Philanthus triangulum. PhTX-433 inhibits several excitatory ligand-gated ion channels, and to improve selectivity two synthetic analogues, PhTX-343 and PhTX-12, were developed. Previous work showed a 22-fold selectivity of PhTX-12 over PhTX-343 for embryonic muscle-type nicotinic acetylcholine receptors (nAChRs) in TE671 cells. We investigated their inhibition of different neuronal nAChR subunit combinations as well as of embryonic muscle receptors expressed in Xenopus oocytes. Whole-cell currents in response to application of acetylcholine alone or co-applied with PhTX analogue were studied by using two-electrode voltage-clamp. α3β4 nAChRs were most sensitive to PhTX-343 (IC50 = 12 nM at −80 mV) with α4β4, α4β2, α3β2, α7 and α1β1γδ being 5, 26, 114, 422 and 992 times less sensitive. In contrast α1β1γδ was most sensitive to PhTX-12 along with α3β4  with α4β4, α4β2, α3β2 and α7 being 3, 3, 26 and 49 times less sensitive. PhTX-343 inhibition was strongly voltage-dependent for all subunit combinations except α7, whereas this was not the case for PhTX-12 for which weak voltage dependence was observed. We conclude that PhTX-343 mainly acts as an open-channel blocker of nAChRs with strong subtype selectivity.Philanthotoxin-433 (PhTX-433) is an active component of the venom from the Egyptian digger wasp,  Philanthus triangulum, used to paralyse insect prey by inhibiting nicotinic acetylcholine receptors (nAChRs) and ionotropic glutamate receptors (iGluRs)13457Philanthotoxin-433 , it has been proposed that the dominant mode of action involves an open-channel blocking mechanism whereby the polyamine tail penetrates deep into the channel pore where the environment is hydrophilic, while the head group interacts with the more hydrophobic outer parts of the pore910Strong receptor selectivity was first realized following the development of an analogue in which the two secondary amine functionalities in PhTX-343 (and PhTX-433) were exchanged for methylene groups thereby generating PhTX-12 . As expe58Interestingly, there is a notable gap in our knowledge of ionotropic receptor inhibition by PhTXs regarding their action on mammalian neuronal-type nAChRs. Only a single study has investigated the effects of PhTX-343 at nAChRs expressed by PC12 cells, showing that it potently antagonised responses to ACh in a voltage-dependent mannerIn the present work, we investigated the inhibitory actions of PhTX-343 and PhTX-12 on some established subtypes of neuronal nAChRs comprising α4β2, α3β4, α7, α4β4 and α3β2, by expression in Xenopus oocytes and voltage clamp recording. Also, we included embryonic muscle-type receptors (α1β1γδ) in our study to facilitate comparison to our previous studies with TE671 cells. We aimed to explore whether PhTXs can be used as subtype-selective inhibitors of nAChRs.ACh was from Sigma. PhTX-343 and PhTX-12 were synthesized as described previously(V253F) and β4(F255V) mutant subunit cDNAs were a kind gift from Dr. Cecilia Borghese, University of Texas at Austin.cDNA clones of rat neuronal nAChR subunits  and mouse muscle subunits  were from the Salk Institute for Biological Studies (Professor Stephen Heinemann). The human α7 and RIC-3 cDNAs were provided by Professor David Sattelle (University College London). The β2Plasmids were linearized and cRNA transcribed using an mMessage mMachine kit (Ambion).Xenopus laevis were supplied by the European Xenopus Resource Centre, University of Portsmouth, UK. Oocytes were treated with collagenase  in Ca2+-free solution  with shaking at 19 °C to defolliculate and remove the connective tissue surrounding the cells. After separation, oocytes were washed 7 times with modified Barth’s solution  and kept at 19 °C in the same solution.Oocytes isolated from mature female Healthy oocytes were injected with cRNA using a Nano-liter Injector . Mixtures of nAChR subunit cRNAs were injected as follows; for heteromeric rat neuronal receptors a 1:1 ratio of α:β at 200 ng/μL; for mouse embryonic muscle a 1:1:1:1 ratio of α:β:γ:δ at 25 ng/μL; human α7 at 100 ng/μL was mixed with RIC-3 at 30 ng/μL. Each oocyte was injected with 50 nL of RNA solution. Injected oocytes were saved in Barth’s solution at 19 °C for two to three days for expression of the target protein. During this time oocytes were regularly checked to remove unhealthy ones.2 and 5 mM HEPES, pH 7.5). Microelectrodes were pulled from borosilicate glass capillaries (Harvard GC150TF-10) using a programmable micropipette puller , and they had resistances between 0.5 and 2.5 MΩ when filled with 3 M KCl. The oocyte was voltage-clamped at holding potentials (VH) between −60 and −100 mV. ACh was consistently used as the agonist, and it was applied without or together with PhTX analogues via an 8-channel perfusion system  for 1 min to allow for equilibration of the current. Currents were recorded to a PC via a digidata 1200 analog-to-digital converter  using WinEDR v3.2.6 Software .Electrophysiological recordings were taken from nAChR-expressing oocytes by two-electrode voltage clamp using a Geneclamp 500 voltage clamp amplifier . An oocyte was placed in the perfusion chamber using a plastic Pasteur pipette and the bath was perfused (~5 mL/min) with fresh Frog Ringer solution . Data were normalized as % control response for PhTX inhibition or % maximum response for ACh agonism. Graphpad Prism 6 was used for data analysis, graph plotting and curve fitting. All plotted points are the mean ± SEM obtained from 4–20 oocytes. Concentration-inhibition and concentration-response curves were used to estimate IC50s and EC50s were compared for significant differences using an extra sum of squares F-test in Graphpad Prism 6.where M is the maximum response and nH is the Hill slope. IC−8 M and 10−3 M (10−2 M for α7) to obtain EC50 values , α3β2 had intermediate sensitivity, while α3β4 and α7 displayed lower sensitivity (EC50s > 100 μM). In all cases EC50 values were slightly lower (1.4- to 6.7-fold) for late current, but a similar pattern of relative sensitivity emerged. These EC50 values demonstrate that, at least for α4β2 and α3β4 where data are available, the dominant subunit stoichiometries are (α4/2)2(β2/4)31650 concentrations of ACh as follows: 10 μM for α4β2, α4β4, and α1β1γδ, 30 μM for α3β2, and 100 μM for α3β4 and α7.Oocytes expressing each subunit combination were exposed to ACh at concentrations between 100 values . Based oXenopus oocytes expressing functional α4β2, α4β4, α3β4, α3β2, α7 and α1β1γδ nAChRs were used to investigate the relative inhibitory potencies of PhTX-343 and PhTX-12. The highest concentration (100 μМ) of each PhTX used in this study was first applied alone (at VH = −80 mV) to oocytes expressing nAChRs, and this did not produce any effect on membrane current. Co-application of PhTX-343 or PhTX-12 with ACh inhibited the current of each subtype of nAChR in a concentration-dependent manner  being 1000 times higher than at α1β1δγ (IC50 = 12 μM) for late current at −80 mV. For PhTX-343 the IC50 values for peak and late current inhibition of α4β4 were roughly 3-fold and 5-fold, respectively, lower than those found for α4β2 nAChRs. Likewise, the potency of PhTX-343 on α3β4 nAChRs was higher by 115-fold on peak current and 137-fold on late current as compared to α3β2 (at −80 mV). To investigate the possible cause of this high affinity of PhTX-343 for β4-containing over β2-containing nAChRs, we compared the amino acid sequence of the M2 domains of both β-subunits ((V253F) or β4(F255V). As expected, for α4β2(V253F) a 2.6-fold reduction (p = 0.049) in late current IC50 was observed as compared to α4β2  as compared to that of α3β4  between the most potent (0.1 μM for α1β1γδ and α3β4) and the least potent (4.9 μM for α7). The ranking order for nAChR affinity based on late current inhibition by PhTX-12 was: α1β1γδ = α3β4 > α4β2 ≈ α4β4 >> α3β2 > α7.PhTX-12 inhibition of nAChRs did not show any clear pattern of dependence on specific α- or β-subunits. Similar to PhTX-343, PhTX-12 was most potent at α3β4 receptors, and it showed low potency at α7 receptors (lowest potency in this case), but in contrast to PhTX-343 it was as potent at muscle-type receptors (α1β1δγ) as α3β4 receptors. However, the range of PhTX-12 IC50 values for PhTX-12. The lowest peak current IC50 value at −80 mV, observed for α4β4 nAChRs, was 1.65 μM, increasing to 60 μM for α3β2 nAChRs. The ranking order of potency in this case was: α4β4 > α4β2 > α3β4 > α1β1γδ > α7 >> α3β2. This reflects a strong time/use dependence for inhibition of α1β1γδ, α3β4 and α3β2 by PhTX-12.On the other hand, nAChR subunit combinations showed a slightly different profile for peak current IC50 values between α4β2 and α4β2(V253F) for peak or late current inhibition ((F255V) there was no difference between the IC50 values for peak current inhibition ((F255V)  .Comparison of the two PhTXs (at −80 mV) with respect to peak current inhibition showed that PhTX-343 was more potent at most of the nAChR subtypes, especially at α3β4 (33-fold), with α1β1δγ being the only exception for which PhTX-12 was slightly more potent. For late current the two toxins were approximately equipotent at α4β2, α3β2 and α7; PhTX-343 was more potent at β4-containing subtypes; PhTX-12 was much more potent at α1β1γδ (119-fold).50 values determined for both PhTXs at two additional holding potentials  on β2-containing nAChRs than on β4-containing receptors, whereas the opposite was found for late current inhibition although this was less pronounced. For example, the peak current IC50 values for PhTX-343 inhibition of α4β2 and α3β2 at −60 mV were decreased by 14- and 18-fold, respectively, at VH −100 mV, whereas only a 3.3- and 3.8-fold decrease in IC50 was observed for α4β4 and α3β4 nAChRs, respectively.PhTX-343 showed strongly voltage-dependent inhibition of heteromeric nAChRs, but it was voltage-independent for homomeric α7 . The ext50 values there was significant voltage dependence, increasing as follows: α4β2 > α3β4 > α3β2 > α7; but in all cases it was weaker than found for PhTX-343, whereas inhibition was voltage-independent for α4β4 and α1β1γδ   . PhTX-34Inhibition of homomeric α7 and muscle-type nAChRs by PhTX-343 was reversible after 6 min washing in frog Ringer solution, while for heteromeric neuronal nAChRs it was irreversible . For theInhibition of nAChRs by PhTX-12 was fully or almost reversible after 6 min. Recovery of the peak current was complete for α3-containing nAChRs, and of the late current for α3β2 and α7. In all other cases 75% (for α4β4 peak current) to 94% (for α3β4 late current) recovery, as compared to the control response, was achieved during the first ACh application after the removal of PhTX-12. Interestingly, the peak current for α3β4 during the first ACh application after removal of PhTX-12 was in fact larger than the control response in 4 out of 6 cells but the combined mean for all cells  was not significantly greater than control (p = 0.217).50 values for peak current in the absence and presence of PhTX-343 for α4β2, α4β4, α3β2, α3β4, α7 and α1β1γδ, as well as for PhTX-12 at α3β4 and α1β1γδ (for which potency was highest) are presented in To further study the mechanism of action by which PhTX-343 and PhTX-12 inhibit acetylcholine-evoked inward current in oocytes expressing nAChRs, we analysed the data obtained from the effects of a single PhTX concentration on acetylcholine concentration-response curves  and 8. T50 in all heteromeric neuronal nAChRs was observed (50s were significantly (p < 0.05) increased (PhTX-343 (1 μM) caused a reduction of the maximum peak response to ACh, and no change in ACh ECobserved . Howeverncreased . For lat50 was increased (p < 0.0001) by the toxin , and we interpret this by F providing an improved interaction with the aromatic moiety of PhTX-343 in the shallow part of the pore, while the polyamine moiety retains interactions with the selectivity filter deeper in the pore. However, the inverse mutation (i.e. F255 → V255) in β4 did not reduce the potency so it is likely that other factors, such as channel open-closed kinetics, may play a part. Similar point mutations in the M2 domain of β-subunits have been shown to change the sensitivity of nAChRs to other blockers such as cocaineResults from our experiments show that inhibition of peak current by PhTX-343 is less effective than inhibition of late current, in agreement with Brier A striking feature of our data was the strong selectivity of PhTX-343 for heteromeric neuronal nAChRs over the muscle-type receptor. It is widely accepted that the pore of the nAChR is lined by eight rings of conserved amino acids which play a role in ion selectivity and conductance. These rings include (from extracellular to intracellular): (1) a charged ring; (2) an outer hydrophobic ring; (3) a valine ring; (4) the equatorial leucine ring (gate); (5) a serine ring; (6) a threonine ring; (7) an intermediate negatively charged ring; and (8) an internal negatively charged ring . We haveThe above proposed mode of binding appears unlikely to have a major impact on PhTX-12 inhibition across the receptors studied here, and indeed PhTX-12 exhibited similar potency at the subunit combinations tested except for α7 and α3β2 receptors that had a lower sensitivity to PhTX-12. These observations may relate to the previous suggestion that PhTX-12 adopts a folded conformation as a result of interaction between the terminal amine and the head region, thereby preventing access to the narrow part of the pore, suggesting binding to an alternative shallow site in the pore resulting in enhanced desensitisation rate822H of −60 mV, −80 mV, and −100 mV, respectively. In comparison, our previous study on human muscle-type nAChRs in TE671 cells showed an 11-fold lower potency of PhTX-12 at this receptor, but a similar 22-fold selectivity over PhTX-343 at −100 mVComparison of the two toxins infers a clear preference for muscle-type nAChR for PhTX-12, while for neuronal nAChR subtypes there are varying degrees of preference for PhTX-343. These results confirm our previous findings that the presence of the secondary amines prevent effective inhibition of muscle-type nAChRs in TE671 cells58In the present work, the rate and extent of recovery from inhibition by PhTX-343 and PhTX-12 was contrasting, and it was subunit-dependent for PhTX-343. Heteromeric neuronal nAChRs showed only poor recovery from inhibition by 10 μM PhTX-343 even after six ACh applications over 36 min. Interestingly, α3β4 receptors recovered better than the other three combinations despite being the most potently inhibited, which may be linked to differences in open-closed channel kinetics that offer a higher probability for binding, but also for dissociation or slightly shallower binding due to F255 in the β4-subunit, as also implied by the lower voltage dependence of inhibition. In contrast, muscle-type nAChRs and homomeric α7 receptors showed almost full recovery upon the first ACh application after 6 min, most likely due to weaker interactions between PhTX-343 and the nAChR pore. On the other hand, recovery from inhibition by PhTX-12 was complete or almost complete for all nAChRs upon the first ACh application after 6 min. The enhanced recovery from inhibition by PhTX-12 may reflect the proposed shallower binding siteet al.N3-Ph-PhTX-343-Lys to nAChRs of Torpedo californica, and even a slight increase was noticed with carbamoylcholine. Likewise, inhibition of human muscle-type nAChRs by PhTX-343 and PhTX-12 was not influenced by the ACh concentrationFrom previous studies, the mechanism of blocking of nAChR inward currents by PhTX analogues has been proposed to be non-competitive. Bixel In conclusion, PhTX-343 potently inhibits ganglionic-type (α3β4) nAChRs with some selectivity over other heteromeric neuronal nAChRs, and with strong selectivity over homomeric α7 and muscle-type nAChRs. In contrast to PhTX-343, PhTX-12 most potently inhibits muscle-type nAChRs but remains a weak inhibitor of homomeric α7 receptors.How to cite this article: Kachel, H. S. et al. Block of nicotinic acetylcholine receptors by philanthotoxins is strongly dependent on their subunit composition. Sci. Rep.6, 38116; doi: 10.1038/srep38116 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Drosophila, Ana2 loads onto the site of procentriole formation ahead of Sas6 in a process that also requires Plk4. However, whereas Plk4 is first recruited to multiple sites around the ring of zone II at the periphery of the centriole, Ana2 is recruited to a single site in telophase before Plk4 becomes finally restricted to this same single site. When we over-ride the auto-destruction of Plk4, it remains localized to multiple sites in the outer ring of the centriole and, if catalytically active, recruits Ana2 to these sites. Thus, it is the active form of Plk4 that promotes Ana2's recruitment to the centriole. We now show that Plk4 phosphorylates Ana2 at a site other than the STAN motif, which lies in a conserved region we term the ANST (ANa2-STil) motif. Mutation of this site, S38, to a non-phosphorylatable residue prevents the procentriole loading of Ana2 and blocks centriole duplication. Thus the initiation of procentriole formation requires Plk4 to first phosphorylate a single serine residue in the ANST motif to promote Ana2's recruitment and, secondly, to phosphorylate four residues in the STAN motif enabling Ana2 to recruit Sas6. We discuss these findings in light of the multiple Plk4 phosphorylation sites on Ana2.The conserved process of centriole duplication requires Plk4 kinase to recruit and promote interactions between Sas6 and Sas5/Ana2/STIL (respective nomenclature of worms/flies/humans). Plk4-mediated phosphorylation of Ana2/STIL in its conserved STAN motif has been shown to promote its interaction with Sas6. However, STAN motif phosphorylation is not required for recruitment of Ana2 to the centriole. Here we show that in  CentrCaenorhabditis elegans, Sas5 and Sas6 [Drosophila and human cells in a process that requires Plk4 to phosphorylate several residues in the conserved STAN motif in the C-terminal part of the respective counterparts of Sas5, Drosophila Ana2 and human STIL [Drosophila, this acts as a binary switch to control Sas6 recruitment to the procentriole as soon as mother and daughter centrioles disengage in late telophase [Drosophila Ana2 into alanine residues prevents recruitment of Sas6 to the site of procentriole formation while still permitting the centriolar recruitment of this mutant variant of Ana2. The process governing the recruitment of Ana2 independently of Sas6 is thus unclear. In human cells, it has been shown that continued activity of Plk4 is required for the recruitment of STIL to the centriole but the molecular basis for this is not understood [Centriole duplication is initiated through an interaction between two conserved proteins first identified in and Sas6 ,14. Dimeand Sas6 ,25. Sas6man STIL –28. In Delophase . Mutatio2.2.1.All cDNA and expression constructs and cloning methods were previously described ,26.2.2.Sas4-F: 5′-GAATTAATACGACTCACTATAGGGAGAATGCAGGAGGCTGGCGAAAGTCC -3′Sas4-R: 5′-GAATTAATACGACTCACTATAGGGAGAGGAGGCTTCATCATCGGCATGAG -3′D.Mel-2 cells  were cultured and treated with dsRNA as described previously . Transfe2.3.Ana2 point-mutations was either as already reported [Generation of reported , or by u2.4.Escherichia coli have been described elsewhere [All recombinant proteins in this study together with the methodology for their expression and purification from lsewhere .2.5.In vitro phosphorylation of 35S-methionine-labelled Ana2 produced by coupled in vitro transcription–translation (IVTT) and of GST-Ana2 proteins on beads were carried out as previously described [escribed .2.6.6 cells were collected from each well 24 h post-transfection and briefly rinsed in PBS. Cells were then lysed in RIPA buffer  on ice for 15 min. Lysates were cleared by centrifugation and supplemented with 1× Lambda-phosphatase buffer and 1× MnCl2 solution provided along with Lambda phosphatase . Two samples (one of Ana2-FLAG + Plk4-NDKD and one of Ana2-FLAG + Plk4-ND) were mock-treated (no phosphatase added), while one sample (Ana2-FLAG + Plk4-ND) was treated with 200 U (0.5 µl) Lambda phosphatase for 30 min at 30°C. All samples were then boiled in Laemmli sample buffer and analysed by immunoblotting.D.Mel-2 cells were co-transfected with pAct5-Ana2-FLAG and either pAct5-Plk4-NDKD (kinase-dead) or pAct5-Plk4-ND (active) in a 12-well plate. Approximately 1 × 102.7.in vitro, or tagged with GFP-, FLAG- or Protein-A and purified from either D.Mel-2 cells or 0–3 h syncytial stage embryos) was digested with trypsin directly on the affinity resin. Twenty per cent (v/v) of the resulting peptide mixture was directly analysed by LC/MS and the remaining 80% enriched for phosphatides using titanium dioxide. Samples were analysed using an Orbitrap-LTQ mass spectrometer (Thermo Fisher Scientific) coupled to a UPLC system (Waters Corporation). Acquired data were searched using the Mascot Search Engine (Matrix Science) against the Drosophila melanogaster database. Phosphorylated peptides identified by Mascot were individually verified by manually inspecting the relevant spectra.Phospho-peptide identification by mass spectrometry was carried out as previously explained . Briefly2.8.The following primary antibodies were used for immuno-fluorescence (IF) or western blotting (WB): rabbit-anti-Ana2   relative to the spindle such that they move towards the spindle-proximal side of the nucleus at the time of the first indication of cytokinetic furrow formation (late telophase). Owing to the adhesion of cells onto the concanavalin A-coated coverslips, the abscission stage of cytokinesis cannot be completed. Thus cells having a long cytokinetic bridge can also be in G1 phase. We classify such cells as in cytokinesis/G1.To deplete centriole components from D.Mel-2 cells for structured illumination microscopy, we carried out RNAi for 5 days (except for 3 days in the case of Plk4 RNAi), as a result of which 30–60% of cells had no centrosomes. Our analysis was then carried out upon cells in which a single centrosome persists showing the defects we document.3.3.1.In our previous study, we followed the centriolar recruitment of both Sas6 and Ana2 relative to the conversion of daughter centrioles to centrosomes monitored by the extension of D-Plp around the periphery of the daughter centriole during progression through mitosis . This reThe above finding led us to ask whether in the normal course of centriole duplication, Ana2 could recruit to the procentriole ahead of Sas6 or whether, alternatively, Ana2 and Sas6 load together. To address this, we co-stained cells to reveal D-Plp, Sas6 and Ana2 and re-examined their relative loading focusing upon cells at the very late stages of anaphase/early telophase using structured-illumination (SIM), super-resolution microscopy. At the earliest stages of centriole disengagement, we were only able to observe single dots of Sas6 staining at the core of mother and daughter centrioles (Zone I ),while a3.2.Drosophila cells of various centriole components by RNAi and used SIM to assess whether Ana2 was present at a single parental site encircled by D-Plp or also at the pre-procentriole site on the periphery of the D-Plp ring. We scored a minimum of 30 centrioles in cells that were in cytokinesis or very early G1, that is at a cell cycle stage at which procentrioles should have been established . During progression through telophase, the ring of Plk4 around the mother and daughter gradually broke down such that by cytokinesis/the following G1, it had resolved into a dot. To relate the distribution of Plk4 to recruitment of Ana2, we assessed the localization of the two proteins with respect to D-Plp in those centrioles in which Ana2 could be observed both at the centriole core/Zone I and at the nascent procentriole (b). We found that at the earlier stages of telophase, centrioles had Plk4 located in a variable number of beads arranged in a ring around the parental centriole, one of which coincided with newly loaded Ana2. Later, in cytokinesis/G1, centrioles had a single Plk4 bead colocalized with Ana2. Thus, Ana2 becomes localized to a single procentriole site ahead of the resolution of Plk4 from its ring-like to dot-like distribution.The requirement for Plk4 for the loading of Ana2 at the centriole led us to examine the relative positions of the two proteins on the centriole at the time of procentriole formation. It has been described that in mammalian cells, Plk4 accumulates in a ring around parental centrioles that resolves into a single dot, reportedly around the time when STIL and Sas6 are recruited for cartwheel formation ,27. We ontrosome a. Duringentriole b. We fou3.4.Drosophila is encoded by slimb [a). We then investigated the effects of over-expressing either kinase active or inactive mutant forms of Plk4-ND. We found that both forms of the ND kinase accumulated ectopically in rings in Zone III of the centrioles in interphase. However, only the active Plk4-ND was able to induce loading of Ana2 to multiple sites, while conversely the kinase dead Plk4-NDKD not only did not induce these ‘rosettes’ but even suppressed the normal loading of Ana2 as a single dot (b). Thus it seems that when Plk4 is continually present as a result of over-riding its auto-destruction, it can accumulate on the entire outer ring of the centriole and, if it is catalytically active, it can recruit Ana2 to multiple sites. Together, these results suggest that there is only one sufficiently active focus of Plk4 that exceeds a threshold for Ana2 loading.Normally, the levels of Plk4 are limited during interphase in both fly and mammalian cells by the SCF ubiquitin-protein-ligase that targets the proteasome-mediated destruction of the kinase by binding to its auto-phosphorylated degron –23. Thisby slimb ,34. We fngle dot b. Thus i3.5.Drosophila Ana2 that when phosphorylated by Plk4 enable the binding and consequent centriole recruitment of Sas6. However, failure to phosphorylate Ana2 at these sites does not prevent its loading to centrioles [Together, the above observations suggested that the Plk4-dependent recruitment of Ana2 to initiate procentriole formation might reflect an ability of Plk4 to phosphorylate Ana2. We have previously described four sites in the STAN motif of ntrioles . As our a; electronic supplementary material, figure S1a). This mobility shift was seen when either FLAG-tagged-Ana2 was introduced into cultured Drosophila cells together with active Plk4 in transient transfections or when Ana2 synthesized in vitro by coupled transcription and translation (Ana2-IVTT) was phosphorylated by Plk4 in vitro (figure 5a). The mobility shift was reversed by Lambda phosphatase treatment indicating that it is a consequence of Ana2's phosphorylation . Moreover, the mobility shift was still observed when the four Plk4-phospho-sites in the STAN motif were mutated to alanine residues indicating that it occurred independently of the mechanism required to recruit Sas6 . We argued that such a dramatic mobility shift should reflect a significant conformational change in Ana2 and hypothesized that this might be required to recruit Ana2 to the centriole. We therefore sought to identify the phosphorylation site(s) responsible for the mobility shift.We had noticed that Ana2 undergoes a profound shift in its electrophoretic mobility following phosphorylation by Plk4 . When we mutated all 21 sites to alanine residues, Ana2 continued to undergo a band shift following phosphorylation by Plk4 (b) indicating that none were responsible for this conformational change. We then used mass spectrometry to identify 12 phospho-sites in Protein-A- or FLAG-tagged Ana2, affinity purified from cultured cells, or in GFP-Ana2, affinity purified from Drosophila embryos . Having mapped the region undergoing the band shift to the N-terminal 280 residues , we mutated the 12 sites to alanine residues and repeated the kinase assay, but none of the changes abolished the band shift . We further narrowed down the region of Ana2 undergoing the band shift to within residues 1 and 140 (c) and then systematically mutated each individual remaining serine or threonine residue within this segment to alanine prior to phosphorylation of the corresponding IVTT peptide by Plk4 (d). This revealed that phosphorylation on serine 38 was responsible for the mobility shift . Suspecting that we had originally failed to detect this modification in our mass spectrometric analysis because it lay within a poorly ionized peptide, we then searched for low abundance ionized fragments within this region following phosphorylation of Ana2 by Plk4. The fragmentation pattern of a peptide extending from residue 13 to 65, which had a phospho-serine residue at position 38 , allowed us to confirm that Ana2 is phosphorylated by Plk4 on serine 38 to change its electrophoretic mobility. Strikingly, this residue lies within a 10-residue motif that is highly conserved from insects to vertebrates (f), which we refer to as the ANST motif.The strategy we adopted to identify the site is primarily presented in the electronic supplementary material, figure S1. Briefly, we first subjected bacterially expressed GST-Ana2 to phosphorylation by recombinant Plk4  by Plk4 b indicat and 140 c and the by Plk4 d. This rty shift d,e. Susptebrates f, which E. coli and immobilized the recombinant proteins on beads for treatment with active or kinase-dead Plk4. We then asked whether S35-labelled Sas6, generated by coupled IVTT, could then bind to the beads. This revealed that Sas6 was able to bind to both wild-type and S38A Ana2 but only after phosphorylation by Plk4 . Thus mutation of S38 in the ANST motif to an amino acid that cannot be phosphorylated does not affect the Plk4-dependent binding of Ana2 to Sas6.Finally, we wished to determine whether phosphorylation of Ana2 on S38 was required for its subsequent interaction with Sas6 that we have previously shown to require Plk4-mediated phosphorylation of its STAN motif . To this3.6.a). We found that the loss of centrosomes resulting from this treatment could be rescued in cells expressing wild-type Ana2 lacking the endogenous UTR but not in cells expressing Ana2-S38A. Therefore, phosphorylation of Ana2 at S38 by Plk4 is required for centriole duplication.To test our hypothesis that the phosphorylation event that induced the mobility shift of Ana2 would be important for the biological function of the protein, we mutated this residue to alanine (Ana2-S38A) and first tested the consequences for centriole duplication. To this end, we used three rounds of RNAi directed against the 3′ UTR of the Ana2 mRNA to deplete the endogenous Ana2 protein and block centriole duplication figures a. We foub). Stable expression of Myc-tagged wild-type Ana2 was able to rescue loading following this treatment to 90% (28/31). By contrast, Myc-tagged Ana2-S38A failed to rescue loading  of Ana2. Moreover, it also had a dominant-negative effect on loading: compare 64% (18/28) loading in control RNAi-treated cells expressing Ana2-S38A to 89% (33/37) loading in control-depleted untransfected cells indicating that Ana2-S38A is a stable protein as confirmed by western blotting . Thus, we conclude that phosphorylation of Ana2 at S38 is required for the loading of Ana2 to permit centriole duplication.We then asked whether S38 phosphorylation was required for the loading of Ana2 onto centrioles. Once again, we used three rounds of RNAi directed against the UTR of Ana2 to downregulate the endogenous protein and found that the proportion of centrioles in which Ana2 was loaded onto the procentriole site on the D-Plp ring was reduced from 89% (33/37) to 46% (14/30) b. Stable4.Drosophila Ana2 at a single serine residue, S38, in what we term the ANST motif in its N-terminal part to enable Ana2 to be recruited to the site of procentriole formation. This occurs ahead of the known requirement of Plk4 to phosphorylate Ana2 upon four residues in the conserved STAN motif in its C-terminal part in order to bind and recruit Sas6 to the pre-procentriole site [Drosophila cells is the phosphorylation of the STAN motif absolutely essential for STIL/Ana2 recruitment [Drosophila cells, the initiation of procentriole formation is, indeed, a two-step process requiring Plk4 to phosphorylate Ana2 at distinct sites (Our study shows that Plk4 phosphorylates ole site . Phosphoole site ,28, wherole site . Howeverruitment ,27. Thesct sites . AlthougOur findings have implications for the mechanisms that regulate the activation of Plk4 and that ultimately restrict it to a single site in normal cell cycle progression. The activation of Plk4 has consequences for its stability as well as for its ability to mediate its cellular functions including centriole duplication. This is because Plk4's destruction is brought about through the auto-phosphorylation of a degron rendering the kinase susceptible to SCF-mediated ubiquitylation and subsequent degradation by the proteasome –23. But Our results also suggest that a threshold level of Plk4 activity might be required to permit Ana2 loading. How this threshold becomes exceeded only at the single Ana2 loading site remains to be determined. Single-site loading of Ana2 could be related to the protection of Plk4 from destruction at the pre-procentriole site. It is possible that Plk4's binding to STIL/Ana2 might protect it from the SCF. This notion finds support from the finding that overexpression of wild-type STIL or a variant lacking the STAN motif can stabilize Plk4 in a ring around the centriole ,35. TherDrosophila, whether it interacts with a different centriole component to allow it to dock and initiate procentriole formation. The conservation of the ANST motif is intriguing as it suggests the possibility, which will be of future interest to test, that Ana2 loading onto the procentriole site might be mediated by a conserved mechanism.If Ana2 is recruited solely through binding to Plk4, then there must be a mechanism that restricts this interaction only to a single site. This could be achieved through a conformational change in one or both partners that could both reinforce and restrict the interaction. In this light, we note that Plk4 appears normally to exist in an autoinhibited state that is relieved through some property of Polo-box 3 suggested to reflect a conformational change resulting from binding a partner protein . The shi


Type 1 Serine/Threonine Kinase Receptors (STKR1) transduce a wide spectrum of biological signals mediated by TGF-β superfamily members. The STKR1 activity is tightly controlled by their regulatory glycine-serine rich (GS) domain adjacent to the kinase domain. Despite decades of studies, it remains unknown how physiological or pathological GS domain modifications are coupled to STKR1 kinase activity. Here, by performing molecular dynamics simulations and free energy calculation of Activin-Like Kinase 2 (ALK2), we found that GS domain phosphorylation, FKBP12 dissociation, and disease mutations all destabilize a D354-R375 salt-bridge, which normally acts as an electrostatic lock to prevent coordination of adenosine triphosphate (ATP) to the catalytic site. We developed a WAFEX-guided principal analysis and unraveled how phosphorylation destabilizes this highly conserved salt-bridge in temporal and physical space. Using current-flow betweenness scores, we identified an allosteric network of residue-residue contacts between the GS domain and the catalytic site that controls the formation and disruption of this salt bridge. Importantly, our novel network analysis approach revealed how certain disease-causing mutations bypass FKBP12-mediated kinase inhibition to produce leaky signaling. We further provide experimental evidence that this salt-bridge lock exists in other STKR1s, and acts as a general safety mechanism in STKR1 to prevent pathological leaky signaling. In summary, our study provides a compelling and unifying allosteric activation mechanism in STKR1 kinases that reconciles a large number of experimental studies and sheds light on a novel therapeutic avenue to target disease-related STKR1 mutants. Kinases play central role in essential physiological process and are attractive therapeutic drug targets. One of the important kinase families is Type 1 Serine/Threonine Kinase Receptors (STKR1), which control gene expression in response to extracellular growth factors. The activities of STKR1 are tightly controlled by their regulatory domain, which is distant from the kinase catalytic site. The underlying molecular mechanism is elucidated here. We identified that formation or disruption of a highly conserved charge-charge interaction located near the ATP binding site, mediates the physiological inhibition or activation of STKR1. We find that the stability of this charge-charge interaction is remotely controlled by interactions propagated from the distant regulatory domain. Several disease-causing mutations are located at the regulatory domain. We demonstrate how those mutations bypass these endogenous STKR1 inhibition mechanisms to produce pathological phenotypes. This study provides a general activation mechanism in STKR1 kinases, thus may benefit understanding the molecular mechanism of diseases and drug development. Here we show how the kinase catalytic site becomes active or inhibited in physiological or pathological situations. Modifications in the regulatory GS domain are propagated toward the catalytic domain where they stabilize or destabilize an inhibitory arginine-aspartate salt bridge positioned next to the ATP binding site. Interestingly, this salt bridge is strictly conserved in all STKR1s but is absent in STKR2s which are constitutively active, indicating the requirement of this electrostatic interaction for kinase inhibition.R206H) revealed in our study may offer a novel strategy to design small molecules specific to leaky mutant receptors without interrupting the WT STKR1 activity, thus minimizing potential drug side effects. In summary, our study provides a compelling and unifying general mechanism for STKR1 regulation, and sheds light on a novel therapeutic avenue for targeting disease-related STKR1 mutants.The salt bridge distance free energy profile reveals three possible states which correspond to the three functional states of the kinase receptors as illustrated in i.e. FKBP12 bound and GS domain unphosphorylated), suggesting that the inactive STKR1 adopts a conformation similar to that of the fully active kinases , and the β-turn between β4 and β5 (residues 273 to 275) were missing and therefore transplanted from PDB ID 3Q4U. The pKa values were calculated using PROPKA. CHARMM-GUI  in the correlation network, with respect to the transmission of motion between a given set of source residues S and target residues T. The following equation describes the procedure for computing the effective flow betweenness score for a given contact pair  given the appropriate pseudo inverse matrix:-1 is the pseudo inverse of the graph laplacian of the correlation resistance network and S and T are the sets of source and target nodes between which motion transfer is being considered. It should be noted that this equation assumes that sets of source and target nodes, S and T, are distinct. If not, the summation must be updated to include only distinct combinations of pairs. Since Laplacian matrices constructed from such graphs are, in general, not directly invertible, the Moore-Penrose pseudo-inverse is used as the effective inverse of the correlation resistance network’s graph laplacian. While the Bozzo and Franceschet paper [Current-flow betweenness provides an alternative scoring metric , 29. Thiet paper  also disIn brief, this method of computing betweenness scores is equivalent to modeling the correlation network as if it were a network of electrical resistors, wherein each edge is represented as a resistor with resistances equal to the reciprocal of the absolute value of that edge’s correlation. The pseudo inverse of the graph laplacian for this network will then represent the relative difference in electrical potential between each pair of nodes. By selecting a set of nodes as the ‘sources’ and another set as the targets, or ‘grounds’, it is then possible to predict the corresponding ‘relative flow’ across each edge as if 1 unit of ‘current’ (e.g. atomic motion) were sent from the source(s) to the target(s). This ‘relative electrical flow’ can serve as a scoring function (betweenness) to rank each edge or node in order of its relative importance with respect to transmission of motion between the source and target nodes. These betweenness scores can then be used to isolate the most relevant sub-networks that transmit motion between either the FKBP binding interface or residue 206, to residue R375 of the R-D saltbridge lock. The betweenness scores and corresponding optimal sub-networks were computed from the correlation and contact map matrices generated by the NetworkView plugin using R and the corresponding igraph  R librarWT, ALK3WT and ALK6WT were purchased from Addgene . Point mutations in ALK2, ALK3 and ALK6 were made in-house using the Q5-Site Directed Mutagenesis Kit from New England Biolabs. Mutagenic oligonucleotides were designed using the NEBaseChanger online tool (New England Biolabs) and purchased from Integrated DNA Technologies. Mutations were confirmed by automated Sanger sequencing (Genewiz).pCDNA3 plasmids harboring ALK23 C2C12/BRE-luc cells (stable mouse C2C12 cells expressing the Id1 promoter-firefly luciferase reporter) were seeded in 96-well plates to culture in the growth medium DMEM/10% FBS without antibiotics. After overnight culture, the cells were transfected with pcDNA3.1(+), human wild type ALK2, ALK2R206H, ALK2R375P, ALK2R375Q, ALK2R375E, wild type ALK6, ALK6R317P, wild type ALK3 and ALK3R401P respectively, along with the Renilla luciferase reporter pRL-TK plasmid. Five hours after transfection, cells were starved in DMEM containing 0.5% FBS for 4 h. Then the cells were untreated or treated with 1μM FK506 (Cayman) or 50 ng/ml BMP6 (R&D) overnight before lysis. Luciferase activities were determined according to the Dual-Luciferase® reporter assay system (Promega) using Renilla for normalization of transfection efficiency. Data are presented as the mean ± S.E.M (standard error), each performed in triplicate. Student’s unpaired t-test was used for two groups of statistical analysis. A p-value <0.05 was considered statistically significant.Transient transfection was performed using Fugene HD transfection reagent (Promega) according to manufacturer’s instructions. Briefly, 7.5 × 10The R scripts used to generate the current flow betweenness scores and sub-optimal networks can be found on GitHub at:https://github.com/LynaLuo-Lab/network_analysis_scripts.S1 FigWT. (b). Center of mass distance between FKBP12 and ALK2 during 300 ns simulations of FKBP12-ALK2WT-Phosp, FKBP12-ALK2R206H, and FKBP12-ALK2Q207E.(a) Salt bridge distance between R375(Cζ) and D354 (Cγ) from a duplicated simulation of FKBP12-ALK2(TIF)Click here for additional data file.S2 FigKa value from each snapshot, light blue dots represent average value, and red dots represent pKa in aqueous solution. Green dashed line indicates physiological pH 7.4. His206 is indicated by red dashed arrow.Dark blue dots represent the p(TIF)Click here for additional data file.S3 FigKa of His206 is plotted against simulation time. Right: a density plot of calculated pKa of His206 during the simulation.The population of His206 protonation state is shown in the top table. The calculated p(TIF)Click here for additional data file.S4 FigWT is shown in teal and ALK2WT-Phosp is shown in orange, taken over trajectory subsets indicated in the previous wavelet analysis figure.FKBP12-ALK2(TIF)Click here for additional data file.S5 FigThis figure was generated by computing silhouette scores for consecutive k-cluster cuts taken from the hierarchical clustering of the frame-wise WAFEX data. Hierarchical clustering was computed using Euclidean distance norms over the log of the wavelet intensities at each frame along with Ward similarity scoring as implemented in the hclust function of the fastcluster package in R.(TIF)Click here for additional data file.S6 FigTop: Clustering performed by eye. Bottom: Automated Hierarchical Clustering cut to 3 clusters. Left: Unphosphorylated ALK2 system with FKBP12 bound. Right: Phosphorylated ALK2 system without FKBP12 bound.(TIF)Click here for additional data file.S7 FigTop: Correlation plot for distance between R375(Cζ)-D354(Cγ) and umbrella restraint center. Middle: Correlation plot for distance between center of mass of hydrogens of non-restrained NH2 moiety of R375 guanidine group to non-restrained oxygen of D354 side-chain carboxylic acid group. Bottom: Correlation plot for distance between center of mass of hydrogens of restrained NH2 moiety of R375 guanidine group and restrained oxygen of D354 side-chain carboxylic acid group, i.e. umbrella restraint center.(TIF)Click here for additional data file.S1 Moviez-axis to emphasize the difference in separation between R375 and steric barrier region of αC helix.Top: Canonical kinase domain, Bot: Rotation of 90° about (MP4)Click here for additional data file.


AUSPEX is a new software tool for the statistical analysis of single-crystal X-ray diffraction data. It can be used to identify problems in the data resulting from the experiment itself, image processing, data scaling or conversion. AUSPEX, a new software tool for experimental X-ray data analysis, is presented. Exploring the behaviour of diffraction intensities and the associated estimated uncertainties facilitates the discovery of underlying problems and can help users to improve their data acquisition and processing in order to obtain better structural models. The program enables users to inspect the distribution of observed intensities (or amplitudes) against resolution as well as the associated estimated uncertainties (sigmas). It is demonstrated how AUSPEX can be used to visually and automatically detect ice-ring artefacts in integrated X-ray diffraction data. Such artefacts can hamper structure determination, but may be difficult to identify from the raw diffraction images produced by modern pixel detectors. The analysis suggests that a significant portion of the data sets deposited in the PDB contain ice-ring artefacts. Furthermore, it is demonstrated how other problems in experimental X-ray data caused, for example, by scaling and data-conversion procedures can be detected by AUSPEX.In this paper,  In crystal structure determination, diffraction data are interpreted using an atomic model of the unit-cell content of the crystal and its lattice symmetry. Consequently, diagnostic tools are important to derive reliable atomic models from diffraction data. In particular, graphical tools provide a fast and convenient representation of data and can reveal problems which might otherwise go unnoticed. As well as being valuable for users, they are also important to methods developers during the design and improvement of algorithms.However, whilst some of the data-processing software for macromolecular crystallography produces plots of averaged reflection intensities against resolution, individual data points for each reflection are not usually shown. Producing the latter is well within the capability of modern computer systems and, as we will demonstrate in this paper, it can reveal artefacts in the data, such as the presence of so-called ice rings, which may otherwise be hidden by averaging.1.2.Ice rings are Debye–Scherrer rings observed at specific resolutions as a result of X-ray diffraction from a multitude of arbitrarily oriented, typically hexagonal or cubic, ice crystals  and hexagonal (Ih) ice exhibit similar interatomic distances and have similar volumetric mass density, and both exist at ambient pressure. Hexagonal ice is the more common of the two forms. Cubic ice can be seen as a metastable form of hexagonal ice, as it occurs only as nanocrystals and with hexagonal stacking  Resolution ranges contaminated by ice diffraction can be omitted  and the background is modelled by a plane, the background estimation may account for an ice ring. However, if an ice ring is narrow or irregular then current background models can fail to account for this diffraction  All integration programs estimate the background under a reflection  Images can be pre-processed  is an important step in macromolecular crystallography  than images collected using earlier detector technologies, leading to shorter exposure times. Consequently, ice rings (and other background-related problems) are hard to identify by visual inspection of single images alone. They become more evident if images are summed together, for example with Fcalc and observed structure-factor amplitudes Fobs (derived from peak integration of the X-ray image) diverge noticeably. Therefore, ice rings are also visible as outliers in plots of the crystallographic R value or similar indicators against resolution.In the presence of an ice ring, the calculated structure-factor amplitudes phenix.xtriage . The observed amplitudes 3.Iobsversus resolution, as shown in Fig. 3Fobs against resolution to identify ice rings in X-ray data sets.In the presence of an ice ring, negative intensities can result from incorrect estimation of the background  and σ(Iobs) versus resolution. A typical output is shown in Fig. 5Fobs) are used instead; if both are present then both sets of plots are generated, which can be useful for the evaluation of conversion to amplitudes. AUSPEX will then automatically detect the presence of ice rings as described in §AUSPEX is written in C++, with input and graphical output managed by a Python wrapper; the Clipper library . 145 had been measured on CCD detectors, 25 on pixel detectors and 16 on image plates. The rest had been measured using other detectors or no detector type was reported. There were no data sets in common with test set A. Plots of 4.2.AUSPEX  were tested with the ‘Anomalous bond length’ feature in the WHAT IF online service  [and plots of Fobs/σ(Fobs)] versus resolution often show clustering around certain values at low resolution. When considering the associated multiplicity values  is.Plots of I has been measured N times, denoted by i, then Ii) are equal, then This is of course because when measurements are summed, and given that these measurements are independent of each other, their variances are summed as well. If the intensity i.e. the number of times a reflection, or its symmetry equivalent, has been measured. Increasing the number of individual measurements contributing to an average results in a lower σ(Iobs) and therefore increases Iobs/σ(Iobs). However, at high resolution the accuracy of the data is usually limited by the crystal as well as the experimental setup  are lost in most conversion methods. Conversion from intensities Iobs to structure-factor amplitudes Fobs is usually performed using the French and Wilson algorithm  with Fobs/σ(Fobs) , where the factor N1/2 accounts for differences in the number of observations per bin, thus stabilizing the score across all resolution ranges. This score essentially measures the departure from the typical shape of the intensity distribution in the given data set, and can be loosely interpreted as a Z-score. This is sensitive to resolution ranges that exhibit sudden sharp changes in the intensity distribution, thus facilitating the detection and assessment of ice rings.For each bin, an ice-detection score is then calculated, Iobs〉 , as shown in Fig. 5S = N1/2(〈Iobs〉s−1 − f) is shown together with the plot of Iobs against resolution for PDB entry 3jqy.Owing to poor background estimation, the presence of ice rings can cause an increase or decrease in the mean intensity 〈5.2.AUSPEX plots, 45 of these contained ice rings, some of which were very weak. This was a similar fraction as found previously by visual inspection of test set B.We selected another test set of 200 random data sets from the PDB for which intensity values had been deposited . By visual inspection of the Of these 45 structures, six had missing data owing to the omission of entire resolution shells from the data.AUSPEX, CTRUNCATE and phenix.xtriage; the results are shown in Table 2CTRUNCATE gives a large number of false positives; phenix.xtriage applies more rigid criteria, resulting in fewer false positives but also more false negatives. AUSPEX performs more consistently in the four categories of false/correct positives and false/correct negatives. The AUSPEX implementation of automatic ice-ring detection is still inferior to the visual inspection of AUSPEX plots.Each of the 200 structures was analysed with 5.3.versus resolution plots. This is a significant fraction which remains relatively consistent even in recent depositions in the PDB, demonstrating that this pathology is generally overlooked, presumably owing to a lack of necessary diagnostics, and that more sophisticated background-determination algorithms are needed to improve intensity estimation.Using this method, a large part of the PDB was evaluated. We found that 19% (5438 out of 28 895) of data sets with intensities deposited were suspected to have contamination owing to ice. This percentage is in keeping with the results from our more limited visual inspection of intensity 5.4.AUSPEX identifies all resolution ranges where ice rings are typically observed for hexagonal or cubic ice (see Table 1S outside a given threshold range (the default is ±5).AUSPEX will not identify Debye–Scherrer rings from sources other than ice. Such rings can be caused, for example, by the crystallization plates used in in situ screening or by sample holders. Since AUSPEX only searches for ice rings in the expected resolution ranges .If there is any doubt over the presence of an ice ring, the plot of the intensity distribution against resolution output by 6.Iobsversus resolution). A similar percentage (19%) was obtained when 28 895 data sets from the PDB for which intensities had been deposited were evaluated with the automatic ice-ring detection implemented in AUSPEX.Even after more than 20 years of specific research to minimize the influence of ice diffraction in macromolecular crystallography  distribution versus resolution as well as automatic ice-ring detection. The automatic ice-ring detection is arguably an improvement over current methods, although visual inspection of AUSPEX plots is presently the most reliable detection method.Optimization of cryoconditions so as to avoid ice rings is hampered by the difficulty in recognizing their presence on diffraction images, in particular images from modern pixel-array detectors, or from scaling statistics. In order to address this problem, a new software tool, The program can be used after scaling to check for data pathology, helping the user to decide whether it is necessary to re-integrate and rescale. It is also useful when looking at data sets that have already been solved in order to check the quality of the data underlying a model.AUSPEX can also be used to investigate the structure and the distribution of errors within crystallographic data sets. The examples given illustrate effects associated with the multiplicity of measurements as well as the conversion from intensities to amplitudes. Although there is little direct evidence to suggest that these effects have a negative influence on structure solution using current software programs, there is clearly scope to improve the estimation of measurement errors in diffraction data (Waterman & Evans, 2010AUSPEX has inspired the development of an improved approach to background estimation that has been implemented in DIALS (Parkhurst et al., 2017Furthermore, CCP4 (Winn et al., 2011The program will be released in the near future as part of 10.1107/S205979831700969X/hi5647sup1.pdfDescription of test sets.. DOI:


The hypothesis that people respond to reminders of mortality with closed-minded, ethnocentric attitudes has received extensive empirical support, largely from research in the Terror Management Theory (TMT) tradition. However, the basic motivational and neural processes that underlie this effect remain largely hypothetical. According to recent neuropsychological theorizing, mortality salience (MS) effects on cultural closed-mindedness may be mediated by activity in the behavioral inhibition system (BIS), which leads to passive avoidance and decreased approach motivation. This should be especially true for people motivated to avoid unfamiliar and potentially threatening stimuli as reflected in a high need for closure (NFC). In two studies involving moderated mediation analyses, people high on trait NFC responded to MS with increased BIS activity (as indicated by EEG and the line bisection task), which is characteristic of inhibited approach motivation. BIS activity, in turn, predicted a reluctance to explore foreign cultures (Study 1) and generalized ethnocentric attitudes (Study 2). In a third study, inhibition was induced directly and caused an increase in ethnocentrism for people high on NFC. Moreover, the effect of the inhibition manipulation × NFC interaction on ethnocentrism was explained by increases in BIS-related affect  at high NFC. To our knowledge, this research is the first to establish an empirical link between very basic, neurally-instantiated inhibitory processes and rather complex, higher-order manifestations of intergroup negativity in response to MS. Our findings contribute to a fuller understanding of the cultural worldview defense phenomenon by illuminating the motivational underpinnings of cultural closed-mindedness in the wake of existential threat. We encapsulate ourselves to avoid death. And life escapes us while we huddle within the defended fortress  MS causes BIS activity, evidenced by increased by greater right-hemispheric frontal asymmetry, (b) BIS activity mediates MS effects on cultural closed-mindedness, and (c) these effects are pronounced among persons with high NFC. Stated differently, we tested the moderated mediational hypothesis that MS effects on cultural closed-mindedness are mediated by BIS activity and are particularly strong for persons with high NFC. These hypotheses were examined by inducing MS (vs. a control topic), measuring NFC, measuring frontal brain asymmetry via the LBT (Study 1) and EEG (Study 2), and measuring cultural closed-mindedness in the form of reluctance to engage with cultural novelty (Study 1) and general ethnocentrism (Study 2). Moreover, in order to substantiate our claim that inhibition is the mediator, we directly manipulated the proposed mediator—inhibition—in Study 3 to test whether inhibition evokes ethnocentric thinking for high NFC persons  persons with high NFC respond with increased BIS activity  to MS, and (b) their increased BIS activity mediates their increased unwillingness to explore foreign cultures.SD = 1.8; range: 19–31). Participant nationalities were 54 German, 18 Austrian, and 2 other. Participants were randomly assigned to one of two conditions, MS (N = 35) or TV salience (N = 39), in a between-subjects design. The study was described as an investigation of personality and visual perception. The study was approved by the ethics committee of the University of Salzburg. All participants signed informed consent, and could withdraw participation at any point, although no participant made use of this option.Seventy-seven students at the University of Salzburg participated in this paper-pencil study, which took place in class, prior to a lecturea priori based on two criteria: First, representativeness of the whole NFC scale, that is, corrected item-total correlations presented by Schlink and Walther (rit = 0.00). The two remaining items were: “I prefer familiar things over unfamiliar and unpredictable ones” and “I don't like unpredictable situations” . A pilot study revealed this short NFC scale to correlate strongly with the conceptually related personal need for structure scale by Neuberg and Newsom  to 6  scale, and averaged to create a single composite score .Following instructions and the baseline LBT measure (described in more detail below), the NFC scale was presented. We measured NFC with three items that were taken from the German version of the NFC scale . In each measure, participants were instructed to mark the perceived center point of eight horizontal lines that ranged from 4 to 6 (102 to 152 mm) in length with their centers offset from each other  to 5 (very much) scale, and averaged to create a single composite score .Passive avoidance of cultural novelty was measured with eight researcher-generated items designed to measure interest in exploring culturally foreign stimuli . Based on the conceptual view that exploration constitutes approach behavior toward novel stimuli  such that avoidance of cultural novelty was especially high under MS vs. control for people high (+1 SD) on NFC . In contrast, there was no conditional indirect effect for low NFC persons, b = −0.15, 95% CI .As per bias-corrected bootstrap procedure using 75000 bootstrap samples . Fifty-five participants held German, 17 Austrian, and 1 another nationality (3 undisclosed). Participants were randomly assigned to one of two conditions, inhibition (N = 36), or control (N = 40), in a between-subjects design. The study was described as an investigation of participants' personalities. The study was approved by the ethics committee of the University of Salzburg. All participants signed informed consent, and could withdraw participation at any point, although no participant made use of this option.Seventy-six students at the University of Salzburg participated in this paper-pencil study, which took place in class, after a lectureM = 3.77, SD = 0.85; r = 0.44, p < 0.001).Because the two-item NFC measure used in Study 1 was shown to be sensitive to MS, we measured NFC with same two items Ethnocentrism was measured in the same way as in Study 2 , anxious (ängstlich), worked up (unruhig), and worried (besorgt), along with the face-valid adjective inhibited . Participants responded on a scale ranging from 1  to 5 (extremely). Finally, participants completed the PANAS , the interaction between the manipulation  and NFC was significant, b = 0.49, SE = 0.21, β = 0.27, t(69) = 2.38, p < 0.02, ΔR2 = 0.07  showed significantly increased ethnocentrism following MS, b = 0.59, SE = 0.24, β = 0.39, t(69) = 2.41, p < 0.05, whereas this was not true for low NFC persons (SD = −1), b = −0.24, SE = 0.25, β = −0.16, t(69) = −0.98, p = 0.33. Thus, our hypotheses were corroborated: inhibition caused ethnocentrism in high NFC persons, whereas low NFC persons were not affected by inhibition in this way.To examine whether high NFC persons respond with increased ethnocentrism to the inhibition recall-task, we used moderated regression analyses= 0.27, t9 = 2.38,M = 3.63, SD = 1.24) than in the control condition , t(35.40) = 9.65, p < 0.001, η2 = 0.63As expected, BIS affect was significantly higher in the inhibition condition  = 49.64, p < 0.001, η2 = 0.43, although BIS affect was highly correlated with negative mood, r = 0.79, p < 0.001. There was no correlation between BIS affect and positive mood, r = −0.16, p = 20.Mood was also affected by inhibition. Negative affect was significantly increased and positive affect was significantly decreased by inhibition, b = 0.44, SE = 0.22, t(63) = 1.95, p = 0.051. Persons with high NFC still showed increased ethnocentrism after inhibition, b = 0.67, SE = 0.31, t(63) = 2.14, p < 0.05, contrary to low NFC persons, b = −0.07, SE = 0.29, t(63) = −0.26, p = 0.80. The PANAS scales did not affect ethnocentrism (ps > 0.21).Likewise, the interactive effect of inhibition and NFC on ethnocentrism was virtually unaltered after controlling for the PANAS subscales, b = 0.17, SE = 0.10, β = 0.32, t(65) = 1.69, p < 0.10, all other ps > 0.22. Moreover, a significant interaction between BIS affect and NFC occurred, b = 0.24, SE = 0.09, β = 0.39, t(65) = 2.73, p < 0.01, ΔR2 = 0.09  = 3.04, p < 0.01, whereas this was not true for low NFC persons, b = −40.05, SE = 0.13, β = −0.11, t(65) = −0.43, p = 0.67. Again, the PANAS scales did not affect ethnocentrism (ps > 0.21). Thus, BIS affect evoked ethnocentrism for high NFC persons in the same way as inhibition, and this effect was independent of changes in mood.For the sake of convergent validity, we explored whether BIS affect elicited ethnocentrism for high NFC persons similar to the inhibition manipulation. We also controlled for the PANAS scales to rule out the possibility that the effect of BIS affect could be explained by mood more generally. There was a marginally significant main effect of BIS affect, These results constitute an important conceptual replication of the relationship between BIS activity and ethnocentric thinking. Manipulating inhibition, the proposed mediator of MS effects on ethnocentrism, provided further support for the proposition that inhibition plays a causal role in generating ethnocentrism following MS. Persons with high NFC responded with increased ethnocentrism to not only induced inhibition but also experiences of BIS-related affect. These results provide important multimethod evidence for the notion that not only perceptual and neural (Studies 1–2) but also cognitive-affective indicators of BIS-mediated inhibition can provoke ethnocentric thinking, and these effects are particularly strong for persons with high NFC.In this paper, we have presented evidence from three studies that shed light on several previously uninvestigated questions that have recently gathered much attention in the existential threat literature  to BIS-mediated inhibition of approach motivation, we used perceptual and neural indicators of frontal asymmetry to gauge BIS activity  and self-expansive growth . The first author of this article was also financially supported by the Doctoral College “Imaging the Mind” of the Austrian Science Fund (FWF-W1233).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Polar seabirds adopt different over-wintering strategies to survive and build condition during the critical winter period. Penguin species either reside at the colony during the winter months or migrate long distances. Tracking studies and survey methods have revealed differences in winter migration routes among penguin species and colonies, dependent on both biotic and abiotic factors present. However, scan sampling methods are rarely used to reveal non-breeding behaviors during winter and little is known about presence at the colony site over this period. Here we show that Adélie penguins on the Yalour Islands in the Western Antarctic Peninsula (WAP) are present year-round at the colony and undergo a mid-winter peak in abundance during winter. We found a negative relationship between daylight hours and penguin abundance when either open water or compact ice conditions were present, suggesting that penguins return to the breeding colony when visibility is lowest for at-sea foraging and when either extreme low or high levels of sea ice exist offshore. In contrast, Adélie penguins breeding in East Antarctica were not observed at the colonies during winter, suggesting that Adélie penguins undergo differential winter strategies in the marginal ice zone on the WAP compared to those in East Antarctica. These results demonstrate that cameras can successfully monitor wildlife year-round in areas that are largely inaccessible during winter. Aptenodytes patagonicus) breed during the summer months, but raise each chick for fourteen months over winter, resulting in an overlap in incubation and chick rearing each summer [Pygoscelis adeliae; Chinstrap penguins, Pygoscelis antarctica; Gentoo penguins, Pygoscelis papua; Macaroni penguin, Eudyptes chrysolophus; Southern Rockhopper Penguin, Eudyptes chrysocome) hatch and raise their chicks to fledgings during summer, yet adopt various overwinter strategies to survive the non-breeding period and build condition prior to breeding the following summer [Extreme seasonality in the Polar Regions has forced many animals to develop adaptations to cope with a dynamic habitat, including a constrained breeding period and resilience during the non-breeding season. At the end of summer, most polar wildlife species enter a non-breeding, winter period with harsh environmental conditions in which individuals must survive while also building condition immediately prior to the next breeding season. Penguins (Spheniscidae) represent a large biomass of seabirds in the Southern Ocean during winter  but occuh summer . Similarg summer .These winter strategies include either long distance migrations  or residence at the colony site  and penguins  and refepenguins ,14,23. Apenguins –27. Subspenguins ,28–30, spenguins , and dynpenguins , may infpenguins  and an ipenguins –34.As an ice-obligate species during winter, recent evidence suggests that Adélie penguin populations have significantly declined along the Western Antarctic Peninsula (WAP) since 1980 ,35–36, aPast studies examining Adélie overwintering behavior have primarily used tracking methods at individual colonies along East Antarctica, the Ross Sea ,23 and aTo further enhance knowledge of the overwintering behavior of Adélie penguins in the WAP and East Antarctica, we observed the presence of Adélie penguins at colony sites during the non-breeding period from camera images taken daily over winter in each of these regions. Cameras were deployed at the Yalour Islands along the WAP and at seven sites near Casey and Mawson stations in East Antarctica. Additionally, the cameras provided high-resolution data on sea ice extent at the Yalour Islands site, allowing us to accurately measure near-shore sea ice and its influence on Adélie penguin presence or absence at the colony site over winter. Using this information, we aimed to answer the following hypotheses:Adélie penguins are present at the Yalour Islands breeding location and colony sites near Casey and Mawson stations throughout winter.The number of adults present increases as sea ice extent diminishes and as daylight hours and daily air temperatures increase throughout the winter across the species’ range.Fieldwork was carried out under permits from the UK Foreign and Commonwealth Office and Australia’s Antarctic Treaty  Act 1980. Our study sites included Adélie penguin breeding sites in the WAP and in East Antarctica . In 2010The east Antarctic study sites comprised seven Adélie penguin breeding sites near Mawson and Casey stations that currently hold around 75,000 breeding pairs , Odbert Island , Shirley Island , and Whitney Point  near Casey Station; Petersen Island , Verner Island , and Welch Island  near Mawson Station; The Yalour Islands camera overlooked three sub-colonies of Adélie penguins. The camera angle was set to view the maximum number of individuals possible while still allocating a large portion of the camera view to the near-shore sea ice. The camera was installed using the techniques described by  with minSimilarly, each of the seven cameras deployed in East Antarctica were installed using the methods described in . Each caCounts were extracted from each image using software developed by . In eachAn Observer’s Guide to Sea Ice [The Yalours camera on the WAP and four of the East Antarctica cameras  overlooked the nearby marine environment enabling the extent and type of near-shore sea ice to be determined. Sea ice extent was determined from the images using the guidelines described in  Sea Ice , origina Sea Ice . Rankingsuncalc function in the RAtmosphere package of R [All the cameras automatically produced metadata on date and time of day, and the Yalours camera also produced temperature data throughout the study period. Daylight hours were extracted using the age of R –50.stats package [glm.nb function, MASS package; [acf function, stats package), we determined that temporal autocorrelation did not occur in our data set and a correlation structure was therefore omitted from the model. Model selection was determined by choosing the model with the lowest ratio of residual deviance to degrees of freedom. Ultimately, the GLM independent variables included an interaction between sea ice type and both photoperiod (aka. daylight hours) and temperature (°C), year, and Julian date . During model selection, the log of the sea ice score (1–10) coefficient was omitted . Lastly, to understand relationships with environmental variables, we examined contrasts by reordering the levels of factors .We used generalized linear models (GLM) to model penguin abundance at occupied colony sites in relation to environmental conditions. To avoid bias due to low daylight hours during winter, and therefore a lower volume of visible counts around the winter solstice, we used only those images taken at 12:00 . All analyses were conducted in R (v 3.0.3) language for statistical computing using the  package , merging package . Due to package; ) to deteWe observed considerable numbers of Adélies at the Yalour Islands colony site during the non-breeding (winter) period compared to those present during the breeding period. When birds were present during the non-breeding (winter) period, their abundance in the cameras’ field of view ranged from 1 to 13 in 2012, 1 to 29 in 2013, and 1 to 9 in 2014.Analysis of images from the East Antarctic sites revealed that Adélies were last seen in photographs at the seven sites between February 28 and March 29 in 2013 and between 4 February and 27 February in 2014. There were no further sightings of penguins at any of the sites until the following October at the beginning of the next breeding season in both years. We therefore considered Adélies as absent from all of the East Antarctic colony sites over the entire non-breeding, winter period.P = 0.015) than open water conditions (P < 0.001), although both sea ice types exhibited a significant relationship with non-breeding (winter) abundance (P = 0.038) was significantly different from photoperiod when open water was present  differed significantly from periods when open water (P = 0.17) or pack ice (P = 0.385) was present and exhibited a positive relationship with penguin abundance  period and each of the environmental variables tested in bundance . The relbundance . In othe(P < 0.001). We also found significant differences between years; penguins were significantly more abundant in 2012 (P = 0.001) than in 2014 (P = 0.007), although there was no significant difference between 2013 and the other two years  season Here, we show that time-lapse cameras can successfully monitor colonial wildlife and near-shore sea ice conditions year-round, which will improve our understanding of winter behavior at colonies in remote regions. By using cameras, we can monitor areas that are logistically difficult to access and increase the replication of monitored sites . MoreoveSubstantial differences exist between East Antarctica and the Western Antarctic Peninsula (WAP) in terms of environmental drivers, particularly related to winter sea ice extent. In East Antarctica, widespread consolidated compact ice exists in many regions throughout the winter months –55. The Our results at the Yalours site support a strong association between winter sea ice and abundance; Adélies were present at the Yalour Islands during the non-breeding period when either compact sea ice or open water conditions were present , Fig 1. Our analysis on Adélies at the Yalours site also supported the idea that winter migrations may be linked with photoperiod (aka. daylight hours). As daylight hours decreased throughout the winter, Adélie non-breeding abundance at the colony increased when either open water or compact sea ice conditions were present offshore. Presence at the colony site during decreased photoperiods may indicate that penguins are resting on land , finding prey directly offshore, and avoiding predation during low light levels –60. AdélOur study highlights the lack of understanding of winter ecology, even though the winter months serve as a critical period of survival. With novel methods of constant effort, we are able to reveal a new mid-winter phase in the annual cycle of a colony at the edge of the species’ regional range, which would otherwise be limited by small sample sizes using survey efforts or tracking approaches. However, it is important to address that there is no way of knowing whether the birds in view during winter are the same as those in view over the summer months, yet it is assumed that many of the birds are present at both times of the year at the colony site based on observations of other Adélie populations in the WAP  and persPygoscelis species along the WAP [It has been proposed that changes in climate over the coming years will increase competition for resources and habitat among the  the WAP . This inS1 Fig(TIFF)Click here for additional data file.S2 Fig(TIFF)Click here for additional data file.S1 Table(CSV)Click here for additional data file.


Plasmodium falciparum is apical membrane antigen 1 (AMA1), which is essential for erythrocyte invasion. The importance of AMA1 as a target of acquired human inhibitory antibodies, their allele specificity and prevalence in populations is unknown, but crucial for vaccine design.The polymorphic nature of many malaria vaccine candidates presents major challenges to achieving highly efficacious vaccines. Presently, there is very little knowledge on the prevalence and patterns of functional immune responses to polymorphic vaccine candidates in populations to guide vaccine design. A leading polymorphic vaccine candidate against blood-stage P. falciparum lines expressing different AMA1 alleles were genetically engineered and used to quantify functional antibodies from two malaria-exposed populations of adults and children. The acquisition of AMA1 antibodies was also detected using enzyme-linked immunosorbent assay (ELISA) and competition ELISA  from the same populations.We found that AMA1 was a major target of naturally acquired invasion-inhibitory antibodies that were highly prevalent in malaria-endemic populations and showed a high degree of allele specificity. Significantly, the prevalence of inhibitory antibodies to different alleles varied substantially within populations and between geographic locations. Inhibitory antibodies to three specific alleles were highly prevalent , identifying them for potential vaccine inclusion. Measurement of antibodies by standard or competition ELISA was not strongly predictive of allele-specific inhibitory antibodies. The patterns of allele-specific functional antibody responses detected with our novel assays may indicate that acquired immunity is elicited towards serotypes that are prevalent in each geographic location.These findings provide new insights into the nature and acquisition of functional immunity to a polymorphic vaccine candidate and strategies to quantify functional immunity in populations to guide rational vaccine design.The online version of this article (doi:10.1186/s12916-016-0691-6) contains supplementary material, which is available to authorized users. Plasmodium falciparum infection was higher for episodes caused by vaccine-like strains compared to vaccine-dissimilar strains [The development of effective vaccines is a major global goal towards achieving malaria control and elimination. However, a major challenge in the development of highly efficacious vaccines is antigen polymorphism, which is an important issue for many leading vaccine candidates. This includes the RTS,S vaccine, which recently completed phase III trials, and vaccines based on apical membrane antigen 1 (AMA1) and merozoite surface protein 2 (MSP2); for all these candidates, vaccine efficacy against malaria or  strains . Current strains –6. Meroz strains , 7. HoweThe merozoite protein apical membrane antigen 1 (AMA1) is a leading polymorphic vaccine candidate that plays an essential role in host cell invasion and is a prominent target of naturally acquired antibodies –9. AMA1 AMA1 is highly polymorphic with more than 200 haplotypes, and reflects the challenges faced in vaccine development of overcoming antigenic diversity to enable highly efficacious vaccines , 30–32. P. falciparum lines expressing one of six antigenically distinct AMA1 alleles that broadly represent global antigenic diversity [To date, there has been no way of quantifying naturally acquired AMA1-specific invasion-inhibitory antibodies in human populations or quantifying the prevalence of allele-specific functional antibodies. Moreover, the prevalence of allele-specific functional antibodies has not been reported for any malaria antigen to date, because of a lack of tools and approaches. In this study we used AMA1 as a model to investigate the patterns, prevalence and nature of acquired functional immunity to a polymorphic vaccine candidate. We developed a novel approach using genetically engineered iversity , 31. TheThe aim of this study was to measure and compare the prevalence and allele specificity of naturally acquired growth inhibitory antibodies to a polymorphic malaria antigen (AMA1) across two geographically distinct locations (Kenya and Papua New Guinea) to inform the design of future vaccines incorporating polymorphic antigens.P. falciparum lines were genetically engineered to express one of six antigenically distinct alleles of AMA1 and were used in in vitro growth inhibition assays (GIAs) to measure the presence of AMA allele-specific functional antibodies in samples from children and adults in Papua New Guinea and adults living in Kenya. Antibodies to AMA1 were also quantified by standard ELISA and competition ELISA. P. falciparum; 26 % P. vivax; 4 % P. malariae; 5 % mixed infections). Serum samples were also collected from adult residents (n = 54) of Nyanza Province, Kenya, an area of high malaria transmission [Blood was collected (in 2004) from adults and children participating in a cross-sectional study in Madang Province, Papua New Guinea (PNG), a region that experiences year-round malaria transmission with seasonal variation , 36. Sersmission . Sera frRabbit antisera against recombinant, refolded AMA1 ectodomains were generated and purified as described , 31. SodP. falciparum and transgenic W2Mef P. falciparum genetically engineered to express one of six different AMA1 alleles were cultured in vitro [P. falciparum (pigmented trophozoite stage) from culture was adjusted to 0.1 % parasitemia and 2 % haematocrit for assay set-up. Human serum samples were tested for growth inhibition at 1/10 dilution in duplicate or triplicate wells in a 96-well format. Parasites were allowed to develop through two cycles of erythrocyte invasion for 72 hours at 37 °C. Parasitemia was evaluated by flow cytometry .This cut-off was selected on the following basis. (1) Variation in the assay was estimated as +/-2 standard deviations around the mean of the Melbourne control sera used in assays; 2 standard deviations was used to define our ‘variation threshold’, and this value was always less than 10 % across assays, as we have found in previous studies [Wild-type W2Mef in vitro . Transgein vitro , while lin vitro . Flow cyin vitro , 38, 39. studies .P. falciparum lines was compared by paired t test, or Wilcoxon’s matched-pairs signed-rank sum test for non-parametric data. Prevalence of AMA1 antibodies or prevalence of specific invasion inhibition was compared using the chi-squared test or Fisher’s exact test. The Mann-Whitney test was used to compare antibody levels. Spearman’s correlation coefficient was used to assess associations between antibodies to different AMA1 alleles measured by ELISA, and between invasion inhibition and AMA1 antibodies.Analyses were performed using GraphPad Prism 5. Invasion inhibition of different P. falciparum isolates from a common parental line (W2Mef) which were genetically engineered to express one of six different AMA1 alleles that broadly represent the antigenic diversity of AMA1, including alleles from our study population [t test P < 0.05, Fig. We generated pulation , 32. We pulation ). Here, P. facliparum lines was also confirmed by Western blot . See Fig. P < 0.0001).We first measured antibodies to recombinant AMA1 in the PNG study population by standard ELISA. The prevalence of AMA1 antibodies was high (>90 %) and similar for each of the six AMA1 alleles that were expressed by the genetically engineered P. falciparum lines by serum antibodies from malaria-exposed individuals provided evidence that AMA1 is a target of naturally acquired invasion-inhibitory antibodies  , and inhibition of W2mef was significantly greater than XIE or Pf2006 . There was no significant difference in prevalence between children and adults for any comparison  , despitP. falciparum line (Fisher’s exact test P = 0.053), and a greater proportion of adults had specific inhibitory activity against one genetically engineered P. falciparum line (P = 0.024).Analysis of the specificity or cross-reactivity of inhibitory antibodies revealed that most serum samples were not broadly cross-inhibitory with few samples inhibiting more than two different AMA1 lines Fig. , with a P. falciparum line (e.g. FVO and W2mef versus FVO only), no difference was observed in antibody level by ELISA  or absolute invasion inhibition (P > 0.7011) , and a similar trend was observed for 3D7 (P = 0.16) but not Pf2006 . These findings suggest that ELISA data are indicative of functional activity to some extent, but they are not strongly or consistently predictive of AMA1-specific inhibitory activity and cannot be relied upon as a surrogate of inhibitory activity.Our analysis considered the relationship between AMA1 antibodies by ELISA and AMA1-specific inhibitory antibodies. AMA1-specific inhibitory antibodies were most prevalent for W2mef and FVO; therefore, these were the main focus of analyses. We found that high antibody responders (defined as greater than the median) to FVO and W2mef, determined by ELISA, had a higher prevalence of specific invasion inhibition of the 006 Fig. .   and amongst samples that did not show differential invasion inhibition (n = 64)   Fig. . Amongst01) Fig. . However01) Fig. , suggestP < 0.0001; Wilcoxon’s matched-pairs signed-rank sum test). Analysis of the cross-reactivity of inhibitory antibodies revealed that most Kenyan serum samples were not broadly cross-inhibitory, with few samples inhibiting more than three different AMA1 lines  and XIE (50 %) AMA1 alleles Fig. . This dines Fig. , as seenP. falciparum lines , and the majority of serum samples (92 %) were positive for each of the six AMA1 alleles tested , no difference was observed in antibody level by ELISA  or absolute invasion inhibition (P > 0.3823). Hence, similar to the results found for PNG sera, the overlap in invasion-inhibitory activity of Kenyan sera is not related to antibody magnitude measured by ELISA. Total growth-inhibitory activity of lines expressing different AMA1 alleles by plasma antibodies was significantly correlated with levels of antibodies to the relevant AMA1 allele measured by ELISA  and was similar for each of the six AMA1 alleles that were expressed by the genetically engineered nes Fig. . Antibodted Fig.  . We found that FVO-specific inhibitory antibodies (relative to 3D7) were not correlated with antibodies to FVO by ELISA . Further analysis by ELISA also found no correlation between antibodies to FVO and FVO-specific inhibitory antibodies relative to HB3 , W2Mef , XIE  or Pf2006 . Hence, while AMA1 antibodies by ELISA are significantly correlated with the total growth-inhibitory capacity of naturally exposed Kenyan sera . There are also no data to suggest that the FVO, W2mef or XIE alleles are inherently more susceptible to inhibitory antibodies, and studies have shown comparable inhibition of different alleles by vaccine-induced antibodies [Analysis of AMA1 sequences suggested that the ability of AMA1 alleles to recruit T cell help is not a differentiating factor in the ability of each AMA1 allele to elicit functional antibody responses. Whilst the number of human major histocompatibility complex (MHC) class II binding peptides predicted to be present in AMA1 varies greatly depending on the human leukocyte antigen (HLA) allele examined, there is no significant difference in the number of MHC class II binding peptides for a given HLA allele across each of the six AMA1 alleles examined in this study  were lower in both populations , 44.Acquired human inhibitory AMA1 antibodies have substantial allele-specific activity, rather than cross-reactive inhibition. The strain-specific nature of human inhibitory antibodies we observed is consistent with the strain-specific efficacy reported in a phase II AMA1 vaccine trial . There wP. falciparum isolates to varying degrees, but may also show some cross-inhibitory activity depending on the alleles being tested [P. falciparum; therefore, allele replacement was the only approach possible to quantify antibodies in functional assays. The predominantly allele-specific nature of AMA1 inhibitory antibodies demonstrated by these data indicates that a multi-allele vaccine approach, or alternate strain-covering approach, will be required for AMA1.Our data are consistent with malaria-exposed individuals acquiring a repertoire of allele-specific invasion-inhibitory antibodies of multiple specificities, rather than cross-inhibitory antibodies. Although ELISA data indicate that humans acquire some cross-reactive antibodies , 21, 32,g tested . The preg tested , 42. It We successfully developed and applied a novel approach to quantify functional immune responses to a polymorphic vaccine candidate in malaria-exposed populations using genetically engineered parasites. Our findings indicate that AMA1 is a key target of invasion-inhibitory antibodies that are largely allele-specific, and that the prevalence of antibodies to different alleles varies substantially within and between populations. These findings indicate that vaccine design, such as the selection of specific alleles for inclusion in a multi-allele vaccine, may need to take account of the different population prevalence of functional antibody serotypes. These findings are broadly relevant to understanding immunity and vaccine design for the many candidate malaria vaccine antigens that have similar issues of antigen diversity, and suggest that further studies are urgently needed to determine the distribution of different serotypes or antigenic variants of leading vaccine antigens, and the patterns of functional immunity to these variants in different populations, to guide rationale vaccine development and achieve maximal efficacy.