Social interactions in classic cognitive games like the ultimatum game or theprisoner's dilemma typically lead to Nash equilibria when multiplecompetitive decision makers with perfect knowledge select optimal strategies.However, in evolutionary game theory it has been shown that Nash equilibria canalso arise as attractors in dynamical systems that can describe, for example,the population dynamics of microorganisms. Similar to such evolutionarydynamics, we find that Nash equilibria arise naturally in motor interactions inwhich players vie for control and try to minimize effort. When confronted withsensorimotor interaction tasks that correspond to the classicalprisoner's dilemma and the rope-pulling game, two-player motorinteractions led predominantly to Nash solutions. In contrast, when a singleplayer took both roles, playing the sensorimotor game bimanually, cooperativesolutions were found. Our methodology opens up a new avenue for the study ofhuman motor interactions within a game theoretic framework, suggesting that thecoupling of motor systems can lead to game theoretic solutions. Human motor interactions range from adversarial activities like judo and armwrestling to more cooperative activities like tandem riding and tango dancing.In this study, we design a new methodology to study human sensorimotorinteractions quantitatively based on game theory. We develop two motor tasksbased on the prisoner's dilemma and the rope-pulling game in which weintroduce an intrinsic cost related to effort rather than the typical monetaryoutcome used in cognitive game theory. We find that continuous motorinteractions converged to game theoretic outcomes similar to the interactiondynamics reported for other dynamical systems in biology ranging in scale frommicroorganisms to population dynamics. Nash equilibrium solutionRiding a tandem, tango dancing, arm wrestling and judo are diverse but familiarexamples of two-player motor interactions. The characteristic feature of suchinteractions is that the two players influence each others behavior through coupledsensorimotor control with continuous action spaces over repeated trials orcontinuously in time. In contrast, two-player interactions considered in classicalgame theory are typically thought to involve cognition in games with discreteactions and discrete time steps for decision-making such as tic-tac-toe, theultimatum game or the prisoner's dilemma Here, we develop continuous sensorimotor versions of the prisoner's dilemmaand the rope-pulling game. In the classical prisoner's dilemma In our continuous sensorimotor version of prisoner's dilemma see , two plaThus, our motor version of the prisoner's dilemma differs from the classicdiscrete version of the game in at least three different aspects. First, actions arecontinuous such that there is a continuous coupling between the two players. Second,reward in terms of money or years is replaced by an implicit cost, that is effort.Third, subjects have to learn their optimal strategy since they are unaware of thestructure of the coupling, i.e. they have incomplete information about the payoffs.We found a clear distinction between the strategies used at the end of a set for theone-player and the two-player conditions. In To investigate the temporal evolution of learning we analyzed the trial-by-trialbehavior of the players averaged across all sets. Initially, in both the one-playerand the two-player conditions, players acted at chance level in their strategy. Later tIn our sensorimotor version of the prisoner's dilemma the cooperative andNash solutions are two extremes of the one-dimensional control variable . Therefore, we designed a motor task based on anothergame, the ‘rope-pulling-game’, which has three additionalfeatures. First, the control variable is two-dimensional and the Nash andcooperative solutions are no longer at the boundaries of the control space. Second,unlike the prisoner's dilemma, where each player can achieve their task(reaching the bar) without paying attention to the strategy of the other player, inthe new task, coordination is required between the players to jointly achieve thetask. Third, the rope-pulling game can be translated into a linear dynamical systemallowing for analytical solutions in terms of feedback policies see . Player In our version of the rope-pulling game, player 1 and 2's task was to move avirtual mass to a fixed target that was equidistant to both players'origin. Again, each participant grasped the handle of the robotic interface and thelocation of the virtual mass was the sum of the . Participants held the handle of the vBOT thatconstrained hand movements to the horizontal plane. The vBOT allowed us to recordthe position of the handle and to generate forces on the hand with a 1 kHz updaterate. Using a projection system we overlaid virtual visual feedback into the planeof the movement All experimental procedures were approved by the Psychology Research EthicsCommittee of the University of Cambridge.x-position was taken as their choice in the game. Duringthe movement, both players experienced a one-dimensional spring attached tothe starting bar. The spring constants depended on the lateral positions x-position wascategorized as cooperate or defect depending on whether Each of the robot handles controlled the position of a cursor in one half ofthe horizontal workspace . The cura matrix , with ina matrix . The parThe position of each robot handle was expressed as a two-dimensional vectorposition where Each game was played by eight pairs of participants and by eight differentparticipants individually. All participants were instructed to achieve thetask as easily as possible. Participants were also told the number of trialsin each set, and the sets were separated during the experiment by shortbreaks. For the two-player game a divider was used to prevent theparticipants seeing the cursor or arm of the other player. In thesingle-player condition subjects saw the same screen that would be displayedto two players in the game condition.Text S1Supplementary Materials(0.15 MB PDF)Click here for additional data file.


Lipid mobilizing factor (250 nM) produced comparable increases in intracellular cyclic AMP in CHOK1 cells transfected with the human β3-adrenoceptor to that obtained with isoprenaline (1 nM). In both cases cyclic AMP production was attenuated by SR59230A confirming that the effect is mediated through a β3-adrenoceptor. A non-linear regression analysis of binding of lipid mobilizing factor to the β3-adrenoceptor showed a high affinity binding site with a Kd value 78±45 nM and a Bmax value (282±1 fmole mg protein−1) comparable with that of other β3-adrenoceptor agonists. These results suggest that lipid mobilizing factor induces lipolysis through binding to a β3-adrenoceptor.Induction of lipolysis in murine white adipocytes, and stimulation of adenylate cyclase in adipocyte plasma membranes, by a tumour-produced lipid mobilizing factor, was attenuated by low concentrations  86, 424–428. DOI: 10.1038/sj/bjc/6600086www.bjcancer.comThe Cancer Research Campaign© 2002  In vivo studies confirmed the ability of LMF to cause selective loss of carcass fat with no change in body water, and a tendency to increase the nonfat mass. LMF was characterized by the ability to stimulate lipolysis directly in isolated adipocytes, as a result of stimulation of adenylate cyclase in a GTP-dependent process  . InductiIn the present study the ability of LMF to interact with the β3-AR has been studied both in white adipocytes and in CHO cells transfected with the human β3-AR.−1. No patient had received radiotherapy or chemotherapy. Urine samples were stored at −20°C in the absence of preservatives prior to use.Urine was collected over a 24 h period from patients with unresectable pancreatic cancer and with a weight loss between 0.5 and 3 kg month32P]-ATP (sp. act. 20 Cimmol−1) and Na [125I] (sp. act. >15 Cimg−1 iodide) were purchased from Amersham Pharmacia Biotech . SR59230A was kindly donated by Dr L Manara of the Research Centre Sanofi Midy, Sanofi Winthrop S.p.A., Milan, Italy.-cyclic AMP  and 20 μg of cyclic AMP-dependent protein kinase  and incubated for 2 h at 4°C. Unbound cyclic AMP was removed by adsorption onto charcoal and the concentration of cyclic AMP in the sample determined by comparison with standard curves using known concentrations of cyclic AMP.CHOK1 cells transfected with the human β3-AR, under the control of hygromycin, together with the b-gal reporter construct, selected for resistance to G418, were a gift from Dr Ian Waddell, Astra Zeneca, Macclesfield, Cheshire, UK. They were grown in Dulbecco's modified Eagles medium (DMEM) supplemented with 2 m125I] (1 mCi per 100 μg protein) for 5 min in 100 μl PBS. LMF (100 μg protein) was then added and the reaction allowed to proceed for 15 min. The iodo-bead was physically removed and free Na[125I] was removed using a Sephadex G25 column eluted with 0.1 M NaI. The [125I] LMF was concentrated using a Microcon microconcentrator with a Mr 10 000 cut-off against PBS.One iodo-bead , washed and dried, was incubated with Na LMF for 60 min at 37°C. The samples were then centrifuged at 13 000 g for 20 min, the supernatant was removed and the radioactivity of the pellet was determined using a Packard Cobra Model 5005 Auto-gamma counter. Binding was analyzed using non-linear regression analysis ).CHOK1 cells transfected with the human β3-AR were lysed by sonication in 0.5 −5–10−7 M) of SR59230A  than LMF (250 nM), suggesting that LMF had a lower affinity for the β3-AR than isoprenaline. The increase in intracellular cyclic AMP produced by both isoprenaline and LMF in CHOK1β3 was attenuated by the non-specific β-AR antagonist propranolol (10 μM), while the effect on LMF, although significant, was less than complete. However, cyclic AMP production by both isoprenaline and LMF was almost completely attenuated by SR59230A, confirming that the action of LMF was mediated through a β3-AR.To investigate this possibility the effect of LMF on cyclic AMP production was determined in CHOK1 cells, which had been transfected with the human β3-AR. The data presented in Figure 2125I and the binding to crude plasma membranes from CHOK1β3 cells was determined. The data is presented in Table 1125I] iodocyanopindolol  has been reported to be significantly increased in weight losing patients with lung (Classical β1 and β2-AR mediate response to noradrenaline released from the SNS. In addition a third β-AR subtype has been identified (reviewed in We have previously shown that cachexia in both mice and humans is associated with LMF production by the tumour and excretion in the urine  have sho


Latimeria) is the extant taxon of a basal sarcopterygian lineage and sister group to tetrapods. Apart from certain apomorphic traits, its morphology is characterized by a high degree of retention of ancestral vertebrate structures and little morphological change. An insight into the molecular evolution that may explain the unchanged character of Latimeria morphology requires the analysis of the expression patterns of developmental regulator genes and their cis-regulatory modules (CRMs).The modern coelacanth (sonic hedgehog (shh) genomic region of Latimeria menadoensis. Several putative enhancers in the Latimeria shh locus have been identified by comparisons to sarcopterygian and actinopterygian extant species. Specific sequence conservation with all known actinopterygian enhancer elements has been detected. However, these elements are selectively missing in more recently diverged actinopterygian and sarcopterygian species. The functionality of the putative Latimeria enhancers was confirmed by reporter gene expression analysis in transient transgenic zebrafish and chick embryos.We describe the comparative and functional analysis of the Latimeria shh CRMs represent the ancestral set of enhancers that have emerged before the split of lobe-finned and ray-finned fishes. In contrast to lineage-specific losses and differentiations in more derived lineages, Latimeria shh enhancers reveal low levels of sequence diversification. High overall sequence conservation of shh conserved noncoding elements (CNE) is consistent with the general trend of high levels of conservation of noncoding DNA in the slowly evolving Latimeria genome. Compensatory changes in cis-regulatory regions argue for a weak correlation of both  sequences were obtained by polymerase chain reaction (PCR) from genomic DNA of Latimeria chalumnae using degenerate primers. The shh-specific paralog was deduced from DNA sequence comparisons and used as a probe to screen a Latimeria menadoensis BAC  library . Repetitive DNA and interspersed elements made it difficult to align the entire BAC clone sequence in a single contig, and correct assembly was verified for the 20-kb region by PCR amplifications. The shh coding portion was deduced from DNA sequence comparisons to mouse and chick shh. Phylogeny reconstruction with shh coding sequences of higher vertebrate species indicated that indeed the Latimeria shh orthologous gene had been sequenced  and medaka (Oryzias latipes). The third peak of conserved sequence found in Latimeria is the region corresponding to ar-B, a floorplate-specific enhancer in zebrafish. It showed similarity with actinopterygian and with marsupial species, but with none of the other sarcopterygians for which genomic data are available. This result suggests that the ar-B enhancer has been lost independently in the different tetrapod lineages. In opossum, the sequence that retains ar-B conservation is interrupted with approximately 230 bp of repetitive DNA. The 5- part of this conservation is further conserved in shh loci of placental mammalian species, such as human . This region was previously shown to be strongly conserved in Latimeria [In summary, atimeria . Therefoshh CNE in the different lineages. We calculated the sum lengths of CNE from pairwise comparisons of two shh genomic regions, spanning from the SFPE1/ar-D upstream enhancer to the shh 3'-UTR . The expression was not considerably higher than background staining and was probably originated from the 0.8-kb proximal shh promoter. Specific reporter gene expression in the anterior floorplate (anterior to the level of the yolk extension) was observed for DNA fragment 1, ranging from position -836 to -1339. The region that is responsible for the ar-D enhancer effect overlaps fully with a CNE that is present in all the other compared sarcopterygian sequences . Latimeria putative enhancer orthologs ar-D, ar-A and ar-B were cloned into the above-mentioned shh minimal promoter constructs, containing GFP (Green Fluorescent Protein) instead of a LacZ reporter. Transient mosaic expression of GFP was measured as read out of reporter construct activity 24 hours after injection of zebrafish zygotes . Potentially, these elements can account for the posteriorly extended expression direction of Latimeria ar-D in the ventral neural tube of zebrafish.To check whether the us study , and thes Figure . The repn Figure . HoweverLatimeria conserved sequence that overlaps with the zebrafish ar-A enhancer drives specific expression in the notochord indistinguishable to the activity of zebrafish ar-A   [shh control plasmid. Electroporation with the Latimeria ar-B enhancer resulted in significantly enhanced GFP expression. However, the expression pattern was not specific to the ventral neural tube. This result suggests that the Latimeria ar-B sequences carry enhancer activity but are either not restricted to the ventral neural tube or are interpreted in a nonspecific fashion in chick. The Latimeria-arD enhancer directed tissue-specific GFP expression in the floorplate, both in hindbrain and in the posterior spinal cord. This result is similar to those observed in transient transgenic zebrafish   and was h Figure .Latimeria conserved enhancers ar-D and ar-A were found to be functional in zebrafish. The Latimeria ar-B enhancer was providing unspecific enhancement of GFP expression in chick, both in the hindbrain and in the posterior neural tube. These results suggest that all three regions are functional enhancers in Latimeria, albeit that experimental verification in Latimeria is currently not feasible.Summarizing, only the predicted shh genomic region of Latimeria menadoensis reveals conservation of all four actinopterygian shh midline enhancers, which indicates an ancestral-like and rather unchanged cis-regulatory architecture of Latimeria shh. Several previous studies with Latimeria enhancers of different loci or genomic regions are in agreement with this interpretation. The analysis of the Latimeria procadherin gene clusters [Latimeria genome as being very stable and having suffered very little from diversifications such as gene duplications or gene conversion events. The characterization of Latimeria HOX clusters revealed a consistently slower diversification of Latimeria CNE with repsect to tetrapods [Latimeria orthologous Otx2 enhancers FM and AM [HoxC8 early enhancer [Otx2 enhancer expression direction was similar to that obtained with skate orthologs, and the HoxC8 early enhancer was described to direct gene expression in mouse, similar to the mouse ortholog. Our study extends the observations cited above to the characterization of a set of proximal regulatory modules within a locus. With availability of the Latimeria genome sequence, a more comprehensive comparative genomic analysis should be possible with a multitude of developmental regulator genes. Such an approach may also reveal how far conservation of cis-regulatory regions at developmental regulator genes is related to its ancient type morphology.The clusters  describeetrapods . The LatM and AM  as well enhancer  revealedar-A, ar-B, ar-C and ar-D in Latimeria and zebrafish reveals preservation of an ancestral set of enhancers that originated before the split between ray-finned and lobe-finned vertebrates . However, there is reason to speculate that the intronic and upstream enhancers are potentially present in such species. Embryonic expression of shh in the embryonic midline has been shown to be strongly conserved in distantly related vertebrate species such as lamprey [So far, there is not enough data available on  lamprey  or dogfi lamprey .shh orthologous genomic regions indicates interesting trends in the conservation of CNE across actinopterygian and sarcopterygian species. A lower overall conservation is observed among teleost species. Less conservation of noncoding DNA in teleost lineages has also been reported previously in DNA sequence comparisons of vertebrate Hox clusters [Latimeria and chick and lower conservation to the mammalian sequences. It indicates that the shh genomic region has stayed remotely constrained across sarcopterygian evolution but has more recently diversified in the mammalian lineage. Comparisons of the conserved blocks of the enhancers ar-A, ar-C and SFPE1/ar-D by relative rate tests [Latimeria. Likewise, the mouse ar-C orthologous enhancer SFPE2 is floorplate-specific and has been reported to be inactive in transgenic zebrafish [Latimeria orthologs [The analysis of total conservation of CNE Figure  in the sclusters , which wclusters . Among ste tests  are in lebrafish . Chick arthologs  showed nrthologs ,34,40.Latimeria orthologous enhancers in transgenic zebrafish and chick provided confirmation of functionality, though it cannot definitively corroborate the endogenous tissue specificity in Latimeria. Although similar, there are qualitative differences in expression patterns generated by the Latimeria ar-D sequence and the 2.4-kb zebrafish shh upstream region that contained the ar-D enhancer. This region directed expression in anterior parts of the floorplate and the ventral brain. The expression of the Latimeria ar-D enhancer in the posterior floorplate can potentially result from enhancer regions that are only conserved among sarcopterygians , characterized in mouse, also revealed to drive reporter gene expression in posterior parts of the floorplate [Latimeria ar-D. The subtle differences in reporter gene expression by orthologous enhancers from different species have also been reported previously and appear to be a common phenomenon [The analysis of expression of putative oorplate . Howeveroorplate  but contenomenon ,34,36,55ar-D activity, Latimeria ar-B proved also to be differently interpreted in zebrafish, compared to its zebrafish homolog. In the transgenic reporter assays employed, Latimeria ar-B did not drive reporter gene expression in zebrafish. However, an unspecific activation of GFP expression by Latimeria ar-B was observed in chick embryos. This suggests that ar-B is a functional cis-regulatory region in the first place. The lack of tissue-specific expression might simply reflect different interpretations of ar-B enhancer elements in zebrafish, chick and Latimeria. We observed conserved sequences among teleost species that map zebrafish ar-B but that are not present in Latimeria , transformed, and single clones from transformations were isolated and prepared. The insert of each clone was sequenced using the primers M13(-20) and M13(reverse) on an ABI PRISM 3100 Genetic Analyzer , using the Big Dye Termination Reaction chemistry (Applied Biosystems). Suitable clone inserts were used as DNA template for the preparation of a 233-bp shh-specific 32P-labeled probe for BAC library screening. 32P-labeling was achieved by random priming [The DNA sequence of  priming .Latimaria menadoensis BAC library VMRC4 [shh-specific BAC clones. One nylon filter set was hybridized overnight in 0.6 M NaCl, 0.02 M ethylenediaminetetraacetic acid (EDTA), 0.2 M Tris pH 8.0, 0.5% sodium dodecyl sulfate (SDS) and 0.05% sodium pyrophosphate at 65°C with the shh-specific probe. The filters were washed twice with 1 × saline-sodium citrate (SSC), 0.1% SDS, once for 30 minutes at room temperature and once for 40 minutes at 37°C. Three shh-positive BAC clones were isolated.The ry VMRC4  was screLarge-scale BAC clone preparation was obtained with the Large Construct Kit . A total of 20 μg of BAC clone DNA in 500 μl TE , pH 8.0, was fragmented by sonication  with 4 × 1-second pulses at 300 W (5-mm microtip). The 2- to 3-kb fraction was isolated from the sheared DNA by preparative gel electrophoresis. DNA fragments were end-polished with Klenow enzyme  and blunt-end ligated into pUC18 vector (Roche). Resulting bacterial clones from transformations were isolated into 384-well plates and grown in 1 ml LB (Luria-Bertani) broth (50 μg/ml ampicillin).2 was sequenced using the shotgun approach. Clone plasmid DNA was prepared manually. The inserts were sequenced directly with the universal primers M13(-20) and M13(reverse) as mentioned above. Sequences were quality trimmed with PHRED [shh sequences and to conserved noncoding elements of shh genomic regions, obtained from the UCSC genome browser [shh coding sequences was performed with RAxML [The subcloned BAC clone 123-Oth PHRED  and asse browser . Contig th RAxML .shh genomic regions of different vertebrate species were extracted with the UCSC genome browser or Ensembl genome browser ). Annotations of genomic regions were done manually by DNA sequence comparisons and repetitive DNA was masked with repeatmasker [The atmasker  using wuFor relative rate tests, alignment blocks were determined as conserved sequences that are found in all compared DNA sequences. Alignment blocks were established with Dialign TX , and relLatimeria shh enhancers were amplified by PCR from BAC clone DNA using specific oligonucleotides  [et al. (1997) [shh upstream DNA regions were obtained with specific oligonucleotides  . Analysi. (1997) . TransieIn vivo experiments with chick embryos were performed by in ovo electroporation. Eggs from White-Leghorn chickens were incubated at 38.5°C in an atmosphere of 70% humidity. Embryos were staged following Hamburger and Hamilton (HH) [et al. (2000) [. (2000) . This me. (2000) . Briefly2HPO4, 2 mM KH2PO4, pH 7.4), 0.1% Tween-20), incubated overnight with a rabbit anti-GFP antibody  and detected using an anti-rabbit Alexa 488-conjugated secondary antibody (Molecular Probes). Images were collected by fluorescence microscopy.Chick embryos were fixed for 4 hours at 4°C in 4% paraformaldehyde in PBS, rinsed, embedded in 5% agarose, 10% sucrose and sectioned in a Leica vibratome . Immunostaining was performed following standard procedures. Briefly, sections were blocked with 10% goat serum in PBS-T : coding sequence; (CNE): conserved non-coding elements.(CRMs): The authors declare that they have no competing interests.Latimeria shh containing BAC clones. YH prepared the Latimeria shh enhancer constructs, and YH and CP performed the Latimeria enhancer transgenic analysis. US and FM provided zebrafish enhancer coinjection analysis. ML, FM, CTA, YH and AM wrote the manuscript.ML, AM, FM and YH designed the experiments. ML, NS and MBB sequenced the BAC clone and analyzed the data. CTA isolated the DNA sequence alignments. Alignments of shh enhancers ar-A, ar-B, ar-C, and ar-D with putative transcription factor binding sites.Click here for filear-B enhancer conservation. VISTA plot of the ar-B specific genomic region. Shuffle-LAGAN alignment, visualized with mVISTA.Click here for fileshh genomic regionVISTA plot of the . VISTA plot with zebrafish shh as reference sequence. Shuffle-LAGAN alignment, visualized with mVISTA. This figure is used for indication of the putative ar-E CNE that is found in zebrafish, medaka and fugu.Click here for fileenhancer blocks. Conserved enhancer sequences used for Relative Rate Tests are presented.Click here for filear-DMapping of zebrafish . The experimental identification of the zebrafish ar-D enhancer is summarized.Click here for fileOligonucleotides. The oligonucleotides for sub-cloning of enhancers and transgenic analysis are listed.Click here for file


Several studies suggest that the α2-AR responsiveness in cells and tissues after chronic epinephrine (EPI) or norepinephrine (NE) exposure may vary, depending on the β-AR activity present there. Recently, we reported that in BE(2)-C human neuroblastoma cells (endogenously expressing α2A- and β2-AR), chronic EPI treatment (300 nM) produced a dramatic β-adrenoceptor-dependent desensitization of the α2A-AR response. The aim of this study is to determine if stable addition of a β2-AR to a second neuroblastoma cell line (SH-SY5Y), that normally expresses only α2A-ARs that are not sensitive to 300 nM EPI exposure, would suddenly render α2A-ARs in that cell line sensitive to treatment with the same EPI concentration.In addition to the regulation of blood pressure, α2-AR subtype expression. Stable clones of SH-SY5Y cells transfected to stably express functional β2-ARs (SHβ2AR4) were selected to compare sensitivity of α2-AR to EPI in the presence or absence of β2-ARs.These studies employed RT-PCR, receptor binding and inhibition of cAMP accumulation to confirm α2-AR heterogeneity. We now report that after transfection of functional β2-AR into SH-SY5Y cells (SHβ2AR4), chronic treatment with modest levels of EPI desensitizes the α2A-AR. This effect results from a β2-AR dependent down-regulation of native α2A-ARs by EPI accompanied by enhanced translocation of GRK2 and GRK3 to the membrane (required for GRK-mediated phosphorylation of agonist-occupied receptors).A series of molecular, biochemical and pharmacological studies indicated that the difference between the cell lines could not be attributed to α2A-AR more susceptible to desensitization with physiological levels of EPI.This study further supports the hypothesis that the presence of the β-AR renders the α Under physiological conditions, norepinephrine and epinephrine  activate the α2-AR along with other members of the AR family, which also includes α1- and β-ARs. The α2- and β-ARs are often co-expressed on the same cell surface. Upon activation by NE and EPI, the independent signals initiated by the α2- and β-ARs often converge to regulate specific physiological endpoints such as insulin release rauwolscine binding to SH-SY5Y cell membranes with higher affinity than prazosin, the α2B/C-selective antagonist -C, NG108-15, OK; CGP-12177 for binding studies as described in Methods. Since BE(2)-C cells express very low levels of β2-AR (Bmax: 18.5 ± 6.2 fmol/mg protein), the SHβ2AR4 cell line that expressed 14.78 ± 4.19 fmol/mg protein of the β2-AR was selected for the subsequent studies. To ensure that the β-ARs were functional, the ability of isoproteranol (ISO) to stimulate cAMP accumulation was assessed  and SHβ2AR4 cells were exposed to vehicle or the indicated concentration of agonist for 16–24 hr. Wt SH-SY5Y cells : 5.2 ± 0.1) and efficacy (Imax (%): 17.0 ± 1.6; P < 0.05 Fig. 2A-AR signal in the wt SH-SY5Y cell line. In contrast, chronic treatment of the β-AR-expressing SHβ2AR4 cells with 300 nM EPI desensitized the α2A-AR signal causing loss of UK14,304 potency (-Log EC50 (M): Vehicle 6.9 ± 0.2; EPI 6.3 ± 0.2) and efficacy (Imax (%): Vehicle 68.2 ± 5.4; EPI 49.3 ± 10.4; p < 0.05; Fig. 2A-ARs and does not alter α2A-AR signaling. We concluded that the difference in α2-AR signaling following EPI treatment between the transfected and wt SH-SY5Y was attributable to the presence of functional β2-ARs, respectively. To ensure that the vector was not responsible for the observed difference between the wt and SHβ2AR4 cells, similar experiments were conducted in SH-SY5Y cells transfected with the vector alone (minus the β2-AR gene). These vector only-expressing clones responded to EPI (300 nM) and NE (1 μM) pretreatments as the parent SH-SY5Y cells did  with the chronic 300 nM EPI treatment. Addition of propranolol blocks EPI-induced α2A-AR desensitization resulting in UK14,304 concentration-response curves indistinguishable from control (-Log EC50 (M) for EPI + Prop 6.7 ± 0.1; Imax (%) for EPI + Prop 67.9 ± 0.4; p < 0.05; Fig. To validate the importance of the β2-AR is functioning properly following catecholamine treatment, we evaluated the ability of ISO to stimulate cAMP accumulation over basal in SHβ2AR4 cells. The β2-AR signal is desensitized following chronic EPI but not NE treatment, consistent with the fact that NE has a low affinity for the β2-AR. Inclusion of propranolol (30 nM) inhibited EPI-induced β2-AR desensitization -C cells, requires β2-AR co-activation since loss of α2A-ARs is prevented when 30 nM propranolol is included with EPI. Down-regulation of the α2A-AR is not observed following chronic activation of α2A-AR alone by 1 μM NE. Further, 300 nM EPI does not alter the expression of α2A-AR in wt SH-SY5Y cells as compared to vehicle-treated cells  consistent with a lack of α2A-AR desensitization. Hence, it can be concluded that chronic EPI treatment induces a loss of α2A-AR response via β2-AR-dependent down-regulation of α2A-ARs in SHβ2AR4, but not in wt SH-SY5Y cells.Our study in BE(2)-C cells suggests that βe α2A-AR ,15. Chro2A-AR desensitization and down-regulation in BE(2)-C cells is mediated via β2-AR-dependent GRK3 up-regulation rauwolscine in SH-SY5Y membrane homogenates with previously reported values, we saw a correlation only with those cells that expressed α2A-ARs (Table 2-AR subtypes involves comparing the prazosin/oxymetazoline (OXY) or OXY/yohimbine affinity ratios . The agfrom  in sterile distilled water. Reaction mixtures were overlayed with mineral oil and subjected to a hot start for 5 min at 95°C. DNA polymerase  was added to each reaction tube after the hot start, and the tubes were subjected to a PCR reaction of 30 cycles in a thermal cycler  for 1 min at 94°C, 1.5 min at 55°C, and 2 min at 72°C with a final elongation step at 72°C for 7 min. Reaction products were separated by electrophoresis through 2% agarose gels and visualized by ethidium bromide staining. PCR products were isolated from the gel using a DNA extraction kit . Identity of the purified PCR products was confirmed by their susceptibility to digestion with restriction enzymes specific for each reaction product : NaCl (137), KCl (5), Na2HPO4 (0.6), KH2PO4 (0.4), NaHCO3 (4), D-glucose (6), MgCl2 (0.5), MgSO4 (0.4) and CaCl2 (1), containing the phosphodiesterase inhibitor IBMX (0.5 mM). In some experiments, antagonists also were included in this step. To prohibit oxidation, sodium ascorbate (0.11 mM) was included when assaying catecholamines. Upon addition of forskolin (10 μM) and agonist, assay tubes were incubated for an additional 10 min at 37°C. Removing the tubes to a boiling water bath for 5 min terminated the assay. All assays were performed in duplicate in a total volume of 0.5 ml. After boiling, samples were centrifuged for 5 min at 14000 × g, and cAMP levels from the supernatant fractions were determined in a [3H]cAMP (0.8 pmol) binding assay as previously described rauwolscine  in a total volume of 1–2 ml in potassium phosphate buffer  containing MgSO4 (5 mM) at 37°C for 45 min. Thereafter, 2 ml Tris-HCl  was added to the homogenate to terminate the binding reaction and the contents of the tubes was filtered over #32 glass fiber filter strips  using a PHD cell harvester . The reaction tubes and the filter strips were rinsed twice with a further 2–3 ml of buffer. Levels of radioactivity were determined by scintillation spectroscopy in a Beckman LS6000 liquid scintillation counter. All assays were performed in triplicate, and specific binding was determined by subtracting the binding in the presence of yohimbine or phentolamine  from the binding in its absence.The level of αd of the ligand for the α2-AR [2-ARs in SHβ2AR4 cell membranes (0.1 – 0.2 mg/mL) were determined using a single concentration (2 nM) of either [3H]rauwolscine or [3H]RX821002 following catecholamine treatment.Previously we have shown that agonist treatments do not alter the Khe α2-AR . Therefo2-AR binding was performed with [3H]CGP-12177. For saturation studies, cell membranes (0.5 mg/mL) were incubated with [3H]CGP-12177 (0.2 to 40 nM) in Tris-HCl buffer  containing MgCl2 (0.5 mM) at 37°C for 30 min. Specific binding was determined by subtracting the binding in the presence and absence of propranolol (1 μM).β3H]rauwolscine was fixed (1 nM), and various (4–9) concentrations of unlabeled drugs were included.Cell membrane fractions were incubated as described above except that the concentration of /[KD of radioligand]. Comparisons between groups were made by two-way Student's t-tests or ANOVA and Tukey's or Dunnett's post hoc test , and groups were considered significantly different if p ≤ 0.05.Kequation  in whichIBMX, 3-isobutyl-1-methylxanthine; HBSS, Hank's balanced salt solution; UK 14,304, 5-Bromo-N--6-quinoxalinamine; ARC-239, 2--piperazin-1-yl)-ethyl-4,4dimethyl-1,3--isoquinolindione, AR, adrenoceptor; ISO, isoproterenol; EPI, epinephrine; NE, norepinephrine; wt, wild-type.2AR4-expressing clones, carried out all chronic treatment experiments, performed the statistical analyses and immunoblotting experiments, and drafted the manuscript. GFA carried out the binding and functional assays characterizing the α2-AR subtype. CDM conducted the molecular analysis studies. DCE participated in the conception and design of the study and helped draft the manuscript. LAS participated in the design and coordination of the molecular studies. KMS conceived of the study, participated in the design and coordination of all experiments and helped draft the manuscript. All authors read and approved the final manuscript.TB-K participated in the design of the study, generated and selected stable SHβ


We have shown that both free fatty acids (FFA)and triglycerides (TG) increase expression ofplasminogen activator inhibitor type 1 (PAI-1)in vivo and in vitro. To determine signalingmechanisms responsible, HepG2 cells wereexposed to FFA, emulsified TG, or the combination.The combination of FFA and TGincreased PAI-1 to a greater extent than eitheragent alone . Cells transfected with PAI-15' flanking region containing the 4G or 5Gpolymorphism displayed similar activity inresponse to FFA, but modestly greater activitywith the 4G polymorphism in response to TG.Deletion analyses demonstrated that FFA andTG induce PAI-1 expression through distinctregions of the promoter. Inhibition of proteinkinase C inhibited the response to FFA but notTG. Accordingly, increased FFA and TG contributeto increased PAI- I through independentmechanisms.


Francisella tularensis, the causative bacterium of tularemia in humans and a potential bioterrorism agent, in macrophages.Autophagy has been shown recently to play an important role in the intracellular survival of several pathogenic bacteria. In this study, we investigated the effect of a novel small-molecule autophagy-inducing agent, AR-12, on the survival of F. tularensis  and F. novicida in macrophages in association with increased bacterial co-localization with autophagosomes. The effect of AR-12 on intracellular F. novicida was fully reversed in the presence of the autophagy inhibitor, 3-methyl adenine or the lysosome inhibitor, chloroquine. Intracellular F. novicida were not susceptible to the inhibitory activity of AR-12 added at 12 h post-infection in THP-1 macrophages, and this lack of susceptibility was independent of the intracellular location of bacteria.Our results show that AR-12 induces autophagy in THP-1 macrophages, as indicated by increased autophagosome formation, and potently inhibits the intracellular survival of F. tularensis can be eradicated by small-molecule agents that target innate immunity.Together, AR-12 represents a proof-of-principle that intracellular  Mycobacterium tuberculosis, Group A Streptococcus pyrogenes and Salmonella typhimurium  in the ER. Inhibition of SERCA will cause Ca2+ efflux from ER to the cytosol, leading to the unfolded protein response and subsequent activation of PERK. Although there is no direct evidence showing that AR-12 can interact with SERCA, the cytosolic [Ca2+] of cancer cells increases immediately after exposure to AR-12 which suggests that AR-12 may inhibit SERCA activity [AR-12 has been reported to inhibit a variety of cellular enzymes, including PDK-1 and P21 activated kinase-1 (PAK-1), and to induce endoplasmic reticulum (ER) stress -39. Amonactivity . IdentifFrancisella activity, and that subsequent screening of a celecoxib-based focused compound library identified Compound 20, a novel analog with multi-fold greater antibacterial activity [F. novicida and F. tularensis  at low μM concentrations. While the intracellular growth of Francisella was also inhibited by Compound 20, this occurred at concentrations that far exceeded those shown for AR-12. Although the antibacterial target of Compound 20 has not been identified, these differences in activity suggest that Compound 20 and AR-12 represent two novel classes of compound with distinct antibacterial mechanisms. These findings highlight the potential value of existing drugs as important sources of lead compounds for the development of novel antibacterial agents.We recently reported that celecoxib, the parent compound of AR-12, also exhibits anti-activity . Unlike The authors declare that they have no competing interests.Francisella tularensis strain Schu S4. SKK helped to design experiment and revised the manuscript. HC helped the assay of intracellular Francisella novicida in macrophage. DW conducted the synthesis of AR-12. JSG provided the ΔABCH strain of F. novicida and helped to design experiments. LSS helped to design experiments and revised the manuscript. CSC conceived of the study and revised the manuscript. All authors read and approved the final manuscript.HCC conceived of the study, designed and performed most experiments, and drafted the manuscript. SS conducted the experiment with virulent


FOXO transcription factors are an important, conserved family of regulators of cellular processes including metabolism, cell-cycle progression, apoptosis and stress resistance. They are required for the efficacy of several of the genetic interventions that modulate lifespan. FOXO activity is regulated by multiple post-translational modifications (PTMs) that affect its subcellular localization, half-life, DNA binding and transcriptional activity. Here, we show how a mathematical modelling approach can be used to simulate the effects, singly and in combination, of these PTMs. Our model is implemented using the Systems Biology Markup Language (SBML), generated by an ancillary program and simulated in a stochastic framework. The use of the ancillary program to generate the SBML is necessary because the possibility that many regulatory PTMs may be added, each independently of the others, means that a large number of chemically distinct forms of the FOXO molecule must be taken into account, and the program is used to generate them. Although the model does not yet include detailed representations of events upstream and downstream of FOXO, we show how it can qualitatively, and in some cases quantitatively, reproduce the known effects of certain treatments that induce various single and multiple PTMs, and allows for a complex spatiotemporal interplay of effects due to the activation of multiple PTM-inducing treatments. Thus, it provides an important framework to integrate current knowledge about the behaviour of FOXO. The approach should be generally applicable to other proteins experiencing multiple regulations. The FOXO  family of transcription factors (TFs) cause changes in gene expression to implement a cellular stress response programme, and an increase in their activity is a consequence of many of the genetic interventions that extend lifespan in model organisms. FOXO transcription factors are active when an organism is fasting, whereas feeding causes the activation of the Insulin- and Insulin-like Signalling (IIS) pathway, and in particular of Akt/PKB, which negatively regulates FOXO TFs by phosphorylations that cause translocation to the cytoplasm, thus suppressing their transcriptional activity. However, the location, synthesis/degradation and transcriptional behaviour can all be modified by signals from a variety of cellular signalling pathways, which are integrated by FOXO as a net response to the total set of modifications , that it undergoes. Integrating this knowledge within an extensible framework would provide a valuable means to organise the information and to explore the role of the modifications.Caenorhabditis elegans the FOXO orthologue is produced from the daf-16 gene (wormbase WBGene00000912). In Drosophila melanogaster, there is again a single dFOXO gene (flybase FBgn0038197). Proteins in the FOXO family are more distantly related to other FOX-domain containing TFs, such as the FOXA family, which themselves have a role in ageing C elegans: MnSOD , FOXO transcription factors are highly evolutionarily conserved in multicellular animals. There are four closely related FOXO paralogues in mammals, FOXO1 (formerly FKHR), FOXO3 , FOXO4 (formerly AFX) and FOXO6 . All are ubiquitously expressed, but FOXO1 is particularly highly expressed in adipose tissue, FOXO3 in the brain, FOXO4 in the heart, and FOXO6 in the developing brain Homo sapiens), mouse (Mus musculus), Xenopus leavis, Dario rerio, Drosophila and C elegans. There are some regions of conservation throughout, especially in the DNA-binding domain (expanded region). Some of the modifiable sites in this domain are highlighted, along with an indication of the enzyme that modifies them. It is clear that there a great many regulators of FOXO , and a great many modifiable sites. The multiple sequence alignment is available as supplementary In FOXO is implicated in many of the interventions that extend lifespan in worms, flies and mice, particularly genetic interventions in the IIS pathway and some protocols of dietary restriction (DR). Overexpression of FOXO itself extends lifespan moderately in flies Many reviews of the behaviour and role of FOXO are available, related to its PTMs To our knowledge FOXO has not been previously the subject of computational modeling, though other transcription factors have, for example the SMAD2/SMAD4 complex It is clear that FOXO has important roles in ageing, nutrient response and stress response pathways; and also that the large number of regulations that it undergoes and their different and often contradictory effects, make it difficult to understand the system. Hence, we believe that it is a good candidate for computational modelling, which, by integrating information from diverse sources and making possible a quantitative description, has the ability to resolve some of these difficulties There are two problems to be faced in modelling the regulation of FOXO. Firstly, it is necessary to choose a model structure that simplifies the large (and still not fully elucidated) set of PTMs, while still retaining enough information to describe all the relevant regulations. Secondly, appropriate parameters must be set for the reactions, even though detailed kinetic measurements are often not available.N FOXO species ; and, in addition, each could be present in the three compartments of the cytoplasm, nucleus or bound to DNA , making 3×2N species in total. Each of these species could be present in a copy number of 0, 1, 2, … at each time point in the simulation. For the set of N = 27 modifications in 8 species, an excessively large number, even before all the reactions to interconvert them are considered. However, simplifications can be made by grouping all PTMs that produce a similar effect on the behaviour of FOXO. This usually means all those made by a particular modifying enzyme or enzymes. So, for example, to a good approximation the three residues modified by Akt  must all be phosphorylated before the effects are seen, which are to enhance export of FOXO from the nucleus to the cytoplasm The first problem is a consequence mainly of the large number of independent PTMs, which lead to a need to include many species to describe all the combinations. This can be appreciated by considering 2O2 (below 100µM)  subsequently, that multiply the “basal” rate, For example, for a species with both Akt and JNK phosphorylations, the effect on translocation is the basal rate×MF due to Akt×MF due to JNK. This is clearly likely to be a fairly gross approximation, but it is necessary to enable the model to be parameterized; its justification will thus be in the empirical validity and usefulness of the model. Of course, as most detailed measurements are made, it will be possible to modify the model to incorporate them.We now turn to the second problem, that of addressing the kinetics of the reactions in which these species are involved. First, consider the reactions involved . A partiFor the transcription reactions represented in A and B represent FOXO species with different PTMs, and the forward and back reactions occur at rates 1v and 2v, catalysed by enzymes aE and bE. The experimental information often amounts to a knowledge of eq(A/B) and some idea of the relaxation time, from an experiment when one of A and B is initially at very low concentration, then the enzyme that produces it is activated. From the rate equations, first with the time derivative equal to zero, and second with one of A or B (near)-zero, it is simple to see that this determines pseudo-first order rate constants, a = kaEak′ and b = kbEbk′. Then, we choose appropriate values for the copy number of aE and bE in the range of 104 to 105 when fully activated  the rates are high  compared to processes like the relocation/degradation/transcriptional output of FOXO. This is true of Akt-phosphorylation We have assumed that at the levels of “high active kinase” , mcg is the cytoplasmic mRNA of gene g, pcg is the cytoplasmic protein corresponding to gene g, and A is a DNA-bound FOXO species. The first- or zero-order rate constants are given in For the transcriptional outputs, the relevant reactions areA python script was written to generate the species and reactions: this script is available as supplementary Simulations were carried out using the gillespie2 stochastic simulator in stand-alone mode and also through BASIS, which provides a web interface to gillespie2 and a database to store the results As we have said above, the kind of equations relating to fractional locations or degrees of up/down regulation do not permit the number of molecules involved to be known exactly. The initial number of FOXO molecules of all species was set to be 1000. This is probably an underestimate, but is the approximate number of consensus FOXO binding sites, GTAAACAA cells having FOXO mostly nuclear or cytoplasmic. Those cells which are experimentally observed to have FOXO both nuclear and cytoplasmic are counted together with those where it is wholly cytoplasmic . In the model the total nuclear FOXO is simply the sum of that in the nuclear and DNA-bound compartments. The basal rate of redistribution is ∼0.1 min−1, to give a half-life of the order of 10 minutes, with the cytoplasmic-nuclear rate slightly higher, and the nuclear-cytoplasmic lower, for unmodified FOXO, to give an equilibrium favouring the nuclear form. The basal rates of DNA binding and unbinding are chosen so that, of the total nuclear FOXO, about two-thirds is DNA-bound and one-third nuclear but not bound to DNA.The first regulation of FOXO to be discovered was its Akt-phosphorylation and translocation between nucleus and cytoplasm. Simply stated, in the absence of Akt-activation, FOXO is primarily nuclear, while activation of Akt, usually by the insulin-signalling pathway, leads to sequestration of FOXO in the cytoplasm. Experimentally, FOXO localization is usually measured as the fraction of cells having FOXO mostly nuclear, mostly cytoplasmic or both nuclear and cytoplasmic, as determined with fluorescent-labelled FOXO. In the current model, the fraction of FOXO in the nuclear or cytoplasmic compartment will be treated as equivalent to the experimentally determined fraction of In 3 in the pre-equilibration level, which is then increased to 2.5×104 to correspond to the IGF stimulation. The experiment lags slightly behind the simulation result at around 5 or 10 minutes, but this is reasonable given that that in the simulation the level of active Akt is set directly whereas in the experiment it must be activated through the IIS pathway, with a delay of the order of a few minutes.In −1, shown in −1. Then, without proteasome inhibition, the amount of FOXO in the cell was quantified with and without insulin stimulation, i.e. with and without Akt activation, over 12h. Although proteasomes are present in the nucleus, it has been convincingly shown that degradation of FOXO occurs only in the cytoplasm −3 min−1 for polyubiquitination, 0.1 min−1 for degradation, and an MF of 3. Taking the copy numbers of the modifiers to be 103 for both the total of ubiquitin ligases (SCF+MDM2) and the proteasome, this gives second order rate constants of 10−6 and 10−4 min−1. With these parameters, Now let us consider the synthesis and degradation of FOXO. This was first studied in detail in experiments on HepG2 cells by Matsuzaki et al 2O2, 200 and 500 µM, that activate CBP/P300 and fully acetylate FOXO4 in 30 minutes . Hence, it is reasonable to choose pseudo-first-order rate constants of ∼0.1 min−1, and we make the choice of the copy number of CBP/P300 and SIRT1 to be 103 when fully activated, and a second order rate constant of 10−4 for each reaction. SIRT1 and CBP/P300 are mostly nuclear Other PTMs significantly modulate the degradation of FOXO. Acetylation is one of these, and is of particular interest because of the suggestion that the FOXO- and histone- deacetylase, SIRT1, may also modulate organism lifepan. The timescale of both acetylation and deacetylation is of the order of 10 minutes when highly activated. This is shown in van der Horst 2004 Another PTM that affects degradation, this time accelerating it, is phosphorylation by IKK. Again, phosphorylation is chosen to be a rapid process in comparison with degradation. The fit in Acetylation also affects transcription, as shown in 5) and mostly deactivated (copy number 100).FOXO may also be modified by AMPK, with modulation of transcription but not subcellular localization JNK-phosphorylation broadly opposes the effects of Akt-phosphorylation, and is discussed in the subsection on scenarios of multiple FOXO regulation below. Other modifications such as CDK2-phosphorylation and monoubiquitination have similar effects, causing alteration of the nuclear-cytoplasmic equilibrium and FOXO-mediated transcription. They can be modelled similarly (data not shown).Several parameters of the model relate to transcription of mRNA, its transport to cytoplasm, translation to protein and and degradation; and also to the degradation of the protein. Lacking absolute quantification of protein or mRNA level, or measurement of mRNA or protein lifetime, we have used generic values (k/X) dX/dk where X is a concentration and k a parameter, were considered. Examples of the detailed results are available as supplementary We have investigated the sensitivity of the concentrations of the species of the model as a function of the parameters, using COPASI 5 when they are active. The PP2A level is 104 molecules, so the PTM that the kinases confer is present in about 80% of the FOXO molecules.Having shown that experimentally-determined responses to single and some multiple regulations can be reproduced by the model, we now move on to study the behaviour of the model in scenarios involving multiple regulations. A simple combination of effects is shown in 2O2 show an interplay between the opposing effects of JNK and Akt; JNK tends to override Akt (as in A–C) but if Akt activity is particularly high it may override a low level of JNK activation 4), while in E it is much lower. It is noteworthy that, with PP2A low and without JNK, a lower level of Akt activation (in E compared to D) is sufficient to drive FOXO to the cytoplasm, but also a lower level of JNK activity suffices to retain it in the nucleus even at a high level of Akt activation (compare the response to JNK = 103). That PP2A, JNK and Akt could all change together is a likely scenario, because oxidative stress tends to inactivate protein phosphatases due to their reliance on a catalytic cysteine that contains an easily oxidised SH group.Experiments in which JNK is activated by Hin-silico experiment was suggested by comments made in Calnan and Brunet Another example is shown in 5). This initial burst of Akt activation causes FOXO translocation to the cytoplasm and transcription almost stops (5 active Akt molecules from t = 540 for the next 720 min (12 hours) ), again causing translocation of FOXO and almost halting transcription of FOXO target genes. This pulse is long enough for the level of FOXO to decline by more than half in the course of it, as a result of polyubiquitination and resultant degradation of the cytoplasmic FOXO. Hence, when the second pulse of high Akt activity ends (at t = 1260) and FOXO returns to the nucleus, the transcription level . With this data, the forward and backward rates of a first order process and its inverse can be estimated, but the multiple constants of more complicated kinetics cannot. Secondly, the simplification of combining multiple phosphorylations into a single collective modification leads naturally to simple kinetics where the FOXO species appears linearly in the rate expression; nevertheless it is likely that more detailed timecourses would show that the kinetics do not follow this kind of behaviour. For example, with multiple regulations, it is to be expected that the fully phosphorylated form will appear as a concave-upwards function of time, such as a cubic function when three phosphorylations occur, in response to a step increase of the enzyme producing it, rather than the linear timecourse of the 1° model. This may often seem to correspond to an apparent initial delay in experiment. As detailed time course data becomes available it may, therefore, be necessary to revise this assumption.Transport into and out of the nucleus has also been represented by simple first order processes with rates dependent on the PTM state, when in fact the process is more complex, involving binding of the FOXO to two 14-3-3 proteins, masking the Nuclear Localization Signal, and its resultant export to the cytoplasm The negative second derivative of the experimental curve of FOXO molecule number against time  suggestsIn general, the kinetics of the phosphatase-catalysed reactions are much less certain that those of the kinase-catalysed reactions. Most of the time they have not been studied in detail. Hence, we have assumed a single “general phosphatase”, assumed to be PP2A, to reverse all the kinase-mediated phosphorylations in the model.When the localization of FOXO alters experimentally in response to changes in PTM, it is experimentally observed that there is a change in the fraction of cells in the population having FOXO primarily nuclear, primarily cytoplasmic, or present in both compartments  and ERK (MAPK1), which may not be synonymous in their effect to those by JNK. It has been shown that ERK and p38 MAPK modify mouse FOXO1 Similar approaches to generating multiple chemical species by enumerating combinations of PTMs have been implemented several times previously (Cellerator C elegans transcription factor SKN-1 (orthologous to NRF TFs in higher organisms) The scripting/rule-based modelling approach to generating reactions is applicable to other proteins that undergo multiple regulations in a similar manner, for example the insulin receptor substrate IRS-1 To summarize, this FOXO model provides a framework into which future measurements can be fitted, and provides a central component for more complex models integrating the multiple upstream pathways and the downstream events that follow from the transcription of the multiple FOXO-regulated genes. We have demonstrated that, even alone, it demonstrates complex spatio-temporal regulations: in particular, the behaviour shown in Dataset S1Multiple sequence alignment of FOXOs from Human, Mouse, Xenopus, Zebrafish, Drosophila and C elegans ).(0.02 MB TXT)Click here for additional data file.Dataset S2Python script used to generate SBML-shorthand for the models in this paper. See comments at the top of the file for usage, software requirements, and how to modify.(0.03 MB TXT)Click here for additional data file.Dataset S3SBML model file for the simulation in (0.06 MB XML)Click here for additional data file.Dataset S4Sensitivity analysis (tab-delimited file) for a similar model to S3.xml (Akt = 1e5 not 2.5e4).(0.02 MB TXT)Click here for additional data file.Dataset S5SBML model file for the simulation in (0.12 MB XML)Click here for additional data file.Dataset S6Sensitivity analysis (tab-delimited file) for model S5.xml.(0.07 MB TXT)Click here for additional data file.Dataset S7SBML model file for the simulation in (0.13 MB XML)Click here for additional data file.Dataset S8Sensitivity analysis (tab-delimited file) for model S7.xml.(0.07 MB TXT)Click here for additional data file.Dataset S9All above supporting information (S1–S8) in single zip archive: multiple sequence alignment of FOXOs , python script, sbml models and sensitivity analyses.(0.07 MB ZIP)Click here for additional data file.


Scincus scincus) is a lizard having the remarkable ability to move through desert sand for significant distances. It is well adapted to living in loose sand by virtue of a combination of morphological and behavioural specializations. We investigated the bodyform of the sandfish using 3D-laserscanning and explored its locomotion in loose desert sand using fast nuclear magnetic resonance (NMR) imaging. The sandfish exhibits an in-plane meandering motion with a frequency of about 3 Hz and an amplitude of about half its body length accompanied by swimming-like (or trotting) movements of its limbs. No torsion of the body was observed, a movement required for a digging-behaviour. Simple calculations based on the Janssen model for granular material related to our findings on bodyform and locomotor behaviour render a local decompaction of the sand surrounding the moving sandfish very likely. Thus the sand locally behaves as a viscous fluid and not as a solid material. In this fluidised sand the sandfish is able to “swim” using its limbs.The sandfish ( ScincusScincus scincus , S. albifasciatus (including S. a. laterimaculatus), S. hemprichii, and S. mitranus. The two last taxa include other previously recognized species in synonymy Scincus s. conirostris and S. mitranus in Saudi Arabia) is not yet well understood and may relate to the grain size of the sandy substrate. The morphological adaptations related to this lifestyle have long been known Scincus (the “sandfish” of the Arabs) in the Saharo-Arabian region The scincid lizard genus Liolaemus species Scincus is unclear.Movement in or under sand is also used by several other lizards and has been independently acquired in several families see . Some ofPhrynocephalus and Phrynosoma, to the so-called “shimmy” burial of Uma, Callisaurus and Cophosaurus. True sand swimming, however, where a lizard with well-developed limbs remains for longer periods below the surface and covers considerable distances with remarkable speed appears to be restricted to the species of Scincus and possibly to the lizard said to be most similar in its sand-swimming behaviour, the gerrhosaurid Angolosaurus. It is tempting to assume that the morphologically different lizards Meroles (Lacertidae) and Eremiascincus (Scincidae) may use a different mode of underground locomotion. It is generally believed that in this locomotory-type propulsive thrust is produced without the use of limbs Angolosaurus, sand diving has been described ScincusThe sand-diving techniques of species with well-developed limbs show much variation and have recently been described and discussed in detail S. scincus using NMR imaging, because, along with the sensory system, the integument In this paper, we test this hypothesis of a serpentiform sand swimming with adpressed limbs by S. scincus. Typical results are depicted in Along with conventional photography, 3D-laserscanning was employed to document the macroscopic morphology of The rapid burying process of an adult sandfish released from the experimenter's hand is documented in To observe the sandfish in the sand, NMR-imaging, adjusted to detect hydrogen atoms, was employed. This was found to be the best method as the dry sand does not yield a hydrogen NMR-signal whereas the tissues of the sandfish give a clear signal yielding a sufficient signal to noise ratio. This enables fast, repetitive imaging which is necessary for the investigation of locomotion within the sand. An image of a sandfish resting beneath about 100 mm of sand is depicted in Using the projection method described in the “In order to quantify the animal's movement in the sand, the longitudinal body axis was documented throughout the NMR-image sequences. The result for the image sequence shown in The sandfish depicted in In order to analyse the movement of distinct parts of the body, the lateral amplitude of the movement of the tip of the head, the mid-part of the body axis and of the tip of the tail were determined. The positions were phase matched. It is clearly evident that the undulations are approximately identical for all parts of the body yielding travelling waves. This can be seen in a schematic drawing of one half-cycle of the sandfish's movement depicted in As will be discussed below, one can estimate that at certain oscillation frequencies granular media like sand could behave more like a fluid than like a solid body. In order to test whether the undulations at the observed frequency result in a so called decompaction (see below), we measured the power consumption which is direct proportional to the forces necessary to move an aluminium rod of a diameter similar to that of a sandfish (∅20 mm) at certain frequencies through sand in a depth of 100 mm. The results are depicted in S. scincus by using fast imaging NMR. We found a meandering in plane body motion accompanied by a “trotting” like movement of the limbs. No torsion of the body, especially of the head with its spatula-shaped snout was observed. This intuitively logical torsional movement was initially assumed to occur Tiliqua rugosai) the limbs are moved forward in loose sand and backward in compacted (compressed) sand. This leads to a net forward thrust; ii) a decompaction of the sand occurs around most of the body of the sandfish.In the present study we demonstrate the mode of movement through sand of the sandfish e∼0.54) to loose packing (pore number e∼1) alone will result in a decrease of the shear strength τ up to a value of 45%, as e·τ is approximately constant in dry quartz sand A complete physical description of the processes in the sand around the “swimming” sandfish is currently under investigation. However, we can estimate the processes taking place in the sand, and do so here by approximating the sandfish problem. Let us first consider the limbs generating thrust. We can only recognise the general characteristics of a trot but cannot distinguish details of the gait pattern i.e. the accelerations the particles experience is above a critical acceleration (the ‘lift-off’ acceleration), the particles will separate and stay separated due to collisions . This is clearly below the frequency of the sandfish's meandering motion in the horizontal plane . Thus, we may expect a local decompaction of the sand which results in the sand locally behaving more like a fluid than a solid body Additionally, the undulatory movement may lead to a decompaction of the sand surrounding the body of the sandfish. This can be roughly approximated according to the Janssen model llisions . AccordiPerforming force measurements in loose sand clearly showed that at a frequency of 3 Hz, a horizontal sinusoidal movement of a cylindrical probe mimicking the geometry of the sandfish movement required the least force. This experimental result is in excellent agreement with the prediction according to the Janssen model.Although the above calculation is a rough estimation based on very simplifying assumptions and neglecting some geometrical restrictions, it is instructive to assume that the undulatory movement of the sandfish results in a local decompaction of the sand. This allows the sandfish to “swim” in this fluid-like granular medium by propelling itself forward by using the limbs, which face low drag during forward movement and high drag during backward movement due to the bending of the body. The drag induced by movement through the decompacted sand is orders of magnitude lower than it would be in compact sand In currently-ongoing work we are attempting to develop a better model for the physical phenomena in the sand around a meandering sandfish based on the NMR-data obtained so far. This might prove useful in two respects. Firstly, we may again a better understanding of the sandfish and other sand-living animals. Secondly, we may be able to learn more about the physics of granular media and some of the means available to effectively decompact these. This might be useful in the engineering sciences in the near future for applications to industrial processing, conveyor technique and handling of grain, sand, powders and the like.Photographic images of the sandfish were taken using a Canon EOS 350D  with the original telephoto lens  attached. The auto exposure setting was used without flash.The digital 3D model of the sandfish was acquired using the following process. Firstly the specimen was scanned from 26 different vantage points using a Minolta VI-900 non-contact laser range digitizer. The scanner casts a moving plane of laser light onto the object to be digitized and captures, for each position of the plane, an image of the laser projection using a CCD camera. The object's shape is then derived from the sequence of observed contours , were also placed symmetrically around the head coil. T1 and T2 relaxation time was due to high concentration of nickel sulphide less than 0.1 ms, meaning, that equilibrium magnetization is reached directly after excitation. No MRI-signal were generated from this additional load since typical echo times for imaging methods and especially the described method is typical beyond 1 ms. The coil was filled with a cylindrical box, 40 cm long, and 22 cm in diameter. The size was chosen to fill the imaging volume of the receiver coil. The lower 60% of the box was filled with “ExoTerra Desert Sand - White” with a grain size of 0.1–1 mm. In the head direction there was an entrance with an attached platform directly above the sand surface. The sandfish was set on the platform. In all cases the animals moved directly into the sand surface, quickly burying themselves. Typical MRI scanners, such as the VISION, require a setup procedure for each measurement. Machine settings for the following imaging experiment rely on these parameters. During setup an object generating MR-signal is required in an isocentric axial plane. Therefore during the setup procedure a 50 ml water-filled centrifuge tube  was placed in the magnet isocenter. This tube was removed before the sandfish measurement was started.The imaging experiments were performed using a 1.5 T whole-body scanner  with a peak gradient amplitude of 25 mT/m and a slew rate of 83 T/m/second. For signal reception, a head coil was used in all measurements. The sensitive area of this coil has a length of approximately 30 cm. Signal transmission was done using the built in body coil. Since the sandfishes, and especially the sand, did not load the body coil sufficiently, additionally four bottles, each filled with 1.5 litres of water containing nickel sulphide  the parameter K = 0.58 and the static friction coefficient for sand was found to be about 0.7 S0 describes the ratio of the height of the sand pile (i.e. the immersion depth of the sandfish) times the perimeter of the pile divided by the cross section of the pile. In our case, if we assume the pile of height 100 mm to be rectangular over the moving sandfish which has a length of 120 mm and is oscillating about 60 mm laterally, we obtain a lift-off acceleration of loΓ≈1.9. Assuming the sandfish diameter 20 mm, the lift-off acceleration will be reached at a frequency for the vertical oscillation of the sand pile of 5 Hz which corresponds to an oscillation of the sandfish in the horizontal plane with 2.5 Hz.According to the theory on the Janssen model a so called decompaction of the granular material, For determination of the force necessary to move a non flexible body of the diameter of a sandfish through loose sand sinusoidally, a 150 mm long aluminium rod with diameter 20 mm was mounted at the end by a tin construction to a linear motor  which was controlled by a E 100 controler (LinMot). The controller was commanded by a PC using the manufacturer's controller software for the E 100. This allowed performing sinusoidal motions of the aluminium rod in bowl of sand at a depth of 100 mm with an amplitude of about 60 mm and defined frequencies ranging from 0.5 Hz to 6 Hz. The force necessary to perform the programmed movement could be directly recorded via the controller by measuring the current consumption over time. This current consumption is, according to the manufacturer, direct proportional to the axial force of the linear motor. The tin construction hardly influenced the results as only the thickness (1 mm) of the tin faced the moving direction. The measuring device is depicted in the supplementary material . The meaFigure S13D-polygonal surface model of a sandfish obtained by 3D-laser scanning. Best seen with quich3D viewer when saved as OBJ-file.(0.40 MB TXT)Click here for additional data file.Figure S2Photograph of the used force measuring apparatus(0.39 MB JPG)Click here for additional data file.Video S1Real time video of the burying behaviour of an adult sandfish observed from above.(1.51 MB AVI)Click here for additional data file.Video S2NMR-imaging sequence of a sand-swimming skink in top view.(1.35 MB MOV)Click here for additional data file.Video S3NMR-imaging sequence of a sand-swimming skink in side view.(1.35 MB MOV)Click here for additional data file.


Two common classes of genomic resources that can be mined are bacterial artificial chromosome (BAC) libraries and expressed sequence tag (EST) libraries.Simple sequence repeats (SSRs) are tandemly repeated sequence motifs common in genomic nucleotide sequence that often harbor significant variation in repeat number. Frequently used as molecular markers, SSRs are increasingly identified via Laupala. SSRs were more densely distributed and contained longer repeat structures in BAC library-derived sequence than in EST library-derived sequence, although neither repeat density nor length was exceptionally elevated despite the relatively large genome size of Laupala. A non-random distribution favoring AT-rich SSRs was observed. Allelic diversity of SSRs was positively correlated with repeat length and was generally higher in AT-rich repeat motifs.288 SSR loci were screened in the rapidly radiating Hawaiian swordtail cricket genus in silico approaches work well for SSR locus identification in both EST and BAC libraries, BAC library sequence and AT-rich repeat motifs are generally superior SSR development resources for most applications.The first large-scale survey of Orthopteran SSR allelic diversity is presented. Selection contributes more strongly to the size and density distributions of SSR loci derived from EST library sequence than from BAC library sequence, although all SSRs likely are subject to similar physical and structural constraints, such as slippage of DNA replication machinery, that may generate increased allelic diversity in AT-rich sequence motifs. Although  Microsatellites, or simple sequence repeats (SSRs), are common features of eukaryotic genomes and can be characterized as generally short, repeated nucleotide sequence elements arrayed in tandem and flanked by non-repetitive regions (reviewed in ). SSRs on SSR loci )es . Two sues . ConseqLaupala, L. kohalensis and L. paranigra, identified in BAC and EST library sequence databases from L. kohalensis. The overall distribution of SSR loci within these genomic libraries is examined in addition to the distribution of allelic richness across all SSR loci to evaluate (1) whether SSR density or mean repeat number is elevated in a species with a relatively large genome size, (2) whether particular SSR motifs are particularly common in library sequence and (3) whether the same motifs are likely to harbor significant allelic variation. We further evaluate the structural properties of the SSR loci associated with elevated numbers of length variants and assess the efficacy of BAC and EST library nucleotide sequences for the development of informative molecular markers.Here, we present a survey of 288 unique SSR loci in two species of X2 = 144, df = 1, p > 0.0001) and SSRs comprised nearly five-fold higher percentage of all sequenced bases in BAC library sequence . Further, SSRs identified in BAC library sequence were comprised of significantly more repeats than were those in EST library sequence (Mann-Whitney test p < 0.0001)  for both the EST and BAC genomic libraries. The analysis could not be extended to include tetra- and pentanucleotide SSR motifs because these comprised less than 2.5% of the dataset and thus were too infrequent to provide adequate statistical power (10 tetranucleotide SSRs in BAC library sequence and 16 tetra- and 2 pentanucleotide SSRs in EST library sequence). The posterior probability distribution was compared to the observed distribution of motifs within SSRs using the chi-square goodness of fit test. In each case, SSRs were found to be significantly non-randomly distributed across motif types , including all ORFs in any reading frame. SSR ORF placement was characterized as being either within an ORF or outside of an ORF. Comparisons using chi-square goodness of fit tests revealed that SSRs from BAC library sequence were randomly distributed with respect to ORFs, while those from EST library sequence were non-random, particularly among trinucleotide SSRs Table . These dL. kohalensis and L. paranigra, 35 failed to amplify entirely and amplification was successful for only a single species in two of the markers screened. Of the remaining 251 loci, 9 loci were scored as having at least one individual with a null allele and were dropped from subsequent analyses. In total, 242 putative SSR loci were assayed for allelic length variation  and a simple count of allele number was used to estimate allelic richness. Mean allelic richness was nearly identical for total SSRs from BAC and EST library sequences . However, distributions of SSR allelic richness within both BAC and EST library sequence is non-random. Allelic richness was unequally distributed across dinucleotide SSR sequence motifs in both libraries  Table , althougAllelic richness is significantly positively correlated with the number of repeats present in genomic library sequence SSRs . Reference sequence repeat number ranged widely in both genomic sequence libraries. Consequently, data were pooled by reference sequence repeat number within each library to increase sample size within each repeat class  and are presented in Figure Laupala kohalensis, consisting of both BAC and EST library databases, for all SSR loci. Of the 736 total SSR loci identified, 288 were screened using four distinct families each from two Laupala species, L. kohalensis and L. paranigra, and characterized according to amplification success and allelic diversity at each locus. In our analysis, we found that identities of SSR loci are far more conserved across these two closely related species  than might have been expected [A ubiquitous feature in the genomes of diverse organisms, simple sequence repeat (SSR) loci, or microsatellites, are frequently overrepresented in eukaryotic genomes relative to total base composition ,40. Furtexpected ,42. DespDrosophila [Drosophila [Laupala is associated with genome size or size of intergenic space in the genome. SSR surveys in the grasshopper Chorthippus biguttulus indicate that the large genome size in this species is associated with elevated repeat length rather than density of SSR loci within the genome [Laupala, where observed repeat lengths and densities were similar to other insect groups [Laupala genome.Consistent with the observation of SSR frequency being positively correlated with genome size and intergenic space ,16, SSR osophila ,44 and sosophila  althoughosophila . SSR locosophila  and an oosophila , it seeme genome ; howevert groups -45. Inten repeats are by far the most numerically dominant of SSR loci in humans and most eukaryotes [Laupala BAC-end sequences are strongly biased toward an excess of (AT)n repeats and generally toward repeats with depressed GC base content. This general trend is similar to that reported among SSR loci in the silkworm, Bombyx mori [(AC)karyotes -49. Contbyx mori ; howeverbyx mori ); howevebyx mori ,51, thisAllelic diversity has been shown to be positively correlated with the length, or repeat number, of SSRs in many organisms ,52,53. BLaupala was nearly equal in SSRs derived from both the BAC and EST libraries, but showed strong effects of SSR base composition. SSRs with low GC content from both libraries were significantly more allele-rich than high GC content repeat motifs. This observation is independent of the numerical abundance of the same repeat motifs, although they may share a mechanistic origin. Different equilibrium microsatellite lengths across species have been attributed to species-specific rates of replication slippage [Laupala replication machinery is intrinsically more likely to suffer slippage during replication of AT-rich motifs, this may explain both the prevalence of these repeat structures within the genome and their diversity across lineages. Similar patterns of base composition-biased allelic diversity have been reported in B. mori [Mean allelic diversity in slippage . If the  B. mori . Previou B. mori ); here, in silico and laboratory-based analyses to evaluate 288 SSR loci in the Hawaiian swordtail cricket genus Laupala, the first such genomic-scale survey in the Orthoptera. Despite a relatively large genome size, SSR loci do not appear to be particularly dense or large in Laupala. SSR loci are significantly more common in BAC library than in EST library sequence and are heterogeneously distributed across all potential base compositions, with a deficit of GC-rich repeat motifs. While many SSR loci can be identified in EST library sequence, the likelihood that selective pressures shape the frequency and diversity of these loci may restrict their utility for certain applications. Allelic diversity of SSR loci is positively correlated with the repeat length found in library sequence and also influenced by repeat base composition. In Laupala, similar physical structural properties of SSRs and the DNA replication machinery likely contribute to the elevated abundance and allelic diversity of AT-rich repeat motifs, suggesting that future screens of Orthopteran molecular markers may benefit by focusing on such SSR motifs. Although allelic diversity profiles are similar in both BAC and EST library-derived SSR loci, the generally higher frequency of SSRs, larger number of repeats within those SSRs, and reduced likelihood of strong selective constraints relative to EST library-derived SSR loci make BAC library sequences far better in silico sources of SSR loci in Laupala and likely other developing model systems as well.Many applications of SSR loci as molecular markers are contingent on the presence of a number of selectively neutral length variant alleles to distinguish species or lineages ,25. HereL. kohalensis were downloaded from the DFCI Cricket Gene Index http://compbio.dfci.harvard.edu/tgi/cgi-bin/tgi/gimain.pl?gudb=Cricket. An additional 1.71 Mb of unpublished BAC-end sequence, also from L. kohalensis were evaluated in parallel . Files were compiled using the program BioEdit , was available for analysis.14363 EST sequences, comprising 10.17 Mb, from http://www.genomics.ceh.ac.uk/msatfinder and all SSRs having repeat motifs of two or greater base pairs and greater than five repeating units were identified. Only simple, perfect repeat motifs were considered; compound and imperfect repeat structures were not included in this analysis. Primers flanking each SSR were designed using Primer3  and all such SSRs were subsequently dropped from the analysis. All primers were designed to produce products ranging in size from 200-350 bp with an optimal Tm of 60°C. Maximum self-complimentarity was set to 8.00 (3.00 at the 3' end) and maximum self-priming was set to 12.00. Program default settings were used for all other parameters.Both genomic libraries were screened for SSR motifs using the Msatfinder script implemented in PERL  was determined using the GenBank Open Reading Frame Finder Laupala were used to screen SSR loci for length variants. The two species, L. kohalensis and L. paranigra, are both from the Kohalensis species group and show less than 0.1% nucleotide sequence divergence at nuclear loci [L. kohalensis male was drawn from each of four lines, collected from two different sampling localities each in the fourth laboratory generation . A single L. paranigra male was drawn from each of four lines, all collected from the same sampling locality , with the first three in the fourth laboratory generation and the final line in the eleventh laboratory generation.Four individuals each from two species of the Hawaiian cricket genus ear loci ,33. A si-1.All individuals were sacrificed by decapitation using a sterile razor. Thoracic and abdominal sections were archived in 100% ethanol, while the head was used for DNA extraction using the DNeasy Blood and Tissue Kit  according to manufacturer's specifications. DNA concentration in each sample was evaluated using a NanoDrop spectrophotometer  and each was diluted to a working stock of 10 ng•nl-1 DyNAzyme II , 0.2 mM dNTPs , 1 μM each forward and reverse primer , and 0.5 ng•μl-1 genomic DNA template were performed in 20 μl volumes. A denaturing step of 2 minutes at 95°C was followed by 35 cycles of 95°C for 30 seconds, 56°C for 30 seconds, and 72°C for one minute and a final 5 minute elongation step at 72°C. Although these conditions did not work for all 288 primer pairs, no attempt was made to optimize conditions for the minority that failed. Primers are presented in Supplementary Table 288 identified SSR loci were screened for repeat variation in the eight animals listed above. PCR reactions containing 1X PCR DyNAzyme II buffer, 0.05 U•μlAll PCR products were evaluated for length variants on 4% 19:1 polyacrylamide:bis-acrylamide gels . Each gel was pre-run for 15 minutes at 60 W prior to loading samples and 100 bp ladder , then run for 3 hours at 55 W. Bands were visualized by silver staining and scored by hand. As precise determination of band size could not be attained, scores reflected number of unique alleles per SSR locus.L. kohalensis and 4 L. paranigra).All statistics were performed using the SPSS11 statistics package . Mann-Whitney U tests were used to test for difference in repeat numbers present across libraries. Chi-square goodness-of-fit tests were used to compare observed SSR distribution within libraries with respect to (A) even distribution of SSRs across all sequence motifs (B) the posterior probability distribution of repeat motifs, (C) an even distribution of SSRs internal and external to identified ORFs, and (D) the posterior probability distribution of ORF inclusion. Posterior probability distributions were calculated separately for each genomic library, BAC and EST, respectively. The posterior probability distribution for SSR motif composition was calculated based on the total frequency of SSR motif within a complete genomic library. Correlation between SSR reference sequence repeat number and allelic richness was performed using a Spearman rank correlation. Distribution of allelic richness with regard to (A) library sequence repeat number, (B) placement within versus outside of ORF sequence, and (C) size of SSR motif (i.e. dinucleotide repeats versus trinucleotide repeats versus tetranucleotide repeats) was evaluated using Kruskal-Wallace tests. Similarly, the posterior probability distribution for SSR placement within ORF regions was based on the distribution of ORF sequence within those library sequences containing identified SSRs. To eliminate the possibility of a sampling bias influencing estimates of allelic richness, any loci with one or more individual failing to amplify were removed from analyses using allelic richness estimates. Consequently, all estimates of allelic richness were based on an equal number of individuals (4 6 base pairs)SSR: Simple Sequence Repeat; ORF: Open Reading Frame; DNA: Deoxyribonucleic Acid; EST: Expressed Sequence Tag; BAC: Bacterial Artificial Chromosome; PCR: Polymerase Chain Reaction; bp: base pairs; Mb: megabases (=10CKE designed the experiment, collected all data, performed data analysis and contributed to writing of the manuscript; KLS contributed to interpretation of results and writing of the manuscript. The final version of this manuscript was approved by both authors.Laupala SSR primer and repeat data from BAC library sequences. Table showing all SSR loci screened in this study for allele number. Includes: SSR sequence identifier, primers, reference sequence repeat number, and observed allele number for SSRs derived from BAC library sequence.Click here for fileLaupala SSR primer and repeat data from EST library sequences. Table showing all SSR loci screened in this study for allele number. Includes: SSR sequence identifier, primers, reference sequence repeat number, and observed allele number for SSRs derived from EST library sequence.Click here for file


C. elegans, where a conserved insulin/insulin-like growth factor signaling (IIS) pathway and the germline both control lifespan by regulating the subcellular localization of the FoxO transcription factor DAF-16. Although the control of FoxO activity through modulation of its subcellular localization is well established, nuclear translocation of FoxO is not sufficient for full FoxO activation, suggesting that undiscovered inputs regulate FoxO activity after its translocation to the nucleus. We have recently discovered a new conserved pathway, the EAK (enhancer-of-akt-1) pathway, which acts in parallel to the Akt/PKB family of serine-threonine kinases to regulate DAF-16/FoxO activity. Whereas mutation of Akt/PKB promotes the nuclear accumulation of DAF-16/FoxO, mutation of eak genes increases nuclear DAF-16/FoxO activity without influencing DAF-16/FoxO subcellular localization. Thus, EAK proteins regulate the activity of nuclear DAF-16/FoxO. Two EAK proteins, EAK-2/HSD-1 and EAK-7, influence C. elegans lifespan and are conserved in mammals. The discovery of the EAK pathway defines a new conserved FoxO regulatory input and may have implications relevant to aging and the pathogenesis of aging-associated diseases.FoxO transcription factors (TFs) extend lifespan in invertebrates and may participate in the control of human longevity. The role of FoxO TFs in lifespan regulation has been studied most extensively in  C. elegans, where reduction of IIS or ablation of the germline extends lifespan in a DAF-16/FoxO-dependent manner [Drosophila [The role of FoxO TFs in lifespan control was first established in t manner . FoxO alosophila ,4. Althoosophila ,6,7, wheosophila ,11,12,13While a direct connection between mammalian FoxO TFs and lifespan control has not yet been established experimentally, recent work in conditional knockout mice supports a role for FoxO TFs in modulating phenotypes that are reminiscent of aging-associated diseases in humans. For example, mice lacking FoxO1, FoxO3, and FoxO4 develop thymic lymphomas and hemangiomas, indicating that FoxO TFs are bonafide tumor suppressors . FurtherC. elegans lifespan modulation by IIS and the germline [daf-16/FoxO null mutants [DAF-16/FoxO is required for germline , as wellgermline . Reducti mutants , indicatIIS and the germline both inhibit DAF-16/FoxO by promoting its cytoplasmic sequestration. When IIS is reduced, or when the germline is ablated, DAF-16/FoxO translocates to the nucleus ,23,24, wHow the germline promotes the cytoplasmic sequestration of DAF-16/FoxO is not entirely understood. Notably, germline ablation extends lifespan in animals with reduced IIS . In animAlthough nuclear localization of DAF-16/FoxO is clearly necessary for DAF-16/FoxO-dependent lifespan extension, multiple lines of evidence indicate that it is not sufficient for full DAF-16/FoxO activation. For example, a DAF-16/FoxO mutant lacking all four canonical Akt/PKB phosphorylation sites localizes to the nucleus but fails to fully extend lifespan ,29. Thisenhance the weak dauer-constitutive phenotype of an akt-1 null mutant (eak screen). We identified 21 independent mutants that define seven eak genes, six of which have been cloned  [7-DA is synthesized by the Rieske oxygenase family member DAF-36 and DAF-9 [4- and Δ7-DA are high-affinity ligands for the nuclear receptor DAF-12 [EAK-2 is allelic to the 3β-hydroxysteroid dehydro-genase family member HSD-1 ,37. HSD- (Δ4-DA) ,37; the nd DAF-9 ,38. Bothr DAF-12 .hsd-1 mutations in the eak screen implicates steroid hormone signaling in the regulation of nuclear DAF-16/FoxO activity. In support of this, we have shown that daf-36 and daf-9mutations also enhance the akt-1 dauer-constitutive phenotype [hsd-1 mutation synergizes with akt-1 mutation to increase DAF-16/FoxO target gene expression, and DAF-16/FoxO target gene expression in hsd-1;akt-1 double mutants requires not only DAF-16/FoxO but also DAF-12 [hsd-1 is expressed exclusively in the two endocrine XXX cells, where daf-9 is also expressed [akt-1 mutation, hsd-1 mutation does not promote nuclear translocation of a functional DAF-16::GFP fusion protein [The identification of henotype . Furthero DAF-12 . Thus, Dxpressed ,37,39. T protein , indicatdaf-36 and daf-9 mutations, which substantially shorten the lifespan of animals lacking a germline [hsd-1 mutation does not affect lifespan extension induced by germline ablation [4- and Δ7-DA and/or the anatomical source of distinct DAs (XXX cells vs. hypodermis and somatic gonad). Alternatively, HSD-1 may participate in the biosynthesis of a different steroid hormone that is required for full lifespan extension in the context of reduced IIS but dispensable for lifespan extension induced by germline ablation.The role of HSD-1 in lifespan control is complex. In wild-type animals, HSD-1 does not have a major impact on adult lifespan. However, in animals with reduced IIS, HSD-1 is required for full lifespan extension . This isgermline ,40, hsd-ablation . This mahsd-1 and other eak single mutants, eak-7 single mutants live approximately 25% longer than wild-type animals, and this lifespan extension requires DAF-16/FoxO as well as the DAF-16/FoxO cofactors HSF-1 and SMK-1 [eak-7 mutation strongly enhances the dauer-constitutive phenotype of akt-1 mutants, its enhancement of lifespan extension in akt-1 mutants is relatively modest [akt-1 mutation may not increase nuclear concentrations of DAF-16/FoxO to the extent that it does in early larval stages, before the germline has proliferated. In accordance with this, eak-7 mutation strongly enhances lifespan extension (by ~50-80%) in animals lacking a germline [In contrast to nd SMK-1 . As is tnd SMK-1 . Notablyy modest . This isy modest , akt-1 mgermline .TBC- and LysM-domain-containing) domain [eak-7 mutation increases steady-state DAF-16/FoxO protein levels in akt-1 mutants without altering daf-16/FoxO transcript levels, suggesting that EAK-7 may modulate DAF-16/FoxO synthesis or turnover [How EAK-7 controls nuclear DAF-16/FoxO activity is unclear. EAK-7 contains a consensus N-myristoylation (N-myr) motif and a TLDc  domain , the fun) domain . Thus, Eturnover . A hypotAlthough studies in mammalian cells have also implicated a regulatory input that controls the activity of FoxO TFs that are localized to the nucleus , the natThe function of the putative mammalian HSD-1 ortholog Sdr42e1 is not known. Sdr42e1 mRNA is expressed in a limited number of mouse tissues, with strikingly high expression in liver , where FIt is noteworthy that the genes encoding human Sdr42e1 and KIAA1609 lie within 2.5 megabases of each other at chromosome 16q23. Copy number variation in this genomic region could affect both genes coordinately, changing baseline EAK pathway activity  accordingly.C. elegans lifespan. Continued investigation of the C. elegans EAK pathway promises to reveal new insights into mechanisms of FoxO regulation. In light of the potential role of FoxO TFs in mammalian aging and the pathogenesis of aging-associated diseases, EAK proteins could emerge as promising targets for the development of new drugs to treat Type 2 diabetes, cancer, and osteoporosis. Studies of Sdr42e1 and KIAA1609 function in mouse models of aging and disease will be especially revealing in this regard.The EAK pathway is a new conserved FoxO regulatory input that acts in parallel to Akt/PKB and the germline to control