The bottleneck of anatase‐type TiO2 remains the water oxidation, that is, the hole transfer reaction from pristine anatase to the aqueous environment. In this work, we report that “grey” (defect engineered) anatase can provide a drastically enhanced lifetime of photogenerated holes, which, in turn, enables an efficient oxidation reaction of water to peroxide via a two‐electron pathway. As a result, a Ni@grey anatase TiO2 catalyst can be constructed with an impressive performance in terms of photocatalytic splitting of neutral water into H2 and a stoichiometric amount of H2O2 without the need of any noble metals or sacrificial agents. The finding of long hole lifetimes in grey anatase opens up a wide spectrum of further photocatalytic applications of this material.Titanium dioxide has been the benchmark semiconductor in photocatalysis for more than 40 years. Full water splitting, that is, decomposing water into H Long‐living photogenerated holes: Grey anatase, in comparison to white anatase, provides a drastically enhanced lifetime for trapped photogenerated holes. This allows to construct a Ni@grey anatase TiO2 catalyst that can photocatalytically split neutral water into H2 and a stoichiometric amount of H2O2 without the need of any noble metals or sacrificial agents.Institute and/or researcher Twitter usernames: @FAU_Germany High costs for noble metals turned the attention to earth‐abundant alternatives with a low overpotential for the H2 catalyst is cost effective and highly efficient: The photoreaction occurs without noble metals and without any sacrificial agent. The catalyst enables photoexcited holes to surface‐oxidize water to peroxide on one hand and allows Ni as a means for electron transfer to activate the catalyst for H2 evolution on the other hand. The basic processes are depicted in Figure 2 catalyst, we loaded Ni(OH)2 onto commercial anatase nanoparticles by a solution impregnation method, followed by hydrogenation at 500 °C in a H2 atmosphere – this hydrogenation step leads to a reduction of Ni species and concomitantly forms the desired reduced grey anatase. For reference, we also included the widely investigated black form of anatase2 reduction was studied on various titania nanostructures namely TiO2 nanotubes and in presence of sacrificial agents showed a strongly enhanced photocatalytic and photoelectrocatalytic performance in comparison to pristine anatase.In this work, we document for the first time that using so‐called grey anatase  can provide, due to a high density of deep trapping states, extremely long‐living photogenerated holes and thus opens up an effective hole‐transfer pathway that allows an efficient oxidation of pure neutral water. As a consequence, also the electron transfer can be mediated with a stable Ni co‐catalyst as outlined below. This Ni@grey anatase TiO2 surface . HRTEM images  lattice planes of anatase and grey anatase. For the Ni particle, the d‐spacing is 0.21 nm and the Fourier transformation results are in line with a (200) d‐spacing of metallic nickel  anatase is that “grey” and “black” anatase show a variation of the density of states distribution (DOS) at the conduction and valence band edge depending on the level of reduction of anatase.s Figure c reveal with the 01 lattic2 was examined as water splitting photocatalyst without the addition of any sacrificial reagents. When using UV light irradiation  we note a substantial photocatalytic production of hydrogen with an H2 evolution rate of ∼700 μmol h− g−1 from neutral water that correlates to a quantum efficiency of 2.3 % .This Ni@grey anatase TiO2 evolution could be detected as a photocatalytic hole transfer product from Ni@grey anatase, but clearly the formation of H2O2 could be identified first qualitatively (using simple H2O2 test stripe (Sensafe Co.) or horseradish peroxidase (HRP)‐based testing2O2 yield over illumination time can be determined by KMnO4 titration2O2 is often reported to be prone to illumination or even to serve as a charge carrier capture agent,2O2 takes place .We then investigated the water oxidation products. In fact, no O2O2 is produced that leads to a H2/H2O2 ratio close to a 1 : 1 stoichiometry . Therefore, the unique water splitting performance observed here must be ascribed to the features of grey titania. It also should be noted that based on X‐ray photoelectron spectroscopy (XPS) result and selected area diffraction (SAED) pattern analysis of the extracted powder after illumination, no significant changes are observed neither in the crystallographic phases nor in the chemistry of the powder . This is also in line with literature data using electron paramagnetic resonance (EPR) that show the defect signatures in grey anatase after catalysis to be virtually the same as the one observed for the initial grey material.As a result, over the entire photocatalytic reaction time, a total amount of H2, grey anatase, and Ni@grey anatase yields free electron lifetimes for all materials in the range of picoseconds (Tables S2 and S3 in the Supporting Information), which is in sound agreement with general literature findings.2.2, 14 ns for grey anatase TiO2 and 3200 ns for Ni@grey anatase TiO2 (Table S3). For the deep‐trapped holes lifetimes show even more pronounced differences; relative to plain anatase TiO2, the deep‐trapped hole lifetime in grey anatase increases from 64 to 820 ns and for Ni@grey anatase TiO2 even to a value of 14.2 μs (Table S3).In order to elucidate the origin of this astonishing feature of grey anatase, we investigated the charge carrier dynamics by in situ femtosecond and nanosecond transient absorption measurements. An overview of the transient absorption spectra and the absorption time profiles for grey titania and Ni@grey titania is compiled in Figure 2 is in line with recent literature on the electronic structure of hydrogenated TiO2, as outlined in Figure S1. An optimized level of reduction affects not only the conduction band and allows facilitated electron transfer2 show a very different valence state distribution (or deep traps) tailing into the bandgap of TiO2.The existence of a high density of deep trapping states for holes in dark anatase TiO2.2 is in line with DFT calculations for hydrogenated material.For the grey anatase sample, in literature, the valence band maximum energy is blue shifted by approximately 0.5 to 0.7 eV towards the vacuum level compared with the typical valence band of anatase TiO2 and grey anatase . A strong PL signal, which is centered at around 520–530 nm, is observed for grey TiO2, while this PL is weak in plain anatase TiO2. The origin of this PL peak in anatase has been investigated in depth by Pallotti et al.2.2O2  for H2 evolution from η=0.85 V vs. RHE for grey anatase TiO2 to η=0.60 V vs. RHE for Ni@grey anatase TiO2. It is noteworthy that after 10 h of UV illumination under open‐circuit potential (OCP), the overpotential for Ni@grey anatase TiO2 is even further reduced to η=0.45 V vs. RHE. This not only shows the effect of Ni as H2 evolution catalyst but also the high stability of the metallic Ni‐cocatalyst under illumination. The OCP potential of the electrode under illumination was measured to be at 0.45 V RHE, i. e. under operating conditions the electrode is in potential range where Ni can effectively catalyze H2 evolution, see Figure To probe the Ni involvement in the water splitting reaction, cathodic electrochemical voltage scans were conducted with films of grey anatase and Ni@grey anatase  nitrate hexahydrate and glycerol (1 : 2 molar ratio) were added to DI water (200 mL) to form an aqueous nickel(II)−glycerol complex. The exact masses of nickel(II) nitrate hexahydrate and glycerol used depended on the target nominal Ni loading. Then, anatase (1 g) was added to the Ni‐complex solution under continuous stirring, and the nickel−glycerol complex was then precipitated on the TiO2 support by a dropwise addition of 0.5 M NaOH until a pH of 12 was reached. The resulting suspension was stirred for further 1 h, then the resulting light green powder (presumably Ni(OH)2@TiO2) was collected by vacuum filtration.We used commercial anatase nanopowder  as a precursor for decoration and hydrogenation. A series of Ni@TiO2 treatments, different Ni loaded TiO2 powders were annealed in hydrogen atmosphere  at different temperatures  for 1 h. These samples show color changes . After the hydrogenation, these samples (i. e. ‘fresh’ samples) were then directly (under Ar atmosphere) transferred, for example, for photocatalytic or electrochemical measurements, to deaerated (Ar bubbled) DI water .For Hλ=325 nm, 55 mW cm−2). The amount of H2 produced was measured using a Varian gas chromatograph with a TCD detector. To prepare suspensions for H2 measurements, 2 mg TiO2 powders  were dispersed in 10 mL of DI water (18 MΩ cm) with ultrasonication for 30 min. During illumination, the suspensions were continuously stirred. To test the stability of catalyst for hydrogen evolution, repeated cycle measurements of hydrogen evolution as photocatalyst over a testing period of 5 days  were carried out. The powder remained the same for each cycle.Photocatalytic hydrogen generation was measured under open circuit conditions from pure neutral DI water using a UV laser , we obtain different H2O2 was qualitatively detected using commercial peroxide check stripes 481015 (Sensafe Co. USA). For each measurement one test stripe was dipped into the solution with constant, gentle back and forth motion for 5 s. Then the test stripe was removed and shaken to remove the excess water. After waiting for 15 s, the color was matched to the color chart provided by the company. Additional confirmation for H2O2 detection was obtained using the ABTS‐HRP method.2O2 was carried out by titration with KMnO4.2SO4. The KMnO4 was added dropwise to the stirred solution until a permanent pink color was apparent. The procedure was verified using standard H2O2 solutions (prepared from H2O2 30 wt% Merck).2 surfaces.2O2.Hλ=258 nm came from the Ti:sapphire laser system described above. The probe pulse , which was generated in a photonic fiber, was synchronized with the femtosecond amplifier. Both measurements were carried out in‐situ using 2 mm quartz cuvettes filled with the suspensions of interest .Carrier life‐time measurements were carried out using femtosecond and nanosecond laser photolysis transient absorption spectroscopy. Femtosecond transient absorption measurements were performed with output from a Ti:sapphire laser system : 775 nm, 1 kHz, and 150 fs FWHM pulses. The excitation wavelength was generated by third harmonic generation (258 nm), pulse widths of <150 fs and energies of 400 nJ per pulse were selected. The transient absorption detection was performed with a transient absorption pump/probe system . Nanosecond transient absorption experiments were performed with an EOS spectrometer (Ultrafast Systems LLC). The pump pulses at The authors declare no conflict of interest.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 demand for allosteric targeting of nuclear receptors is high, but examples are limited, and structural information is scarce. The retinoic acid‐related orphan receptor gamma t (RORγt) is an important transcriptional regulator for the differentiation of T helper 17 cells for which the first, and some of the most promising, examples of allosteric nuclear receptor modulation have been reported and structurally proven. In a 2015 patent, filed by the pharmaceutical company Glenmark, a new class of small molecules was reported that act as potent inverse agonists for RORγt. A compound library around the central thienopyrazole scaffold captured a clear structure‐activity relationship, but the binding mechanism of this new class of RORγt modulators has not been elucidated. Using a combination of biochemical and X‐ray crystallography studies, here the allosteric mechanism for the inverse agonism for the most potent compound, classified in the patent as “example 13”, is reported, providing a strongly desired additional example of allosteric nuclear receptor targeting. Example 13: Inhibition of the RAR‐related orphan receptor gamma t (RORγt) nuclear receptor has proven to be a novel approach for targeting autoimmune diseases. Recently, RORγt was shown to feature an allosteric pocket. Using X‐ray crystallography, we now show that compound 13, a promising thienopyrazole analogue of previously reported RORγt modulators, exerts its inverse agonism by binding to this allosteric pocket. Nuclear receptors (NRs) are a family of ligand‐dependent transcription factors that constitute an important class of drug target.MRL‐871 . To determine if compound 13 has an allosteric mode of action analogous to MRL‐871, we performed a competition TR‐FRET assay. Compound 13 titrations were performed in the presence of increasing concentrations of the well‐characterized orthosteric agonist cholesterol .13 suggests that the binding mode of 13 is independent to that of cholesterol and therefore that 13 likely binds to an allosteric pocket on RORγt. Next, we sought evidence to prove that compound 13 was indeed binding to the same allosteric site as MRL‐871. For this, we performed a ligand displacement assay using a previously reported MRL‐871‐derived fluorescent probe that is known to bind the previously published allosteric site. Compound 13 effectively displaced the probe in a dose‐dependent manner  of human RORγt was used for this study; the region surrounding this native cysteine is important for crystal packing of RORγt in complex with allosteric inverse agonists.13 were co‐crystallized and crystals grew as hexagonal bipyramids overnight using sitting‐drop vapor diffusion. The data collection and refinement statistics of the crystal are provided in Table S1. The crystal structure (PDB: 6TLM) shows the complete LBD of RORγt and reveals that compound 13 indeed binds in the anticipated allosteric pocket and that the orthosteric pocket is devoid of ligand .13 and FM26 induce a fold of the loop between helix 11 and 12 that has F498 in the “flipped‐out” conformation. In contrast to compound 13, the pyrrole of FM26 makes an additional interaction with the backbone carbonyl groups of L353 and K354, which can explain the higher affinity of FM26 for RORγt (IC50=264±23 nM).To now unambiguously confirm the binding mode of compound 50 values of compound 13, FM26 and MRL‐871 would suggest that steric bulk toward helix 4 does not favor the affinity of the allosteric modulator for RORγt . MSD's patent application showed that bulky substitutions on the 4 and 5 position of the indazole moiety also resulted in a reduced affinity for the receptor.Comparison of the IC13 was shown to bind to an allosteric pocket of RORγt. The binding mode of compound 13 is similar to MRL‐871 and the recently reported FM26, with small but notable differences. The structural data imply that the lower affinity of 13 for RORγt relates to additional bulk on the thienopyrazole core pointing to RORγt helix 4, which leads to changes in the overall protein fold. Such changes are likely to affect the dynamics of the protein and stability of the specific fold. The new structural data expand the collection of crystallized ligands binding to allosteric binding pockets on NRs, and specifically on RORγt. The resulting new insights will aid in the understanding of reported compounds classes, potentially also addressing the same allosteric RORγt pocket and in the development of new compound classes with more diverse chemotypes or optimized pharmacodynamics profile.In summary, using X‐ray crystallography and supported by biochemical studies, Glenmark's compound 13 and the biochemical assays have been described earlier.The synthesis of compound The expression and purification of RORγtC455H‐LBD were performed as described earlier.13 was dissolved in 50 % DMSO and 50 % EtOH to a final concentration of 20 mM. One equivalent of compound 13 was added to the RORγt protein solution and the mixture was placed on ice. After 1 hour incubation, centrifugation at 20.000 RCF for 20 minutes at 4 °C was used to remove protein and ligand precipitate. Sitting‐drop vapor diffusion was used to generate crystals using 800 nL of the protein‐ligand solution and 400 nL crystallization solution (1.6 M (NH4)2SO4 and 0.1 M Tris (at pH 8.5)). Crystals grew as hexagonal bipyramids to their final size overnight. Because the crystallization solution was not cryogenic, the crystal was briefly transferred to a cryo‐solution (1.6 M (NH4)2SO4, 0.1 M Tris, 25 % glycerol and 200 μM compound 13 (at pH 8.5)) before being flash‐cooled. The crystal was measured at the i24 microfocus beamline of the Diamond Light Source . Initial data processing was performed using the CCP4i2 suite (version 7.0.078).FM26 (PDB: 6SAL) as a search model for molecular replacement, PHASER was used to phase the data and ligand restraints were generated using AceDRG.Compound The authors declare no conflict of interest.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.


However, its reaction results in a large voltage hysteresis due to the structural degradation arising from an oxygen loss. Herein, an interesting P2‐type Mn‐based compound exhibits a distinct two‐phase behavior preserving a high‐potential anionic redox (≈4.2 V vs Na+/Na) even during the subsequent cycling. Through a systematic series of experimental characterizations and theoretical calculations, the anionic redox reaction originating from O 2p‐electron and the reversible unmixing of Na‐rich and Na‐poor phases are confirmed in detail. In light of the combined study, a critical role of the anion‐redox‐induced two‐phase reaction in the positive‐negative point of view is demonstrated, suggesting a rational design principle considering the phase separation and lattice mismatch. Furthermore, these results provide an exciting approach for utilizing the high‐voltage feature in Mn‐based layered cathode materials that are charge‐compensated by an anionic redox reaction.A breakthrough utilizing an anionic redox reaction (O 0.60Li0.20Mn0.80O2 undergoes a reversible two‐phase reaction at high voltage (≈4.2V vs Na+/Na), in which oxygen ion contributes the charge compensation. The detailed reaction mechanism is thoroughly investigated through experimental and theoretical analyses including in situ XRD and XAS observations. The combined study provides a rational material design for the reversible two‐phase reaction.P2‐type Na Therefore, enormous research efforts have focused mainly on modifying TM ions to increase the energy density of cathode materials. However, this strategy is limited by the amount of TM ions in the compounds.In conventional transition metal (TM) layered oxide cathode materials for lithium‐ion batteries (LIBs) and sodium‐ion batteries (SIBs), charge compensation during the intercalation/deintercalation process relies on changes in the oxidation states of the transition metal ions.2, 3 Thanks to these findings, a new paradigm of anionic redox reactions for charge compensation has led to improved capacity of Li‐rich layered oxide compounds via the introduction of another redox center for cathode materials. Among them, Li‐rich layered manganese‐based oxides, xLi2MnO3∙(1‐x)LiTMO2, are mainstream classes, and they are composed of two parts: a typical layered oxide (LiTMO2) and a Li‐rich layered oxide (Li2MnO3). The structure of Li2MnO3, which is also denoted as Li[Li1/3Mn2/3]O2, is similar to that of typical LiTMO2, but 1/3 of the Mn ions are replaced by Li ions, resulting in a structural distortion from a hexagonal system to a monoclinic system induced by the in‐plane honeycomb‐type ordering where the Li ion is surrounded by six Mn ions. In earlier studies of Li‐rich layered oxides, Li2MnO3 was recognized as electrochemically inactive due to the stable electron configuration of the initial Mn4+ (t2g3eg0), which is not feasible to further oxidation in an octahedral geometry. However, Li2MnO3 exhibits a considerable charge capacity with a long plateau upon extraction of Li+ at the high voltage at 4.5 V (vs Li+/Li), involving the oxidation reaction of the lattice oxygen ions in Li2MnO3. The mechanism of the oxygen redox reaction in Li‐rich layered oxide compounds is related to the unhybridized O 2p orbitals in the LiLi‐O‐LiTM configuration (where a LiLi ion is located in the Li layer and the other (LiTM) is located in the TM layer) because of the relatively small overlap with the Li 2s orbital.In recent years, it has been reported that oxygen ions can reversibly change their oxidation states, as opposed to the conventional concept of stable oxide ions with a constant oxidation number of −2.5 Than suggested a possible contribution of lattice oxygen ions to a charge compensation reaction to account for unusual excess capacity of P2‐type Na0.78Ni0.23Mn0.69O2 during the first charge process. More recently, a possible anionic redox activity in Na[Li1/3Mn2/3]O2 by first‐principle calculations was predicted; as a result, the initial charge capacity (≈150 mAh g−1) with a large plateau at 4.2 V (vs Na+/Na) of the synthesized Na‐deficient Na0.76Li0.25Mn0.75O2 phase relies solely on the oxygen redox activity. And, it was demonstrated that a lone pair of O 2p‐electrons (unhybridized O 2p‐electron) in the linear Na—O—Li configuration participates in the charge compensation during charging. Furthermore, Yabuuchi et al. reported that a P2‐type Na2/3[Mg0.28Mn0.72]O2 without alkali–metal ions in a transition metal layer exhibited a large reversible capacity beyond the theoretical capacity via only Mn3+/4+ redox reactions. Maitra et al. found that the excess capacity originated from the oxygen redox reaction of P2‐type Na2/3[Mg0.28Mn0.72]O2. This research interest in the anionic redox reaction at the high voltage has triggered discovery of various high‐energy‐density SIB cathode materials to date.In addition, the oxygen redox reaction in SIB layered oxide cathode materials has been intensively studied in recent years. An earlier study12 sug On the other hand, among these oxygen redox active materials, Na0.6[Li0.2Mn0.8]O2 with a P3‐type layered structure shows an unusual flat discharge plateau at 4.1 V (vs Na+/Na), as reported by Du et al. This result is clearly distinguishable from the solid‐solution behavior of P2‐type Nax[LiyMnz]O2 compounds despite the similar [LiyMnz]O2 slab structure. Rong et al. also investigated a charge compensation mechanism in P3‐Na0.6[Li0.2Mn0.8]O2 that relies solely on an anionic redox reaction exhibiting the reversible charge–discharge plateau in the voltage range of 3.5–4.5 V (vs Na+/Na) using a spectroscopic study, and identified the shortening of the O—O distance related to peroxo‐like dimers using the neutron pair distribution function. Recently, House et al. reported that the low voltage hysteresis of anionic redox reaction could be achieved by controlling superstructure between Li and Mn ions. By comparing two P2‐type ribbon‐ordered Na0.60Li0.20Mn0.80O2 and honeycomb‐ordered Na0.75Li0.25Mn0.75O2, they suggested that the ribbon‐type Li/Mn ordering inhibits disorder of the inherent patterning along the ab plane that causes voltage hysteresis by changing Mn coordination of On− ions.In the abovementioned studies, the anionic (oxygen) redox active layered oxide compounds exhibit a characteristic long charge plateau at the high voltage (4.2 V) during the first charge process, leading to high‐energy‐density cathode materials for SIBs. Subsequently, they show a large voltage hysteresis with loss of the plateau in the following discharge process, resulting in poor energy efficiency.16 On 2−/On− are observed upon cycling; and, how to innovate the unstable cycle retention arising from the anion‐redox‐induced two‐phase reaction lacks systematic studies so far. In addition, an in‐depth understanding of the two‐phase reaction is highly required to use the full potential of anion‐redox features because the unmixing reaction accompanied by huge structural variations for SIBs is a dominantly deteriorative point to the electrochemical cycle performance. Therefore, in this work, various experimental analyses combined with first‐principle calculations were performed to provide a comprehensive understanding of the anion‐redox‐induced two‐phase reaction at the high‐voltage (≈4.2 V vs Na+/Na) in the P2‐type Na0.60Li0.20Mn0.80O2 (NLMO). In addition, this study suggests rational design principles for enabling a practical use with a stable cycle feature of the Mn‐based layered oxide for SIBs. Our electrochemical charge–discharge profiles of NLMO unambiguously show the highly reversibility of two‐phase reaction induced by the anionic redox reaction of lattice O2−/On− at the high‐voltage. Using in situ synchrotron X‐ray diffraction (XRD) measurement, we clarify the unmixing phase reactions exhibiting the newly discovered Z phase evolution resulting from the original P2‐phase of NLMO during the charge/discharge process, and discovery the involvement of anionic redox reactions to compensate the charge‐imbalance caused by the Na‐extraction in the compound through ex situ soft X‐ray absorption spectroscopy (XAS) and X‐ray photoelectron spectroscopy (XPS). From the first‐principle calculation results, the detailed qualitative and quantitative calculations of Mn 3d‐electron and O 2p‐electron theoretically explain the oxygen redox mechanism without the further oxidation of Mn4+ to Mn5+ in the high‐redox‐potential of NLMO. Furthermore, the thermodynamic mixing entalpy energy clearly shows the distinct two‐phase reaction with the calculated desodiation potential during the charge process. Considering our concrete understandings on the anion‐redox‐induced unmixing reaction of Na‐rich and Na‐poor phases, reducing the degree of phase separation through the increase of vacancy‐solubility during the two‐phase reaction, and alleviating the volume shrinkage accompanied by the lattice mismatch between the separated phases are suggested to rationally use the highly reversible anion‐based redox reaction featuring high‐energy‐density coupled with stable cycle retention performance in Mn‐based layerd cathode materials for SIBs.Although the reversible anion‐based redox reactions with the small voltage polarization at the high‐voltage range during the first charge/discharge process have successfully occurred, severe decreases of the initial large anionic capacity originating from O22.1xMnO2 (S. G. P63/mmc) as shown in Figure S1a, Supporting Information. The lattice parameters of NLMO are refined as a = 2.8492(1) and c = 11.1372(6). The corresponding P2‐type model is illustrated in Figure S1b, Supporting Information, where P indicates that Na ions are located at the prismatic sites between the edge‐sharing MnO2 layers, and the following number, 2, means that two MnO2 layers (AB and BA layers) are repeated along the c‐axis. In addition, the minor Bragg peaks at 12.8°, 20.0°, 21.3°, 26.6°, and 29.1°, which are not originated from the P2‐type structure, are consistent with peaks of the ribbon‐type Li/Mn ordering model (S. G. P21/c) proposed in the previous report. The atomic composition of NLMO was evaluated as 0.594: 0.194: 0.806 (Na: Li: Mn) by using ICP‐AES. Figure S2, Supporting Information, presents the field emission scanning electron microscopy (FE‐SEM) image of synthesized powder, in which the sample has a homogeneous atomic distribution of Na, O, and Mn without the aggregation of the elements. The galvanostatic charge/discharge profiles of NLMO are shown in Figure+ extraction. The initial charge capacity of NLMO up to 4.4 V is 105 mAh g−1, which corresponds to the extraction of approximately 0.36 Na+ per formula unit from its lattice. During the following discharge process, the high‐voltage plateau was reversibly maintained until ≈0.25 Na+ ions per formula unit (74 mAh g−1) were inserted into its lattice between 3.5 and 4.4 V. Since further oxidation of Mn4+ are not feasible at octahedral symmetry, the high voltage plateau is expected from reversible oxygen redox reaction. The corresponding charge compensation mechanism will be discussed later section. When discharged to 2 V, the Na+ content of NLMO returned to 0.6, which is identical to the initial sodium content. Moreover, NLMO exhibits a mild sloped profile between 2 and 1.5 V for additional Na+ insertion (0.2 Na+ per formula unit), which is expected to be associated with Mn4+ reduction.P2‐type NLMO powder was synthesized via a solid‐state reaction. The major Bragg peaks of NLMO are clearly indexed as a P2‐type layered Nas report.24 The2.2Figure+ extraction is characterized by a low‐crystalline structure derived from a considerable amount of stacking faults, reflected as very broad peaks. In particular, the peak broadening caused by stacking faults is more pronounced in the (002) diffraction peak than in the (100) diffraction peak, indicating relatively well‐preserved TMO2 planes and a severe loss of P2‐type stacking. This structural feature of the new phase is crystallographically similar to the highly desodiated P2‐type layered oxide materials referred to as the “Z” phase. Therefore, we will refer to this new phase as the Z phase for simplicity.To investigate the detailed structural evolution of NLMO upon a galvanostatic charge/discharge test, in situ synchrotron XRD measurement was carried out during the first cycle, as shown in  This migration is further confirmed by the evolution of X‐ray absorption near edge structure (XANES) Mn pre‐edge peaks during charge . In addition, the new (100) diffraction peaks of the Z phase were split into two peaks at 27.0° and 27.2° with smaller d‐spacing values than that of NLMO, indicating that the Z phase also contracted along the ab plane , NLMO shows solid‐solution behavior as can be seen in Figure In Figure 25 Thie Figure . When di2.3Figure2,3‐edge are relatively intense and very sensitive to the oxidation state of the metal owing to the electric dipole‐allowed 2p → 3d transition. Figure 2,3‐edge spectra measured at various charged and discharged states corresponding to the points in Figure 3‐edge at 643.1 eV and L2‐edge at 653.8 eV, are caused by the respective electronic transitions from the 2p3/2 and 2p1/2 core levels to an unoccupied 3d level. Moreover, the split L3‐edge feature of pristine state is highly consistent with that of MnO2 in a previous report. The peak position of the Mn L2,3‐edge did not change throughout the first charge process, indicating that the tetravalent Mn4+ of the pristine state did not contribute to the charge compensation due to the difficulty in further oxidation of Mn4+ at the octahedral site. In addition, during the subsequent discharge process, this oxidation state was almost constant until the end of the oxygen redox plateau  on the lattice oxygen of the crystalline network.To confirm the charge compensation mechanism of NLMO, ex situ soft XAS (sXAS) at the Mn L‐edge spectra and Ar etching assisted ex situ XPS at the O 1s spectra were employed for cationic and anionic redox couple, respectively Figure3. Alth2O3. Thus, in this region, the reduction reaction from Mn4+ to Mn3+ contributed to the charge compensation. These results are well consistent with in situ Mn K‐edge XANES results, in which the Mn K‐edge spectra gradually shifted to a lower energy level after the high voltage plateau (from No. 9 to No. 12), as shown in Figure S3.However, NLMO exhibited a significant shift in its peak position to a lower energy level when discharged at 2 V, and this was accompanied by a change in the peak shape, which has no split feature similar to Mn2O3.29 Thu Figure S4 illustrates the enlarged pre‐edge region corresponding to the Mn K‐edge XANES spectra of pristine, charged (4.4 V), and discharged (1.5 V) states of NLMO. The pre‐edge peaks in the Mn K‐edge spectra consist of two distinct peaks at 6,541 eV due to the electric transition from the 1s orbital to the t2g orbitals, and 6,543 eV due to the electric transition from the 1s orbitals to the eg orbitals. During the charge process of NLMO, the intensity of the pre‐edge peaks increased, as shown in Figure S4. This increased intensity at high voltage supports the migration of a fraction of Mn ions in the TM layer into the tetrahedral sites of the Na layer. During the subsequent discharge process, the pre‐peak intensity again decreased at 1.5 V. However, the pre‐peak intensity did not completely return to its original state. These results indicate that the metal migration is only partially reversible during the two‐phase reaction and the residual Mn ions remains at the tetrahedral sites.Additionally, in the K‐edge XANES spectrum of transition metals, the pre‐edge peaks are strongly related to the coordination number; the four‐coordinate state has a larger pre‐peak intensity than the six‐coordinate state.30 Fig2− anions from the crystalline network, whereas other peaks at higher BEs are ascribed to contamination by surface oxygen deposits. Upon charging, new three peaks were evolved besides the lattice oxygen (O2−) peak. The two major peaks at 532.3 and 533.8 eV are attributed to CEI components, which correspond to carbonates/O—C=O and C—O in O—C=O, respectively. Another minor peak at 531.1 eV was introduced to account for the asymmetry of the lattice oxygen peak, which has an increased BE of ≈1 eV compared to that of the lattice oxygen peak. This extra peak is attributed to the oxidized oxygen On− (n < 2) formed by the oxidation of lattice oxygen during charging at high voltage, according to the previous reports. From these observations, it is confirmed that the lattice oxygen of NLMO solely contributes the charge compensation upon charging without any participation of the Mn ion oxidation. The relative intensity of the On− peak gradually increased up to the end of charging as compared to the lattice oxygen (O2−) peak. Subsequently, the intensity of On− peak gradually decreased over the high‐voltage plateau region where discharging proceeded, indicating that oxygen reversibly reduced during discharge. Therefore, the charge compensation of NLMO is contributed by the On−/O2− redox reaction in the high‐voltage plateau region and Mn4+/Mn3+ redox reaction in the sloped region, respectively.To examine the contribution of oxygen redox to the charge compensation of NLMO, we carried out ex situ XPS at the O 1s region, as shown in Figure ectively.31 Ano2.4Figurex) in NLMO over the charge range (0.375 ≤ x ≤ 1.0). Based on the crystal field theory (CFT), Figure x = 0.375 presents a conventional charge order of Mn with the oxidation state of 4+ at high‐spin states (t2g3eg0), similar with the pristine NLMO sample. In general, a further oxidation of Mn4+ to Mn5+ is not preferential at the octahedral site coordinated with six oxygen in the perspective of energetics in CFT; therefore, the stabilized electronic configuration of Mn4+ predicts that the redox activity from NLMO would be generated by the anionic redox reaction of O2−/On−, because some O 2p‐electron are located in higher energy states as compared with the stabilized Mn 3d‐electron in lower energy states. Figure 4+ electronic configuration, whereas an oxidation behavior of O 2p‐electron is observed over the charge range. These qualitative analyses imply that the anion species participate in the compensation of charge‐imbalance when Na is extracted. For a quantitatively direct investigation of the redox mechanism, we calculated the net charges, calculated by the Bader charge method, of Mn and O during the Na‐extraction  in NLMO over the full range for 0.0 ≤ x ≤ 1.0 so that it is to investigate in detail a thermodynamic phase‐stability during the Na‐extraction  For a better understanding, the lowest‐energy phases at each ratio of □/Na were determined to be ground  and pseudoground  states, as depicted in Figure x = 0.5 and x = 0.875 in NLMO indicating a distinct two‐phase reaction, which is consistent with the experimentally observed two‐phase XRD pattern and electrochemical charge profile. In the viewpoint of thermodynamics, a □‐solubility into NLMO decreases for 0.5 ≤ x ≤ 0.875; that is, a relative phase‐stability NLMO decreases with varying □ content (x) during the charge process. These thermodynamic phase‐instability originate from the anion‐based redox reaction of O2−/On− when Na is extracted from the oxides in the charge range. In addition, a further Na‐extraction from x = 0.875 to x = 1.0 leads to the thermodynamically drastic decrease of phase‐stability resulting in significant irreversibility of capacity, and voltage‐drop in the first cycle, which is not favorable region to gain electrochemical high‐performance upon cycling. Using the thermodynamic mixing enthalpy values with varying □ content (x), we calculated desodiation potentials of NLMO during the charge process /Na atomic configurations as a function of □ content  for NLMO as a promising cathode material for SIBs, the reversible capacity of two‐phase reaction originating from the anion‐based reaction of O2−/On− gradually decreases upon charge/discharge cycling. Thus, despite the stable capacity retention of NLMO, it suffers from a gradual voltage decay accompanied by a reduction of high voltage plateau resulting in a sloped discharge curve similar to various anionic redox active layered material, which would be correlated with the gradual structural rearrangement due to the accumulation of Mn ions at the tetrahedral of Na layer during the subsequent cycles. Therefore, clarified insights are required to enable high‐energy‐density with a cycle stability of NLMO upon cycling. First, from the perspective of thermodynamics, the two‐phase reaction in the high‐voltage region generates a severe phase separation into an electrochemically active Na‐rich and inactive Na‐poor phases owing to the thermodynamic instability in a spinodal decomposition region. Second, from the chemomechanics viewpoint, a drastic volumetric shrinkage accompanied by the thermodynamic phase separation would result in inter‐ and intra‐granular cracks derived from mismatch strains between separated phases and an acceleration of the evolution of the phase separation reaction. Finally, for a rational use of the highly reversible two‐phase reaction, occurring the high‐voltage at ≈4.2 V (vs Na+/Na), induced by the anion‐redox‐reaction upon cycling, we suggest that A‐pillared NLMO cathode materials  exhibiting i) a reduction of the degree of phase separation based on the increase of □‐solubility into the pillared layered oxide and ii) a physical suppression of the extreme structural deformation upon cycling.Although our experimental and theoretical findings have showed the highly reversible two‐phase reaction induced by the anionic redox reaction in the high‐voltage (≈ 4.2 V vs Na3+/Na) in NLMO cathode material was explored using various experimental analyses such as in situ synchrotron XRD, ex situ soft XAS, and XPS and first‐principle calculations considering cation‐anion‐combined electronic structures and thermodynamics. The electrochemical charge‐discharge profile of P2‐type NLMO showed the preserved high‐potential plateau during the charge and subsequent discharge processes, which was understood by the measurements of in situ synchrotron XRD patterns presenting the newly evolved Z‐phase (Na‐poor) coexisting with the original P2‐phase (Na‐rich) in the structural perspective. From the various spectroscopic investigations, the anionic redox reaction induced by the lattice O2−/On− during the thermodynamic unmixing reaction in the oxide was confirmed without any further oxidation of Mn4+ to Mn5+. These experimental results were in detail elucidated by the combined electronic structures of Mn 3d‐electron and O 2p‐electron based on the energetics of CFT, the thermodynamic phase‐stability considering the binary mixture of fully sodated and desodiated phases, and the theoretically calculated desodiation potentials. In spite of the surprising reversibility of oxygen redox activity exhibiting the high (de)sodiation potential for high‐energy‐density cathode materials, the two‐phase reaction induced by the oxygen‐redox showed the drastic decrease of reversible anion‐capacity upon cycling. Therefore, we believe that a breakthrough approach to maintain the reversible anion‐capacity with substantial cyclability may be easily found from the thermodyanamic and chemomechanical perspectives such as i) lowering a spinodal decomposition barrier closely related to the vacancy‐solubility and ii) reducing the severe volumetric strain coupled with the residual strain caused by the separated phases for Mn‐based layered cathode materials in SIBs. Furthermore, this work illustrates an intriguing material design concept for the direct utilization of the high‐voltage redox plateau in anionic redox‐active layered compounds for rechargeable battery research fields.The highly reversible anion‐redox‐induced two‐phase reaction operating at the high voltage , Li2CO3 (Alfa Aesar), and Mn2O3 (Sigma Aldrich) as the metallic precursors. Accurate stoichiometric amounts of the precursor powders, without excess Na or Li sources, were homogeneously mixed via ball milling with acetone. After drying at 65 °C for 2 h, the powder mixture was heat‐treated in air at 900 °C for 10 h and was naturally allowed to cool to room temperature. To avoid unfavorable side reactions with the ambient atmosphere and moisture, the synthesized powder was transferred to an argon‐filled glove box after the heat treatment.P2‐type Naα radiation (λ = 1.5406 Å) in the 2ϑ range from 10° to 100°. The Rietveld refinement of XRD pattern was carried out using general structure analysis system software. The schematic illustration of P2‐type layered structure was obtained using visualization for electronic and structural analysis software. Inductively coupled plasma atomic emission spectroscopy (ICP‐AES) was performed to obtain the atomic composition of each sample using OPTIMA 8300 . FE‐SEM images were collected using AURIGA .XRD patterns of the sample powders were collected using a SmartLab  with Cu Ksoftware.34 The0.6Li0.2Mn0.8O2 was prepared by homogeneously mixing the active material powder, Super P, and polyvinylidene fluoride in N‐methylpyrrolidone in a weight ratio of 7:2:1. This slurry was cast on an Al foil and dried at 120 °C for 10 h in a vacuum. The thoroughly dried composite electrode was roll‐pressed and transferred to the argon‐filled glove box. For the electrochemical evaluations, 2032‐type coin cells were assembled, in which a single sodium metal disk (Sigma Aldrich) was used as the counter and reference electrodes, and 1 m NaClO4 in ethylene carbonate (EC)/propylene carbonate (PC) (1:1 v/v) with 2 wt% fluoroethylene carbonate as an additive was used as the electrolyte. Glass fiber (Whatman GF‐A) was used as a separator. Galvanostatic charge/discharge cycles at various current densities were performed at 25 °C on a WBCS3000S battery cycler . The cells were cycled at a current density of 10 mA g−1 between 1.5 and 4.4 V (vs Na+/Na). All the voltage units (V) in this work indicate the measured voltages using sodium metal as the counter electrode (V vs Na+/Na).Slurry containing the synthesized P2‐type Na0.60Li0.20Mn0.80O2. XRD patterns were obtained at intervals of approximately 5 min. The 2D diffraction images were radially integrated using Fit2D software.In situ synchrotron XRD measurement was performed on the beamline 3D at Pohang Light Source II (PLS II), Korea. To use the transmission measurement mode, a customized 2032‐type coin cell with a hole in the center was employed. Data were collected on a Mar 345 image plate  with incident wavelength of 1.1273 Å for P2‐type Nasoftware.36 Li2MnO3, Mn2O3, and MnO2 powders were used as references. Ex situ soft X‐ray absorption spectroscopy (ex situ sXAS) measurements  were carried out on beamline 4D at PLS II. After careful disassembly of the P2‐type Na0.6Li0.2Mn0.8O2 cells with different charged/discharged states in an argon‐filled glove box, the electrodes were rinsed with a pure propylene carbonate solvent several times over to remove any residual Na salts and dried in a vacuum chamber of the glove box. To avoid exposure to air, an argon‐filled laboratory‐made vessel was used for transferring the samples into the ultra‐high‐vacuum (<10−9 Torr) chamber of the synchrotron X‐ray equipment. The absorption spectra were collected with the PEY mode, which has a probing depth of ≈1 nm.In situ X‐ray absorption spectroscopy (in situ XAS) measurement was performed on the beamline 8C at Pohang Light Source II (PLS II), Korea. The incident beam was tapered by an in‐vacuum undulator, and a Si(111) double‐crystal monochromator was used to eliminate higher‐order harmonics. Similar to the in situ synchrotron XRD measurements, the customized 2032‐type coin cell was used for applying a galvanostatic charge/discharge test. Mn K‐edge spectra were collected under the transmission mode using gas ionization chambers to measure the incident and transmitted X‐ray intensities, and a Mn foil was used as an internal reference for the photon energy calibration. Data analysis was carried out using an Athena software package.37 Li2α (1486.8 eV) as the X‐ray source. Samples of the P2‐type Na0.6Li0.2Mn0.8O2 cells for XPS measurements were prepared in the same manner with ex situ sXAS. To avoid air exposure, coin cells were disassembled in Ar‐filled glove box, which was directly connected to XPS sample chamber. Each sample was analyzed after etching using Ar ion beam for about 60 s. Peaks were recorded with a constant pass energy of 20 eV. The binding energy scale was calibrated using the C 1s core peak at 284.6 eV from the Super P, which was used in the preparation of the electrodes. XPS O 1s spectra were analyzed using a Shirley‐type background and 70% Gaussian/30% Lorentzian line shapes. The peak positions and areas were optimized by a weighted least‐squares method.XPS measurements were carried out using Sigma Probe  with Al K To more accurately describe the Na+, the Na pseudopotential including one 3s‐ and six 2p‐electrons and the standard Mn potential were used. In the perspective of typical computational parameters, the Monkhorst‐Pack approach was covered to sample the k‐points in the reciprocal space with 4 × 4 × 2 meshes; in addition, a cut‐off energy of 400 eV was dealt in all calculations. To obtain the thermodynamic energies and the electronic structures in the ground states, the cell parameters and atomic coordinates were fully relaxed. The thermodynamic formation energy of mixing enthalpy considering binary mixtures of the fully (de)sodiated phases and the equilibrium average voltage during desodiation were calculated on the basis of the well‐developed model.Based on a plane wave set with projector augmented wave pseudopotentials, a density functional theory method implemented in the Vienna ab initio simulation package was employed to conduct all theoretical calculations. The generalized gradient approximation utilizing the Perdew–Burke–Ernzerhof model was used for the exchange‐correlation functional with spin‐polarized conditions. In light of the strong interaction between the 3d‐band of transition metals and the 2p‐band of ligands in crystal frameworks, a Hubbard‐type U correction applied to the pseudopotentials in all calculations was used, and the corresponding value of Mn was taken from previous reports.38 To The authors declare no conflict of interest.Supporting InformationClick here for additional data file.


We used CRF1 and CRF2 receptor transfected Cos7 cells to assess the ability of CRF and urocortin (UCN) peptides to activate cAMP, inositol monophosphate (IP1), and extracellular signal‐regulated kinase 1/2 signaling and determined the ability of antagonists to block agonist‐stimulated cAMP and IP1 accumulation. The ability of RAMPs to interact with CRF receptors was also examined. At the CRF1 receptor, CRF and UCN1 activated signaling in the same manner. However, at the CRF2 receptor, UCN1 and UCN2 displayed similar signaling profiles, whereas CRF and UCN3 displayed bias away from IP1 accumulation over cAMP. The antagonist potency was dependent on the receptor, agonist, and signaling pathway. CRF1 and CRF2 receptors had no effect on RAMP1 or RAMP2 surface expression. The presence of biased agonism and agonist‐dependent antagonism at the CRF receptors offers new avenues for developing drugs tailored to activate a specific signaling pathway or block a specific agonist. Our findings suggest that the already complex CRF receptor pharmacology may be underappreciated and requires further investigation.The corticotropin‐releasing factor (CRF) receptors represent potential drug targets for the treatment of anxiety, stress, and other disorders. However, it is not known if endogenous CRF receptor agonists display biased signaling, how effective CRF receptor antagonists are at blocking different agonists and signaling pathways or how receptor activity‐modifying proteins (RAMPs) effect these processes. This study aimed to address this by investigating agonist and antagonist action at CRF CLRcalcitonin receptor‐like receptorCRFcorticotropin releasing factorCTRcalcitonin receptorERK1/2extracellular signal‐regulated kinase 1/2GPCRG protein‐coupled receptor1IPinositol monophosphateRAMPreceptor activity‐modifying proteinUCNurocortin1The neuropeptide, corticotropin releasing factor (CRF) is a member of the secretin peptide family.CRF is best characterized for its roles in stress and anxiety.1 and CRF2. The CRF1 receptor binds CRF and UCN1 with high affinity, whereas the CRF2 receptor can bind CRF, UCN1, UCN2, and UCN3 with high affinity.1 antagonists which have been explored in clinical trials to treat anxiety, depression and addiction.1 receptor antagonists are reportedly safe and clinical trials are ongoing, whereas other CRF receptor antagonists have been discontinued due to a lack of efficacy.1 and CRF2 receptors have functional splice variants and have been reported to form heterodimers with a receptor activity‐modifying protein (RAMP).The CRF peptide family can bind two G protein‐coupled receptors (GPCRs); CRFThe activation or specific inhibition of a discrete signaling pathway can be associated with a biological event, driven by biased ligands. Such molecules can activate the same receptor, giving rise to a different pattern of signaling and potentially different biological outcomes.In order to enable further exploitation of CRF receptor‐mediated signaling pathways in drug discovery, we profiled several signaling pathways in response to the endogenous peptides CRF and the three UCN peptides. Moreover we investigated how effectively CRF receptor antagonists block CRF‐ or UCN1‐stimulated receptor signaling.22.1(9‐41), and astressin2B were purchased from the American Peptide Company. CP‐376,395 was purchased from Tocris Bioscience.Human CRF (CRF) and human urocortin 1 (UCN1) were purchased from the American Peptide Company or Bachem. Human urocortin 2 (UCN2), human urocortin 3 (UCN3), α‐helical CRF2.21α and hCRF2α (GenBank accession numbers AY457172 and AY449734) were obtained from the cDNA Resource Centre (Bloomsburg University) and are referred to as CRF1 and CRF2 in this manuscript. Hemagglutinin (HA) epitope‐tagged human Calcitonin (CT) receptor (CT(a) splice variant) and myc‐tagged human RAMP1 were used as described previously.Plasmids containing hCRF2.32. For transfection, cells were counted  and plated at a density of 20 000 cells per well in 96‐well plates. The cells were transiently transfected 48 hours prior to assaying using polyethylenimine (PEI), as described previously.Cos7, HEK‐293S, and HEK‐293T cells were cultured as described previously.2.41α or hCRF2α receptors was measured using the LANCE cAMP detection assay kit  as described previously.cAMP accumulation in Cos7 cells transfected with hCRF2.5The AlphaLISA Surefire Ultra extracellular signal‐regulated kinase 1/2 (ERK1/2) phosphorylation (pERK1/2) assay kit  was used to measure pERK1/2 in cell lysates after agonist stimulation. For these assays 20% FBS was used as a positive control. Following peptide stimulation at 37°C for 0‐30 minutes with 100 nmol/L agonist (time courses) or 7 minutes with media alone or 1 pmol/L to 1 µmol/L agonist, media was removed and 20 µL of lysis buffer added to each well. Plates were shaken at room temperature for 10 minutes. Each sample was then transferred to a 384‐well optiplate and pERK1/2 measurement performed as per the manufacturer's instructions. Samples were read using an Envision plate reader (PerkinElmer).2.61), a by‐product of IP3 produced after receptor‐mediated Gαq activation in Cos7 and HEK‐293S cells. Briefly, culture media was removed and replaced with DMEM containing 0.1% BSA for 30 minutes at 37°C. Cells were then incubated with media containing 50 mmol/L lithium chloride (LiCl) for an additional 0‐120 minutes (time courses) or 90 minutes at 37°C in the presence or absence of an antagonist. Agonist concentrations of 1 pmol/L to 10 µmol/L were used for agonist only assays or 10 pmol/L to 10 µmol/L concentrations for antagonist experiments. After cell stimulation, 14 µL of stimulation buffer was added to each well. Three microlitres of each detection antibody were added in turn to the plate and incubated at room temperature for 1 hour. Fifteen microliters of sample was then transferred to a 384‐well optiplate and read by an Envision plate reader (PerkinElmer). IP1 concentrations were calculated from a standard curve generated in duplicate.The IP‐one Gq assay kit (Cisbio) was used to quantify accumulated myo‐inositol‐1‐phosphate  was included on each assay plate in each independent experiment. The requirement for multiple concentrations of agonist/antagonist to be made‐up by a single operator for individual assays resulted in blinding not being feasible. All group sizes were designed to be equal at n = 5 independent experiments. However, when F tests performed on individual experiments indicated that a single curve could fit to both agonist and antagonist curves or no agonist concentration‐response curve could be fitted to the data, neither pBK nor pEC50 values could be determined, respectively. Therefore, no statistical comparisons were performed and experiments were curtailed at n = 3‐4 individual experiments. For antagonism of UCN1‐mediated IP1 accumulation by CP‐376,395 at the CRF2 receptor, one additional experiment was performed. All data were plotted and analyzed using GraphPad Prism 6.0 or 7.0 (GraphPad Software Inc). Data points are the mean ± standard error of the mean (SEM) from n separate experiments, combined.In all experiments the position of peptides and antagonists (pharmacology) or transfected receptors (ELISAs) on assay plates was randomized during each independent experiment, which are independent biological replicates. In all cases, duplicate, triplicate, or quadruplicate technical replicates were conducted for each independent experiment. For transfected cell experiments, each independent experiment involved plating cells from a distinct passage, separate transient transfection and separate signaling or ELISA assays, constituting experimental n. For ERK1/2 and IP2.9F tests were performed to determine if the Hill slope was significantly from one (GraphPad Prism). When the Hill slope was not significantly different from one the curves were constrained to one and pEC50 values obtained. When the Hill slope was significantly different from one, this parameter was unconstrained. To combine the data, maximal responses (Emax) were determined and the data expressed as a percentage of the Emax obtained for matched UCN1 on the same assay plate. For pERK1/2 time course assays, the data were normalized to the response from 20% FBS conducted in parallel. Data normalization was necessary due to variation introduced by transient receptor transfection.For agonist signaling assays data were fitted with a four‐parameter logistic equation. 2.10Operational Model for Bias, GraphPad Prism 7.0 was used to determine Log(τ/KA). The Hill slope was determined using the procedure outlined for agonist assays. Thus, the Hill slope was constrained to one for cAMP and IP1 assays and unconstrained for pERK1/2, the Emax parameter was shared or set to 100 if the fit was ambiguous, to reflect the reference ligand response. ΔLog(τ/KA) was then calculated by subtracting the reference ligand Log(τ/KA) from each test ligand Log(τ/Ka). ΔΔLog(τ/KA) ratios were determined by comparing the ΔLog(τ/KA) for each signaling pathway to the reference signaling pathway (cAMP). The bias factor for each ligand was defined as the inverse log of the ΔΔLog(τ/KA) for a given ligand.Agonist signaling bias was calculated as published previously.2.11BK antagonist potency values were calculated using pEC50 values from concentration response curves of agonist alone, or agonist in the presence of one or three different antagonist concentrations. Initially, F tests were performed to determine if both the agonist alone and agonist in the presence of antagonist data sets could be fitted using a single curve. When a single curve did not fit all data sets, pBK values were calculated. When the Emax in the presence of antagonist was not significantly different from the agonist alone curve (F test), the data were analyzed using global Schild analysis for competitive antagonists (Graphpad Prism). F tests were then performed to determine if the Schild slope was significantly from one. When the Schild slope was not significantly different from one, this parameter was constrained to one and antagonist pBK values were obtained. When the Emax in the presence of antagonist was significantly different from the agonist alone curve (F test), the method of Gaddum for an insurmountable or non‐competitive antagonist was used to determine antagonist potency.25 and EC75 for antagonist curves were plotted on a double reciprocal plot to create a linear regression. The resulting slope was then used to calculate the antagonist BK when substituted into the equation BK = [B]/(slope − 1)  curve and Emin was set to 0%. When the Hill slope of the control curve was equal to one, n was set to one. All other parameters were shared between all data sets. The β value was constrained to 0 when initial fits reported an ambiguous value which was near 0. The CRF2 data sets used a single antagonist concentration and therefore could not be fitted to the operational model of allosterism.For antagonist assays, p2.12To compare the cell surface expression of RAMP1 and 2 between receptors, the data were normalized to the maximum surface expression generated by CLR and RAMP1 or 2 because CLR gives reproducibly high surface expression of both RAMP1 and RAMP2.2.1350 and pBK values were averaged from separate biological replicates  to generate mean values. For signaling data, pEC50 and pBK which are log values and assumed to be normally distributed, significant differences were determined using parametric tests. When two values were compared, an un‐paired two‐tailed Student's t test was used. When more than two values were compared, a one‐way ANOVA with post hoc Dunnett's test was used. For cell surface expression of RAMP1 and RAMP2 (ELISAs), the mean normalized surface expression from individual experiments were combined. Significant differences were determined using one‐way ANOVA with post hoc Dunnett's test. In all cases statistical significance was defined as P < .05.The data and statistical analysis comply with the recommendations on experimental design and analysis in pharmacology.33.11 and CRF2 receptors was characterized in transiently transfected cells by determining the ability of their endogenous ligands to activate different intracellular signaling pathways. We selected three signaling molecules for interrogation; cAMP, IP3 (via IP1) and ERK1/2. cAMP and IP1 are important signaling molecules for Gαs and Gαq signaling, respectively, and pERK1/2 is reportedly important for downstream effects of CRF.1 accumulation to conduct concentration‐response experiments at CRF receptors. The data suggested that in response to CRF and UCN1, 7 minutes for pERK1/2 and 90 minutes for IP1 accumulation were the optimal time points for both receptors  method1 accumulation relative to UCN1 at the CRF1 receptor.Concentration‐response experiments at the CRFn Figure ; Table 1Table τ/KA method1 receptor, the CRF2 receptor displayed greater variation in ligand responses between pathways. CRF, UCN1, UCN2, and UCN3 all produced concentration‐dependent increases in cAMP, pERK1/2, and IP1 accumulation at this receptor .In comparison to the CRF3.21 accumulation by CRF and UCN3 relative to UCN1 at the CRF2 receptor indicated that the activation of these receptors is more complex than is currently appreciated. To further understand CRF receptor signaling behavior, the ability of antagonists to block CRF, and in some experiments, UCN1‐mediated cAMP and IP1 accumulation at CRF receptors were investigated. Three antagonists were selected; α‐helical CRF(9‐41), astressin2B, and CP‐376,395.50 format assays. Although these types of assays give a snapshot of antagonist activity, they do not have the depth of Schild analysis, which can highlight additional molecule behavior, such as partial agonism and insurmountable antagonism. Thus, where possible, we elected to undertake Schild‐style analysis.Overall, the signaling behavior and identification of biased signaling for cAMP over IP3.3(9‐41), has been reported as a competitive antagonist of CRF and UCN1 at both CRF1 and CRF2 receptors.(9‐41) at the CRF1 receptor  also weakly stimulated cAMP accumulation with an Emax of 14.8% indicating that it can act as a weak partial agonist of this receptor  at the CRF1 receptor has previously been reported.(9‐41), global Schild analysis fitted the data well. The Schild slope was not significantly different from one and was therefore constrained to one. α‐helical CRF(9‐41) antagonized CRF at the CRF1 receptor with a pBK of 6.77  was approximately 10‐fold more potent at antagonizing CRF‐induced cAMP accumulation at the CRF2 receptor, compared to the CRF1 receptor  at the CRF2 receptor  antagonized CRF at the CRF2 receptor with a pBK of 7.73   than UCN1‐mediated (pBK of 8.44) cAMP signaling  and astressin2B, which are larger peptide antagonists, CP‐376,395 displayed no evidence of partial agonism. CP‐376,395 effectively antagonized both CRF and UCN1‐mediated cAMP accumulation at the CRF1 receptor  than UCN1‐mediated (pBK of 5.34) cAMP accumulation  than UCN1 (α of 0.03) activity. The allosteric model suggested that CP‐376,395 had a small effect on agonist efficacy for CRF (β of 0.63) and UCN1 (β of 0.63). Conversely, F tests performed on the non‐linear fits indicated that there was no significant difference in Emax values. Antagonism of CRF and UCN1‐mediated IP1 accumulation was also examined at the CRF1 receptor  than UCN1‐mediated (pBK of 6.67) IP1 accumulation at the CRF1 receptor  compared to UCN1 (α of 0.15) activity. However due to apparent high negative co‐operatively, β values could not be determined for the effect of CP‐376,395 on either CRF‐ and UCN1‐mediated IP1 accumulation and were assumed to be ~0.CP‐376,395 is a small molecule antagonist reported to be selective for the CRFceptor.9‐1 and astceptor.9‐1 and astd Figure . CP‐376,1 receptor, antagonist activity was compared at the CRF2 receptor. One hundred micromolar CP‐376,395 had no effect on either CRF or UCN1‐mediated cAMP accumulation at the CRF2 receptor  were also examined in parallel. In Cos7 cells, the cell surface expression of myc‐tagged RAMP1 was significantly increased in the presence of the CLR, CTR, and VPAC1 receptors. However, no change in RAMP1 surface expression was observed with CRF1 and CRF2 receptors ‐dependent antagonism and apparent pathway‐dependent non‐competitive antagonism or negative allosteric modulation. The peptide antagonists, α‐helical CRF(9‐41) and astressin2B, displayed similar antagonist potency to previous reports.(9‐41) and astressin2B displayed weak partial agonism at the CRF1 and the CRF2 receptors, respectively. This phenomenon may have resulted in the antagonist determination being inaccurate and potentially over‐estimated. The appearance of partial agonism is perhaps unsurprising as several antagonists derived by truncating the endogenous peptide for class B GPCRs display this property and partial agonism has previously been reported for α‐helical CRF(9‐41) in a receptor dependent manner.Profiling antagonist activity at the CRF2B and CP‐376,395. The results indicated that UCN1 was antagonized less potently when compared to CRF. This relationship was evident for the CRF1 receptor, however, at the CRF2 receptor the weaker potency of CRF and CP‐376,395 resulted in this being difficult to confirm. Most prior studies have used binding assays or functional IC50 style approaches and typically a single agonist to define antagonism and therefore may not have been able to detect such a difference. However, this apparent agonist‐dependent antagonism may have profound implications for drug‐discovery. If complete blockade of UCN1 activity is required for full efficacy of an antagonist drug, then CRF would not be the appropriate agonist to use in screening campaigns as this would over‐estimate effectiveness. Furthermore, this observation may open up opportunities to develop antagonists capable of specifically blocking a CRF receptor mediated physiological response due to one peptide, without altering physiological responses caused by other ligands. Agonist‐dependent effects should be carefully considered for future studies of CRF receptors.In this research, CRF and UCN1 were both utilized to define the antagonist properties of astressinEmax, have been reported for several small molecule CRF1 receptor antagonists.1 receptor, non‐competitive antagonism for the blockade of cAMP accumulation is reportedly directly correlated with a slow dissociation or off‐rate for small molecule antagonists.1 accumulation. We cannot rule out the possibility that this discrepancy is due to CP‐376,395 not reaching binding equilibrium during the time course of the cAMP assay, although a preliminary cAMP antagonism experiment suggested that a 75 minute pre‐incubation did not increase antagonism or result in a reduction of Emax. The differences in antagonist behavior between signaling pathways may be contributed to by the phenomenon of receptor reserve. Relatively high levels of receptor expression can mask a drop in Emax if maximum activity only requires a small proportion of receptors to be activated. It is possible that in transiently transfected Cos7 cells, CRF receptors have weaker efficacy for the activation of IP1 responses compared to cAMP, resulting in cAMP responses potentially being resistant to antagonists that reduce Emax. It is interesting to note that a similar difference was observed for the blockade of UCN1, where antalarmin was a competitive antagonist of Gαs and a non‐competitive antagonist of Gαi activity.Non‐competitive or insurmountable antagonist‐like behaviors which display a reduction in 1β receptor with RAMP2, which enhanced Gαq coupling, resulting in increased Ca2+ mobilization.1α and CRF1β, but not CRF2β, interact with RAMP2 and was supported by prior in vivo data from RAMP2−/− mouse models, which display a weaker plasma ACTH response to CRF.2α receptors. Surprisingly, in the current study, neither CRF1α nor CRF2α increased RAMP1 or RAMP2 cell surface expression in the three cell lines tested. However, the current study is somewhat in agreement with a recent report, where the CRF1 receptor only weakly interacted with RAMP2 and the CRF2 receptor did not interact with either RAMP1 or RAMP2.1 receptor signaling. The reasons for the discrepancy in the effect of CFR1α on RAMP2 between studies is not clear. However, this may relate to differences between the expression level of RAMP2 or CFR1α between studies and the capacity to detect weak or uncommon interactions. We also tested in parallel three other class B GPCRs that had previously been shown to interact with RAMPs; the glucagon, VPAC1 and PAC1 receptors.1 receptor, which is reported to interact with all three RAMPs,1 receptor was reported to translocate all three RAMPs to the cell surface,1 only interacted with RAMP2 or RAMP3.1 receptors, translocation of RAMP1 to the cell surface is more robust than for RAMP2. Inconsistencies between studies have also previously been reported for the glucagon receptor.The most compelling data for RAMP‐GPCR interactions centers on the class B GPCRs; CLR and CTR. CLR is an obligate heterodimer, requiring RAMPs for function and both CLR and CTR have been co‐localized with RAMP1 in rat and human tissues.There is significant unmet clinical need in the treatment of stress and anxiety. Despite setbacks, the CRF receptors remain a tantalizing target for the development of new therapeutics for stress and anxiety and may have utility in other disorders, due to the wealth of information that exists. Our observations of biased agonism and agonist‐dependent antagonism illustrate some of the complexity involved in understanding the CRF receptor family and offer new avenues for developing drugs. New medicines could be tailored to activate a specific signaling pathway or block a specific agonist through a CRF receptor. Based on our findings we propose that the already complex pharmacology associated with the CRF receptors may be underappreciated and requires further investigation.4.1http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY,Key protein targets and ligands in this article are hyperlinked to corresponding entries in The authors declare no conflicts of interest.The study was conceived and designed by CSW and DLH. ZT designed, performed the majority of experiments, and analyzed the data. PW and CSW performed some antagonist experiments. CSW, DLH and ZT drafted the manuscript. All authors approved the manuscript.Fig S1‐S6Click here for additional data file.


The outer shell of the complex is capped by a combination of pyridine molecules and bromide ions. Magnetic data, measured at temperatures as low as 0.4 K and fields up to 35 T, reveal competing antiferromagnetic exchange interactions; DFT calculations showing that the magnitudes of the coupling constants are highly dependent on both the Fe‐O‐Fe angles and Fe−O distances. The simplicity of the synthetic methodology, and the structural similarity between [Fe34], bulk iron oxides, previous FeIII–oxo cages, and polyoxometalates (POMs), hints that much larger molecular FeIII oxides can be made.The dissolution of anhydrous iron bromide in a mixture of pyridine and acetonitrile, in the presence of an organic amine, results in the formation of an [Fe Fe Maiden: An [Fe34] molecular metal oxide consists of an [Fe4] tetrahedron encapsulated in a [Fe18] truncated tetrahedron, face‐capped by four [Fe3] triangles, and is characterised by alternate layers of tetrahedral and octahedral Fe ions linked by oxide and hydroxide anions. There appears to be no chemical reason why such species cannot form, and their synthesis would help bridge the “gap” between the fields of molecular magnetism (where the vast majority of molecules have nuclearities less than twenty) and POM chemistry (where complexes can be an order of magnitude larger), producing species with a myriad of potentially interesting physical properties, with applications in chemistry, physics, materials science, biology and medicine.It is interesting to note the enormous size difference between the largest polyoxometalates (POMs), most commonly constructed from high oxidation state, diamagnetic metal ions,17] complex in particular hints at a potentially successful route to the synthesis of such species. It is made very simply by dissolving anhydrous FeX3  in wet pyridine .III34(μ4‐O)4(μ3‐O)34(μ2‐OH)12Br12(py)18]Br2 (1)  metal ions in the inner tetrahedron and the outer triangles are all tetrahedral and the (eighteen) Fe ions in the truncated tetrahedron are all octahedral. The presence of tetrahedral‐octahedral–tetrahedral “shells” of metal ions is as found in the [Fe13] and [Fe17] complexes  O2− ions . Each face‐capping, oxo‐centred [Fe3] triangle is connected to the [FeIII18] truncated tetrahedron via six μ3‐O2− ions. The remaining twelve μ2‐OH− ions link the metal ions situated on the triangular faces on the truncated tetrahedron. Each of the octahedral metal ions in the [Fe18] truncated tetrahedron and tetrahedral metal ions in the face capping [Fe3] triangles have their coordination geometries completed through the presence of a pyridine (py) molecule and a bromide, respectively. The Br counter anions are associated with the triangular faces of the truncated tetrahedron with Br⋅⋅⋅(μ2‐)O distances in the range 3.21–3.48 Å, and the py C‐atoms on a neighbouring molecule . The other prevalent intermolecular interactions occur between adjacent Br ions and py molecules .Compound by FeIII triangle1 strongly hint at rather large, competing, antiferromagnetic exchange interactions between the Fe centres. The susceptibility data  is well below the Curie constant expected for thirty four uncoupled FeIII ions (150 cm3 K mol−1). As temperature is decreased the value of χT first increases to a broad maximum of ≈75 cm3 K mol−1 centred around T=150 K, before dropping slowly to a value of ≈70 cm3 K mol−1 at T=50 K. Below this temperature the value drops significantly, and is strongly field dependent. Magnetisation (M) data  before first plateauing and then increasing in a more linear fashion to B=7 T where M≈15 μB. This linear like increase is continued in the B=7–35 T field range .Magnetic measurements of a Figure  b show ae Figure  c, whereχT data, we investigated a quantum model where we assume a low‐lying level structure similar to a zero‐field split total spin S=11 with g fixed at g=2.04, as obtained from HFEPR measurements . This effective model fits both χT vs. T and M vs. B . While the absolute numbers in this effective model should be taken with a pinch of salt, they do hint at the presence of non‐neglibile anisotropy. We also expect that the true low‐energy spectrum contains numerous additional  spin states, and in the absence of any out‐of‐phase χ′′ signals in the ac susceptibility, that may not be of perfect easy axis character.A simple explanation of the temperature and field dependence of the magnetic data is not straightforward. Since the cluster is much too large for a quantum calculation in a spin model such as the Heisenberg model we resort to two approximations. Classical Monte Carlo calculations (CMC) of a classical Heisenberg model, that often deliver accurate results in the temperature and field regions where quantum effects are of minor importance, that is, at elevated temperatures compared to the exchange couplings,1 we have employed a DFT methodology (B3LYP/TZVP) known to yield excellent numerical estimates of J values.III ions (J1) connected by μ3‐O2− ions; (ii) inner tetrahedral and outer octahedral FeIII ions (J2) connected by μ3/μ4‐O2− ions; (iii) outer octahedral FeIII ions (J3) connected by μ3‐O2− ions; (iv) outer tetrahedral FeIII ions (J4) connected by μ3‐O2− ions; and (v) outer tetrahedral and outer octahedral FeIII ions (J5) connected by μ3‐O2− ions (Scheme S1). The calculated J values are listed in Table In order to estimate the magnitude of the magnetic exchange interactions in J pairs (Tables S7–S11), which show a direct correlation between the number of orbital interactions and the magnitude of the antiferromagnetic exchange. For example, for J3 only two dominant overlaps (dxy||dxy and dx2-y2||dxy) are detected leading to the smallest calculated J value (−15.7 cm−1), whereas there are seven different, large interactions for J5, resulting in the largest J value (−68.2 cm−1). Note that in the latter, the dxz orbital of the tetrahedral FeIII ion is found to overlap significantly with all the d‐orbitals of the octahedral FeIII ion, with the exception of the dxy orbital.Spin density data are provided in Figures S13–S17 and Tables S2–S6. We have computed the overlap integrals for all 3 produces an [FeIII34] complex, double the size of the cage produced in wet py/MeCN alone, [FeIII17]. The molecule, whose structure describes an [Fe4] tetrahedron encapsulated in a [Fe18] truncated tetrahedron, face‐capped by four [Fe3] triangles, is characterised by alternate layers of tetrahedral and octahedral Fe ions linked by oxide and hydroxide anions. Magnetic measurements reveal relatively strong, competing AF exchange interactions between the FeIII ions, with DFT calculations suggesting a direct correlation between the number of orbital interactions and the magnitude of the AF exchange. The simplicity of the synthetic procedure and the structural similary of [Fe34] to bulk iron oxides such as magnetite and maghemite , and to much larger POMs of high oxidation state, diamagnetic metal ions such as V, W and Mo, hints that a diverse family of novel FeIII molecular metal oxide structures awaits discovery. This, in turn, suggests an exciting route to the bottom‐up formation of molecular metal oxide “nanoparticles” with a raft of potential applications.In conclusion, the addition of an organic amine  to a wet py/MeCN solution of FeBrThe authors declare no conflict of interest.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.


However, the specific mechanisms of action are still under debate and intense effort is ongoing to improve our understanding of the crucial steps involved in the mechanisms of AβOs toxicity. We report the development of a mathematical model describing a proposed mechanism by which stimulation of Phospholipase C (PLC) by iAβO, triggers production of IP3 with consequent abnormal release of Ca2+ from the endoplasmic reticulum (ER) through activation of IP3 receptor (IP3R) Ca2+ channels. After validating the model using experimental data, we quantify the effects of intracellular rise in iAβOs on model solutions. Our model validates a dose-dependent influence of iAβOs on IP3-mediated Ca2+ signaling. We investigate Ca2+ signaling patterns for small and large iAβOs doses and study the role of various parameters on Ca2+ signals. Uncertainty quantification and partial rank correlation coefficients are used to better understand how the model behaves under various parameter regimes. Our model predicts that iAβO alter IP3R sensitivity to IP3 for large doses. Our analysis also shows that the upstream production of IP3 can influence Aβ-driven solution patterns in a dose-dependent manner. Model results illustrate and confirm the detrimental impact of iAβOs on IP3 signaling.Alzheimer’s disease (AD) is a devastating illness affecting over 40 million people worldwide. Intraneuronal rise of amyloid beta in its oligomeric forms (iA In AD, iAβOs are now believed to play a major role in the early phase of the disease as their intracellular rise correlates well with the symptoms of AD [βOs have been found to be predictive of cognitive status at death among patients with AD [2+ elevation with the concomitant increase of iAβOs observed in neurons during the progression of the AD [βOs on the normal functioning of the IP3-signaling pathway has been indicated as a potential mechanism responsible for alteration of the Ca2+ homeostasis in AD neurons.Ca neurons . Accordiechanism . Althougms of AD . More ge with AD . Variousf the AD . Among tβ42Os is responsible for the overproduction of IP3 and consequent rise of cytosolic Ca2+ observed in cells exposed to iAβ42Os [β may cause cytosolic Ca2+ rise by a mixed mechanisms of PLC-dependent and independent manner [β42Os on intracellular Ca2+ fluxes have previously been investigated by developing a computational model to study important intracellular Ca2+ pathways in normal and in iAβ42Os affected conditions [3 production processes were incorporated in the model. Here, we have illustrated a possible mechanistic way for how iAβ42Os triggers IP3 overproduction with consequent rise in cytosolic Ca2+ by including some mechanisms of upstream IP3 production in the model. Specifically, we pinpoint two main possible sites of action for iAβ42Os to interact in the cascade of events resulting from stimulation of G-protein in the plasma membrane to the release of Ca2+ from the ER.We and others have suggested that a G-protein mediated activation of PLC by iA iAβ42Os , 18. Mort manner , 16, 55.nditions . Howeverβ42Os act on IP3Rs in the generation of Aβ-induced Ca2+ signaling events. The results of the model are consistent with this for the small doses parameters. However, the model also suggests that iAβOs may in-fact be directly affecting the IP3Rs when large doses are introduced. The analysis illustrated in 2+ signaling in the presence of iAβ42Os as changes to IP3Rs occur. The persistent increase of iAβ42Os may alter the sensitivity of IP3Rs to Ca2+ over time. For large doses of iAβ42Os, IP3Rs may become more sensitive to low- or sub-threshold IP3 levels and in turn trigger local and global Ca2+ signaling events. The fact that the parameter k−4 appears to play a major role in the decay of observed Ca2+ signals singles out the potential that iAβ42Os do act on the IP3R itself, at least for large doses. Our model suggests the need for further investigation on the relationship between iAβ42Os and the sensitivity of IP3Rs to IP3 levels.In our previous study , we arguβ42Os on IP3 signaling mechanisms that does not necessarily depend on the choice of the IP3R model. When a saturating binding rate model for the IP3R model is used  2+ signaling, our model also provides a way to control for and test various therapeutic strategies in a modeling environment. For example, to mimic the intrinsically slow accumulation of Aβ seen in the pathology of AD, Aβ can be introduced very slowly into the model and solutions simulated accordingly. We can then introduce artificial agonists or antagonists that affect G-protein activation and PLC function to see how they affect Ca2+ signals over various timescales. Using the model to better understand what happens to Ca2+ regulation in these simulations can directly influence and suggest how one could control Ca2+ signaling in the presence of Aβ, and more generally, various AD environments.Because of the complex cross-talk nature of Ca2+ signaling in the presence of increasing concentrations of iAβ42Os. In in vivo environments, it may be the case that in the early phase of AD, slowly accumulating levels of iAβOs remain relatively small. Under such conditions, the small doses model may be better suited than the large doses model. Regardless, our model development and analysis suggests that increasing the amount of iAβ42Os present in the cell can have a pervasive impact on numerous cellular mechanisms.The results of this study suggest the need for two different dose-dependent models to incorporate changes in cellular CaBuilding computational models can help provide a better understanding for the complex cross-talk between various signaling mechanisms within neurons, something difficult to establish with current experimental capabilities. Through further analysis and development, researchers can use the model to formulate novel experimental procedures and eventually suggest new therapies for treating AD.2+ concentration are associated with changes in fluorescence through the equationKD is a dissociation constant, fmax is the intensity of the dye at maximum Ca2+ concentration, Rf = fmax/fmin is the indicator’s dynamic range with fmin being the intensity at minimum Ca2+ concentration, δfmax is the saturation of the Ca2+ indicator, fm = fmax/f0, and c0 is the resting Ca2+ concentration. The values of KD and Rf are associated with attributes of the indicator in a particular cellular environment [KD and the dynamic range of Rf may vary batch to batch and should be estimated using a similar protocol and cellular cytoplasmic domain [According to Maravall et al. (2000) , changesironment  and as sironment . Howeverc domain , 59.δfmax is the key parameter needed for establishing the conversion from fluorescence to Ca2+ concentrations. When we fix the initial Ca2+ concentration, c0, we can estimate δfmax usingKD and Rf are known. In practice, δfmax can be used to estimate the unknown resting Ca2+ concentration by inverting the relationship in In attained  and KD a2+ concentrations only requires obtaining values for fm, Rf, and δfmax during experimental procedure. However, these values are often not reported in favor of the traditional δf fluorescence measurements and extracting them from reported changes in fluorescence ratio, or establishing their values a posteriori, can be challenging. As such, in order to complete a conversion for data given in terms of δf we approximate a number of parameters. Since both KD and Rf depend on indicator properties, they can be approximated for a variety of indicators. Based on the experiment in [KD ≈ 0.2−0.5 μM and that Rf has a dynamic range Rf ≈ 85−100 and note that uncertainties in Rf have minimal affect on δfmax is plotted as a function of KD and Rf when c0 = 0.01 μM and c0 = 0.05 μM in A and B, respectively.With Eqs iment in , we assuRf has little effect on the value of δfmax. This is consistent with the idea that for indicators with a large dynamic range, the exact value of Rf is insignificant [KD is often assumed to be between 0.25 and 0.5 μM but some studies suggest that KD may have much greater range [As can be seen from nificant . For inder range , 61.2+ concentration of c0 = 0.05 μM and set KD = 0.3 μM and Rf = 100. Because we have no previous knowledge for the value of fm, we consider a range fm ≈ 1−100 where the exact value depends on the ratio of the maximal intensity and the resting intensity. Using these values, we plot the corresponding Ca2+ concentrations from the fluorescence data in [fm. β at concentrations of a = 3 μg/ml, a = 10 μg/ml, and a = 30 μg/ml, respectively. In fm ranging from fm = 1 to fm = 100 (black) with n = 11 . The maximum value is also highlighted for each conversion plot (circle) and provides the peak time for the three Aβ levels.Without loss of generality, here we consider a basal Ca data in  for vari2+ concentrations, fm and the range of δfmax between 6 and 20. These three dimensional plots allow us to better understand the impact of the conversion parameters on the maximum values of the fluorescence data in [fm or c0, a true conversion from fluorescence to concentration is elusive. However, in all the profiles illustrated, each conversion does capture the changes in amplitude and latency to peak time observed experimentally as levels of Aβ are increased.To study the impact of the conversion to Ca data in . Again,