Halophila ovalis leaves. Fluorescence images demonstrated Diuron affected the entire leaf surface evenly and responses were not influenced by isolating leaves from the plant. The optimum exposure duration was 24 h, by which time the inhibition of effective quantum yield of PSII (∆F/Fm’) was highest and no deterioration of photosystems was evident in control leaves. The inhibition of ∆F/Fm’ by Diuron in isolated H. ovalis leaves was identical to both potted and hydroponically grown plants (with leaves remaining attached to rhizomes), indicating similar reductions in photosynthetic activity in these acute well-plate assays. The sensitivity of the assay was not influenced by irradiance (range tested 40 to 400 μmol photons m-2 s-1). High irradiance, however, caused photo-oxidative stress in H. ovalis and this generally impacted in an additive or sub-additive way with Diuron to damage PSII. The bioassay using isolated leaves is more rapid, uses far less biological material and does not rely on specialised aquarium facilities in comparison with assays using potted plants. The development and validation of this sensitive bioassay will be useful to reliably screen and monitor the phytotoxicity of existing and emerging PSII herbicides and contribute to risk assessments and water quality guideline development in the future.Photosystem II (PSII) herbicides have been detected in nearshore tropical waters such as those of the Great Barrier Reef and may add to the pressure posed by runoff containing sediments and nutrients to threatened seagrass habitats. There is a growing number of studies into the potential effects of herbicides on seagrass, generally using large experimental setups with potted plants. Here we describe the successful development of an acute 12-well plate phytotoxicity assay for the PSII herbicide Diuron using isolated  A dose response curve was plotted using inhibition data at 24 h exposure. A four parameter logistic curve was fitted to each data set separately (Sigmaplot 11.0) [50 values and corresponding confidence intervals were determined from each curve by applying standard curve analysis. The probability that midpoints (IC50s) generated by the logistic curves were statistically different was tested by applying the F test in Graph Pad Prism V 6.0. IC50s were considered different when p < 0.05 and the post-hoc results presented for each comparison in the relevant Results sections.Photoinhibition (inhibition % relative to solvent control) was calculated from treatment data as Inhibition (%) = [(Yot 11.0) . IC50 va-2s-1 was maintained. Single leaf well plate exposures were performed as described above.The photobiological response of single leaves to Diuron in 12-well plate format exposures was compared with intact potted plants (including sediment to 4.5 cm depth) and hydroponic plants (whole plants in 0.45 μm filtered seawater without sediments or nutrients). While potted plants most closely represent the natural state, hydroponic plants represent an intermediate or bare state where the entire root-rhizome system is intact but not submerged in sediments. Potted plant samples were contained in 500 ml experimental pots (13.5 9.8 4.5 cm). The hydroponic and potted arrangements were placed in 6 l indoor glass aquaria . A constH. ovalis, the seagrass was exposed to 40, 100, 200 and 400 μmol photons m-2 s-1 with varying concentrations of Diuron (0–100 μg l-1). This experiment utilised the well plate bioassay with isolated horizontal leaves and dose-response curves for inhibition of effective and maximum fluorescence yields were developed. Inhibition was calculated in two ways: (i) in comparison to the yield of the solvent control at the relevant irradiance and (ii) in comparison to the yield of the solvent control at the lowest irradiance.To assess how irradiance and Diuron affected the photosynthetic efficiency of F/Fm’ and Fv/Fm for additivity of effects was calculated by applying the Independent Acion (IA) equation to the inhibition data: Pp = P(L) + P(D) – P(L) × P(D) [p is the predicted additive effect of both variables tested; P(L) is the effect of light intensity in the absence of Diuron and P(D) is the effect of Diuron at the control light level, 40 μmol photons m-2 s-1. Both P(L) and P(D) are derived from raw data means. Expected combined effect of both light intensity and Diuron exposure were calculated for seven herbicide and four light combinations and plotted against the measured inhibition data relative to the 40 μmol photons m-2 s-1 light intensity treatment solvent control .Expected inhibition of both Δ) × P(D) ,36. HereFv/Fm greater than 0.65). Damaged leaves and/or those exhibiting low Fv/Fm values deteriorated over 24 h in well plates resulting in low and often erratic fluorescence response.A series of preliminary experiments indicated that consistent maximum quantum yield measurements were only possible following careful pre-screening of the leaves  of 44 μmol photons m-2 s-1 was relatively low, indicating that the seagrass maintained in the indoor aquarium at 280 μmol photons m-2s-1 for upright leaves followed by horizontal acclimation for the assay at 100 μmol photons m-2s-1 were adapted to relatively low light. The rETR reached a maximum of 19 μmol electrons m-2s-1. These measurements allowed us to choose four irradiances for subsequent experiments that represented light limiting (40 μmol photons m-2 s-1), saturated (100 μmol photons m-2 s-1) and two higher irradiances representing irradiance-stressed conditions at 200 and 400 μmol photons m-2 s-1 where illumination exceeded the photosynthetic capacity of the seagrass (leading to elevated photoinhibition).A rapid increase in rETR was observed during the light limiting region of the curve (α = 0.43). The minimum saturating irradiance .The isolated o Diuron . There wFv/Fm from 0.71 ± 0.01 (SE) to 0.69 ± 0.01 (less than 3%) in solvent controls indicating limited deterioration or additional photoinhibitory stress over 24 h. In this experiment 10 μg l-1 Diuron inhibited ΔF/Fm’ by 82% ± 2% (SE) after 24 h relative to solvent control leaves. The uptake of Diuron was rapid with 95% maximum inhibition of ΔF/Fm’ (toxicity) reached after 12 h and steady state toxicity between 18 and 24 h (Leaves selected by pre-screening (above) exhibited little reduction in and 24 h , indicatΔF/Fm’ by Diuron was similar in each case  more sensitive to Diuron. The Diuron concentrations that inhibited ∆F/Fm’ by 10% and 50% (IC10 and IC50) can be found in Fv/Fm in H. ovalis by Diuron was consistent between techniques . The complete inhibition of Fv/Fm was not reached over this range of exposures potentially due to incomplete binding of Diuron to the binding sites of PSII.Dose-response curves obtained from different PAM fluorometers indicated that although the inhibition of ach case  that theF/Fm’ in H. ovalis leaves in 12-well plates was identical to whole potted or hydroponic plants, with IC10 values ranging from 0.53 to 1.0 and IC50 values ranging from 3.0 to 3.5 μg l-1 . However, Diuron caused more inhibition of Fv/Fm in well plate leaves which exhibited IC50 for Fv/Fm of 4.2 μg l-1 compared with 11 and 16 μg l-1 for potted and hydroponic arrangements respectively .The effect of Diuron on ∆F/Fm’ relative to solvent controls at the corresponding light intensity was relatively consistent across herbicide exposures . Similar results were observed for inhibition of Fv/Fm by Diuron when measured against the controls at corresponding light intensities .The inhibition of ∆xposures . Althougensities , Table 2F/Fm’ relative to the solvent control measurements was far greater under increased illumination compared to those taken under 40 μmol photons m-2 s-1  against measured inhibitions (The mixture model of Independent Action (IA) was applied to ∆ibitions  revealedFv/Fm at four light intensities and seven Diuron concentrations , which non-destructively measures change in the efficiency of PSII and will be useful to rapidly and reliably screen exposure to existing and emerging PSII herbicides. Fluorescence images demonstrated that Diuron affected PSII across the entire leaf surface evenly and measurements were not affected by isolating leaves from the plant. The inhibition of ∆F/Fm’ (but not Fv/Fm) by Diuron in isolated H. ovalis leaves was identical to potted and hydroponic plants, following the optimum exposure duration of 24 h. The sensitivity of isolated H. ovalis leaves to Diuron was also consistent (i.e. IC50 and IC10 were similar) with other potted seagrasses  in a study that proposed revision of the environmental guidelines based on toxicity responses to four herbicides [H. ovalis. Irradiance acted in an additive or sub-additive way with Diuron to impact on PSII thus highlighting the need for understanding how interacting environmental factors could influence screening procedures, as well as seagrass sensitivity to herbicides under variable environmental conditions.An acute 12-well plate phytotoxicity assay using isolated rbicides . This siFv/Fm) in solvent controls and confirming there was <3% decrease over the exposure period. The fluorescence images also demonstrated that Diuron was acting on PSII evenly across leaves. Previous studies have shown very fine-scale differences in absorptivity across seagrass leaves [F/Fm’ (which is minimised as it is based on a ratio of fluorescence emissions). Ralph et al. [F/Fm’ values due to either chronic oxidative damage or differences in light adaptation over the length of a leaf. In the current study these differences were minimised by choosing young leaves and pre-screening for leaves with high photosynthetic efficiencies. Importantly the images demonstrated that the herbicide did not flood through the vascular system but entered the leaf evenly across the semi-permeable cell walls. This is consistent with a previous study which demonstrated that the PSII herbicide Atrazine was preferentially transported through the leaves rather than via the root-rhizome complex [F/Fm’ by ~12 h was consistent between the isolated leaf well-plate format and potted H. uninervis and Z. muelleri [H. uninervis and Z. muelleri showed no additional inhibition of ∆F/Fm’ or Fv/Fm by Diuron between 24 and 72 h [The main challenge in developing a well-plate phytotoxicity assay for isolated seagrass leaves was to ensure that the photosystems in the leaves were not degraded during the exposure period and that the exposure was sufficiently long for the leaves to reach steady-state response to the herbicide. The integrity of the leaves was confirmed in two related ways. Firstly, fluorescence images across the leaf surface using I-PAM showed that in uncontaminated seawater there was little difference in fluorescence response over the 24 h . This was leaves  but thish et al.  also dem complex . The uptmuelleri . An optiand 72 h .F/Fm’) by Diuron in isolated leaves using the 12-well plates was identical to inhibition in intact plants (both hydroponic and potted). The narrow range of IC50s (-1) demonstrated not only the consistency of the method with larger, more time-consuming alternatives, but also supported our case that the isolated leaves are a valid alternative for acute phytotoxicity tests and supported previous studies that herbicide toxicity in seagrass is via leaf, not root-complex uptake [10 and IC50 values for ∆F/Fm’ in isolated H. ovalis leaves were also similar to those reported for the effects of Diuron on ∆F/Fm’ in potted H. uninervis and Z. muelleri over 24 and 72 h [F/Fm’, there was good agreement in assay results for the measurement of inhibition of leaves using the two PAM instruments . However, it seems that the horizontal leaves at 100 μmol photons m-2s-1 may have been under greater photooxidative pressure (lower Fv/Fm) using these conditions [F/Fm’), but highlights the influence of irradiance on PSII by herbicides and may differ between isolated leaves and intact plants.The inhibition of photosynthetic activity   under different irradiances . This result shows that although the irradiance spanned an order of magnitude that the relative inhibition of ∆F/Fm’ was not appreciably influenced, further highlighting the robust nature of this endpoint.As discussed above, the sensitivity of aquatic plants to PSII herbicides can be influenced by irradiance. Photosynthetic activity increases with irradiance (light-limited phase) until saturating irradiances are reached and ∆d phase) , and ene reduced . Indeed  reduced  demonstr-2 s-1 far exceeded the photosynthetic capacity of these leaves [Fv/Fm by 400 μmol photons m-2 s-1 relative to control values at the lowest illumination, even at low herbicide concentrations  and maximum quantum yield (Fv/Fm) to PSII in seagrass is likely to be applicable to a variety of PSII herbicides under a broad range of conditions such as reduced salinity and temperature extremes. It does not rely on elaborate experimental aquarium systems to maintain potted plants and takes far less time, resources and biological material to deliver reliable ecotoxicology data. Application of this assay has the potential to provide management agencies and regulators with considerably more toxicological data for the development of improved risk assessments and water quality guidelines.While fluorescence well-plate assays have been used widely and effectively to study the impacts of PSII herbicides on microalgae ,46, only the GBR . These r the GBR . The hig the GBR . H. oval species ,24,49, a


Neuromyelitis optica (NMO) is an autoimmune demyelinating disease of the central nervous system in which binding of anti-aquaporin-4 (AQP4) autoantibodies (NMO-IgG) to astrocytes causes complement-dependent cytotoxicity (CDC) and inflammation resulting in oligodendrocyte and neuronal injury. There is compelling evidence for a central role of complement in NMO pathogenesis. Here, we evaluated the potential of C1-esterase inhibitor (C1-inh) for complement-targeted therapy of NMO. C1-inh is an anti-inflammatory plasma protein with serine protease inhibition activity that has a broad range of biological activities on the contact , coagulation, fibrinolytic and complement systems. C1-inh is approved for therapy of hereditary angioedema (HAE) and has been studied in a small safety trial in acute NMO relapses (NCT 01759602). In vitro assays of NMO-IgG-dependent CDC showed C1-inh inhibition of human and rat complement, but with predicted minimal complement inhibition activity at a dose of 2000 units in humans. Inhibition of complement by C1-inh was potentiated by ∼10-fold by polysulfated macromolecules including heparin and dextran sulfate. In rats, intravenous C1-inh at a dose 30-fold greater than that approved to treat HAE inhibited serum complement activity by <5%, even when supplemented with heparin. Also, high-dose C1-inh did not reduce pathology in a rat model of NMO produced by intracerebral injection of NMO-IgG. Therefore, although C1r and C1s are targets of C1-inh, our in vitro data with human serum and in vivo data in rats suggest that the complement inhibition activity of C1-inh in serum is too low to confer clinical benefit in NMO. Neuromyelitis optica (NMO) is autoimmune disease of the central nervous system in which inflammatory demyelinating lesions cause optic neuritis and transverse myelitis There is strong evidence for a central role of complement in NMO pathogenesis and hence for the utility of complement-targeted therapeutics. Inflammatory lesions in human NMO show prominent vasculocentric deposition of activated complement Though further clinical evaluation of eculizumab in NMO is awaited, there is interest in the development of alternative complement-targeted therapeutics in NMO as eculizumab is very costly and associated with significant infectious complications including meningococcal meningitis 2/95% air. Recombinant monoclonal NMO antibody rAb-53 (referred to as NMO-IgG) was generated from clonally expanded plasma blasts from cerebrospinal fluid of NMO patients and purified, as described Chinese hamster ovary (CHO) cells stably expressing M23-AQP4 were used, as described Lewis rats were purchased from Charles River Lab . Experiments were done using weight-matched rats (250–350 g). Protocols were approved by Institutional Animal Care and Use Committee (IACUC) at the University of California San Francisco (Project Number: AN89748). All surgery was performed under ketamine/xylazine anesthesia, and analgesics were used post-operatively to minimize pain.M23-AQP4-expressing CHO cells were plated onto 96-well microplates  at 25,000 cells/well and grown for 24 h. Cells were washed with phosphate-buffered saline (PBS) and incubated at room temperature for 60 min with NMO-IgG or purified IgG from NMO patient serum in live cell buffer (PBS containing 6 mM glucose and 1 mM sodium pyruvate) containing pooled normal human complement serum  with or without C1-inh  in a final volume of 50 µl. Some experiments were done with rat serum, prepared from freshly obtained rat blood and then frozen, in place of the human complement serum. In some experiments, a solution containing pooled normal human complement serum was incubated with C1-inh for specified times before addition to the CHO cells. To measure cytotoxicity, cells were washed twice with PBS, and incubated with 50 µl of a 20% Alamar Blue solution (Invitrogen) for 45 min at 27°C. Cytotoxicity was measured from resorufin fluorescence using a TECAN Infinite M1000 plate reader  (excitation/emission 560/590 nm).Adult rats (250–350 g) were injected by tail vein with C1-inh (600 units/kg) in saline in a total volume of 500 µl. Blood was collected through the tail vein at 0.25, 0.5, 1, 2, 4, 6, and 8 h, left for 30 min at room temperature to allow clotting, and centrifuged for 10 min at 3000 g, 4°C. Serum was collected and bioassayed for CDC by addition of 2% rat serum and 1 µg/ml NMO-IgG to M23-AQP4-expressing CHO cells for 60 min. Cytotoxicity was measured by the Alamar Blue assay. In some experiments heparin (1.5 mg/kg) was injected by tail vein with or without C1-inh (600 units/kg) in saline in a total volume of 500 µl.2) of immunonegative area.A rat intracerebral injection was used, as described Data are presented as mean ± S.E. Comparisons between two groups were performed using Student's t-test.To study C1-inh inhibition of NMO-IgG-dependent CDC, cytotoxicity was assayed in CHO cells expressing AQP4 after incubation with NMO-IgG and human complement, as diagrammed in 50 at 10% human complement by ten to extrapolate to undiluted serum), may be required to produce 50% inhibition of CDC with undiluted (100%) human serum. Given the equivalence of 1 unit/mL C1-inh to 2.5 µM, a 2000 unit dose of C1-inh administrated to a human with 5000 mL blood volume could produce a maximum increase of serum C1-inh of 0.4 units/mL (or 1 µM), much less than 70 µM. The inset to Because C1-inh action involves blocking the classical complement pathway by irreversible inactivation of enzymes C1r and C1s in the C1 complex Based on the in vitro data showing weak C1-inh complement inhibition, we attempted to augment and potentiate the complement inhibition action of C1-inh using macromolecules, such as heparin, that inhibit complement directly To investigate the complement inhibition activity of C1-inh in rats, C1-inh was administered intravenously, and serum obtained at different times was assayed for complement activity. Initial studies done using doses used to treat HAE in humans (20 units/kg) did not show inhibition of complement activity in rat serum. We therefore increased the C1-inh dose by 30-fold (to 600 units/kg). However, no significant inhibition of rat serum complement activity was found at any time after C1-inh administration at this high dose . The preGiven the results above showing that heparin and related polysulfated macromolecules can potentiate the inhibitory effect of C1-inh on CDC in vitro, we administered rats high-dose C1-inh and heparin together. However, as shown in We used an established rat model of NMO to test the in vivo efficacy of C1-inh. Though the lack of complement inhibition in rat serum would predict no significant clinical benefit in a rat model of NMO, the complement data do not rule out other actions of C1-inh that might be beneficial in NMO. In our model NMO pathology in brain is produced by a single intracerebral injection of NMO-IgG, which results in characteristic pathological features of NMO including astrocyte damage (loss of AQP4 and GFAP immunoreactivity), inflammation and demyelination by 5 days The complement system is an attractive target for therapy of NMO, as complement-dependent astrocyte cytotoxicity is an early initiating event in the neuroinflammation and demyelination that occurs in NMO. Selective inhibition of early steps in the classical complement pathway is of particular interest because NMO pathogenesis involves NMO-IgG complement effector function in which the antibody Fc region binds C1q to initiate complement activation. We found previously that CDC requires physical clustering of NMO-IgG on astrocytes We found here that C1-inh produced modest inhibition of NMO-IgG-dependent CDC in vitro, and that intravenous administration of large amounts of C1-inh in rats did not inhibit rat complement activity nor did it reduce brain pathology in a rat model of NMO. The studies here were done using rats rather than mice because of the weak activity of the mouse classical complement pathway The recently completed C1-inh safety trial (NCT 01759620) involved intravenous administration of CINRYZ, 2000 units per day for 3 days, in NMO patients undergoing an acute exacerbation Given the weak complement inhibition activity of C1-inh, as a possible strategy to potentiate its activity, we investigated the efficacy of polysulfated macromolecules, including heparin, dextran sulfate, and fucoidan. Assays of NMO-IgG-dependent CDC in vitro showed that each of the three polysulfated macromolecules enhanced the inhibitory effect of C1-inh, with ∼10-fold potentiation produced by heparin and dextran sulfate. However, administration of C1-inh and heparin to rats showed no C1-inh -dependent serum complement inhibition.Though modest C1-inh inhibition of human and rat complement were found in in vitro assays, no significant inhibition of serum complement activity was found in rats in vivo, even after high-dose intravenous C1-inh administration, which translated to lack of efficacy in a rat model of NMO. There are several possible reasons for the lack of C1-inh efficacy on vivo. Whole blood contains many substrates of C1-inh, in addition to C1r and C1s, that are present relatively large amounts, including mannan-binding lectin-associated proteins (MASP-1 and MASP-2), kallikrein, FXIIa and plasmin, which compete for inactivation by C1-inh. C1-inh is a member of a suicidal family of inhibitors, in which a covalent complex is irreversibly formed between C1-inh and its protease target In conclusion, our in vitro and rat data in NMO models suggest that the complement inhibition activity of C1-inh in serum is far too low to produce significant complement inhibition and hence therapeutic benefit in NMO. Notwithstanding the similar activities of rat and human complement, and the similar C1-inh inhibition efficacy, studies done in experimental rat models should be extrapolated to humans with caution. Definitive evaluation of the potential efficacy of C1-inh in human NMO will require controlled clinical trials.


This view seemed consistent with long-standing difficulty in detecting any reaction intermediates at the Qo site. New perspective on this issue is now offered by recent, independent reports on detection of SQ in this site. Each of the described SQs seems to have different spectroscopic properties leaving space for various interpretations and mechanistic considerations. Here, we comparatively reflect on those properties and their consequences on the SQ stabilization, the involvement of SQ in catalytic reactions, including proton transfers, and the reactivity of SQ with oxygen associated with superoxide generation activity of the Qo site.The two-electron ubiquinol oxidation or ubiquinone reduction typically involves semiquinone (SQ) intermediates. Natural engineering of ubiquinone binding sites of bioenergetic enzymes secures that SQ is sufficiently stabilized, so that it does not leave the site to membranous environment before full oxidation/reduction is completed. The ubiquinol oxidation Q The enzyme couples electron transfer between ubiquinone/ubiquinol and cytochrome c with proton translocation1 across the membrane. Typically, the transfer of electrons from ubiquinol to cytochrome c contributes to generation of protonmotive force used for adenosine triphosphate synthesis . Howeveiquinone ,4.o site) directs electrons into two separate cofactor chains. One electron is used to reduce cytochrome c1 via electron transfer through the Rieske cluster (FeS) and haem c1 in one cofactor chain (the c-chain), whereas the other electron is transferred across the membrane to the Qi site via two haems b (haem bL and bH of the b-chain).The translocation of protons across the membrane involves two types of ubiquinone-binding sites facing opposite sides of the membrane: one site oxidizes ubiquinol, whereas the other reduces ubiquinone . The joib and the other cytochrome c, was introduced by Wikström & Berden in 1972 , t, to siteres) still remained in the presence of this inhibitor. SQres shared some of the characteristics of stigmatellin-sensitive signal which was assigned as SQo. Both SQo and SQres signals were broader than the signal of SQi and both showed similar power-saturation profiles. On the other hand, addition of exogenous relaxation enhancer (Ni2+ ions) suggested that the SQres was more exposed to the aqueous phase. For that reason, SQres was assigned to non-enzymatic oxidation of DBH2 in solution. However, as the experiment was performed in the absence of oxygen, this oxidation could not have been associated with O2. Rather, one can envisage that SQres formation might have been a result of a comproportionation. SQres exhibited different proton electron nuclear double resonance (ENDOR) spectrum from the SQ chemically induced in buffer (SQchem). At the same time, SQo signal was reported to have indistinguishable CW EPR spectrum from the chemically produced SQchem. Both SQo and SQres showed decreased amplitudes (greater than 10-fold) in the presence of molecular oxygen. The signals were not seen in the bc1 subcomplex .o, SQres and SQchem) were in the anionic form. This was inferred from the observation that hyperfine coupling constant of five-methyl group to the SQ electron spin in all three cases was different from the values characteristic for protonated/neutral SQs. A contribution of central line in SQo and SQres ENDOR spectra was different from that found in the spectrum of SQchem which was taken as indication that both SQo and SQres are located in the environment of lower proton concentration comparing with the aqueous phase of the SQchem environment. Electron spin echo envelope modulation (ESEEM) spectra showed no indications that SQs form hydrogen bonds with amide group of polypeptide chain nor histidine residues. Importantly, the properties of SQo, including power saturation behaviour, did not reveal signs of dipolar magnetic interactions between SQo and neighbouring paramagnetic cofactors of the Qo site, such as reduced FeS or oxidized haem bL. This, together with the confusing, in our view, properties of SQo versus SQres and problematic initial state of the enzyme raise concern about the origin of the signals.Analysis of proton ENDOR spectra indicated that all three types of SQs (i.e. SQo trapped using a method similar to that described previously [c was added to provide oxidizing power to the c-chain and initiate the reactions in the Qo site (c). While the width of new EPR signal of SQo was similar to that reported previously, the relaxation properties were clearly different. The spin echo of SQo decayed (2p-ESEEM experiment) much faster in comparison with SQchem signal in buffer which indicated that this time, unlike the previous case, the SQo interacted with fast-relaxing paramagnetic species. The authors concluded that the paramagnetic species that affect SQo are haems nearest to SQo which, based on simulations, were proposed to be either two haems bL  or haem bL and haem c1 . However, no spectroscopic data verifying the oxidation state of haems were provided, nor relaxation rates for haems used in simulations, which are crucial parameters in determining distances by the use of relaxation enhancement [In 2013, Kramer and co-workers  describeeviously , except  Qo site c. While ancement ,48. The o site inhibitors was not reported and it was not shown whether this new SQ signal disappears in the control mutants with inactive Qo site. The overall shape of SQo proton ENDOR spectrum was similar to those reported previously for SQo and SQres indicating that SQo was deprotonated. Nevertheless, the splitting of doublet signals flanking the distant protons peak in ENDOR spectra was clearly larger than previously reported [While the new SQ signal was generally sensitive to stigmatellin, around 30% of the signal was still observed in CW EPR spectra in the presence of substoichiometric concentration of this inhibitor. The sensitivity to other Qreported  implyingo was not hydrogen-bonded to the protein. Comparison of bacterial and yeast cytochrome bc1 did not reveal any spectral differences which indicated that SQo in both cases is the same chemical species trapped in similar environment.The analysis of 4p-ESEEM spectra combined with the lack of the signal of nitrogen in 2p-ESEEM indicated that SQo that emerged from ESEEM and ENDOR data led the authors to propose a model of ‘electrostatic cage’ trapping deprotonated SQo. In this model, SQ is destabilized by lack of specific binding through hydrogen bonds or salt bridges. Insulating dielectric cage blocks the proton uptake back to SQo which secures that it does not leave the site. At the same time, the cage is supposed to prevent escape of any superoxide anion (or SQ) formed in the site. However, the destabilized SQo is proposed to conserve sufficient redox energy to reduce haem bL which seems difficult to reconcile with the statement that SQo interacts paramagnetically with the oxidized haem bL. Furthermore, it is important to bear in mind that in photosynthetic reaction centres a similar concept of low dielectric gate around the SQ binding site was introduced to rationalize high stability of SQ, because the contributions from electrostatic energy and hydrogen bonds were not enough to explain SQ stabilization [The properties of SQlization .5.o site [o formed at this site. The first signal at g = 1.94 was assigned as one of the transitions originating from the spin–spin exchange of two unpaired electron spins: one coming from SQo and the other from the reduced Rieske cluster (a). The second transition near g = 2.0 corresponded to the population of SQo for which the spin–spin exchange did not exist or was too weak to be resolved (b). Both populations were observed in samples of isolated, antimycin-inhibited cytochrome bc1 of R. capsulatus exposed to substrates, DBH2 and oxidized cytochrome c, under aerobic conditions. The changes in the amplitudes for these two signals  during the catalytic turnover can be divided into two time regions. In the first (earlier) region, the amplitudes increase until they reach maximum, whereas in the second (later) region, the amplitudes progressively decrease to zero at the time point when the system reaches equilibrium.In 2013, our group reported a discovery of two EPR transitions associated with the activity of the Qo site . Those t cluster a. The seresolved b. Both po-site-specific inhibitors (including myxothiazol and various synthetic strobilurins). Both signals were not observed in specific mutants that disabled activity of the Qo site (such as cytb:G158 W) [bc-subcomplex [o site for prolonged time), the signal amplitude was higher compared with the native protein. More recent experiments indicate that both signals can also be generated under anaerobic conditions and that the characteristic g = 1.94 can be observed in native chromatophore membranes of R. capsulatus [Both signals were sensitive to stigmatellin and several other Q:G158 W) ,50 or thbcomplex . Moreovepsulatus .o reflect two configurations of the Qo site. The spin–spin exchange (g = 1.94) by its nature has a clear distance constraint and can take place only when SQo and Rieske cluster are in proximity, as shown in figure 3a. In this configuration, a formation of a hydrogen bond between histidine residue coordinating Rieske cluster and ubiquinone molecule is possible. At larger distances (figure 3b) or upon breaking the putative hydrogen bond between SQo and histidine ligand, spin–spin exchange disappears and SQo becomes detected as a separate free radical species having a signal near g = 2.0. Nevertheless, in this case, SQo exhibited unusually fast relaxation compared with the relaxation of chemically generated SQ in buffer , which was expected given that the SQo is located in proximity to fast-relaxing paramagnetic metal centres of the Qo site: oxidized haem bL [o manifested itself in significant homogeneous broadening of the EPR lines (both at X and Q band), the inability to saturate it with microwave power, and the presence of a Leigh effect (decrease in amplitude without apparent line broadening upon decrease of temperature). All these specific properties differentiated this SQo from the radical signals described in [We proposed that the two populations of SQ haem bL  and/or r haem bL . The fasribed in –35.o were incorporated to the model of electronic bifurcation of the Qo site. We envisaged that the SQo–FeS (g = 1.94) form might represent an initial step of ubiquinol oxidation when oxidized FeS withdraws an electron from ubiquinol. This state evolves into the state where SQo and reduced FeS exist as separate identities  before reduction of haem bL by SQo takes place to complete the oxidation of QH2 at this site.The two populations of SQ6.2+ (double protonated SQ) is very high [2 to make transfer of the first electron possible. Accordingly, in the ubiquinol oxidation at the Qo site, a release of one or two protons is often considered to be a step initiating the entire reaction [o site, the detected SQo intermediates offer interesting new insights into this issue.The process of uptake and release of protons is an inherent part of redox chemistry of ubiquinones. As the energy of the SQHery high , at leasreaction ,54,55. Wg = 2.0 are believed to be in a deprotonated/anionic form. Thus, it is plausible to expect that these SQs are relevant to a state having the two protons already released . However, the spin–spin exchange state (g = 1.94), which most likely involves the hydrogen bond between histidine ligand of Rieske cluster and ubiquinone molecule, could represent a state before the proton release. For this state, at least two scenarios are possible.The radicals with typical g = 1.94; figure 4b). This model, however, requires that histidine residue is maintained by the enzyme in a deprotonated form before it reacts with ubiquinol, which, as discussed in [The first scenario would accommodate an early model of proton pathway which proposed that initially deprotonated histidine ligand of Rieske undergoes protonation upon formation of hydrogen bond with ubiquinone molecule to subsequently withdraw the first proton from ubiquinone ,56. Thisussed in ,57, is do site can directly accept protons from ubiquinol upon its oxidation [o and FeS cluster (c). Unlike the first scenario, this model allows the hydrogen-bonded configuration for spin–spin exchange to be assembled independently of ubiquinol proton stripping events.The attractive alternative emerges from recent MD simulations which indicate that water molecules in the Qxidation . In this cluster c. Unlike7.Ks for this reaction is often referred to as the stability constant for SQ which depends on the redox potentials of Q/SQ and SQ/QH2 pairs :EQ/SQ and E2SQ/QH couples at pH 7 are −230 and +190 mV in bulk solution [Ks is around 10−8. When concentrations of both Q and QH2 are, for example 100 μM, then equilibrium concentration of SQ− is approximately 30 nM, which is at the lower limit of concentration needed to detect this species by EPR spectroscopy.Quinones in solution under equilibrium undergo comproportionation (reverse of disproportionation) reaction according to the scheme ,58:The eKs has a strict sense when considering comproportionation/disproportionation of Q/SQ/QH2 triad under equilibrium in solution but it is often used to describe the stability of SQ that can be formed within the Qo sites of cytochrome bc1 even though species formed in the catalytic sites are insulated from bulk solution and they are unable to disproportionate directly [o has been considered as highly unstable with the low stability constant Ks of the order of 10−7 or less [o detections which report signals in the range from 1% up to 17% of the total Qo sites. Cape et al. reported that SQo occupies 0.01–0.1 Qo sites per monomer [bc1 and QH2, it is possible to calculate the value of Ks around 10−2. Similar calculations performed by Sarewicz et al. give the estimated Ks of the order of 10−2.6 [Ks that is larger than 10−2) [Ks suggest some kind of stabilization of SQo in comparison with bulk solutions. We emphasize, however, a potential difficulty in describing stability of SQo using the Ks parameter, because all reported SQo signals in cytochrome bc1 were detected under non-equilibrium conditions for which the Ks parameter defining thermodynamic equilibrium may not be valid.The directly . Since o or less ,42,59,60 monomer . Given tf 10−2.6 . These van 10−2) . Such reo intermediates summarized above it appears as if different SQ species for the Qo site have been reported. A feature that unites all these reports is the observation that SQo cannot be detected in cytochrome bc1 unless the Qi site is inhibited by antimycin or haem bH is knocked-out by mutation. This effectively impedes re-oxidation of haem bL through the path involving haem bH/Qi. It thus appears that the state with reduced haem bL is required as condition for increasing probability of trapping SQo. In reversibly operating Qo site, SQo can, in principle, be formed in two ways and both indeed require reduced haem bL as an initiation [bL prevents electron transfer from SQo to haem bL after oxidized FeS initially withdraws one electron from QH2 forming SQo. In a semireverse reaction, reduced haem bL initiates SQo formation by electron transfer to Q. In this context, the properties of SQo in [o was formed along with reduced haem bL pointing towards the semiforward reaction scheme. On the other hand, the properties of SQo in [bL which points towards the semireverse reaction scheme. This mechanism is also supported by the observation that the rate of superoxide generation has a bell-shaped dependence on Q/QH2 ratio [Considering the properties and conditions of trapping  [bL at the time when FeS cluster occupies positions remote from the Qo site and is unable to immediately engage in electron transfer reaction with SQo. This increases the probability of reaction of SQo with oxygen , as indeed implicated experimentally [Interestingly, the semireverse reaction has recently been considered as the one leading to formation of SQ Qo site –65. In tc-chain) . It is tmentally ,65,68.o with oxygen implies that anaerobic conditions should promote trapping SQo. The reports of detection of SQo signals under anaerobic condition follow this expectation [o could not have been observed under aerobic conditions [o in the presence of molecular oxygen, but those experiments were performed using freeze-quenched samples of cytochrome bc1 non-inhibited by antimycin [g = 1.94 and g = 2.0) concerned aerobic conditions [o (spin–spin coupled to the Rieske cluster) detected under these conditions suggest that SQo is not as highly reactive with oxygen as commonly presumed at least in the presence of the spin exchange. In addition, high levels of SQo were observed in +2Ala mutant, which does not produce any detectable superoxide [o and FeS (g = 1.94) might be protective against superoxide generation.The presumed high reactivity of SQectation –35. In onditions . There wntimycin . Howevernditions . The relperoxide , implyin8.Ks of SQo has traditionally been used to explain the long-standing difficulty in experimental detection of SQo. We now seem to face the opposite situation where several seemingly different SQo intermediates have been exposed. The differences concern both the properties of SQo species and the experimental conditions used to trap the SQo intermediates. In our view, this certainly does not make it easier for a general reader to follow the progress in understanding the mechanism of ubiquinol oxidation at the Qo site as it leaves space for various interpretations and mechanistic considerations that at this stage do not seem to converge into one generally accepted model of action. It remains to be seen whether the detected SQo signals represent the same intermediate of the Qo site, or rather reflect different states of the reaction scheme. Are all of them truly associated with the operation of the Qo site? What is the role of haem bL in the formation of SQo and superoxide production? How does the intermonomer electron transfer between the two haems bL influence these reactions? The available set of data on SQs provides now a framework for further studies in which various hypotheses can be critically examined and verified. Hopefully, this will lead to the formulation of the integrated model of the Qo site catalysis and its involvement in superoxide generation.The assumption about extremely low


Linum usitatissium belonging to the Linaceae family. They are appreciated for its antioxidant properties and may be add to different foods.Flaxseeds are the seeds of the plant A 44-year-old man presented with dyspnoea and generalized urticaria after the ingestion of a yogurt with flaxseed. Clinical history revealed an anaphylactic reaction after nuts consumption and an oral allergy syndrome after peach ingestion. He had a history of moderate allergic rhinoconjunctivitis and mild intermittent asthma.Prick-prick carried out with flaxseed showed a strong positivity. Skin prick test to inhaled allergens were positive to mites, dog and cat danders, plain tree and grasses. Skin prick test to food allergens were positive to almond, hazelnut, peanut, chestnut, walnut, mustard, lentils, soybeans, rice, oat, corn and lupine. Total IgE was: 291 UI/ml. Specific IgE  to flaxseed was 2.68 kU peach 7.65 kU/L, peanut 2.01 kU / L , lettuce 0.92 kU / L , lupin 3.76 kU / L. Corn IgE 2.71 kU / L , Pru p3 7.62 kU / L , Cor a8 0.68 kU / L. ISAC, ImmunoCAP was positive to Fel d1, Phl p1, Der f1, Der f2, Der p1, Der p2, Pla a3, Ara h9, Cor a8, Jug r3, Pru p3 and Art v3. ISAC performed after inhibit patients's serum (ISAC inhibition) with a complete flaxseed extract  did not reduced the positivity to non-specific lipid transfer proteins (nsLTP). ISAC inhibition with a complete mite extract  reduces exclusively the signal of mites  in >83%.An immunoblot performed with flaxseed extracts (soluble and liposoluble), showed the presence of an IgE-binding band of about 18 kDa in the liposoluble fraction and a 20 kDa band in the soluble one.invivo and in vitro tests. The ISAC inhibition rejects the nsLTP as the allergen responsible of this hypersensitivity, the allergen of 18 KDa detected in the liposoluble fraction of flaxseed could correspond to an oleosin. Flaxseed allergy has rarely been reported and to the best of our knowledge this is the first report where an oleosin could be the responsible of flaxseed allergy.We present a case of anaphylaxis after eating a yogurt containing flaxseed, in a patient polysensitized to inhaled allergens and nsLTP. IgE-mediated hypersensitivity has been demonstrated by Written informed consent was obtained from the patient for publication of this abstract and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.


RAF kinases are key players in the MAPK signaling pathway and are important targets for personalized cancer therapy. RAF dimerization is part of the physiological activation mechanism, together with phosphorylation, and is known to convey resistance to RAF inhibitors. Herein, molecular dynamics simulations are used to show that phosphorylation of a key N‐terminal acidic (NtA) motif facilitates RAF dimerization by introducing several interprotomer salt bridges between the αC‐helix and charged residues upstream of the NtA motif. Additionally, we show that the R‐spine of RAF interacts with a conserved Trp residue in the vicinity of the NtA motif, connecting the active sites of two protomers and thereby modulating the cooperative interactions in the RAF dimer. Our findings provide a first structure‐based mechanism for the auto‐transactivation of RAF and could be generally applicable to other kinases, opening new pathways for overcoming dimerization‐related drug resistance. RAF kinases connect the Ras GTPase to activation of the MEK‐ERK pathway. This pathway regulates many fundamental cellular functions, including cell proliferation, and is dysregulated in approximately 50 % of human cancers.Based on recently available crystallographic data for RAF . Remarkably, the C‐terminal end of the αC‐helix is neutral in most kinases, whereas in RAFs it carries a +3 charge. Moreover, mutations of these charged residues impair kinase activityOur simulations also reveal that the phosphorylated NtA motif is connected to the active site via the R‐spinePrevious experiments showed that mutation of a conserved Trp residue  impedes transactivation and impairs the dimerization of BRAF.Importantly, our results indicate phosphorylation‐induced large‐scale structural changes in RAF dimers, whereas the unphosphorylated dimers remain structurally similar to their starting structures, in agreement with the crystallographic structures. Only when all phosphorylated residues, including the two activation‐loop residues near V600, are present in the simulated system, we observe large changes between the universally conserved HRD and DFG motifs Figure . These cIn summary, our results show that phosphorylation of the NtA motif promotes dimerization through several interprotomer salt bridges formed between the NtA motif of one of the protomers and the positively charged C‐terminal end of the αC‐helix of the other protomer. They further reveal the importance of the conserved tryptophan residue located at the N‐terminal end of the kinase domain (W450 for BRAF), which plays a crucial role by connecting the R‐spines of the two protomers. This suggests a cooperative interprotomer interaction that is mediated by salt bridges involving the phosphorylated NtA motif. The direct interaction of the R‐spines via W450 explains why mutation of W450 abolishes the transactivation of RAF dimers.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


The kinase Bub1 functions in the spindle assembly checkpoint (SAC) and in chromosome congression, but the role of its catalytic activity remains controversial. Here, we use two novel Bub1 inhibitors, BAY-320 and BAY-524, to demonstrate potent Bub1 kinase inhibition both in vitro and in intact cells. Then, we compared the cellular phenotypes of Bub1 kinase inhibition in HeLa and RPE1 cells with those of protein depletion, indicative of catalytic or scaffolding functions, respectively. Bub1 inhibition affected chromosome association of Shugoshin and the chromosomal passenger complex (CPC), without abolishing global Aurora B function. Consequently, inhibition of Bub1 kinase impaired chromosome arm resolution but exerted only minor effects on mitotic progression or SAC function. Importantly, BAY-320 and BAY-524 treatment sensitized cells to low doses of Paclitaxel, impairing both chromosome segregation and cell proliferation. These findings are relevant to our understanding of Bub1 kinase function and the prospects of targeting Bub1 for therapeutic applications.DOI:http://dx.doi.org/10.7554/eLife.12187.001 The DNA in our cells is packaged into structures called chromosomes. When a cell divides, these chromosomes need to be copied and then correctly separated so that both daughter cells have a full set of genetic information. Errors in separating chromosomes can lead to the death of cells, birth defects or contribute to the development of cancer.Chromosomes are separated by an array of protein fibers called the mitotic spindle. A surveillance mechanism known as the spindle assembly checkpoint prevents the cell from dividing until all the chromosomes have properly attached to the spindle. A protein called Bub1 is a central element of the SAC. However, it was not clear whether Bub1 works primarily as an enzyme or as a scaffolding protein.Baron, von Schubert et al. characterized two new molecules that inhibit Bub1’s enzyme activity and used them to investigate what role the enzyme plays in the spindle assembly checkpoint in human cells. The experiments compared the effects of these inhibitors to the effects of other molecules that block the production of Bub1. Baron, von Schubert et al.’s findings suggest that Bub1 works primarily as a scaffolding protein, but that the enzyme activity is required for optimal performance.Further experiments show that when the molecules that inhibit the Bub1 enzyme are combined with paclitaxel – a widely used therapeutic drug – cancer cells have more difficulties in separating their chromosomes and divide less often. The new inhibitors used by Baron, von Schubert et al. will be useful for future studies of this protein in different situations. Furthermore, these molecules may have the potential to be used as anti-cancer therapies in combination with other drugs.DOI:http://dx.doi.org/10.7554/eLife.12187.002 During eukaryotic cell division, the spindle assembly checkpoint (SAC) contributes to ensure the accuracy of chromosome segregation. This evolutionarily conserved surveillance mechanism monitors the status of kinetochore (KT)-microtubule (MT) interactions and delays anaphase onset until all chromosomes have undergone bipolar attachment to the spindle. Both KT-MT interactions and SAC activity are regulated by several KT-associated protein kinases, including Aurora B, Monopolar spindle 1 (Mps1) and Budding uninhibited by benzimidazoles 1 (Bub1) . SAC actThe serine/threonine kinase Bub1 is one of the first proteins to accumulate at unattached kinetochores . Its recSo far, only few substrates of Bub1 have been identified. Best characterized is the phosphorylation of histone H2A on threonine 120 (T120) . PhosphoSaccharomyces cerevisiae  and HeLa cells, and phospho-histone H2A-T120 staining at kinetochores was monitored by immunofluorescence  and in-cNext, we set out to directly compare the impact of Bub1 kinase inhibition with the previously reported consequences of Bub1 depletion  or genetOne of the most interesting effects of Bub1 depletion described so far relates to sister chromatid cohesion . In partIn addition to preserving sister chromatid cohesion, Sgo1 and Sgo2 play important roles in the recruitment of the CPC, comprising Aurora B kinase . This prTo examine the impact of Bub1 inhibition on the catalytic activity of Aurora B at both centromeres and chromosome arms, we next monitored phosphorylation of CENP-A Ser7  and histWhile inhibition of Bub1 by BAY-320 or BAY-524 or inhibition of Haspin by 5-Iodotubercidin  similarlTo quantify CPC localization over chromosome arms, analysis of fixed cells proved inadequate. We therefore used an RPE1 cell line expressing one endogenous allele of Aurora B tagged with EGFP  to monitConsidering the role of Aurora B kinase in the regulation of KT-MT interactions and SAC signaling, the above results raised the question of what contributions Bub1 activity might possibly make to chromosome congression and/or the SAC. Although our initial analyses had not revealed a major impact of BAY-320 or BAY-524 on the overall timing of mitotic progression , we consDepletion of Bub1 is known to weaken SAC signaling in human cells . To testThe association of Bub1 with unattached KTs is dynamic , raisingAs a further read-out for the effects of Bub1 inhibition on SAC activity, we used live cell imaging to monitor the responses of nocodazole-arrested HeLa and RPE1 cells to BAY-320 or BAY-524 and compared these to the responses seen in Bub1-depleted cells . Over a Importantly, we also compared the requirements for Bub1 activity and Bub1 protein in a cellular background in which SAC activity was partially compromised by the treatment of HeLa or RPE1 cells with a low dose of Reversine, a widely used inhibitor of the SAC kinase Mps1 . In agreTo analyze the impact of Bub1 inhibition on chromosome alignment, we treated cells with the Eg5 inhibitor Monastrol  and thenversus diploid cells . HoweveClarification of the role of Bub1 activity in mitosis has previously been hampered by the absence of specific inhibitors. While genetic or siRNA-mediated depletion experiments generally suffer from poor temporal resolution, small molecule inhibitors offer a unique opportunity for acute kinase inactivation. The only previously described inhibitor of Bub1 is the bulky ATP analog 2OH-BNPP1 , but neiThe major conclusion emerging from the present study is that the overall impact of Bub1 inhibition on mitotic progression is surprisingly mild, clearly less severe than the impact of Bub1 depletion. This reinforces the notion that the requirement for Bub1 during chromosome congression and segregation primarily reflects a scaffolding function . It is dWe show that Bub1 inhibition by BAY-320 or BAY-524 results in loss of Shugoshin and CPC subunits proteins from centromeres/KTs, and that CPC levels at these locations are further reduced upon simultaneous inhibition of Bub1 and Haspin, in line with the established roles of these kinases in the phosphorylation of histones H2A-T120 and H3-T3, respectively . FurtherIt may appear surprising that the persistent chromosome arm cohesion observed in Bub1 inhibited cells did not markedly prolong mitotic timing . HoweverInhibition of Bub1 kinase activity did not significantly reduce the KT recruitment of Mad1, Mad2 and BubR1 and barely affected the ability of nocodazole-treated cells to maintain a SAC arrest. Even when SAC activity was compromised by partial inhibition of the SAC kinase Mps1, Bub1 inhibition triggered only minor weakening of SAC signaling. In striking contrast, Bub1 depletion produced a drastic weakening of the SAC in this sensitized background. For comparison, combined inhibition of Mps1 and either Plk1 or Aurora B resulted in a complete SAC shutdown and immediate mitotic exit, in line with previous results . CollectInhibition of SAC kinases has emerged as a potentially attractive strategy to kill tumor cells . SeveralBAY-320 and BAY-524 were synthesized as described previously . For bioInhibitory activities BAY-320 and BAY-524 towards Bub1 in presence of 2 mM ATP were quantified as previously published . A time-BAY-320 was screenedin vitro, at 10 µM and 10 µM ATP, against a panel of 222 kinases using the Eurofins kinase profiler screen (Millipore). In addition, BAY-320 was screened, at 300 and 1000 nM, in an active site-directed competition-binding assay measuring 403 human kinases .3VO4, 1 mM β-glycerophosphate, 1 mM NaF and complete protease inhibitor (Roche)). Lysates were cleared by centrifugation for 15 min at 21,000 g, 4°C, and LAP-Bub1 proteins isolated by a 2 hr incubation with S-protein-agarose . Beads were washed six times in lysis buffer containing increasing concentrations of NaCl  and three times in kinase buffer . The bead-bound LAP-Bub1 was then aliquoted and used for kinase assays in 30-μl reaction volumes. Kinase reactions were carried out at 30°C in kinase buffer in the presence of 100 μM ATP, 5 μCi γ-32P-ATP, 1 μg recombinant histone H2A  as substrate, and serial dilutions of Bub1 inhibitors. The reactions were stopped after 30 min by the addition of sample buffer and heating to 95°C. The samples were then resolved by SDS-PAGE and visualized by autoradiography and Western blotting.HEK 293T cells were transfected with plasmids coding for LAP-tagged Bub1 wild-type (WT) or the K821R kinase-dead (KD) mutant  . After iHeLa S3 cells, HeLa S3 cells expressing histone H2B-GFP , HeLa KyTransient transfections of HEK293T cells with plasmids and small interfering RNA (siRNA) duplexes were performed using TransIT-LT1 transfection reagent  and Oligofectamine (Invitrogen), respectively, according to manufacturers protocols. The following siRNA duplex oligonucleotides were used: siGl2 CGTACGCGGAATACTTCGA , siBub1 HeLa S3 or RPE1 cells were incubated with kinase inhibitors or depleted of the indicated proteins for 48 hr. Cell suspensions were then fixed with 70% ice-cold ethanol and incubated with 0.2 mg/ml RNase (Sigma-Aldrich) and 5 μg/ml propidium iodide (Sigma-Aldrich). Cellular DNA content was determined by flow cytometry using FACSCanto II  and FlowJo  instruments.g, 4°C, and proteins were resolved by SDS-PAGE and analyzed by Western blotting.Cells extracts were prepared on ice for 30 min in Tris lysis buffer  and phosphatase inhibitor cocktails . Lysates were cleared by centrifugation for 15 min at 21,000 g, 4°C, 10 min) and washed three times with histone lysis buffer. After an additional wash with Tris-EDTA , the nuclear pellet was incubated for 2 hr in 0.4 M HCl at 4°C. After high-speed centrifugation of the sample, 6 volumes of acetone were added to the supernatant, followed by overnight incubation at -20°C. Histones were collected by centrifugation, washed with acetone, air-dried and resolved by SDS-PAGE.HeLa S3 cells were as described above and mitotic cells were collected by shake-off. The cells were the washed with cold PBS and lysed at 4°C for 30 min using histone lysis buffer . Nuclei were collected by centrifugation  or Cy5-conjugated donkey antibodies . For Western blotting, signals were detected using HRP-conjugated anti-mouse or anti-rabbit antibodies .Antibodies used for Western blotting: anti-Bub1  or ab900, anti-IN50 value was then determined by curve fitting (using a four parameter fit).HeLa cells were seeded into 96-well plates for 5 hr at 37°C (ca. 25’000 cells/well). Then, cells were treated with Nocodazole for 16 hr and varying concentrations of test compounds for 1 hr. The cells were fixed, washed and blocked with buffer before incubating with the primary antibody  overnight at 2–8°C. After washing, secondary IRDye-labeled antibody mix with cell stains was added for 1 hr and washed again. Plates were scanned with a LiCor Odyssey Infrared Imager at 800 nm for P-H2A and at 700 nm for Draq5/Sapphire, a cell stain. The signal ratio (800/700 nm) for cells treated only with Nocodazole was set to 100% and the corresponding ratio for untreated cells to 0%. The IC2, 4% formaldehyde) or methanol at -20°C (for CENP-A pS7), respectively. Images of randomly selected cell were acquired as z-stacks using a DeltaVision microscope  on an Olympus IX71 base , equipped with a Plan Apochromat N 60x/NA1.42 oil immersion objective (Olympus) and a CoolSNAP HQ2 camera (Photometrics). Serial optical sections were deconvolved and projected using SoftWorx software . Images were quantified as previously described  in a climate-controlled environment. Images were acquired at multiple positions at indicated time intervals. MetaMorph 7.7 software  was used for acquisition and processing of data. FRET, FRAP, and high sensitivity microscopy (monitoring endogenously EGFP-tagged proteins) experiments were carried out using a spinning disk confocal system (Intelligent Imaging Innovations) based on a Zeiss Axio Observer stand equipped with a Photometric Evolve 512 back-illuminated EMCCD camera, 63x/NA1.4 plan apochromat objective and diode lasers and run by SlideBook software. FRET analyses were carried out by excitation with a 440 nm diode laser and by recording of CFP  and YFP  fluorescence emission in z-stacks. Background-corrected FRET ratios  were calculated in ImageJ using the Ratio Plus plugin. FRAP analysis of EGFP-Bub1 was performed with a 488 diode laser on one KT pair per cell. Overall bleaching was corrected using the signal intensities at a cytoplasmic region not targeted for photobleaching (average of the first 4 frames). Fluorescence recovery half-times and plateaus were determined by non-linear curve fitting based on a one-phase association in Prism software (GraphPad).Asynchronous cell cultures  were plated on 6-well plates . After 7 days of proliferation in the presence of the indicated drugs, the cells were fixed with ice-cold methanol at -20°C and stained with 0.1% Cresyl Violet according to standard procedures. Dried culture plates were scanned and intensities measured using ImageJ after black-and-white-conversion and inversion of the images.50 values were determined by means of a 4-parameter fit after normalization of measurement data to vehicle (DMSO)-treated cells (=100%) and readings taken immediately before compound addition (=0%). IC50 isobolograms were plotted with the actual concentrations of the two compounds on the x- and y-axis, and the combination index (CI) was calculated according to the median-effect model of Chou-Talalay (BAY-320 plus Paclitaxel combination studies were conducted with HeLa and NCI-H1299 cells. Cells were plated into 384-well plates at 600 (HeLa) or 700 (NCI-H1299) cells per well. After 24 hr, cells were treated with BAY-320  and Paclitaxel  for single compound treatments, and in nine different fixed-ratio combinations of BAY-320 (D1) and Paclitaxel (D2) . Cell viability was assessed after 96 hr exposure, using the Cell Titre-Glo Luminescent Cell Viability Assay (Promega). IC-Talalay . A CI ofBUB1) gene were amplified from RPE1 cell genomic DNA. Targeting constructs allowing the insertion of an EGFP tag C-terminal to Bub1 were assembled by 4-piece ligation in a NotI-digested pAAV vector. Recombinant adenovirus-associated virus (rAAV) particles were generated as previously described . The evaluation has been overseen by the Reviewing Editor and Ivan Dikic as the Senior Editor.Thank you for submitting your work entitled "Probing the catalytic functions of Bub1 kinase using the small molecule inhibitors BAY-320 and BAY-524" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:In this study the authors have developed small molecule inhibitors that specifically inhibit the Bub1 kinase. They demonstrate the specificity of their inhibitors in vitro on a panel of kinases, and in vivo using phosphorylation of Histone H2A threonine 120 as a read out. They show that inhibiting Bub1 causes mild effects on the timing of mitosis and on chromosome congression, but causes persistent cohesion of chromosomes arms concomitant with the chromosome passenger complex remaining on the arms. Although the effects of the Bub1 kinase inhibitors alone only mildly affect mitosis, they have a synergistic effect with low doses of taxol.Essential revisions:All three reviewers recognised the quality of the study, and that specific Bub1 kinase inhibitors would be very useful research tools. There were two major experimental concerns, however:1) More evidence for the specificity of the compounds should be presented. At present this mostly consists of a patent and kinase assays on a panel of kinases.2) Are the effects of the compounds in cells specific to inhibiting Bub1? The authors are using a relatively high concentration of the inhibitors , and the toxic concentration is 10 μM. Moreover, there is still some residual H2A-T120 phosphorylation , which sPlease address these concerns by providing further experimental data. For example, what are the phenotypes of cells treated with the inhibitors in which Bub1 has been depleted and rescued with a kinase-dead construct? If the inhibitors are specific to Bub1 there should be no further effect of the inhibitors.In addition to this experimental concern, the reviewers suggest some re-writing is required to clarify what is currently known about the function of Bub1 kinase activity. One reviewer makes the following suggestions:“I would suggest that much of the general introductory material in paragraphs 1 and 2 be condensed. Also, the last paragraph that summarises the results can be replaced by a single sentence, e.g. ‘To address the role of Bub1 kinase activity we characterised two novel inhibitors BAY-320 and BAY-524’. Condensing this text will then allow the authors to provide more detail with regards to what is known about Bub1. For example, in the Results they describe Sgo1, that H2A is a substrate, the link with Haspin etc.; this should all be in the Introduction. Also, what is the prior art regarding Bub1's kinase activity? They say it is controversial but I don't think that is sufficient. Indeed, there is literature that supports the authors’ claims and this should be included. By way of an example, Perera at al deleted Bub1 in MEFs and added back kinase-dead mutants showing that a catalytic mutant or a mutant lacking the entire kinase domain restored BubR1 localisation, SAC function and chromosome alignment but failed to restore Sgo1 localisation. Klebig et al. described similar findings using an RNAi-complementation assay. Similarly, McGuinness et al. showed that in meiosis I, a Bub1 mutant lacking the entire kinase domain restored SAC function. There are other primary papers that should be cited here, e.g. describing work in various model systems. NB, as alluded to above, the authors could highlight the technical challenges with complementation assays as a means of justifying the use of small molecule inhibitors.” Essential revisions:All three reviewers recognised the quality of the study, and that specific Bub1 kinase inhibitors would be very useful research tools. There were two major experimental concerns, however:1) More evidence for the specificity of the compounds should be presented. At present this mostly consists of a patent and kinase assays on a panel of kinases.In the original manuscript we had reported the results of inhibition assays on a panel of 222 protein kinases. To further document the specificity and selectivity of the Bub1 inhibitors we have now performed two additional experiments:Firstly, using BAY-320 we have conducted an inhibitor-binding assay (DiscoveRx assay) on a vast panel of human kinases, including mitotic kinases. As we show in the new 50 value of 379 +/- 156 nM for BAY-320 (at cellular levels of ATP), in reasonable agreement with the IC50 determined by in vitro phosphorylation at 2mM ATP (680 nM).Secondly, we have generated dose-response curves using an “in-cell western assay” detecting phospho-Thr 120 signals in mitotically arrested HeLa cells see new . This asAs with all small molecules, definitive proof of selectivity in a cellular context is nearly impossible to obtain. However, we emphasize that both BAY-320 and BAY-524 have gone through extensive validation in the course of drug development at Bayer; this, combined with the absence of any unpredicted phenotypic effects, makes us highly confident of the selectivity of these compounds.2) Are the effects of the compounds in cells specific to inhibiting Bub1? The authors are using a relatively high concentration of the inhibitors , and the toxic concentration is 10 μM. Moreover, there is still some residual H2A-T120 phosphorylation , which sPlease address these concerns by providing further experimental data. For example, what are the phenotypes of cells treated with the inhibitors in which Bub1 has been depleted and rescued with a kinase-dead construct? If the inhibitors are specific to Bub1 there should be no further effect of the inhibitors.As we stated above (see response to concern No. 1), dose-response curves obtained from “in-cell western” assays show maximal reduction of Thr120 phosphorylation between 3.0 and 10.0 μM, a concentration range used in key experiments throughout this study. These new results are in full agreement with the data shown in To further document the specificity of the phenotypes produced by BAY-B320 and BAY-B524 treatment, we have included additional data to complement the results shown in From a more general perspective, we emphasize that the two Bub1 inhibitors did not trigger any phenotypes that could not also be observed in response to Bub1 depletion, arguing strongly against off-target effects. Thus, we are confident that our observations specifically reflect Bub1 inhibition.We have also carefully considered the suggested rescue experiment, but in the end decided against attempting it. Although such an experiment appears plausible at first glance, it would be technically very difficult and, in our opinion, not necessarily conclusive. Any additional effect produced by Bub1 inhibition in a cell depleted of wild-type Bub1  could in fact be attributed to inhibition of residual, depletion-resistant wild-type Bub1.In addition to this experimental concern, the reviewers suggest some re-writing is required to clarify what is currently known about the function of Bub1 kinase activity. One reviewer makes the following suggestions:“I would suggest that much of the general introductory material in paragraphs 1 and 2 be condensed. Also, the last paragraph that summarises the results can be replaced by a single sentence, e.g. ‘To address the role of Bub1 kinase activity we characterised two novel inhibitors BAY-320 and BAY-524’. Condensing this text will then allow the authors to provide more detail with regards to what is known about Bub1. For example, in the Results they describe Sgo1, that H2A is a substrate, the link with Haspin etc.; this should all be in the Introduction. Also, what is the prior art regarding Bub1's kinase activity? They say it is controversial but I don't think that is sufficient. Indeed, there is literature that supports the authors’ claims and this should be included. By way of an example, Perera at al deleted Bub1 in MEFs and added back kinase-dead mutants showing that a catalytic mutant or a mutant lacking the entire kinase domain restored BubR1 localisation, SAC function and chromosome alignment but failed to restore Sgo1 localisation. Klebig et al. described similar findings using an RNAi-complementation assay. Similarly, McGuinness et al. showed that in meiosis I, a Bub1 mutant lacking the entire kinase domain restored SAC function. There are other primary papers that should be cited here, e.g. describing work in various model systems.”We have re-written major parts of the Introduction and Results sections. In particular, we have shortened the opening paragraphs of the Introduction, moved introductory material from the former Results section, and discussed prior studies on the role of Bub1 activity in more detail. We are grateful for these referee suggestions and feel that the text of our revised manuscript has gained in clarity.“NB, as alluded to above, the authors could highlight the technical challenges with complementation assays as a means of justifying the use of small molecule inhibitors.”To address this comment, we have added a sentence on technical challenges of genetic studies and advantages of small molecule inhibitors (Discussion).


Urosaurus ornatus lizards that exhibit a discrete dewlap colour polymorphism that reflects alternative mating tactics. We found blue morphs specialize on prey at higher trophic levels, yellow males display plasticity in trophic and morphological attributes and orange males are trophic generalists. Our results also demonstrate that morph diet differences are enhanced in resource-limited habitats. We conclude that discrete behavioural morphs may also diverge in morphology and trophic niche. Jointly, these processes may enhance speciation rates in colour polymorphic taxa.Discrete colour morphs associated with alternative mating tactics are assumed to be ecologically equivalent. Yet suites of behaviours linked with reproduction can also favour habitat segregation and exploitation of different prey among morphs. By contrast, trophic polymorphisms are usually attributed to morphs exhibiting habitat or prey selectivity. An alternative hypothesis is that habitat variation generates a trophic polymorphism driven by differences in morph reproductive behaviour, the spatial dispersion of morphs in a landscape and their exposure to different prey types. In this scenario, morphs are allowed to vary in habitat or diet selectivity  as they do in behaviour, rather than being assumed to exhibit equivalent levels of ecological specialization. We test this hypothesis using male  Colour Apart from behavioural differences, morphs are assumed to have similar ecological roles. This assumption is counterintuitive, because behavioural dissimilarities among morphs have been linked to differences in their spatial dispersion within a landscape, the size of their territories or home ranges and the quality of those used habitats –11. For Trophic polymorphisms have been documented in a diverse array of taxa ,14. Alth3) versus grasses (C4). The distinction between C3 and C4 vegetation types is based in part on both metabolic differences including the nature of the first stable compound during carbon fixation , as well as clear differences in isotopic signature [3-based) versus grassy (C4-based) energetic pathways. Although C3-based pathways may be preferred over C4-based pathways owing to their higher protein and nitrogen content [3 vegetation, morphs should exhibit reduced spatial overlap given the abundance of their preferred prey in those habitats. Moreover, all morphs should also be capable of exploiting C3-based trophic pathways, regardless of any differences in their prey selectivity or social behaviour. By contrast, C4-dominated habitats would favour both greater spatial overlap and trophic dissimilarity among morphs. Because morphs differ in mating and territorial behaviour, we also predict that only aggressive morphs should monopolize preferred C3 dietary resources when they are limiting [Alternatively, morph differences in behaviour may coincide with differences in both habitat and prey selectivity among them, but the actual observed degree of trophic differentiation among morphs may depend on resource availability. Under this combination of the first two mechanisms, variation in habitat characteristics  and prey diversity (number of distinct prey types), morph behaviour and the spatial dispersion of morphs interact to shape a trophic polymorphism. Moreover, this hypothesis also allows morphs to differ in their foraging and microhabitat selection tactics  as they do in behaviour, rather than being assumed to all exhibit the same (or similar) level of habitat or prey specialization. Here, we envision a scenario for a polymorphic lizard in which colour morphs occupy habitats that vary in structural elements such as the relative dominance of woody vegetation , to differ predictably in carbon isotopic composition [3 versus C4 vegetation) and consumer trophic level (nitrogen), which have been assimilated over timescales varying from weeks to years [The ability to describe a trophic polymorphism as well as understand the mechanisms underlying differences in trophic niche occupancy among morphs requires data on their resource-use patterns across contrasting habitats. That is, do morphs select a unique suite of prey types across habitats independent of changes in prey availability and/or habitat quality between habitats? By contrast, do morphs consume prey types in proportion to their availability at a given site? These patterns in trophic niche variation are not captured by estimating diet properties in a single site or by addressing either habitat or prey selectivity alone. In this study, we adopt an integrative approach that capitalizes on the use of stable isotope methods to test whether male colour morphs of the tree lizard,  ornatus , exhibitposition , and varposition . Stable to years . As a reU. ornatus populations in a semi-arid grassland region of southeastern Arizona. We selected two sites that contrast in habitat structure and C3 and C4 base-resource availability owing to prescribed burning. Prior to the 1920s, periodic fires ignited by lightning strikes maintained the semi-arid grassland environments. However, prolonged fire suppression resulted in an invasion of the grassland ecosystem by trees and shrubs. Recent management practices reinstated burns to mimic historical processes and reduce recruitment of tree and shrubby vegetation (C3) in favour of grass (C4) propagation [U. ornatus morphs, as well as in the diversity of prey types supported at each site [We sampled pagation . As a repagation . This vaach site .U. ornatus [U. ornatus colour morphs that exhibit alternative mating tactics also diverge in trophic niche occupancy.Finally, we also consider whether morphological variation among the colour morphs plays a role in their diet selection. In particular, colour morphs in other lizard species differ in head shape ,27, whic ornatus , variati2.U. ornatus) at our site are arboreal and establish home ranges in trees within semi-arid grasslands, including oaks (Quercus sp.) and mesquite (Prosopis sp.). Male U. ornatus from many populations are characterized by a discrete polymorphism in dewlap coloration, which is associated with alternative mating tactics [U. ornatus colour morphs occur in syntopy within these sites.Tree lizards , and or tactics ,31. We q2.1.U. ornatus were collected from within the LB (n = 22) and HB (n = 18) sites via noose-pole, and all capture points were georeferenced using a portable GPS unit. Lizard surveys were conducted daily between 08.00 and 18.00 h to ensure sampling accounted for the daily activity period of lizards at the study sites. We evaluated whether male morphs selected microhabitat characteristics that differed from the available habitat by sampling within the home range of each lizard (=resource use) and also at non-capture points (=resource availability) established along a grid within each site. These capture points are representative of the resources used by U. ornatus, because at this point the territories and spacing patterns among males have stabilized at our sites [U. ornatus: they accounted for the full range of available microhabitat types at each site . At each point (use or available), we sampled within 100 m2 (approx. 5.6 m radius) plots , because the mean territory of U. ornatus is less than this size [Male ur sites . Each grhis size ,33.2 frames were placed randomly within each 100 m2 sampling plot. Within each frame, we estimated the per cent cover of vegetation  and structural features , woody debris and leaf litter). All cover variables were estimated to the nearest 5%. The averages of the coverage data based on the four frames from each plot were retained for analysis. We also recorded distance to the nearest tree from the centre of the plot (nearest 0.1 m). We collected vegetation samples  within each plot for isotopic analysis and stored these samples in separate plastic bags within a freezer bag.Four 1 mca 09.00–11.00 h) and 2 h during the afternoon (ca 13.00–15.00 h) for each sweep-net day. This method captured representative arthropods from every major group identified by Asplund [We determined the available arthropod diversity at each site by systematically sweep netting each study site for 4 h per day over a 2 day period at each site . We netted for approximately 2 h during the morning . We pla2.2.For each male lizard captured, we recorded the colour pattern of the dewlap, mass, as well as snout–vent length (SVL), head width (HW) and jaw length (JL). Mass was measured to the nearest 0.1 g using a spring scale, and SVL, HW and JL were measured to the nearest 0.1 mm using callipers. We toe-clipped all males to prevent recapture . The claws from these toes were retained for stable isotope analysis prior to release at their capture sites.2.3.Patterns in trophic polymorphisms are often based on point estimates derived from prey items identified by stomach flushing or behavioural observations. Such estimates represent a short-term characterization of prey consumption by an organism over a period of hours. We suggest that trophic variation characterized using stable isotope methods represents a more inclusive estimate of individual diet ,37.http://stableisotopefacility.ucdavis.edu) for analysis of δ13C and δ15N values using a PDZ Europa ANCA-GSL elemental analyser interfaced to a PDZ Europa 20–20 isotope ratio mass spectrometer . The error deviation for δ13C was 0.2‰ and 0.3‰ for δ15N. All isotopic values generated by this analysis are expressed in standard per-mil delta (δ) notation,X = δ13C or δ15N and R = 13C/12C or 15N/14N, respectively. Our experimental design allowed calculation of δ15N and δ13C values from n ≥ 3 replicate samples of each vegetation type and arthropod consumer type per site.All tissue samples  were stored in a freezer at −20°C until the end of the study. We then oven-dried samples at 60°C for 48 h, and then ground and homogenized the dried samples separately. We weighed and transferred all dried samples into separate 5 × 8 mm tin capsules . We pooled some arthropod samples by family, because of their small mass, to generate enough tissue for isotopic processing . These samples were then sent to the University of California Davis Stable Isotope Facility  between the sites using chi-square statistics.We applied Sorenson's D coefficient to presence–absence data of arthropod families to determine the dissimilarity between sites in arthropod composition . The valp > 0.4; spatial segregation, interaction p > 0.7).We used a multinomial test to compare morph frequencies between the sites. We assessed head shape variation among the male morphs at each site using a multivariate analysis of covariance (MANCOVA) with morph and site as fixed factors, SVL as a covariate, and HW and JL as response variables. We analysed the magnitude of the spatial segregation among morphs at each site using separate MANCOVAs with morph as a fixed factor, distance to the nearest tree as a covariate, and normalized latitude and longitude of each lizard capture point as response variables. Here, normalization refers to centring of the coordinate data to a mean of zero and unit standard deviation. We included tree distance as a covariate, because the distribution of this species may be influenced by tree density. Prior to running these MANCOVAs, we ensured homogeneity of all regression slopes  into three types: grass, forb or shrub (including trees). We used a MANOVA to test for differences in the stable isotope values of vegetation with site and vegetation type as factors and δ13C and δ15N values as the response variables. Following Gratton & Denno [3 herbivores , C4 herbivores (most grasshoppers), non-spider predators , and spiders. We tested for differences in the carbon and nitrogen stable isotope values of each of these consumer types between the two sites using a MANOVA with site and consumer type as factors and δ13C and δ15N values as the response variables.We noted that shrubs and trees exhibited high overlap in isotopic delta-space, but significant differences occurred between these types and grasses and forbs. We divided the individual plants sampled at each site  as lizard parameters in our mixing models. We used published discrimination factors for the four arthropod consumer types in these models . We tested for differences in the trophic niche of each morph using a MANCOVA containing site and morph as fixed factors, SVL as a covariate, and δ13C and δ15N values as response variables. We detected no significant interaction between either site or morph and SVL in this model (both p > 0.7). We used correlation tests to explore relationships between head shape and diet (isotopic nitrogen data). Finally, we only report Bonferroni-adjusted p-values (padj) for significant post hoc pairwise comparisons.Prior to analysing our lizard diet isotopic data, we estimated the relationship between lizard body condition  and ondition ,48. We d3.3.1.F1,62 = 10.27, p = 0.001; electronic supplementary material, table S2). The LB and HB sites were characterized by variation in structural features of the habitat  and vegetation cover . The HB site had higher grass and rock cover, more leaf litter cover, but also had less bare ground cover and fewer forbs than the LB site . The structural habitat and vegetation cover at male capture points differed from resource availability points . We detected an interaction between site and plot type . Specifically, the difference in habitat characteristics between lizard capture localities and availability points was greater at the HB site . Male microhabitats were characterized by higher tree density, greater amounts of leaf litter, fewer rocks and greater forb cover than resource availability points . Male morphs used microhabitats with similar characteristics at both sites .The LB and HB sites differed in habitat structure and vegetation cover . We captured a higher diversity of non-spider predators and spiders at the LB compared with the HB site .We collected a higher diversity of arthropods at the family level at the LB site  differed from C3 . The δ13C and δ15N values of vegetation differed by site and type only . The vegetation at the LB site is characterized by a depletion in δ13C and δ15N values in relation to the HB site . The differences in isotopic values between the sites is supported by the higher proportional cover of C3 vegetation at the LB site  compared with greater C4 vegetation cover at the HB site .Overall, isotopic carbon signatures of Cδ13C and δ15N values of arthropods differed by consumer type but not by site , with blue males less common at the HB site . Male lizards were larger in body size at the HB site . Head shape differed by morph but not by site . Blue morph males had longer jaws than either yellow or orange males , and yellow males had longer jaws than orange males . Moreover, the patterns of head shape variation among the morphs also differed by site . The jaws of yellow males at the HB site were longer than those of yellow males at the LB site (padj = 0.003), but similar in size to those of blue males at the HB site (padj = 0.28). There was no effect of body size on head shape . Finally, we detected only spatial segregation among male morphs at the LB site .Morph frequencies differed between the sites , but primarily exploited forbs at the HB site (4 herbivores were more consistent in resource use across sites (more than 54% reliance on grasses at both sites). Non-spider predators in the HB site preferred C3 herbivores over C4 herbivores (69% versus 31%), which contrasts with their preference in the LB for C4 herbivores overall (74%). Spiders maintained consistent diets across sites and favoured C4 herbivores and non-spider predators over C3 herbivores (approx. 15% reliance on C3 herbivores at both sites).Overall, C HB site . C4 herbThe trophic niche of blue and orange tree lizards at the LB site was characterized by a higher consumption of spiders . There was no effect of body size on diet . The lack of interaction between site and morph is because of the high similarity of prey δ13C values between both sites (i.e. padj < 0.001), but yellow males in the HB site foraged at a higher trophic level than yellow males in the LB site (padj = 0.001). In terms of head shape, JL positively correlated with diet (isotopic nitrogen data) in our overall sample . Likewise, the shift in diet by yellow males at the HB site corresponded with their variation in head shape .The diet of lizards captured at the HB site is enriched in both tes i.e. . Yellow 4.U. ornatus colour morphs may feed at different trophic levels and exhibit greater spatial overlap in resource-limited habitats [Our results revealed a trophic niche polymorphism in male tree lizards that coincided with the colour morphs. The patterns emerging from our analysis support the hypothesis that habitat variation contributes to the relative spatial segregation of morphs and the spatial variation in diversity of available prey that shape trophic divergence. Variation in diet among morphs has long been considered to play a key role in the speciation process , especiahabitats . Our curhabitats , and thePodarcis melisellensis [Podarcis muralis), morph-specific differences in diet based on analysis of faecal pellets are attributed to potential spatial segregation of morphs within a shared habitat [Trophic polymorphisms are expected to evolve via one of two avenues. Trophic divergence may evolve in sympatry when morphs vary in their ability to exploit different prey types . In Podaellensis , dietaryellensis . Alternaellensis ,50. In I habitat . HoweverP. melisellensis morphs , would ems, see . This meems, see ,29,52–54ems, see  and Karpems, see  to explaems, see  are needThe extent of the trophic polymorphism depends on variation in habitat quality. For example, consistent dietary differences among morphs in sympatry across different habitats should only be expected to arise if they are an outcome of similar mechanisms operating within each morph, such as selectivity for specific prey types, regardless of shifts in prey availability among habitats. However, if morphs differ in their mechanisms of prey selection, as they do in mating behaviour, then habitat variation should influence their trophic niche differences. In high-quality habitats, dietary overlap among morphs may arise, because prey and microhabitat resources are abundant. By contrast, poorer-quality habitats (e.g. burned areas) with limited resources would instead favour ecological segregation of morphs based on their differences in aggressive behaviour, potentially enhancing their trophic niche differences. These considerations emphasize the need for trophic niche data across contrasting habitats in order to draw conclusions regarding the nature of a potential trophic polymorphism.The patterns of diet and morphological divergence among the morphs at our study sites coincide with their behavioural traits. Among males inhabiting the LB site, blue and orange morphs have a higher percentage of predatory arthropods in their diet, whereas yellow males consume all arthropod types at a similar frequency. Greater tree and shrub cover, and higher predatory arthropod diversity, is common at sites that are burned infrequently ,55. GreaU. ornatus are also contingent on behavioural asymmetries among the male morphs, as our mixing model results support that morphs also differ in their degree of diet specialization. Dominant blue males usurp high-quality patches and exhibit the greatest dietary specialization at both sites. Orange males are nomadic and may encounter a broader range of prey types as they move through the habitat. Our isotopic data for orange males suggest they are trophic generalists as their diet tracks changes in prey availability between the sites. By contrast, yellow males exhibit a plastic dietary niche that involves a shift in diet between sites that is unrelated to changes in prey availability. In addition, their differences in resource use were also linked to head shape morphology: blue males had longer heads than the other morphs, and yellow males in the HB site had longer heads than yellow males in the LB site. The shift in diet by yellow males is probably associated with their satellite behavioural tactic , coupled with the decrease in frequency of dominant blue males at the HB site . In addsee also . In anotat study . Studiesat study ,27. Thusat study , morph-s5.U. ornatus are coincident with their colour polymorphism and associated with increased morphological diversity (in terms of head shape). Collectively, these findings support a role for trophic polymorphism in enhancing rates of ecological divergence in colour polymorphic taxa [Studies of the factors maintaining colour polymorphism within a species have generally focused on the fitness consequences of individual variation in mating behaviour, with scant attention towards the potential ecological consequences of differences in morph behaviour. By contrast, trophic polymorphisms are attributed to divergent natural selection associated with either microhabitat or prey selectivity alone. The results of our study suggest an alternative mechanism for the generation of a trophic polymorphism: diet differences among morphs may be a consequence of both socially mediated sexual selection and natural selection mechanisms. The differences in diet and foraging tactics we document in hic taxa ,49.