PGE2 has a wide variety of effects within the airway, including modulation of inflammation, sensory nerve activation and airway tone. However, the effect of PGE2 on airway MVL and the receptor/s that mediate this have not been described.Airway microvascular leak (MVL) involves the extravasation of proteins from post‐capillary venules into surrounding tissue. MVL is a cardinal sign of inflammation and an important feature of airway inflammatory diseases such as asthma. PGEEvans Blue dye was used as a marker of airway MVL, and selective EP receptor agonists and antagonists were used alongside EP receptor‐deficient mice to define the receptor subtype involved.2 induced significant airway MVL in mice and guinea pigs. A significant reduction in PGE2‐induced MVL was demonstrated in Ptger2−/− and Ptger4−/− mice and in wild‐type mice pretreated simultaneously with EP2 (PF‐04418948) and EP4 (ER‐819762) receptor antagonists. In a model of allergic asthma, an increase in airway levels of PGE2 was associated with a rise in MVL; this change was absent in Ptger2−/− and Ptger4−/−mice.PGE2 is a key mediator produced by the lung and has widespread effects according to the EP receptor activated. Airway MVL represents a response to injury and under ‘disease’ conditions is a prominent feature of airway inflammation. The data presented highlight a key role for EP2 and EP4 receptors in MVL induced by PGE2.PGE BAFLbronchiolar lavage fluidCOPDchronic obstructive pulmonary diseaseIPAintrapulmonary airwaysMVLmicrovascular leakageOVAovalbuminet al., et al., et al., et al., et al., et al., et al., et al., et al., Airway microvascular leakage (MVL) and plasma exudation represent classical features in the pathogenesis of various respiratory diseases, including asthma and chronic obstructive pulmonary disease (COPD)  and plasma of asthma patients . Homozygous breeding pairs of mice genetically modified to disrupt one of the following genes: Ptger1 (EP1), Ptger2 (EP2) and Ptger3 (EP3)  mice do not survive on the C57BL/6 background because of patent ductus arteriosus  Act of 1986 and the ARRIVE guidelines  and male Dunkin Hartley guinea pigs (250–350 g) were purchased from Harlan . Mice breathe spontaneously under urethane and so were therefore not artificially ventilated. Depth of anaesthesia was assessed using the pedal and corneal reflex. Once surgical anaesthesia was reached, the jugular veins were then exposed following a midline incision over the thorax. The administration of substances i.v. was achieved by passing the injection needle through the pectoralis major as this helped prevent bleeding on withdrawal. Animals received Evans Blue dye (20 mg·kg−1) followed by the specific mediator or its vehicle 1 min later (4 mL·kg−1). The doses and timings used were those shown to be effective in previous studies within the group and from the literature , the thoracic cavity was opened and a small incision was made in the left ventricle and also the left atrium of the heart. A cannula was then inserted into the left ventricle and the systemic circulation perfused with sterile saline (0.9%) at a pressure of approximately 100 mmHg. This was continued until the perfusate ran clear (2–5 min). The purpose of this was to remove any intravascular dye. The heart, lungs and oesophagus were removed en bloc and then the trachea and lungs separated from the heart and oesophagus. The oesophagus and bladder were taken for the initial studies and used as non‐airway, reference tissues. The trachea was isolated by cutting just above the bifurcation of the bronchi and the larynx removed. The parenchyma was then carefully scraped off using a scalpel to reveal the intrapulmonary airways (IPA). The trachea, the bronchi and IPA, the oesophagus and the bladder were then all weighed, and the wet tissue weight was recorded. Each tissue was then incubated in 120 μL of formamide at 37.5°C for at least 18 h to facilitate the extraction of Evans Blue dye. The concentration of Evans Blue extracted from each tissue was determined by light absorbance at 620 nm using a spectrophotomer; 100 μL of formamide was removed from each Eppendorf and pipetted into a 96‐well plate alongside a standard curve of Evans Blue in formamide . The concentration was then calculated by interpolation from the standard curve and expressed as ng·mg−1 of tissue. End points were assessed by a different operator than the experimental part of the study.Apart for the data generated in the various mouse knockout line, animals were randomised as regards the treatment groups. Animals were anaesthetized with i.p. urethane  and 5‐HT (10 mg·kg−1 at 4 mL·kg−1) as a positive control or vehicle . Thirty minutes after administration, Evans Blue extravasation was measured. A non‐selective COX inhibitor, diclofenac (30 mg·kg−1 in 10 mL·kg−1)  or PGE2 (3 mg·kg−1) in 50 μL. Once surgical anaesthesia was reached, mice were then given Evans Blue and killed 30 min later.A dose–response curve to PGE2‐induced airway MVL as they more closely resemble humans in terms of their respiratory anatomy (lobing and branching) and physiology (mediator release). Male Dunkin–Hartley guinea pigs (300–600 g) were anaesthetized with urethane (2 g·kg−1 i.p. of a 25% solution). Once surgical anaesthesia was reached, animals received Evans Blue (20 mg·mL−1 at 1 mL·kg−1 i.v.) and then 1 min later vehicle , PGE2 (3 mg·kg−1) or 5‐HT (1 mg·kg−1), all i.v. at 1 mL kg−1; 5 min after 5‐HT or 30 min after PGE2, the extravasation of Evans Blue was measured.Guinea pigs were chosen as a second (larger) species to assess PGE2 (3 mg·kg−1) or vehicle  used. Additionally, the response to 5‐HT in EP receptor knockouts (ptger2−/− and ptger4−/−) and their wild types was investigated to ensure that their lack of response to PGE2 was not due to an overall disruption in MVL. A submaximal dose of 5‐HT (1 mg·kg−1) was used as a known inducer of MVL.The same MVL protocol was used as before with a single submaximal dose of PGEet al., et al., et al., 1), ONO‐AE1‐259 (EP2), ONO‐AE‐248 (EP3), ONO‐AE1‐329 (EP4) or PGE2 (3 mg·kg−1) i.v. at 4 mL·kg−1 or vehicle .To parallel the effects seen in the EP receptor‐deficient mice, the response to selective EP agonists was studied. Following Evans Blue administration, animals were given selective EP receptor agonists (Okada 2 agonist) was performed to establish a suitable dose to use. The doses used were 0.1, 0.3, 1 and 3 mg·kg−1 (i.v. at 4 mL·kg−1). The EP2 antagonist, PF‐04418948, was used in subsequent studies and was dosed at 3, 10, 30 or 100 mg·kg−1 (10 mL·kg−1 i.p.). A vehicle control was also included . Mice were dosed with PF‐04418948 . Next, a dose–response curve to ONO‐AE1‐329 (EP4 agonist) was carried out using 0.1, 0.3, 1 and 3 mg·kg−1 (i.v. at 4 mL·kg−1). The EP4 antagonist, ER‐819762 . A vehicle control was also included . Mice were dosed with ER‐819762 or vehicle 1 h before ONO‐AE1‐329 administration (3 mg·kg−1 in 4 mL·kg−1 i.v.).Selective receptor antagonists were used to confirm data generated with selective agonists. Initially, a dose response to ONO‐AE1‐259 (EP−1 at 10 mL·kg−1 i.p.) or ER‐819762 (30 mg·kg−1 at 10 mL·kg−1 i.p.) or vehicle  1 h before PGE2 administration (3 mg·kg−1 at 4 mL·kg−1 i.v.). Additionally, in a separate study, mice were dosed with both PF‐04418948 and ER‐819762 simultaneously 1 h before PGE2.Mice were dosed with PF‐04418948 ) to the sample at a ratio of 2.5 to 1 mL, vortexing and then adding 2.5 mL of hexane. After vortexing and centrifugation, lipids were recovered in the upper hexane layer. The samples were then re‐extracted by addition of an equal volume of hexane. The combined hexane layers were dried and analysed for free or esterified PGs using LC‐MS/MS. Lipids were separated on a C18 Spherisorb ODS2, 5 μm, 150 × 4.6 mm column  using a gradient of 50–90% B over 10 min  with a flow rate of 1 mL·min−1. Products were quantified by LC‐MS/MS electrospray ionization on a Sciex 4000 Q‐Trap using parent‐to‐daughter transitions of m/z 351.2 [M − H]− to m/z 271 for PGE2 and m/z 355.2 to 275.3 for PGE2‐d4 with declustering potential of −55 and collision energy of −26 V. Products were identified and quantified using standards run in parallel under the same conditions.Mice were sensitized with ovalbumin (OVA)  on days 0 and 14 and on days 24, 25 and 26 mice were intranasally challenged with saline or OVA (50 μg in 50 μL) under isoflurane anaesthesia. PGEptger2−/− and ptger4−/−) mice.Based on these data, MVL was measured at 2 and 24 h after the final intranasal challenge. In a subsequent study, allergy‐induced MVL was compared in wild‐type and EP receptor knockout . The EP4 antagonist, ER‐819762, was a gift from Eisai . The EP1 receptor agonist (ONO‐D1‐004), the EP2 receptor agonist (ONO‐AE1‐259), the EP3 receptor agonist (ONO‐AE‐248) and the EP4 receptor agonist (ONO‐AE1‐329) were gifts from Ono Pharmaceuticals . PGE2 was purchased from Cayman Europe . Both antagonists were made up in 0.5% methylcellulose, 0.2% Tween 80 in saline (or water for p.o. administration). PGE2 and all agonists were made up in 1% ethanol in saline. All other chemicals and reagents were from Sigma Aldrich .The EPpost‐hoc comparisons were performed by Dunn's multiple comparison test, comparing selected columns to a control. Additionally, an unpaired t‐test and/or non‐parametric Mann–Whitney test, with two‐tailed P values, was carried out, where appropriate, to determine statistical significance between two groups. Differences were considered statistically significant if P < 0.05. The figures have been graphically presented on a range‐specific axis. The data and statistical analysis comply with British Journal of Pharmacology guidelines  induced dose‐dependent MVL in both the trachea and the bronchi and IPA , respectively]. Diclofenac, a nonselective COX inhibitor, was administered prior to PGE2 administration to block the production and release of endogenous prostanoids to ensure that they were not influencing baseline or PGE2‐induced airway leak. The data indicate that diclofenac had no effect on baseline or PGE2‐induced leak in the trachea or bronchi and IPA  was administered and EB leak measured after 30 min. Topical PGE2 induced a similar MVL response to that seen with i.v administration  induced significant MVL in the trachea, bronchi and IPAs was comparable with 5‐HT  on MVL was compared in wild type and mice deficient in individual EP receptors (Ptger1–4−/−). The data demonstrated a significant increase in MVL in wild‐type, Ptger1−/− and Ptger3−/− mice in response to PGE2  and ONO‐AE1‐329 (EP4) both significantly increased MVL in both the trachea and bronchi and IPA of mice, while ONO‐D1‐004 (EP1) and ONO‐AE‐248 (EP3) had little or no effect on MVL in either trachea or bronchi and IPA  produced a dose‐dependent increase in MVL. Similar to previous studies 3 mg·kg−1 induced a significant increase in MVL in the trachea (data not shown). This dose was therefore used in subsequent antagonist studies. The EP2 receptor antagonist, PF‐04418948 (i.p.), evoked a dose‐dependent inhibition with a dose of 30 mg·kg−1 showing a significant inhibition of EP2‐induced MVL  produced a dose‐dependent increase in MVL with a significant increase in airway MVL at 3 mg·kg−1 (data not shown). The EP4 receptor antagonist, ER‐819762 (i.p.), was able to significantly inhibit MVL induced by ONO‐AE1‐329  and ER‐819762 (10 mg·kg−1 i.p.) were selected for further study.The EPMVL Fig. A, B. ONO2‐induced MVL in mouse airways was investigated. Initially, each antagonist was dosed separately; however, as demonstrated in Fig. 2‐induced airway MVL. When both antagonists were given simultaneously, there was a significant reduction in PGE2‐induced MVL in the trachea and in the bronchi and IPA  responsible. This is an important step in understanding the biological profile of EP receptors given that PGE2 is a key mediator produced by the lung in health and in patients with lung disease.Airway microvascular leak is a cardinal sign of inflammation and is a prominent feature of both asthma and COPD. Various inflammatory mediators are capable of inducing this effect following an inflammatory insult to the airways. These inflammatory mediators act upon the endothelium of post‐capillary venules to open intercellular gap junctions. PGE2 in the airways of mice. PGE2 caused a dose‐dependent increase in MVL in the trachea and bronchi and IPA of mice, and in addition, blocking the production of endogenous prostanoids with the COX inhibitor diclofenac did not impact on PGE2‐induced MVL. We also observed a measurable and significant increase in MVL following an intranasal dose of PGE2, indicating that the effects we are seeing are not specific to the route of administration. Furthermore, we did not detect any MVL in non‐airway, reference tissues, oesophagus and bladder, suggesting that in our model system, i.v. administration of PGE2 mainly impacts on the airways. As the majority of research into airway MVL has been carried out in larger species, including the rat and the guinea pig, we wanted to rule out that what was being observed with PGE2 was not exclusive to the mouse. In the guinea pig, PGE2 produced a large, substantial increase in airway MVL that was comparable to that seen with 5‐HT. Both the mouse and the guinea pig produced similar profiles in the magnitude of their responses, demonstrating that the effect of PGE2 is translatable between species.Initial experiments were focused on investigating the effect of exogenous PGE2 induced MVL in the mouse, we wanted to determine which of the EP receptors were central to this response. Differing effects of PGE2 have been demonstrated in vivo, and these, often opposing, effects can be attributed to the activation of different receptor subtypes (EP1–4) with different signalling characteristics , ONO‐AE1‐259 (EP2), ONO‐AE‐248 (EP3) and ONO‐AE1‐329 (EP4), and the data clearly demonstrated that, similar to PGE2, the EP2 and EP4 agonists significantly increase airway MVL. This confirmed that activation of these EP receptors mediated MVL in mouse airways, while activation of EP1 and EP3 receptors produced no significant increase. Interestingly, others, using an in vitro system with human microvascular endothelial cells, have reported that PGE2 can strengthen endothelial barrier function via the EP4 receptor (Birukova et al., Konyaet al., in vivo model or the permeability characteristics of an intact microvascular endothelium.Having shown that PGE2 and EP4 receptor antagonists that are potent and selective for their respective receptor subtype have emerged and can be used to confirm data generated in developmental knockout mouse strains. Previously, AH6809 had been used by most investigators to examine EP2 receptor events, but this also antagonizes EP1 and DP1 receptors (Jones et al., et al., et al. (2 receptor antagonist, PF‐04418948, which was further characterised by our laboratory (Birrell et al., 2 agonist, ONO‐AE1‐259. In 2010, Chen et al. described a novel EP4 receptor antagonist, ER‐819762, that was highly selective for EP4 receptors in a number of different in vitro assays as well as being effective in both mouse and rat models of arthritis and chronic pain. Similarly, ER‐819762 produced a dose‐dependent inhibition of EP4‐induced MVL. However, initial experiments demonstrated that neither antagonist alone was able to inhibit PGE2‐mediated airway MVL. Interestingly, when both antagonists were administered simultaneously, at the same doses and time points, a decrease in PGE2‐induced MVL was evident, with a significant inhibition seen in the trachea and in the bronchi and IPA. This emphasized the possibility for a dual role for EP2 and EP4 receptors in mediating PGE2‐induced MVL responses. Little research has been published on the role of prostanoid receptors of the EP subtype in airway MVL, and so, the data presented herein demonstrate an EP receptor activity profile, which will aid in further understanding its role in airway inflammation.In recent years, novel EP2 and EP4 receptor signalling have been implicated in acute skin inflammation by enhancing blood flow in the micro‐environment (Kabashima et al., 2‐induced MVL may be caused by vasodilatation and an increase in blood flow to the airways. There is considerable evidence indicating that EP2 and EP4 receptors are pivotal in mediating the vasodepressor actions in mouse and human vascular preparations (Kennedy et al., et al., et al., et al., et al., There is, however, some information available from other organ systems implicating various EP receptors in MVL by evoking vasodilatation and increasing blood flow, an effect that is particularly evident in the skin. In this regard, both EPet al., et al., et al., et al., 2 induced airway MVL via the EP2 and EP4 receptors, its role in a murine respiratory model of airway inflammation was then investigated. An increase in PGE2 was measureable in lavage fluid 2 and 6 h but not 24 h after OVA challenge. Others have reported increases in PGE2 levels in their asthma models (Herrerias et al., et al., 2 levels in that we could detect an increase at 2 h post‐challenge but not 24 h later. Furthermore, as with the exogenous PGE2 experiments, it appears that allergen‐induced MVL is via the activity of PGE2 on EP2 and EP4 receptors.It has been well‐documented that microvascular leak and oedema are prominent features of allergic airway diseases, such as asthma, with high concentrations of plasma proteins being present in airway secretions from acute asthmatic patients (Meerschaert 2 and EP4 agonists caused almost identical increases in airway MVL and, in the knockout mice, responses to PGE2 were almost completely abolished in both EP2 and EP4 receptor knockouts. Moreover, the selective receptor antagonists required simultaneous dosing to block the stimulatory effects of PGE2. This suggests that EP2 and EP4 may be functionally interdependent. A similar effect has been reported in human adipocytes and breast cancer cells with both receptors playing a role in PGE2‐induced induction of aromatase and inhibition of either receptor attenuating the effects of PGE2 (Subbaramaiah et al., 2 and EP4 receptors form heterodimers, which has been illustrated in Fig. 2 or EP4 receptors, PGE2 is unable to induce MVL. Furthermore, when EP2 and EP4 antagonists were administered alone, when both functional receptors are present, PGE2 still induced significant MVL. Only when both receptors were antagonized simultaneously was PGE2‐induced leak attenuated.An interesting observation from the data presented here was that both EPet al., et al., et al. (et al. (1 receptors and β2 adrenoceptors. However, more investigations are obviously needed to establish whether this is occurring between the EP2 and EP4 receptors in this in vivo model.There is increasing evidence that GPCRs are able to heterodimerize and that this interaction can affect the function of both receptors (McGraw 2 is a key mediator produced by the lung and has widespread effects according to the EP receptor that is activated. Airway MVL represents a response to injury and under ‘disease’ conditions is a prominent feature of airway inflammation. The data presented here highlight a key role for PGE2 and the EP2 and EP4 receptors. These findings are novel and make an exciting addition to the expanding repertoire of effects mediated by EP receptor activation.PGEM. A. B., M. G. B., S. A. M., M. G. and V. O. D. devised the experiments; V. C. J., M. A. B. and M. G. B. wrote the manuscript; V. C. J., S. A. M., S. R. C. and M. G. performed the experiments.The authors declare no conflicts of interest.


In adult muscles the αγ‐ is replaced by the αε‐site. Quantal EPSCs (qEPSCs) were elicited in adult muscles by depolarization pulses and recorded through a perfused macropatch electrode. One to 200 nmol L−1 CTx reduced amplitudes and decay time constants of qEPSCs, but increased their rise times. CTx block at the αδ binding sites was incomplete: The qEPSCs still contained long bursts from not yet blocked receptors, whereas their average decay time constants were reduced by a short burst component generated by ACh binding to the αε‐site. Two nanomolar CTx applied for 3 h reduced the amplitudes of qEPSCs to less than half with a constant slope. The equilibrium concentration of the block is below 1 nmol L−1 and lower than that of embryonic receptors. CTx‐block increased in proportion to CTx concentrations (average rate 2 × 104 s−1·mol−1 L). Thus, the reactions of ‘embryonic’ and of adult nicotinic receptors to block by CTx are qualitatively the same. – The study of the effects of higher CTx concentrations or of longer periods of application of CTx was limited by presynaptic effects of CTx. Even low CTx concentrations severely reduced the release of quanta by activating presynaptic M2 receptors at a maximal rate of 6 × 105 s−1·mol−1 L. When this dominant inhibition was prevented by blocking the M2 receptors with methoctramine, activation of M1 receptors was unmasked and facilitated release.In ‘embryonic’ nicotinic receptors, low CTx concentrations are known to block only the  αδ binding site of adult nicotinic receptors, very small and short quantal synaptic currents (qEPSCs) are generated by binding of ACh quanta at the αε‐site, This is very similar to the effects of CTx at embryonic receptors where the short qEPSCs are generated by binding at the αγ site. CTx also activates presynaptic muscarinic M1 and M2 receptors.When CTx blocks the  The two binding sites for ACh are between the α and the γ or ε subunits and between the α and the δ subunits (Karlin Vertebrate muscles are activated by the release of quanta of acetylcholine (ACh) from the motor nerve terminal. ACh molecules bind to nicotinic receptors in the muscle membrane that intermittently open channels, and current flow depolarizes the membrane. The postsynaptic nicotinic ACh receptors are pentamers of closely related subunits that are arranged around a central pore which can open to pass channel currents γαβδα in s Karlin .αγ‐ and the αδ‐sites of patch‐clamped embryonic receptors. Binding of agonist to the αδ site generated single openings of on average 6 μs duration. Binding to the αγ‐site elicited single 62 μs openings, but also short, on average 0.75 ms bursts of openings. Simultaneous binding to both sites elicited the familiar long bursts of channel openings.While the generation of long bursts of channel currents seemed to be understood, it has been possible only recently to relate several types of openings to the binding of agonists at one of the single binding sites of the embryonic nicotinic receptors. Stock et al.  have comα‐conotoxin M1 (for short CTx), block neuromuscular transmission. CTx has been shown to bind primarily to the αδ interface of the nicotinic receptor : NaCl, 108; KCl, 3.5; CaCl2, 1.5; MgCl2, 0.7; NaHCO3, 26; NaH2PO4, 1.7; Na‐gluconate, 10; glucose, 5.5; saccharose, 7.6 (pH adjusted to 7.4 adding NaOH), tetrodotoxin (TTX), 0.2 μmol L−1. It was held at 20°C by driving it through a thermostated heat exchanger. The study employed 51 mice and recorded successfully from 35 preparations. It was approved ethically by the respective authorities.Experiments were performed on about 20‐day‐old balb C mice that were reared in the animal department of the Institute. They were killed by cervical dislocation and decapitation. The diaphragms were excised rapidly, and hemidiaphragms were pinned down in a narrow Perspex chamber, lighted through a glass bottom and were superfused rapidly. The superfusate was saturated with 95% Oμm internal tip diameter. The tip was perfused with a solution containing (mmol L−1): NaCl, 162; KCl, 5.3; CaCl2, 2; MgCl2, 1; NaH2PO4, 0.67; Hepes, 15: glucose, 5.6. pH was adjusted with NaOH to 7.4 and 0.2 μmol L−1 TTX was added to prevent excitations. The fluid at the tip was exchanged several 100 times per second.Excitatory postsynaptic currents (EPSCs) were recorded by means of a macropatch pipette with a 10 α‐conotoxin M1 (CTx) and the blockers of muscarinic receptors M1 and M2, pirenzepine and methoctramine, respectively.Both the perfusate of the electrode and the superfusate of the bath could separately be switched to drug containing solutions; we used −1. The tip of the pipette was lowered to touch the surface of the muscle causing a slight indentation. Negative pressure of about 0.01 bar at the tip of the pipette improved the seal toward the solution outside. While applying depolarizing pulses, the tip was moved slowly over the surface of the muscle until signals from a superficial endplate were picked up. The position was optimized to obtain maximal amplitudes and minimal rise times of quantal EPSCs (qEPSCs). For further details of the recording conditions see Dudel , a rise time of 0.36 ± 0.07 ms and a time constant of decay of 2.35 ± 0.26 ms. The qEPSCs in Fig. −1 CTx had been applied for 85 min, the amplitude of qEPSCs was on average maximally depressed to 0.41 ± 0.084 nA (N = 284), and its rise time increased to 0.46 ± 0.11 ms. The samples in Fig. αδ‐site of the receptors obviously was quite incomplete: most qEPSCs in Fig. In the experiment of Fig. The high‐resolution recordings in Fig. −1 CTx was applied for 10 min, and the amplitude of quantal postsynaptic currents (qEPSCs) was reduced from 1.4 nA to 0.24 nA. As in all such CTx applications, the effect outlasted the application considerably, here by 12 min. This delay is not entirely due to slow outwash of CTx: during the recordings presented in Fig. A typical time course of the effects of low CTx concentrations is shown in Fig. After the peak depression of the amplitudes of the qEPSCs  dropped from 0.47 to 0.24 quanta/pulse at minute 11, when the qEPSC amplitude still measured 1039 nA. Thus with the employed CTx concentrations and their relatively short periods of application postsynaptic inhibition does not cause the observed inhibitions of quantal release.It might be argued that the described presynaptic inhibition by CTx is really a postsynaptic one, a reduction in some qEPSC amplitudes to a level that could not be resolved. In the experiment of Figs A well‐known depression of release by the agonist ACh (autoinhibition) is mediated by binding to muscarinic M2 autoreceptors . The initial facilitation in Fig. The experiment of Fig. −1 CTx was added to the perfusate, and release about doubled again within 10 min, presumably due to the activation of M1 receptors by CTx. This conclusion is supported by the experiment in Fig. At 30 min, 50 nmol Lμmol L−1 methoctramine reduced the amplitudes of the postsynaptic currents from 1.6 nA to 1.2 nA, and simultaneously shortened the time constants of decay of the qEPSCs from 1.74 ms to 1.0 ms . The block of the αδ binding sites of the nicotinic receptors certainly never was complete. The decay time constant of qEPSCs corresponds to the burst duration  tubocurarine ‐tubocurarine has a similar rate of binding to the open channel and shortens qEPSCs accordingly, but the rate of unbinding is only 0.8 s−1 (Bufler et al. Could CTx also shorten the decay of qEPSCs by open channel block? In the classical open channel block of nicotinic receptors, high ACh concentrations blocked the open channel with very short intermitted closings that prolonged the duration of bursts (Neher and Steinbach 6 s−1·mol L−1. At 2 × 10−7 mol L−1, the highest concentration of CTx used here, the binding rate to the open channel would be 0.2 s−1, that is, block after 5 sec – there is no open channel block by CTx.For a possible open channel block by CTx, one has to consider the rate of binding to the open channel and the CTx concentration. The rate of binding remains unknown, but it should be lower than the rates for procaine or tubocurarine. CTx is a much larger and more rigid molecule than the latter, and one may doubt whether CTx would fit into the channel at all. Let us assume a rate of binding of CTx to the open channel of 10× 10−4 mol L−1, binding to the open channel would occur after 5 ms.Even with the highest CTx concentration used in Stock et al. , 2 × 10−CTx is a very potent poison at pre and postsynaptic receptors in the nervous system. Possibly it does not only affect the motor synapses of vertebrates, but also other synaptic systems with nicotinic and/or muscarinic receptors.The group of substances that appear in this study, ACh, CTx, methoctramine, muscarine, nicotine, procaine, and tubocurarine, all bind to some ACh receptor. It is not surprising that their specificity for binding to a certain receptor type is limited. For instance, in Fig. We thank Drs. Manfred Heckmann and Stephan Hallermann for comments on the manuscript, and Mrs. Hongbo Jia, Mrs. Christine Karrer and Mrs. Frauke Köhler for technical assistance.None declared.


An expansion of polyglutamine (polyQ) sequence in ataxin‐3 protein causes spinocerebellar ataxia type 3, an inherited neurodegenerative disorder. The crystal structure of the polyQ‐containing carboxy‐terminal fragment of human ataxin‐3 was solved at 2.2‐Å resolution. The Atxn3 carboxy‐terminal fragment including 14 glutamine residues adopts both random coil and α‐helical conformations in the crystal structure. The polyQ sequence in α‐helical structure is stabilized by intrahelical hydrogen bonds mediated by glutamine side chains. The intrahelical hydrogen‐bond interactions between glutamine side chains along the axis of the polyQ α‐helix stabilize the secondary structure. Analysis of this structure furthers our understanding of the polyQ‐structural characteristics that likely underlie the pathogenesis of polyQ‐expansion disorders. HttHuntingtinJDJosephin domainMBPmaltose‐binding proteinMJD/SCA3Machado‐Joseph Disease/Spinocerebellar Ataxia Type 3polyQpolyglutamineUIMubiquitin‐interacting motifATXN3 gene 2AGA, 1YZB, 2DOS) 2KLZ) Machado–Joseph Disease, or Spinocerebellar Ataxia Type 3 (MJD/SCA3), is an inherited autosomal‐dominant disease of the brain that is genetically associated with CAG triplet expansion to over 55 repeats in the In this study we focused on structural analysis of the carboxy‐terminus of Atxn3 using X‐ray crystallography techniques. We determined the crystal structure of the Atxn3 C‐terminal region containing a polyQ repeat region with 13 glutamines interrupted by a single lysine. Both random coil and α‐helix conformations were observed in the protein crystals that diffracted at 2.2 Å resolution. Our analysis revealed an intrahelical hydrogen‐bonding pattern connecting glutamine side chains. These findings are consistent with results from our previous crystallographic studies of the Huntingtin (Htt) polyQ region NotI and PstI sites of the pMAL vector encoding the maltose‐binding protein (MBP) with a modified carboxy‐terminal tri‐alanine linker Escherichia coli BL21(DE3) strain. A single colony carrying pMAL‐Atxn3‐C plasmid was inoculated into 10 mL of LB medium supplemented with ampicillin overnight at 37 °C with shaking. The culture was transferred into 1 L of LB containing ampicillin, and cells were cultivated until absorbance at 600 nm was 0.8. Protein synthesis was induced by the addition of IPTG  for 16 h at 16 °C with vigorous shaking. Then cells were harvested by centrifugation  and were resuspended in 50 mL of lysis buffer . Cells were disrupted by sonication . Lysate was centrifuged  and the supernatant was applied onto 3 mL of amylose resin (NEB) pre‐equilibrated with equilibration buffer . After washing, protein was eluted with elution buffer . MBP‐Atxn3‐C was concentrated and applied onto the gel‐filtration column Superdex75 16/60  pre‐equilibrated with gel‐filtration buffer  at 1 mL·min−1 flow rate. Fractions containing MBP‐Atxn3‐C fusion were pooled and concentrated to approximately 10 mg·mL−1 using Amicon centrifugal filters . The resulting protein was at least 95% pure as shown by Coomassie staining of a SDS/PAGE. Purified MBP‐Atxn3‐C was crystallized using the hanging drop vapor diffusion technique without removing either MBP or the C‐terminal tag. Single crystals were obtained in the following crystallization conditions: 25% polyethylene glycol monomethyl ether (5K), 1.0 m sodium acetate, 0.1 m imidazole, pH 8.0, 0.1 m zinc acetate  and 24% polyethylene glycol monomethyl ether (5K), 0.9 m sodium acetate, 0.06 m imidazole pH 8.0, 0.1 m zinc acetate . No additional cryoprotection steps were undertaken due to the high polyethylene glycol concentration in the crystallization drop. Crystals were flash‐cooled in liquid nitrogen, and the complete diffraction datasets for crystals C1 and C2, which had different shapes, were collected at the 19ID beamline of Advanced Photon Source Synchrotron. These raw diffraction data were indexed and scaled using hkl‐2000 software A fragment of human Atxn3 (amino acids 278–329) containing a 14‐amino acid tract with 13 glutamines interrupted by a single lysine was amplified by polymerase chain reaction and cloned into phaser software 1ANF) were used for Atxn3‐C structure model. Structure determination was performed separately for each crystal. Electron density in the region corresponding to MBP was well defined, whereas the initial map of the Atxn3‐C region was of poor quality. The complete maps were obtained for MBP region, and partial maps were obtained for the Atxn3‐C region. The map derived from the C1 crystal dataset allowed only limited determination of Atxn3‐C structure due to intrinsic disorder of entire Atxn3‐C region. Maps derived from C2 crystals were of better quality and enabled more extensive model building. In the process of the model building and refinement R(work), R(free) improved and the electron density of the Atxn3‐C region became clearer. The Cα backbone was built first, and side chains in the Atxn3‐C region were assigned in the later stages of refinement. Crystallographic parameters for C1 and C2 crystals and final refinement statistics are listed in Table coot software refmac5 program molprobity software pisa software phenix, hbond server and secondary structure was assigned with DSSP algorithm Phasing was performed by the molecular replacement method in To crystallize the carboxy‐terminal fragment of Atxn3, we adopted the crystallization approach used successfully in our previous crystallographic studies of the polyQ region of Htt Htt‐Ex1)  1A) to fStructures of MBP‐Atxn3‐C in C1 and C2 crystals were solved by molecular replacement with the MBP structure as a search model  and   or between i and i + 3 (one case) Table ; Fig. 3.In the two molecules composing the asymmetric units of the C2 crystal, the glutamine side chains adopt slightly different conformations. The major difference is that in one the Q393···Q397 bond is present and in the other it is not. We calculated electron density maps with side chains omitted for both units and calculated a real‐space correlation coefficient between the map and the model Fig. B and B‐f3IOR, 3IOT, 3IOU, 3IOV, 3IOW, 3IO4, 3IO6 and 4FE8, 4FEB, 4FEC, 4FED) In our previous structural studies of Htt, we observed both α‐helix and a random coil conformation of the polyQ region . Structures of the apo‐forms of other anti‐polyQ antibodies also suggest that they recognize a similar extended polyQ conformation  In our analysis of Atxn3‐C, one cannot ignore the influence of MBP and crystallization effect. The stabilizing effect of MBP on the α‐helix may be due to both solvation effects and molecular interactions. In the well‐defined region from Q385 to Q394 in the C2 crystal MBP has a caging effect. MBP limits solvent access and favors intramolecular interactions between glutamines. Existing structural and biochemical data support the idea that polyQ itself is capable of forming an α‐helix. For example, a similar helical structure was observed in the polyQ region of Htt‐Ex 1 without stabilizing interactions with other regions of the crystallized protein Intramolecular stabilization of a polyQ α‐helix was previously proposed et al. the region of the Atxn3 sequence from L281 to K291 was predicted to be a part of an α‐helix in agreement with our crystallographic data. In comparison to solution study on the synthetic polyQ peptides and native/pathogenic Htt exon I, the studies found the polyQ in solution exists in an equilibrium between random coil and alpha‐helix (estimated helix content 40–75%) The mechanism of polyQ aggregation is not apparent from our findings because the current research was focused on the polyQ tract of nonpathogenic length. It has been proposed that the polyQ α‐helix is involved in helix‐helix interactions that lead to pathogenic fibril‐aggregation in neurons In the MBP‐Atxn3‐C crystal structure, glutamine residues form multiple coordinated hydrogen bonds along the polyQ helix and limited interactions with MBP. Interestingly, Perutz initially proposed a model of polyQ known as a ‘polar zipper’ model Here, we observed that the polyQ regions of Atxn3 adopt random coil and α‐helical conformations in two separate crystal structures. The presence of both random coil and α‐helical conformations in the polyQ region is consistent with the result from our previous crystallographic studies of Htt‐Ex1 MWK designed the experiments. MWK performed the crystallization and data collection experiments. VZ and MWK processed and analyzed the data. VZ, MWK, AK, IB wrote the final manuscript. All authors read and approved the final manuscript.


Zebrafish is increasingly used as an animal model to study the effects of environmental nuclear receptors (NRs) ligands. As most of these compounds have only been tested on human NRs, it is necessary to measure their effects on zebrafish NRs. Estrogen receptors (ER) α and β and peroxysome proliferator activated receptor (PPAR) γ are main targets of environmental disrupting compounds (EDCs). In humans there are two distinct nuclear ERs (hERα and hERβ), whereas the zebrafish genome encodes three ERs, zfERα, zfERβ1, and zfERβ2. Only one isoform of PPARγ is expressed in both humans and zebrafish. In this review, we described reporter cell lines that we established to study the interaction of EDCs with human and zebrafish ERs and PPARγ. Using these cell lines, we observed that zfERs are thermo-sensitive while zfPPARγ is not. We also showed significant differences in the ability of environmental and synthetic ligands to modulate activation of zfERs and zfPPARγ in comparison to hERs and hPPARγ. Some environmental estrogens  which are hER panagonists displayed greater potency for zfERα as compared to zfERβs. hERβ selective agonists  also displayed zfERα selectivity. Among hERα selective synthetic agonists, 16α-LE2 was the most zfERα selective compound. Almost all zfPPARγ environmental ligands  displayed similar affinity for human and zebrafish PPARγ while pharmaceutical hPPARγ agonists like thiazolidones are not recognized by zfPPARγ. Altogether, our studies show that all hERs and hPPARγ ligands do not control in a similar manner the transcriptional activity of zfERs and zfPPARγ and point out that care has to be taken in transposing the results obtained using the zebrafish as a model for human physiopathology. Human nuclear hormone receptors (NHRs) are a family of 48 transcription factors, many of which have been shown to be activated by ligands. NHRs regulate cognate gene networks involved in key physiological functions such as cell growth and differentiation, development, homeostasis, or metabolism  destabilization of corepressor or chaperone interfaces, (2) exposure of nuclear localization signals to allow nuclear translocation and DNA binding of cytoplasmic receptors, and (3) recruitment of coactivators triggering gene transcription through chromatin remodeling and activation of the general transcription machinery. The crystal structures of many NHR LBDs have been determined, revealing a conserved core of 12 α-helices (H1–H12) and a short two-stranded antiparallel β-sheet (S1 and S2) arranged into a three-layered sandwich fold. This arrangement generates a mostly hydrophobic cavity in the lower half of the domain, which can accommodate the cognate ligand. In all hormone-bound LBD structures, the ligand-binding pocket (LBP) is sealed by helix H12. This conformation is specifically induced by the binding of hormones or synthetic agonists and is referred to as the “active conformation” because it allows the dissociation of corepressors and favors the recruitment of transcriptional coactivators  causing reproductive, developmental, metabolic, or neurological diseases as well as hormone-related cancers . Both hERs are widely distributed throughout the body, displaying distinct but overlapping expression patterns in a variety of tissues  and ERβ   are increasingly being used as model species to study in vivo effects of EDCs estrogens for zebrafish and human ER isoforms, establishing the fact that a direct translation of (anti)estrogenic effects (activities or potencies) from mammals to zebrafish is not possible. Although none of the tested compounds specifically activated either zebrafish or human ERs, transcriptional activities toward human and zebrafish ERs need to be studied.Natural (E2) and pharmaceutical (EE2) estrogens display similar affinities for hERs and zfERs. Some environmental estrogens  with similar affinity for hERs preferentially activated zfERα rather than zfERβ s. Other environmental estrogens  with similar affinity for hERs displayed slightly higher affinity for zfERα and zfERβ2 than for zfERβ1. Benzophenone 2 and phytoestrogens  which have higher affinity for hERβ than for hERα also displayed slightly higher affinity for zfERα and zfERβ2 than for zfERβ1. Finally, hERβ selective synthetic compounds  preferentially activated zfERα compared to zfERβs. On the contrary and similar to hERs, the synthetic compound 16α-E2, which has 1000-fold more selectivity for hERα   and pharmaceuticals 8bv-E2, DPN) to activate preferentially the zfERβ isoforms is explained by the mutation of a critical amino acid involved in genistein binding in hERβ. In all zfERs, the position homologous to hERβ M336 is occupied, as in hERα, by a leucine residue (Figure , DPN to in vitro and in vivo through induction of the differentiation of preadipocytes of the fibroblastic lineage into mature adipocytes (Grun and Blumberg, PPARs are involved in the regulation of glucose, lipid, and cholesterol metabolism in response to fatty acids and their derivatives, eicosanoids, and drugs used in the treatment of hyperlipidemia and diabetes. The human PPAR subfamily contains three members known as hPPARα, hPPARβ, and hPPARγ. Each hPPAR subtype shows a distinct tissue distribution and ligand preference. hPPARγ is highly expressed in adipose tissue and is a central regulator of lipid storage and adipocyte gene expression and differentiation (Tontonoz et al., in vivo effects of EDCs on PPARγ (Lyche et al., Like for ERs, zebrafish begin to be used as model species to study HG5LN-hPPARγ and -zfPPARγ reporter cell lines were established in a similar way (Seimandi et al., s and PPARγ depends on the halogenation degree of BPA analogs. The bulkier are brominated BPA analogs, the greater is their capability to activate PPARγ and the weaker is their estrogenic potential (Riu et al., Screening of environmental and pharmaceutical ligands in the HG5LN-zfPPARγ cell lines showed that known hPPARγ ligands are not always able to induce transcriptional activity of zebrafish PPARγ (Riu et al., Comparison of human and zebrafish PPARγ sequences reveals several residue differences which could explain the differential ligand specificity of the various species Figure . In partStructural and biophysical studies revealed that TBT binds to both hRXR and hPPARγ through formation of a covalent bond between the tin atom and the sulfur atom of cysteine residues located in the LBP of both receptors (le Maire et al., We have shown above that there are clear differences between the activity of various EDCs for zebrafish and human ERs and PPARs, demonstrating that a direct translation of effects from mammals to zebrafish is not possible. The differences revealed in this study, in terms of transcriptional activities toward human and zebrafish ERs and PPARs, highlight the need to take into account the species of origin when assessing the potency of chemicals. This is particularly important with regard to EDCs screening for hazard assessment since at the present time established test guidelines are only based on human cell lines expressing human nuclear receptors.in vitro cell lines expressing zebrafish nuclear receptors can serve as useful screening tools to address nuclear receptor potency of chemicals for fish models. Hence, an initial screening should be followed up with an NR-subtype specific analysis using both human and zebrafish NRs to elucidate the full spectrum of NR-mediated EDCs effects.To this end, such The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


These results reveal the ability of the alpha particle to partially repair the damage generated by the heavy recoil nuclei highlighting the radiation resistance of nuclear glass and the difficulty to accurately simulate its behaviour by single ion beam irradiations.Safe management of high level nuclear waste is a worldwide significant issue for which vitrification has been selected by many countries. There exists a crucial need for improving our understanding of the ageing of the glass under irradiation. While external irradiation by ions provides a rapid simulation of damage induced by alpha decays, short lived actinide doping is more representative of the reality. Here, we report radiological NMR experiments to compare the damage in International Simplified Glass (ISG) when irradiated by these two methods. In the 0.1 mole percent  Calorimetric study of 244Cm doped glass18α/g and 0 to 1.4 × 1018α/g, respectively.The actinide doping approach, despite being more realistic, limits the number of structural characterization techniques that can be deployed, simply due to the radioactivity level of the sample. Among spectroscopic techniques, solid state NMR is certainly the one that has seen many major achievements in the last few decades and is nowadays considered as a cutting-edge method for the elucidation of glass structure2111B MAS NMR spectra, presented in 4, at 0 ppm and three coordinated boron, BO3, at 10 ppm4 and BO3 units can be applied to the spectra of ISG-Cm glasses to extract the BO3 and BO4 populations, using two components for each speciation . MQMAS spectroscopy did not revealed the formation of highly coordinated species in ISG-Cm glass , as recently found in external heavy ion irradiation studies27Al MAS NMR spectrum of the annealed glass and those of the damaged glass (αD doses of 4.4 and 5.8 × 1018α/g)  is very similar to those of the ISG-Cm-damaged glass with an initial αD dose of 4.4  × 1018α/g and which increased to 5.8 × 1018α/g over a 10 months time period. No significant spectral evolution was observed in going from 4.4  × 1018α/g to 5.8  × 1018α/g indicating a saturated damaged state at 1.4 × 1018α/g already. Because MQMAS spectroscopy do not show any evidence of highly coordinated species, the width increase from the pristine state arises mainly from an increase of the local electric field gradient (EFG) and can have several origins, i.e. an increase of the NMR parameter distribution width either related to a change in the charge compensator of the AlO4 tetrahedra (here Ca++ or Na+)4 tetrahedra. In such oxide glass compositions, the Na ions are considered as the main charge compensators of aluminum tetrahedral (peak position at −20 ppm). It is also known that the charge compensation by Ca ions (i.e. Na substitution) induces a very important increase of the width of Al spectra4 units is one of the probable mechanism to explain the increase of the width of 27Al MAS NMR spectrum, as the ballistic disordering is known to induce significant atomic displacements and that Na ions are the most displaced atoms in the glassy structure4 tetrahedra and replaced by Ca ions. If the increase of the global disorder around AlO4 tetrahedra due to the global damage of the glassy network could certainly contribute to the width increase, as no such broadening was observed for the other former cations in the glass; here boron and silicon; we believe that change of charge compensator is the main mechanism for explaining this width increase.The 4 population versus αD dose. A regular evolution of the structure with dose with a saturation phenomenon occurring at around 2 × 1018α/g is found, and can be fitted with a direct impact modela = 1-exp(-viDα), where fa is the damage volume fraction, vi is the damage volume per event (g) and Dα is the alpha decay dose per gram. Such an evolution indicates that only a single alpha decay event is enough to induce a full transformation of the material to the saturated damaged state. This model is usually used in glassy materials to fit the evolution of the density23 associated to an individual αD event was extracted which is in quite good agreement with those obtained (~300 nm3) by fitting evolution of macroscopic or structural parameters of other actinides doped glasses79327Al local EFG) are qualitatively similar to those observed in glasses submitted to external heavy ion irradiation (1 to 7 MeV Au ions or 92 MeV Xe ions)6244Cm doping compared to +16% by Xe or +13% by Au ions. Similarly, 27Al MAS NMR data from ISG-Cm only exhibit four coordinated Al atoms whereas around 4 and 2% (8 and 4%) of penta and hexa coordinated aluminum atoms were measured in Xe  irradiated ISG glass629Si and 23Na spectra contrary to the results obtained on Xe and Au ISG irradiated glass6244Cm into 240Pu, a difference between the damage efficiency of Au (1 to 7 MeV)/Xe 92MeV ions and 244Cm alpha decay RN or an annealing effect of AP on the damaged state generated by RN. During the eight years of storage of ISG-Cm glass, around 25% of the Cm atoms have been transmuted to Pu atoms. So the chemical effect associated to the transmutation of Cm into Pu concerns only 0.025at%, whose role in the glass structure is quite similar (modifier role with coordination number between 7 and 8)244Cm alpha decays. But it was shown by Raman spectroscopy, micro hardness and swelling that swift heavy ions with electronic energy loss at least greater than 4 keV/nm (case of Xe irradiation) cause a significant damage level which is in good qualitative and quantitative agreement with the damage from the ballistic collisions, the case of heavy RN of 244Cm alpha decays244Cm alpha decays, which mainly undergo nuclear and electronic collisions respectively. Indeed, competition between these two energy loss mechanisms and its impact on the global damage level has been addressed recently in a sodium borosilicate glass using double ion beam irradiations1064 ↔ BO3 + NBO to the right1029These structural modifications  was fabricated in the Atalante laboratory whose composition (molar %) is SiO2 60.02, B2O3 15.96, Na2O 12.61, Al2O3 3.81, CaO 5.69, ZrO2 1.71, Cm2O3 0.13, PuO2 0.07. This glass is considered as a good surrogate to the French R7T7 industrial glass and was chosen by the international community as an international glass standard to compare the long term behavior methodologies developed worldwide18α/g and one from the same glass, but annealed at 873K for 2 h and then cooled down slowly (1K/min) to fully recover the irradiation induced damage. These two powders were then characterized periodically during which their αD dose evolved from 4.4 to 5.8 × 1018α/g (10 months) and 0 to 1.4 × 1018α/g (22 months), respectively.In 2007, one batch of et al.et al.27Al, 11B and 23Na spectra were acquired with single pulse excitation of short lengths  to ensure quantitatively and with recycle delays ensuring a full relaxation of the nuclear spins . Multiple Quantum MAS (MQMAS) spectra were collected using the Z-filter pulse sequence (for 27Al) or the RIACT (II) pulse sequence for 23Na and 11B29Si, the CPMG pulse sequence has been employed, coadding echoes, followed by Gaussian apodization of 100 Hz before Fourier Transform Data were processed and fitted with an in-house written codeAll spectra of the ISG-Cm glasses were collected on a 400WB Bruker Avance III spectrometer operating at a magnetic field of 9.4T with spinning frequencies from 40 to 50 kHz using 1.3mm outer diameter rotors (30mg of powder for each glass sample was packed in rotors). In an earlier approach developed by Farnan For purpose of comparison, spectra collected on the pristine and heavy Ion (Xe and Au) irradiated ISG glasses are also presented. They were acquired on a conventional 500WB Bruker Avance II spectrometer operating at a magnetic field of 11.75~T as described in ref. How to cite this article: Charpentier, T. et al. Self-healing capacity of nuclear glass observed by NMR spectroscopy. Sci. Rep. 6, 25499; doi: 10.1038/srep25499 (2016).


A remarkable phenomenon in spatiotemporal dynamical systems is chimera state, where the structurally and dynamically identical oscillators in a coupled networked system spontaneously break into two groups, one exhibiting coherent motion and another incoherent. This phenomenon was typically studied in the setting of non-local coupling configurations. We ask what can happen to chimera states under systematic changes to the network structure when links are removed from the network in an orderly fashion but the local coupling topology remains invariant with respect to an index shift. We find the emergence of multicluster chimera states. Remarkably, as a parameter characterizing the amount of link removal is increased, chimera states of distinct numbers of clusters emerge and persist in different parameter regions. We develop a phenomenological theory, based on enhanced or reduced interactions among oscillators in different spatial groups, to explain why chimera states of certain numbers of clusters occur in certain parameter regions. The theoretical prediction agrees well with numerics. The collective behaviors of systems of coupled oscillators have been a topic of continuous interest12578910111213141516171819202122232425262728293031323334353637383940781063417181411272878422330243344467While a chimera state is commonly referred to as the situation where two dynamically distinct states coexist in different regions of the physical space, in certain particular settings more than two coexisting states can occur, e.g., in systems with time delay112728m) of clusters emerge, i.e., become stable, and then disappear (become unstable). An interesting phenomenon is that, certain m-cluster chimera states can undergo a period-doubling like bifurcation to states with 2m clusters. We propose a phenomenological theory, based on the intuitive idea of mutual enhancement among oscillator subsets exhibiting similar dynamical behaviors in space, to explain the “bifurcation” behavior of chimera states with distinct spatiotemporal patterns. The theory predicts correctly key features such as the emergent order of m-cluster chimera states, the corresponding region of the topology parameter, and the possible m values for the occurrence of cluster doubling. Our results imply that multicluster chimera states can occur in non-locally coupled oscillator networks more commonly than previously thought.In this paper, we demonstrate that multicluster chimera states can occur commonly in the “traditional” setting of Kuramoto networks of phase coupled oscillators, without the need to impose special dynamical features on time delay, phase lag, or special coupling function differing from that associated with the classical Kuramoto model. In fact, perturbing the coupling configuration23N non-locally coupled, identical phase oscillators with periodic boundary condition (the ring configuration). The system is mathematically described asWe consider a one-dimensional network of ϕ(xi) is the phase of the ith oscillator at position xi. For convenience, we choose the range of the spatial variable to be . Since the oscillators are identical, the natural velocity and phase lag parameter, ω and α, respectively, are chosen to be constants that do not depend on the spatial location of the oscillator. Without loss of generality, we set ω = 0 and choose α  <  π/2. The kernel G(xi − xj) = [1 + Acos(xi − xj)]/(2π) is a non-negative even function that characterizes the non-local coupling among all the oscillators. The quantity cij is the ij th element of the N × N coupling matrix C, where Cij = 1 if there is coupling from the jth oscillator to the i th oscillator, and Cij = 0 indicates the absence of such coupling. We systematically remove certain fraction of links from every node, while ensuring that all nodes remain identical and structurally indistinguishable. To do this we introduce a tunable topological parameter η = 2L/N , the fraction of neighbors removed for any given oscillator, where L denotes the number of removed links from each side of the node. We have Cij = 0 for j = i − L, . . . , i + L. The connection pattern of a node after link removal is shown in where Z, definedi asThe network dynamics can be characterized by the following complex order parameter θ = ϕ − Ωt, and Ω denotes the velocity of the oscillators in the coherent subset when a chimera state emerges. Theoretical insights into the chimera states can be obtained by resorting to the continuum limit N → ∞ to reduce the system to one described by PDE42f that satisfies the continuity equationwhere the phase of the oscillator is written as v is phase velocityf can be expressed in terms of Fourier series expansion asand c.c.” stands for the complex conjugate of the previous term, and the nth coefficient is the nth power of some function h that effectively characterizes the state of the system. The time evolution of h associated with the order parameter Z is4243where “G(x − x′) is a piecewise-smooth coupling functionwhere η fraction of couplings. The modulus of the order parameter, taking into account the deletion of the nearby R, we can identify the emergence of multiple cluster chimera states, where each cluster corresponds to a coherent group of oscillators. m-cluster chimera states for different intervals of η, which indicates that the emergence of the chimera-state patterns is robust with respect to reasonable variations of these parameters. In the simulations, the system parameters are A = 0.995 and α = 1.39, and the initial condition is generated8ϕ(x) = 6r exp(−0.76x2), where r is a random variable uniformly distributed in . In fact, the results obtained from direct simulations of N → ∞ agree with each other with similar spatiotemporal patterns. As shown in the inset of η values differs by orders of magnitude. For clarity, we use different color bars to distinguish the magnitudes of the spatiotemporal patterns in different panels. As η is increased, the number m of clusters undergoes changes from 4 to 3 (or 3&6), to 5 (or 5&10), to 7, and to 9, etc. Here the 3&6 state (or the 5&10 state) is a state that switches between 3-cluster and 6-cluster (or between 5-cluster and 10-cluster) chimera behaviors.Numerically, we observe a variety of rich phenomena when links are systematically removed. In particular, using the real order parameter To better understand the impact of multicluster chimera states on global coherence of the system, we calculate the average order parameter m , with k being an arbitrary positive integer. The piecewise smooth coupling function G(x) employed in our present work, however, consists of an infinite number of harmonics. In this case, it is not clear whether a mathematical theory can be developed to analyze the pattern formation process. Because of this difficulty, we resort to developing a phenomenological theory, in which the value of I(η) effectively determines, in a self-consistent manner, the emergence of m-cluster patterns in the continuum limit, as exemplified in I(η) takes into account only the coarse-grained configuration of multi-cluster patterns formed due to the mutual interactions between oscillators in distinct spatial regions.Since the coupling structure of the oscillator system, as controlled by the parameter To demonstrate the general applicability of our mutual-enhancement theory with respect to the choice of different coupling kernels, we have studied the case of normalized exponential coupling kernelxi and xj run from 0 to 1 with periodic boundary conditionI(η) from η, 1], while η values  are shown in where Furthermore, we have studied the case of rectangular coupling kernelxi and xj run from −1 to 1 with periodic boundary condition, and I(η) and a number of typical spatiotemporal patterns are shown in γ = 0.6 and 0.8, respectively. The results are essentially the same as those for the case of sinusoidal coupling function, demonstrating the general applicability of our mutual-enhancement theory.where How to cite this article: Yao, N. et al. Emergence of multicluster chimera states. Sci. Rep.5, 12988; doi: 10.1038/srep12988 (2015).


Quantification of H2O2 level in tissue fractions in presence of H2O2 metabolizing enzymes may always provide an incorrect result. A modification is proposed for the spectrofluorimetric determination of H2O2 in homovanillic acid (HVA) oxidation method. The modification was included to precipitate biological samples with cold trichloroacetic acid  followed by its neutralization with K2HPO4 before the fluorimetric estimation of H2O2 is performed. TCA was used to precipitate the protein portions contained in the tissue fractions. After employing the above modification, it was observed that H2O2 content in tissue samples was ≥2 fold higher than the content observed in unmodified method. Minimum 2 h incubation of samples in reaction mixture was required for completion of the reaction. The stability of the HVA dimer as reaction product was found to be >12 h. The method was validated by using known concentrations of H2O2 and catalase enzyme that quenches H2O2 as substrate. This method can be used efficiently to determine more accurate tissue H2O2 level without using internal standard and multiple samples can be processed at a time with additional low cost reagents such as TCA and K2HPO4.Hydrogen peroxide (H Its level therefore is used as a major biomarker in multiple investigations  were run to check any background fluorescence of the reaction mixtures. Time kinetics was performed to determine the optimum time period for completion of the reaction at which all the H2O2 molecules were supposed to consume by the reaction system. Since the aim of the experiment was to precipitate the tissue samples with TCA, therefore, the required concentration of H2O2 was precipitated in TCA  and then was neutralized with 6 M K2HPO4 (3.2 mL 6 M K2HPO4 with 15 mL H2O2 in 5% TCA) to keep the pH of the mixture within 7.50 ± 0.04. Then the fluorescence of the above TCA treated H2O2 was determined. H2O2 was also incubated with CAT enzyme (5–20 U) for different time periods (5–20 min) to confirm the role of H2O2 in the reaction system. After incubation with or without CAT, H2O2 samples were precipitated with TCA (5%) and then neutralized with required volume of K2HPO4 to keep the pH ~7.5. The reaction product of HVA dimer in both TCA treated and nontreated H2O2 samples was kept for different time periods (2–60 h) to check the stability of the HVA dimer product. Finally, the method was employed in light dependent superoxide generating system. The above was done to check H2O2 content by incubating the mixture with SOD enzyme which coverts superoxide anions to H2O2. Both tris and phosphate buffers were used to study the effects of buffers on the assay system. And there was no significant change in fluorescence pattern observed with either of the buffers. Therefore, the assay was carried out in phosphate buffer.Reaction mixture for the calibration curve of H Scylla serrata contain variable amount of H2O2 and CAT  were run to check any background fluorescence of the reaction mixtures. In the above step, both TCA precipitated and nonprecipitated samples were used.It was observed that tissues of the crab and CAT . Also tiize H2O2 . Along wnomenons , tissuest-test at P < 0.05 level was used to compare the values between TCA precipitated and control samples. One-way ANOVA followed by Duncan New Multiple range test was performed to validate the statistical difference between different time point groups of either TCA treated or control samples at P < 0.05.Data are presented as mean (± S.D.) of three samples each having technical triplicates. Student's 2O2 was used in log phase concentrations from 1 μM to 10, 000 μM  was linear when incubated for 1 h. However, the time required for the saturation of the reaction with the above assay composition appeared to be more than 1 h  was determined. It was observed that around 2 h was needed to quench maximum number of the H2O2 molecules present in the reaction mixture with 125 μM HVA and 0.1 U mL−1 HRP  followed by neutralization with K2HPO4 was not only similar but also was almost the same with that of their corresponding control samples  of catalase enzyme for different (0–20 min) time periods  higher amount of H2O2 was observed than TCA non-treated samples  using biochemical methods can be a challenge. A modified method using HVA oxidation principle is proposed to measure the exact H2O2 level in tissues. Homovanillic acid is a nonfluorophore compound which is converted to a highly fluorescent compound after oxidation in HRP-H2O2 system  before it is being completed. Most of the biological samples especially tissue fractions contain several enzymes such as CAT and several peroxidase and oxidase enzymes which can metabolize H2O2. During tissue fractionation and incubation steps for H2O2 determination in such samples, the possibility of getting inconsistent result cannot be ruled out. Therefore, the assay system can measure more accurate H2O2 content in the sample if only the H2O2 metabolizing enzymes are removed from the reaction mixture and the reaction mixture gets sufficient time period for completion. In this communication, a small but important modification in HVA oxidation method to estimate H2O2 in biological samples is reported. This was achieved by precipitating tissue fractions with ice cold TCA (5%) and neutralization with K2HPO4 followed by H2O2 quantification. The time kinetics of the reaction to determine the optimum time required to complete the reaction between all H2O2 and HVA molecules in the system was also investigated.Higations . H2O2 meications . H2O2 is2 system . This pr samples . In all n to 1 h , 6, 18. 2O2 samples with CAT treatment suggest the specificity of the reaction by H2O2 only. Time kinetics study suggests that minimum 2 h incubation time of assay mixture was required (1 to 25 μM range of H2O2 or sample with 125 μM HVA and 0.1 U mL−1 HRP) to complete the reaction. However, one can reduce the time of incubation by increasing the HRP and HVA concentration [2O2 and tissue samples was found to be stable for more than 60 h with only loss of 10% of fluorescence intensity. However, up to 12 h, the loss was less than 2% and statistically was not significant than the value observed after 2 h incubation. Thus, reaction product can be kept conveniently in the dark for at least half a day for quantification of H2O2.Decreased florescence values that were observed for Hntration , 19. HVA2O2. The above assay mixture that generates superoxide radical under light was incubated with SOD enzyme. It was expected that H2O2 could be measured and the value could be correlated with the generated superoxide radical level. However, no significant fluorescent value was obtained in the process for H2O2 in the above system. It may be due to the breakdown of the produced H2O2 under high fluorescent light.An attempt was made to measure superoxide radical  by employing this method. Light mediated riboflavin reduction method was employed for superoxide generation. SOD enzyme converts superoxide radicals to H2O2 content in the tissues of an invertebrate containing most of the H2O2 metabolizing enzymes (the mud crab S. serrata) was measured. It was observed that around ≥2 folds H2O2 was lost in muscle, hepatopancreas, and gills of the crab during tissue processing and incubation time for H2O2 determination. Recently, it is described that activities of CAT and glutathione peroxidase, the two major H2O2 catalyzing enzymes in this crab, are tissue-specific [2O2 in the crabs during H2O2 determination without TCA precipitation. Therefore, the loss of H2O2 during measurement may vary in different organs of an organism and in the same organ of different organisms due to the difference in H2O2 contents and H2O2 metabolizing enzymes.Finally, Hspecific . This ma2HPO4 (6 M) was not evaluated. So, the neutralization of TCA treated samples to study the final pH must be done prior to any assay. The method can also be applied to study other metabolites in tissue fractions where specific enzymes interfere in assaying a particular metabolite. However, before adapting TCA precipitation method to quantify a particular metabolite, its assay system must be checked in similar way with TCA and the pure metabolite. This modification method can be used in multiple studies where tissue H2O2 level is used as an important index [One of the limitations of this method was that tissues were not directly homogenized in acids such as metaphosphoric acid which is used to remove enzymes from tissue fractions. TCA is an aggressive acid and, therefore, homogenization of tissues directly in TCA is not recommended. Neutralization of TCA  treated samples was needed to keep the pH between 7 and 8 because HRP has optimum activity within this pH range. Therefore, the exact stoichiometry of the neutralization reaction between TCA  and Knt index . The opt2O2 assay system. However, it seems that there is a very less chance for the interference of phosphate ion on the assay system as no difference was observed in TCA treated and untreated positive controls, that is, pure H2O2 samples, because, in TCA treated pure H2O2 samples, high concentrations of phosphate ions would be there since 6 M K2HPO4 was used to neutralize it. Although higher fluorescence values were observed in the present study with tris buffer than phosphate, it was statistically not significant (data not given). Nevertheless, a negative control by using excess catalase in the tissue sample prior to TCA precipitation would enhance the accuracy of the method. However, no fluorescence was observed in the samples when intentionally the stored (at 4°C for 1-2) tissue fractions were used for H2O2 content measurement (data not given). The reason(s) could be contributed to the H2O2 scavenging activity of the tissue fractions due to multiple reasons including acceleration of Fenton chemistry and/or utilization of the H2O2 by the presence of internal catalase and other H2O2 catabolizing enzymes.It is claimed that phosphate ion or buffer, if used, may interfere in the fluorescence assay system . HoweverμM range) such as quercetin, capsaicin, and α-tocopherol but not in presence of the reduced glutathione. Therefore, the presence of the above phenolic antioxidants in μM range to interfere in the HRP mediated HVA dimer production by H2O2 in crab samples is very low. Also for the above additional function of HRP, it needs a proper stoichiometry between HRP and the phenolic antioxidants (in μM range) in the tissue fractions. Phenolic antioxidants are more specifically observed in plant tissues. On the other hand, the specificity of the HRP mediated HVA dimer production can also be evaluated from the basal level of the fluorescence values that were obtained for the blank (assay mixture having no tissue fractions), from samples having tissue fractions and HVA only, and from positive controls. The specificity of the HRP mediated HVA dimer production by H2O2 is also reported by several authors [Shirasaka et al.  noticed  authors , 14 and,2O2 during processing of tissue samples in order to measure more accurate tissue H2O2 content in samples. Further, several samples can be processed simultaneously for H2O2 determination and the end product conveniently can be measured within 12 h.TCA precipitates proteins in biological samples. It is concluded that TCA precipitation can be employed to prevent internal loss of H


Here, the environmental factors controlling stomatal behaviour of leaf-level carbon and water exchange were examined across the evolutionary continuum from C3 to C4 photosynthesis at current (400 μmol mol–1) and low (280 μmol mol–1) atmospheric CO2 conditions. To this aim, a stomatal optimization model was further developed to describe the evolutionary continuum from C3 to C4 species within a unified framework. Data on C3, three categories of C3–C4 intermediates, and C4Flaveria species were used to parameterize the stomatal model, including parameters for the marginal water use efficiency and the efficiency of the CO2-concentrating mechanism (or C4 pump); these two parameters are interpreted as traits reflecting the stomatal and photosynthetic adjustments during the C3 to C4 transformation. Neither the marginal water use efficiency nor the C4 pump strength changed significantly from C3 to early C3–C4 intermediate stages, but both traits significantly increased between early C3–C4 intermediates and the C4-like intermediates with an operational C4 cycle. At low CO2, net photosynthetic rates showed continuous increases from a C3 state, across the intermediates and towards C4 photosynthesis, but only C4-like intermediates and C4 species  had higher water use efficiencies than C3Flaveria. The results demonstrate that both the marginal water use efficiency and the C4 pump strength increase in C4Flaveria to improve their photosynthesis and water use efficiency compared with C3 species. These findings emphasize that the advantage of the early intermediate stages is predominantly carbon based, not water related.C This additional cycle results in high CO2 concentrations around Rubisco, thus suppressing the enzyme’s oxygenase function and nearly eliminating photorespiration and the associated carbon and energetic costs.While only 3% of the world’s terrestrial plant species use the C4 photosynthesis has evolved independently at least 66 times in lineages throughout the plant kingdom and, in some of these lines, there are intermediate species that are neither C3 nor fully C4  have confirmed that C3 photosynthesis is the basal state and C4 photosynthesis is more derived; these analyses also place photosynthetic intermediate species as evolutionary intermediates to these two photosynthetic types  from near 1000 ppm to ~400 ppm ~30 million years ago (mya), where the predominant benefit from a CO2-concentrating mechanism would have been enhanced net CO2 fixation rates through suppression of photorespiration   than are found in C3 species  and marginal WUE , where q is the temperature coefficient for that parameter (0.074 for Kc and 0.015 for Ko) and Tl is leaf temperature. Vc,max was adjusted as Vc,max=Vc,max25exp[0.088(Tl−25)]1+exp[0.29(Tl−41)], where Vc,max25 is the maximum carboxylation rate at 25 °C. The Γ* values for each of the five photosynthetic types were approximated using averaged values of Γ (the CO2 compensation point) from Flaveria species in Rd=0.015Vc,max) and can be ignored (J(αpεmQ) needed in determining a1=J and a2=Jk2/requires an estimate of the product αpεm . The value for αp was set at 0.8 , it follows that the measured A are consistent with the estimate J=0.08Q, which gives an Anet of 40 μmol m–2 s–1. This evidence supports the assumption that αpεm is stable across photosynthetic types. A more rigorous parameterization would require direct observations of αpεm or reliable Vc,max–Jmax relationships across the C3–C4 continuum.To parameterize the above model for , kcat) . The Mic ignored . BecauseAnet(ci) curve] measured under saturating light for Flaveria species were taken from ci the slope of the Anet(ci) is approximately CE=Vc,maxη/Kcair, so that η=CEKcair/Vc,max.Therefore, knowledge of Γ, Kcair, Kc, and Ko allowed an estimate of η for each Flaveria species.Carboxylation efficiencies  In our re-analysis of the 3, and Type I and Type II intermediates using the estimated changes in η  was accompanied by a quadrupling of λ compared with that used to characterize the data for Type I and II intermediates. Thus, the stomatal optimization approach could be successfully used to capture key changes in the measured relationship between Anet and gs across the C3–C4 spectrum using the estimated η values, but only when the marginal WUE of the C4-like and C4 species was modelled to be 4-fold greater than that of the C3 species 2. Therefore, the increase in λ is generated by a decline in ci (where ca is assumed to be 400 μmol mol–1). This finding suggests that the increase in C4 WUEi values, the increase in λ, and the decline in ci (as expected by the presence of a C4 pump) are all interconnected and predicted from the proposed stomatal optimization model.While it might,  η as in , but wit η as in . This re species . This in4 photosynthesis evolved under low ca, with the transition in Flaveria occurring within the last 3 million years (Anet and WUEi was further investigated under both current (400 μmol mol–1) and low CO2 concentrations  elevated Anet in C3 species by 80% due to greater substrate availability  relationship, as indicated by minor differences between filled and open symbols. A Type I intermediate has a 15% higher Anet than a C3 species, which could provide a competitive edge to the intermediate in a low CO2 environment; a similar jump in Anet is seen for each photosynthetic group along the evolutionary trajectory, in agreement with a recently proposed smoothly increasing fitness landscape for C4 evolution  than C3 congeners  between C3, and Type I and Type II intermediates could be maintained constant, but λ had to be quadrupled to model C4-like and C4Flaveria . When leaf-level fluxes were modelled at low CO2, there was no evidence for a greater WUEi in the C3–C4 intermediates (compared with a C3Flaveria) until they developed a full C4 cycle. However, the model results suggest a steady increase in net carbon fixation rates across the C3 to C4 photosynthetic range. While this implies that carbon, not water, was the main driving pressure for the early steps of C4 evolution in this genus, the increase in λ indicates that there was a fundamental shift over the evolution of C4 photosynthesis between the relative costs of carbon and water, resulting in higher carbon costs of water losses.Using a stomatal optimization approach, the full range of CJXB online.Supplementary data are available at Table S1. Parameter table.