Norwegian Meteorological Institute.Ecological and chronological data acquired in the project are found in the Supplementary Information (Supplementary Datasets Supplementary InformationDataset S1Dataset S2Dataset S3


Salmo trutta, in oligotrophic streams with different population densities; to determine subpopulation site fidelity, 5,195 conspecifics from 14 subpopulations were individually tagged during spring and autumn. During a 7‐year‐long field study, we tested the hypothesis that brown trout individuals from subpopulations with high site fidelity would display lower movement. The hypothesis was supported, and reduced movement was further related to high subpopulation density in association with high slope indicating the physical environment‐influenced movement. The probability of contact between individuals increased with subpopulation site fidelity and subpopulation density. No influence of food abundance on brown trout movement was found. Furthermore, increased body size predicted higher movement (and vice versa). The least movement occurred during the day and during the full moons. Our study tended to show that individuals reused preferred areas and needed less movement to exploit available resources.The movement of individuals within preferred areas is reduced by a high availability of food and information about its distribution, while high number of competitors promotes increased movement. Experienced animals use information about social and physical environment to improve resources exploitation, tended to maintain positions within the preferred areas and reuse the environment that is often referred to as site fidelity. In this study, radio‐telemetry was used to observe the movements of 98 adult brown trout,  We performed observations throughout the diel cycle as the movement of brown trout varies over 24 hr, and movement peaks have been reported during the day , and its headwaters consist of mountainous oligotrophic streams with pristine morphologies. Twenty representative sampling sites distributed across the entire studied catchment area were selected based on maps, field visits, and knowledge/restrictions of the national park management to maximize the representativeness of the stream characteristics; however, final site selection was also influenced by the access granted by the national park. All sampling sites were wadable with substrate dominated by pebble and gravel; the average conductivity was 20.36 μS/cm (range 8.5–40 μS/cm), and the average flow varied from 0.01 to 2 m3/s along the longitudinal gradient .The study was carried out in the headwaters of the Otava River, located in Šumava National Park, Czech Republic. The overall catchment area of the studied streams, including the two main tributaries of the Otava River, Vydra and Křemelná, is approximately 224 square km Figure . The Otat Figure  that ana2.2LS, mm), weighed , and individually tagged on the lower left jaw using VIA tags . Specimens of insufficient size for individual tagging were marked using VIE tags . Throughout the study, 5,195 brown trout were tagged  and released at the site of their capture. Regarding the welfare of the study animals, a single‐pass electrofishing method was used, which is considered sufficient for determining the abundance of brown trout in mountain headwater streams  over the 7‐year period from October 2005 to October 2011 , and weighed (g). The types of transmitters available from the manufacturer  differed over the duration of our study. Thus, six types of coded radio transmitters , with mean operational life of 33 days (range 21–43 days), an average weight of 0.74 g in the air (range of 0.26–1.8 g), an average width of 6.36 mm (range of 5–8.2 mm), and an average length of 14.5 mm (range of 10–19 mm), were used throughout the study. Radio transmitters were implanted into the body cavities of brown trout through a midventral incision that was closed with three separate stitches using sterile, braided, absorbable sutures (Ethicon Coated Vicryl). The mass of the transmitter never exceeded 2% of the body mass of the fish  from eleven sampling sites that were identical to those from which the population data were obtained Figure . Altoget Winter, . The fisAt all study sites, tracking series were carried out weekly until the end of transmitter battery life. Tracking predominantly occurred in June (87% of data) with some observations made in late May. Once all the fish in a particular tracking series were positioned, most (depending on the tracking conditions) were observed over two subsequent 24‐hr tracking cycles, resulting in 5,520 individual fish positions. In a 24‐hr cycle, fish positions were determined once during each 3‐hr period . The time between measurements varied slightly depending on the tracking conditions (3 hr ± 30 min). The fish were positioned with the help of a GPS unit  using a radio receiver (Lotek SRX_400 receiver firmware version W31) and a three‐element Yagi antenna equipped with a compass. Compass bearings were taken on the transmitter direction from locations positioned with the help of a GPS unit. A computer program was developed to obtain fish position coordinates and plot them on a map using the biangulation method proposed by White and Garrott . The acc2.4Habitat variables were measured using the following devices: water temperature (°C) and conductivity ; light intensity ; and lunar phase . Throughout the study, all the above‐mentioned variables were measured once every 3 hr on the days when fish were tracked. River slopes (%) were measured using a Pulse Total Station  at each sampling site , when the tracking series were conducted, and the area used for calculating each “subpopulation density” were obtained by pooling all the trout and the areas, respectively, of the sampling sites integrated at a given spatial scale . The data  were log10 transformed prior to the LMM analyses when necessary to improve their fit to the normal distribution of the model . Fixed effects and their interactions that were not statistically significant are not discussed further. The differences between classification variables defined in the CLASS statement were tested with t tests , and a Tukey–Kramer adjustment was used for multiple comparisons. The degrees of freedom were calculated using the Kenward–Roger method  with random factors (PROC MIXED) in the SAS software package  in relation to length difference, subpopulation site fidelity, light interval, moon phase, weight difference, slope, subpopulation density, subpopulation site fidelity ratio, water temperature, conductivity, and their possible two‐way interactions. We applied an analysis of repeated measurements based on the generalized estimating equation (GEE) approach , while distances varied from 0 to 1,509 m (mean 221 m). Brown trout displayed contact in 4% of all pairwise observations. Among the aquatic invertebrates used for the food availability analyses, individuals belonging to the orders 5 m, whilF1,117 = 31.88, p < .0001). There was also an interaction between subpopulation site fidelity and river slope , indicating brown trout movement decreased when slope steepened and site fidelity increased . All variables in the final model improved its fit . Models with excluded variables describing site fidelity had the second and third highest ∆ AIC values (∆ AIC 40 for the model with excluded interaction of river slope and subpopulation site fidelity and ∆ AIC 27.7 for the model with excluded subpopulation site fidelity ratio), suggesting the overall importance of site fidelity in brown trout movement explanation. Nevertheless, the probability that a subpopulation had a high site fidelity ratio was not influenced by the aquatic insect assemblage  and during the full moon . Movement increased with fish body size .Brown trout movement decreased with increasing subpopulation density . The probability of contact further increased with increasing subpopulation density  and site fidelity . The interaction between subpopulation site fidelity and river slope  confirmed that the probability of contact increased with subpopulation site fidelity, while the influence of river slope was not possible to determine . The probability of contact was the highest during the day  and during the waning moon, that is, the first moon phase after the full moon .The probability of contact increased as the size between two individuals became more similar  showed higher movement and lower food intake compared with residents, and they experienced more attacks, indicating that relocating to a new territory induces higher energy costs, including those associated with stress, as well as decreased energy intake  during competition for shelter; that is, to acquire available resources, intruders increased their movement more than residents  were more active in areas with a potentially higher predation risk  showed more motivation to search for shelter than larger conspecifics  nurturing offspring displayed increased activity during a full moon, but solitary individuals did not (Penteriani, Kuparinen, Delgado, Lourenco, & Campioni, The results presented here agree with former studies observing brown trout feeding activity over a 24‐hr cycle Elliott, , with pe4.5Presented results indicate that changes in distance between radio‐tagged individuals were not random showing a higher probability of contact between individuals in subpopulations with high site fidelity and density. The long‐term relation to preferred areas, that is, site fidelity forms a social population structure displayed by lower spatial requirements and reduced agonistic interactions Stamps, . The inf5The findings presented here illustrate the ability of animals to alter movement based on social and physical conditions and the tendency to reuse preferred areas. Brown trout showed reduced movement in subpopulations with high site fidelity and density and within the high‐slope stretches. The results tended to show the influence of site fidelity on the social structure of the brown trout subpopulation, and the spatial requirements and energy costs of their movement supported the reuse of preferred areas. This study also revealed the reduced movement of brown trout in the wild was correlated with the full moon and daylight indicating the influence of physical conditions on the spatial distribution of individuals. Behavioral ecology research is mostly performed under experimental conditions in laboratories, but there are examples that show considerable differences in activity patterns of the same species or even the same individual between the laboratory and the natural environment (Calisi & Bentley, None declared.Ondřej Slavík conceived and designed the experiments, performed the experiments, wrote the paper, and reviewed drafts of the paper. Pavel Horký conceived and designed the experiments, performed the experiments, analyzed the data, prepared figures or tables, contributed materials/analysis tools, and reviewed drafts of the paper. Matúš Maciak analyzed the data, developed the statistical model, contributed materials/analysis tools, and prepared figures. Petra Horká analyzed the data and reviewed drafts of the paper. Iva Langrová analyzed the data and reviewed drafts of the paper. Click here for additional data file. Click here for additional data file. Click here for additional data file. Click here for additional data file.


Cdc55 phosphatase counteracts Cdk phosphorylation during interphase and delays phosphorylation of late Cdk substrates. PP2ACdc55 specifically counteracts phosphorylation on threonine residues, and consequently, we find that threonine-directed phosphorylation occurs late in the cell cycle. Furthermore, the late phosphorylation of a model substrate, Ndd1, depends on threonine identity of its Cdk target sites. Our results support a model in which Cdk-counteracting phosphatases contribute to cell-cycle ordering by imposing Cdk thresholds. They also unveil a regulatory principle based on the phosphoacceptor amino acid, which is likely to apply to signaling pathways beyond cell-cycle control.In the quantitative model of cell-cycle control, progression from G1 through S phase and into mitosis is ordered by thresholds of increasing cyclin-dependent kinase (Cdk) activity. How such thresholds are read out by substrates that respond with the correct phosphorylation timing is not known. Here, using the budding yeast model, we show that the abundant PP2A  •Cdc55 counteracts Cdk phosphorylation during the cell cycleThe phosphatase PP2A•It does so specifically on threonine, but not serine, phosphorylation sites•Consequently, threonine phosphorylation is a late event in the cell cycle•Cdc55 contributes to setting the timing of mitosisThereby, PP2A Cdc55 counteracts and thereby delays cell-cycle phosphorylation by cyclin-dependent kinase specifically on threonine, but not serine, phosphorylation sites. This reveals an ordering principle that might be also relevant in other signaling networks.The eukaryotic cell cycle comprises a robustly ordered series of phosphorylation events. Godfrey et al. show that the phosphatase PP2A The cell-division cycle is made up of an ordered series of events that ensure faithful replication and accurate partitioning of the genetic material. A major impetus for cell-cycle progression stems from the phosphorylation of numerous target proteins by the master cyclin-dependent kinase (Cdk) . Cdk subWhile cyclin specificity provides an apparently straightforward ordering principle, cell-cycle progression is remarkably resilient to its abolition. For example, the ordering of S phase and mitosis remains uncompromised if S phase cyclins are replaced by mitotic cyclins in both budding yeast and vertebrates . In anotCdc55 phosphatase to the dynamics of cell-cycle phosphorylation. We find that PP2ACdc55 indeed counteracts and thereby delays Cdk phosphorylation, but only on a subset of substrates. These are enriched for threonines as the phosphoacceptor sites, consistent with the known biochemical PP2A substrate preference. Our results portray cell-cycle phosphorylation as a dynamic process in which the steady-state level of phosphorylation is under control of the changing activity ratio of Cdk and its counteracting phosphatases.If Cdk activity is chemically inhibited at various cell-cycle stages, Cdk substrate phosphorylation is rapidly lost . This suCdc55 phosphatase to the regulation of Cdk substrate phosphorylation during the budding yeast cell cycle. PP2ACdc55 and its orthologs in vertebrates have been studied for their impact on cell-cycle progression by controlling inhibitory Cdk tyrosine phosphorylation . Without inhibitory Cdk phosphorylation, swe1Δ cells enter mitosis slightly earlier than wild-type cells but show otherwise undisturbed ordering of cell-cycle events  or full  detected with a less than 2-fold difference between the two labeling conditions see the . We therCdc55 shows phosphorylation site preferences when selecting its substrates. To this end, we assigned each phosphosite to one or more phosphorylation site consensus motifs. We then performed a 2D annotation enrichment analysis, which tests for the enrichment of motifs at either high or low SILAC ratios, based on their rank . Together, these results reveal that phosphothreonines are preferentially kept underphosphorylated by PP2ACdc55 and that this correlates with their late phosphorylation during the cell cycle.In a complementary analysis, we subdivided all Cdk sites into early, intermediate, late, or stable categories depending on the time they reached their maximal level of phosphorylation. Early sites included known early Cdk targets , while late sites included known late substrates , thus validating our assignments. The averaged phosphorylation kinetics of each group over time is shown in Cdc55 delays Ndd1 phosphorylation by targeting threonines, PP2ACdc55 impacted on the phosphorylation timing of Ndd1-10S to a smaller degree when compared to wild-type Ndd1  was substantially reduced by cdc55 deletion .Further information and requests for reagents may be directed to, and will be fulfilled by, the corresponding author, Frank Uhlmann , that was integrated to replace endogenous NDD1. Yeast cultures were grown in rich YP medium supplemented with 2% glucose or with 2% raffinose + 2% galactose (Epitope tagging of endogenous genes and gene deletions were performed by gene targeting using polymerase chain reaction (PCR) products . Cdc55 oIplac128 , includialactose , if not alactose .Protein extracts for western blotting were prepared following cell fixation using trichloroacetic acid, as described by , and anaIndirect immunofluorescence was performed on formaldehyde-fixed cells using the α-Tub1 antibody and Alexa Fluor 594-coupled secondary antibody listed in the 13C6]arginine and [13C6]lysine, respectively  under vacuum and dried. To ensure complete digestion, peptides were further digested using Lys-C in 10% acetonitrile, 50 mM ammonium bicarbonate , followed by trypsin digestion . Digested proteins were then desalted again and dried.1 mg of protein sample was reduced by 5 mM dithiothreitol (DTT) , alkylated with 10 mM iodoacetamide  and quenched with 7.5 mM DTT. Samples were then diluted with 50 mM ammonium bicarbonate to reduce the urea concentration to < 2 M, prior to trypsin digestion . Peptides were then desalted using a C2) was carried out as follows. Dried peptide mixtures were re-suspended in 1 M glycolic acid + 80% acetonitrile + 5% trifluoroacetic acid, sonicated (10 min) and added to titanium dioxide beads (5:1 (w/w) beads:protein). The beads were washed using 80% acetonitrile + 1% trifluoroacetic acid, followed by 10% acetonitrile + 0.2% trifluoroacetic acid, and dried under vacuum centrifugation. Phosphopeptides were eluted from the beads by adding 1% ammonium hydroxide followed by 5% ammonium hydroxide, and dried by vacuum centrifugation. Dried phosphopeptides were re-suspended in 100 μL of 1% trifluoroacetic acid and sonicated (15 min). A C18 membrane was packed into a 200 μL pipette tip and washed using methanol and equilibrated with 1% trifluoroacetic acid. The peptides were loaded onto the Stage Tip and washed with 1% trifluoroacetic acid followed by elution with 80% acetonitrile + 5% trifluoroacetic acid. The eluted peptides were again dried by vacuum centrifugation.Phosphopeptide enrichment using titanium dioxide (TiO18 column) with one activation method per run: collision induced dissociation (CID), multi-stage activation (MSA) and higher energy collision dissociation (HCD).An LTQ-Orbitrap Velos was used for data acquisition. Phosphopeptide mixtures were re-suspended in 35 μL 0.1% trifluoroacetic acid and injected three times (10 μL per injection). Each run consisted of a 3 hr gradient elution  was added as a variable modification, ‘Filter labeled amino acids’ was deselected, re-quantify was selected with the instruction to keep low-scoring versions of identified peptides within parameter groups and match between runs was selected. MaxQuant output files were imported into Perseus (version 1.4.0.2) and the normalized heavy-to-light (H:L) ratios were used for all subsequent analyses. Data were also imported into R for statistical testing and data visualization.MaxQuant (version 1.3.0.5) was used for all data processing. The data were searched against a UniProt extracted 2-test across the time categories presented in 2D annotation enrichment analysis  was perfCdc55, S. cerevisiae strains were grown in ‘heavy’ or ‘light’ SILAC medium and mixtures were prepared according an experimental design table, shown as cdc55Δ strain was grown in ‘heavy’ and the wild-type in ‘light’ medium were considered the forward (F) conditions. The mixtures in which the cdc55Δ strain was grown in ‘light’ and the wild-type in ‘heavy’ medium were considered the reverse (R) conditions. The same strain was used in the ‘heavy’ and ‘light’ conditions for each of the control mixtures that are shown in the table diagonal. The expected heavy/light (H/L) ratios for phosphorylation sites in the control mixtures are 1.0  and the ratio variability is a measure of the reproducibility of the experiment. The total number of phosphorylation sites identified (1% FDR) in the 12 SILAC experiments was 5949. The total number of phosphorylation sites quantified for each individual mixture is shown in each cell of For the experiment in 2 ratio versus log10 intensity plot. For each of the control experiments, over 90% of the data points were contained within a two-fold change interval . Raw data, all MaxQuant output files, and MaxQuant output Phospho(STY) have been deposited.Abridged tables of the mass spectrometry data are contained in M.G. and F.U. conceived the study, M.G. performed most experiments, S.A.T. and M.K. contributed the Ndd1 phosphorylation acceptor site analysis and performed additional experiments during the revisions, A.J. and A.P.S. performed the phosphoproteome analyses, and M.G. and F.U. wrote the manuscript with input from all authors.


We recently found that S131 of Akt2 (homologous to the well established CK2 target S129 of Akt1) is not phosphorylated by CK2 either in vitro or in vivo, although the consensus sequence recognized by CK2 (S/T-x-x-E/D/pS/pT) is conserved in it. Here, by exploiting synthetic peptides, in cell transfection experiments, and computational analysis, we show that a single sequence element, a T at position n+1, hampers phosphorylation, causing an α-helix structure organization which prevents the recognition of its own consensus by CK2. Our results highlight the role of negative determinants as crucial modulators of CK2 targeting and corroborate the concept that Akt1 and Akt2 display isoform specific features. Experiments with synthetic peptides suggest that Akt2 S131 could be phosphorylated by kinases of the Plk (Polo-like kinase) family, which are insensitive to the presence of the n+1 T. The low phylogenetic conservation of the Akt2 sequence around S131, as opposed to the extremely well-conserved Akt1 homologous sequence, would indicate a dominant positive role in the selective pressure only for the Akt1 phosphoacceptor site committed to undergo phosphorylation by CK2. By contrast, Akt2 S131 may mediate the response to specific physio/pathological conditions, being consequently shielded against basal CK2 targeting.CK2 is a pleiotropic S/T protein kinase (formerly known as  CK2 is an acidophylic S/T protein kinase that phosphorylates hundreds of proteins involved in almost all cellular processes . The phoin vitro and in vivo. Their sequences, retrieved in databases such as Phosphosite-plus (http://www.phosphosite.org/) or phosphoElm (http://phospho.elm.eu.org/), allowed the generation of weblogos  (5000–10000 cpm/pmol). Assays were stopped by spotting onto phosphocellulose filters. Filters were washed four times in 0.5% orthophosphoric acid before counting.Phosphorylation of synthetic peptides was performed with recombinant CK2 α33P]ATP (~8000 cpm/pmol), in a total volume of 20 μl. Reactions were stopped by the addition of Laemmli loading buffer. Samples were boiled for 5 min, run on 11% SDS/PAGE and blotted on Immobilon-P membranes (Millipore); radioactive bands were detected and quantified by digital autoradiography .Full length Akt phosphorylation experiments were performed on HA-Akt immunoprecipitated from overexpressing cells, incubated for 20 min at 30°C with 0.1 μg of recombinant CK2 α in the presence of a phosphorylation reaction mixture consisting of 25 mM Tris–HCl, pH 7.5, 10 mM MgCl2, 10 μM [γ-33P]ATP (2000-6000 cpm/pmol). Assays were stopped by spotting onto phosphocellulose filters. Filters were washed four times in 0.5% orthophosphoric acid before scintillation counting.1.5 μg of purified recombinant-PLK3 were incubated with the substrate peptides for 10 min at 37°C in 20 μl of kinase assay buffer containing 20 mM Tris-HCl pH 7.5, 10 mM MgCl2, 5mM DTT (dithiothreitol), 20 μM -ATP: as shown in Fig 3B only the latter underwent detectable phospho-radiolabeling by the kinase.Its in vitro phosphorylation by CK2 was also demonstrated by immunoprecipitation of Akt2 from cells expressing either the wt kinase or its T132G mutant, followed by incubation with CK2 and [γFig 3C, confirmed that the mutation of T132 to G, evoking the Akt2 phosphorylation at Ser131, is also accompanied by an increased phosphorylation of T308, compared to wt Akt2.Since we have previously demonstrated that S129 phosphorylation promotes a higher Akt1 activity, at least in part due to a higher level of T308 phosphorylation , we inveOnce established that the sharply different susceptibility to CK2 phosphorylation of Akt2 and Akt1 is conferred by a threonyl residue adjacent to the target Serine in Akt2, the question we wanted to answer was why this feature has such a detrimental effect on Akt2 S131 phosphorylation, although all the acidic determinants for the site recognition by CK2 are still there.Figs . The structure prediction analysis of the peptides  shows an α-helix organization for Akt2, while, on the contrary, the Akt1 peptide is entirely random coiled. This is consistent with the presence in +1 of a G, which displays low propensity to form α-helix, due to its high conformational flexibility. Indeed, the replacement of this G with T, as in the Akt1GT peptide, restores the α-helix organization, while the substitution of T in Akt2 with other residues  prevents it.To address this question, an in silico study was performed grounded on peptides secondary structure prediction, protein-protein docking analysis, and molecular dynamics simulation : in the case of Akt1 peptide, 64.8% of the most energetically favorable complexes are compatible with a catalytic interaction between the target S129 and CK2 catalytic center (D156). On the contrary, the Akt2 peptide is docked close to the CK2 substrate binding site only in the 3.2% of the final sampling, and S131 is not correctly oriented against D156. Interestingly, the Akt1GT peptide displays a docking sampling percentage similar to Akt2 peptide (2.9%), confirming the major negative role of the T in +1. Consistently, the Akt2 peptide variants where T was replaced by G, A or S displayed docking sampling percentages similar to that observed for Akt1 peptide .When the Akt1, Akt2, and Akt1GT peptide models were docked against CK2α crystal structure (PDB code: 3Q04) through a protein-protein docking approach, we observed different percentage of docking sampling : in the case of Akt1, the peptide is lounged into CK2 substrate binding site with Akt1 S129 correctly oriented towards CK2 catalytic site (2.8 Å). Moreover, the glutamic residues downstream to the phosphorylatable S129  interact through electrostatic interactions with CK2α residues K74, K77, the well known lysines implicated in the recognition of CK2 substrates at downstream position [position . On the . Lucubrating about this intriguing coincidence, we reasoned that survival of this phosphoacceptor site might reflect the intervention of another kinase, different from CK2, and, unlike CK2, committed to the phosphorylation of the linker region of both Akt1 and 2 under special circumstances. Pertinent to this may be two observations: firstly, a reduced yet still detectable phosphorylation of Akt1 S129 is occurring in C2C12 cells in which the whole CK2 catalytic activity has been knocked out by the CRISPR/Cas9 procedure [Tables 2). An acidic residue in this position is welcome by CK2 but not required for its site recognition, while it may be of crucial relevance for the targeting by other kinases, especially those of the Polo-like kinase (PLK) subfamily [Figs 1A and 1B), the opposite is true of PLK3 . Consistently, PLK3 does not perceive T in n+1 as a negative determinant, since its introduction instead of G in Akt1, or its replacement with G in Akt2, have no significant effect .From the biochemical data and the bioinformatics analysis presented above, it appears that an individual residue, T132 replaced for its homologous residue in Akt1, G130, is responsible alone for local structural alterations that make Akt2 much less prone to CK2 phosphorylation as compared to Akt1. Intriguingly, however, this happens without any loss of the typical signature of CK2 sites conferred by a number of acidic residues which are conserved around the target serine in Akt2 as well as in Akt1, as also nicely illustrated by the observation that the phospho site specific antibody that recognizes Akt1 pS129 also recognizes Akt2 pS131 rocedure , disclosubfamily . Based oThis makes PLKs plausible candidates as phosphorylating agents of Akt2, although for the time being the phosphorylation of Akt2 at S131 remains a matter of conjecture.Fig 2). The T to G mutation restores phosphorylation not only of the peptide but also of the full length Akt2, both in vitro and in cell .The finding of a differential phosphorylation by CK2 of Akt1 and Akt2  was hardFig 4).The in silico analysis for structure peptides prediction and docking towards CK2α provides a rationale for this finding by showing that a T at n+1  promotes an α-helix organization at the downstream side of the consensus, hampering the interaction between the crucial acidic side chain at position n+3 and the key residues of the CK2 active site committed to its binding  or not (-). In all of them the glutamic acid in +3 is preserved, while it is evident that the lack of significant phosphorylation correlates with the presence of a T in +1 position. This rises the question whether putative CK2 sites with the same motif (S-T-x-E/D) are bona fide targets of this kinase. In the Phosphosite.org page for CK2 substrates (http://www.phosphosite.org/), albeit uncommon, this motif is present in 12 site . It could be interesting to verify whether CK2 is their actual phosphorylating agent; in this case, the negative effect of a threonyl residue adjacent to the C-terminal side of the target serine, as observed in the case of Akt2 S131, has to be considered sequence-sensitive. Otherwise, we have to assume that kinases other than CK2, such as PLKs, are likely involved.The first practical relevance of our observation is a warning against cursory identification of CK2 sites, just based on the presence of an acidic determinant in +3. In the second place, our findings definitely confirm that human Akt2 is not targeted by CK2. The present demonstration that S131 phosphorylation is prevented by a simple sequence element, makes it unlikely the occurrence of physiological conditions able to overcome this impasse. Akt1 and Akt2 are the two main ubiquitous isoforms of Akt; they share the same structural organization and mechanism of activation, and their functions are largely overlapped. However, they display also distinctive features and substrates, which are particularly relevant in explaining their isoform-specific roles –38; theiTable 1) is consistent with a dominant role of this CK2 phosphorylation site in Akt1. We have already shown that S129 phosphorylation, besides upregulating active Akt1 [The absolute conservation of Akt1 S129 and surrounding sequence , suggesting that whenever present, its phosphorylation does not critically impinge on the selective pressure process. The CK2 consensus is in fact conserved, but in several organisms (underlined in Table 2), notably human, other primates, rodents, and elephant, its efficacy is abrogated by a T at position n+1. Rather, the possibility that kinases other than CK2 can phosphorylate this Akt2 site deserves attention, and major candidates are PLK kinases. Like CK2 also PLKs are acidophylic, but their consensus is mainly dictated by upstream determinants [Fig 5. Consistently the motif D-x-S, of crucial relevance for S131 recognition by PLKs, is conserved in Akt2s from all organisms. To the best of our knowledge, Akt2 S131 has never been found phosphorylated in literature or reported in phospho-sites database , and this suggests a sporadic occurrence of its phosphorylation. Nevertheless, it should be considered that, at variance with CK2, PLKs are not constitutively active: they play specific roles in mitotic entry and cell cycle, and they are controlled by complex mechanisms involving phosphorylation, proteolysis and transcription, depending on the biological context [Conversely, the Akt2 sequence around S131 is more variable (rminants , and it  context . Thus, t