The Ir in the SrCo0.9Ir0.1O3-δ catalyst shows an extremely high intrinsic activity as reflected from its high turnover frequency, which is more than two orders of magnitude higher than that of IrO2. During the electrochemical cycling, a surface reconstruction, with Sr and Co leaching, over SrCo0.9Ir0.1O3-δ occurs. Such reconstructed surface region, likely contains a high amount of structural domains with corner-shared and under-coordinated IrOx octahedrons, is responsible for the observed high activity.Exploring robust catalysts for water oxidation in acidic electrolyte is challenging due to the limited material choice. Iridium (Ir) is the only active element with a high resistance to the acid corrosion during water electrolysis. However, Ir is rare, and its large-scale application could only be possible if the intrinsic activity of Ir could be greatly enhanced. Here, a pseudo-cubic SrCo While water splitting could provide a green means to store energy, there are few materials that can sustain high water oxidation half-reaction rates in acidic electrolytes. Here, authors design a perovskite oxide that generates high performance under-coordinated iridium sites during electrocatalysis. However, due to the intermittent availability, the storage of the electric energy by these sources becomes pivotal. The conversion of the electricity into hydrogen fuel is expected to be one of the most efficient storage ways due to the high energy density of hydrogen fuel. Water electrolysis is a promising strategy to realize such energy conversion. The alkaline condition has been popular for industrial water electrolysis, while the acidic electrolyte condition is not. This is mainly due to the limited choices of robust anode catalysts in acidic condition during the oxygen evolution reaction (OER). In fact, the acidic condition offers a high proton concentration and thus enables much faster reaction kinetics of hydrogen production3. An ideal anode catalyst in acid should exhibit both high catalytic activity and high tolerance to the severe acidic corrosion at a high anodic potential. To date, the iridium oxides have been found high in stability in harsh acidic environment5. Due to the high cost and scarcity of Ir, great efforts have been made to develop novel Ir-based materials for water oxidation with high efficiency, e.g., a high activity normalized to the material surface area (intrinsic activity) and, more importantly, a high turnover frequency (TOF) for Ir11.The electricity from renewable energy sources, such as wind and solar power, has been showing a gradual proportional increase in global energy infrastructure15. The perovskite is a type of oxide with a general formula of ABO3, where A represents alkaline-earth-metal or lanthanide and B represents active transition metals. Among these perovskite catalysts, a SrIrO3 perovskite, with reconstructed surface was found with the highest intrinsic activity  for catalyzing OER in acidic environment to date13. Nevertheless, the synthesis of such SrIrO3 catalysts requires special tools or conditions, such as a pulse laser deposition (PLD) equipment16 or a high pressure (~5 GP) for solid state synthesis17, which could hinder the practical application of such catalyst. In addition, the key surface reconstruction step in this PLD-SrIrO3 is likely time-consuming because a ~30 h electrochemical activation process is required.Recently, several Ir-based perovskites were reported as highly active catalysts for OER0.9Ir0.1O3−δ material with an orthorhombic structure. In this design, the Ir and Co co-share the octahedral site and all octahedrons are corner shared. To form a comparison, we investigate a monoclinic SrIrO3 (m-SrIrO3) perovskite, which adopts a highly distorted 6H BaTiO3 structure with mixed corner-shared and face-shared IrO6 octahedrons19. We show that the intrinsic activity (TOF) of Ir from SrCo0.9Ir0.1O3−δ is approximately two orders of magnitude higher than the m-SrIrO3 and more than one order of magnitude higher than the benchmark PLD-SrIrO3. The Sr and Co leach from SrCo0.9Ir0.1O3−δ surface during electrochemical tests. As a result, a highly active surface IrOx layer likely contains a high amount of corner-shared and under-coordinated IrOx octahedrons without long-range ordering, and is responsible for the observed superior activity of SrCo0.9Ir0.1O3−δ.Here, we design a pseudo-cubic SrCo3, pseudo-cubic SrCo0.9Ir0.1O3−δ, and m-SrIrO3. In PLD-SrIrO3 and pseudo-cubic SrCo0.9Ir0.1O3−δ, all IrO6 octahedrons are corner-shared. Nevertheless, in m-SrIrO3, corner-shared and face-shared IrO6 octahedrons are alternatively arranged. Both m-SrIrO3 and SrCo0.9Ir0.1O3−δ samples were synthesized with a classical solid state method, which is easily accessible for large scale preparation. Figure 3 and SrCo0.9Ir0.1O3−δ samples. The corresponding Brunauer–Emmett–Teller (BET) surface area for these two samples are also presented. The SrCo0.9Ir0.1O3−δ has a lower BET surface area due to its relative large particles. A m-SrIrO3 with a space group (SG) of C 2/c and a SrCo0.9Ir0.1O3−δ with SG of P nma are confirmed based on their X-ray diffraction (XRD) patterns  images are taken from white square box regions. The measured interplanar distances from inverse FFT images correspond well with the results from XRD refinement, which are in brackets. Selected area electron diffraction (SAED) patterns are also presented. All patterns can be well indexed based on the XRD refinement results, indicating the formation of m-SrIrO3 and SrCo0.9Ir0.1O3−δ with high purity.Figure 0.9Ir0.1O3−δ, m-SrIrO3 and commercial IrO2 for the OER were then evaluated in a perchloric acid solution . The measured specific activity , representing the intrinsic activity of a catalyst20, is shown in Fig. 3 prepared with PLD method from literature is also presented for comparison13. The calculated specific activity of IrO2 is similar to the previously reported values7. A higher specific activity, approximately one order of magnitude higher than that of IrO2, is obtained from the m-SrIrO3. For instance, at a potential of 1.5 V (vs. RHE), a specific current density of ~1 × 10−2 mA cm−2 and ~0.2 mA cm−2 is recorded for IrO2 and m-SrIrO3, respectively. However, such activity is much inferior to that of the PLD-SrIrO3. Moreover, different from the reported PLD-SrIrO3 catalyst, the m-SrIrO3 did not show a gradually increased activity during the successive test . This activity difference indicates that the pseudo-cubic structure is a key factor to determine the intrinsic activity of SrIrO3. On the other hand, regardless of the low Ir atom ratio in the lattice, the specific activity of SrCo0.9Ir0.1O3−δ is more than one order of magnitude higher than that from m-SrIrO3 and comparable with the reported initial activity of PLD-SrIrO3. Interestingly, such specific activity is just 2–4 times lower than the final specific activity of PLD-SrIrO3, which hints the intrinsic activity of Ir over the surface of SrCo0.9Ir0.1O3−δ is higher than that of Ir over the surface of PLD-SrIrO3. We caution that the Co in SrCo0.9Ir0.1O3−δ does not contribute to the observed superior activity of SrCo0.9Ir0.1O3−δ for its high solubility in the acidic electrolyte . Figure 2, m-SrIrO3, and SrCo0.9Ir0.1O3−δ. Three cases, including Ir at outermost surfaces (upper limit), of Ir from reconstructed surface regions  and all Ir from the bulk (lower limit), were considered for TOF calculations. Calculation details are presented in Methods. Among them, the TOF of Ir by considering the effect of surface reconstruction should be the most reliable for comparing the intrinsic activity of Ir from different materials. From Fig. 0.9Ir0.1O3−δ is found with a high TOF of 2.56 ± 0.15 s−1, which is more than 10 times and ~75 times higher than PLD-SrIrO3 (~0.2 s−1) and m-SrIrO3 (0.034 ± 0.001 s−1), respectively. It confirmed that the observed superior activity of SrCo0.9Ir0.1O3−δ is attributed to the formation of highly active surface Ir species.The activities of SrCo2, it has been reported with a high resistance to acidic corrosion21. However, it is unlikely that m-SrIrO3 and SrCo0.9Ir0.1O3−δ can keep their original structure in strong acidic condition due to the thermodynamic instability of Sr and Co. The surface structure evolution of m-SrIrO3 and SrCo0.9Ir0.1O3−δ is then evaluated by HRTEM. Moreover, since the performance of both SrCo0.9Ir0.1O3−δ and m-SrIrO3 become stable in a few cycles (within 5 cycles), the surface structures of these two materials after 5 cycles were also investigated. The HRTEM images from pristine and cycled m-SrIrO3 are presented in Fig. 3, while a surface reconstruction from cycled m-SrIrO3 is observed with a depth of approximately 5 nm. By comparing the FFT images from bulk and surface, such reconstructed surface is likely amorphous with no long-range order. We caution that nanoparticles (~2 nm) emerge from the bulk of the m-SrIrO3 after a short-term electron beam illumination, and such nanoparticles were determined to be Ir  cycles. The XPS of Sr_3d from the m-SrIrO3 are shown in Fig. d spectra are normalized based on corresponding Ir contents. In Fig. d signal is observed from cycled m-SrIrO3, indicating a Sr leaching during the test. Moreover, although the Sr_3d signal from pristine m-SrIrO3 and cycled m-SrIrO3 can both be fit with two doublets, their profile is totally different. Specifically, both doublets in the pristine m-SrIrO3 should be mainly related to the Sr from m-SrIrO3 lattice as two low binding energy of 130.55 and 131.81 eV for the 3d5/2 peak are obtained24. In addition, a relative larger full width at half maximum (FWHM) for the second doublet (marked in green) indicates it also includes a small amount of surface Sr components, such as SrO, SrCO3, and Sr(OH)2, with higher binding energies (above 133 eV)25. In the cycled m-SrIrO3, the first doublet (marked in yellow) with a low binding energy of 131.15 eV should still be related to lattice Sr. More surface Sr components seem formed because the second doublet shifts to a higher binding energy of 132.62 eV for the 3d5/2 peak. In combination with the HRTEM results, the increased amount of surface Sr components is likely related to the leached bulk Sr, which re-deposits on the electrode surface. Figure d and Co_2p from pristine and cycled SrCo0.9Ir0.1O3−δ samples. The fitting parameters for Sr_3d spectra are listed in Supplementary Table d from pristine SrCo0.9Ir0.1O3−δ sample, only one doublet at 131.83 eV for the 3d5/2 peak is observed, which should be mainly related to lattice Sr. Importantly, a severe Sr leaching is observed from the cycled SrCo0.9Ir0.1O3−δ sample as the Sr_3d signal is almost invisible just after 5 CV cycles (time of duration: ~10 min). Similarly, a fast cobalt leaching is observed from the surface of cycled SrCo0.9Ir0.1O3−δ because the XPS signal of Co_2p from cycled SrCo0.9Ir0.1O3−δ sample is almost invisible. As summarized in Fig. 3. In the reported PLD-SrIrO3 catalyst, approximately 75% Sr is expected to leach from its surface after 30 min of electrochemical OER testing13. Although the leaching degree of Sr from the surface of m-SrIrO3 is slightly lower than that in PLD-SrIrO3, considering the much inferior activity of m-SrIrO3, the initial pseudo-cubic structure with corner-shared IrO6 octahedron is critical for a highly active Ir site after the cation leaching. This corresponds well with the previous calculation results13, which found the reconstructed surface with a structure similar to IrO3 or anatase IrO2 is active to catalyze OER. Different from m- and PLD-SrIrO3, the surface region of SrCo0.9Ir0.1O3−δ loses approximately 92% Sr + Co after 5 CV cycles. This Sr and Co leaching in acid from SrCo0.9Ir0.1O3−δ likely causes the obvious surface reconstruction on SrCo0.9Ir0.1O3−δ. More importantly, we can confirm the formed amorphous surface Ir-related species after Sr and Co leaching are responsible for the measured activity of m-SrIrO3 and SrCo0.9Ir0.1O3−δ.The X-ray photoelectron spectroscopy (XPS) was then performed to study the surface chemical states in m-SrIrO2, m-SrIrO3, and SrCo0.9Ir0.1O3−δ were investigated by XPS to gain more information about the Ir in reconstructed surfaces and the Ir_4f spectra from the three samples before and after electrochemical tests are given in Fig. f spectra from the IrO2 are studied first. Distinctive asymmetric tail can be observed from the peaks in the two spectra and is reported related to the screening response of 5d conduction electrons26. Based on the previous studies, both Ir_4f spectra can be fitted with two doublets and an additional single satellite peak. The fitting results are listed in Supplementary Table f7/2 peak with a binding energy of 61.61 and 61.63 eV is found from the first doublet (marked in yellow) in the pristine IrO2 and the cycled IrO2, respectively. The observed binding energies are within the reported ranges for Ir_4f7/2 peak in IrO2 (61–62 eV). The second wide doublet (marked in green), with an energy shift of approximately 1 eV as compared with the first doublet, should be related to the shake-up satellites of the first doublet27. The spin–orbit splitting in both doublets is 2.98–2.99 eV, which is also close to the reported values29. The additional single satellite at 67.6 eV for pristine IrO2 or at 67.76 eV for the cycled IrO2 is related to the localized non-bonding states28. From the fitting results, the Ir 4f spectra from the IrO2 is almost unchanged after the electrochemical cycling, confirming a highly stable surface structure of IrO2 during OER in acidic environment.Ir from IrOf spectra from the m-SrIrO3 were also fit to explore the change of surface Ir after electrochemical cycling. The fitting parameters are listed in Supplementary Table 3, the Ir_4f7/2 peak from the first doublet located at a low binding energy of 60.33 eV, which is generally considered as the signal of Ir metal30. However, Ir metal is unlikely to exist in the sample as a long-term high temperature calcination is required in the solid state method for sample preparation. Considering the Ir in the m-SrIrO3 is 4+ for charge neutrality, it is rational to define this first doublet comes from Ir4+. Moreover, in the previous studies, the Ir_4f7/2 peak of Ir4+ from PLD-SrIrO3 is found with a binding energy located in between 61.8 and 62.6 eV13. The much lower binding energy observed in the m-SrIrO3 indicates the XPS signal of Ir is strongly related to its local structural environment. The second doublet has a binding energy ~1.4 eV higher than that of the first doublet. This energy shift between two doublets is similar to the cases observed in rutile IrO2 and PLD-SrIrO3, indicating the second doublet should be shake-up satellites. After electrochemical cycling, apparent shifts of the two doublets in the Ir_4f spectra to higher binding energy are observed. The Ir_4f7/2 in the first doublet is at 61.2 eV, which is approximately 0.9 eV higher than that in pristine m-SrIrO3. This moderate energy shift is different from the case observed in PLD-SrIrO3, in which the Ir_4f7/2 in the first doublet is almost unchanged irrespective of the Sr leaching during electrochemical testing13. The Sr leaching in m-SrIrO3 probably influences the chemical state of Ir and thus certain SrxIrOyHz (x < 1) phase(s) may form, considering a high amount of Sr remains in the surface. The Ir_4f7/2 peak from the second doublet has a high binding energy of 63.35 eV, which, however, cannot be solely treated as the shake-up satellites because an energy shift of approximately 2.2 eV, relative to the first doublet, is observed. Such energy shift is much larger than those (1–1.6 eV) observed in rutile IrO2, PLD-SrIrO3 as well as the pristine m-SrIrO3. The unusual energy shift of this second doublet hints the formed surface is very complex and multiple phases can exist.The Ir_4f spectra from the pristine SrCo0.9Ir0.1O3−δ is different from those observed in rutile IrO2 or SrIrO3 phases. The fitting parameters are listed in Supplementary Table f7/2 peak at approximately 59.7 eV for the first doublet. This Ir_4f7/2 peak with a large FWHM likely represents the mixed Ir4/5+ in SrCo0.9Ir0.1O3−δ since a low binding energy is also observed in the pristine m-SrIrO3 phase. The second doublet with a binding energy ~2 eV higher than the first doublet cannot be solely treated as the shake-up satellite. Certain Ir specie(s) from SrCo0.9Ir0.1O3−δ surface can exist, and its identification is beyond the scope of this study. After electrochemical cycling, the Ir_4f spectra from SrCo0.9Ir0.1O3−δ can be well fit with two doublets. A binding energy of 61.0 eV is observed for the Ir_4f7/2 peak in the first doublet. Such peak position is more than 1 eV higher than that in pristine SrCo0.9Ir0.1O3−δ, but ~0.6 eV lower than that in rutile IrO2. The second doublet in cycled SrCo0.9Ir0.1O3−δ shifts ~1.5 eV to higher binding energy, which then can be related to the shake-up satellites. As a result, certain IrOxHy phase may form over the SrCo0.9Ir0.1O3−δ surface as Sr and Co have leached out. Previously, some amorphous IrOx phases have also been prepared for a higher activity than crystallized rutile IrO232. However, the Ir_4f spectra from amorphous IrOx and crystalline rutile IrO2 always have similar peak positions and profiles33. Moreover, the influence of Sr on Ir_4f in XPS can be excluded since there is almost no Sr in the surface region after five cycles , Co2+/3+ (~1 V), and Ir4+/5+ (~1.2 V), can be observed at the first cycle36. In the following cycles, the Co peak disappeared and only the two peaks belonging to Ir become remarkable, which is probably due to the dissolution of Co as well as the formation of IrOxHy species in the surface region. The intensities of the two Ir-related oxidation peaks simultaneously increased, which should be related to the increased degree of surface reconstruction. Interestingly, the surface reconstruction process likely stopped after 5000 cycles since the 5,000th cycle almost overlapped with the 10,000th cycle. The peak at ~1.2 V indicates the oxidation of Ir4+ to Ir5+ is greatly facilitated over the SrCo0.9Ir0.1O3−δ surface. For m-SrIrO3, two oxidation peaks, representing Ir3+/4+ (~0.8 V) and Ir4+/5+ (~1.2 V), can be observed. However, unlike SrCo0.9Ir0.1O3−δ, the redox of Ir3+/Ir4+ is dominant on m-SrIrO3. As for Ir metal, one distinctive oxidation peak at ~1 V is observed after ~1000 cycles, indicating the formation of surface Ir hydrous oxide. Such broad oxidation peak likely due to successive oxidation of Ir3+ to Ir5+35. By comparing the CVs of these three catalysts, one can conclude that they give different Ir-based amorphous phase(s) by cycling. The formation of different Ir-related phase(s) over these catalysts is also indicated by corresponding XPS results . This may also explain the inferior activity of m-SrIrO3 in our study. In fact, for an amorphous IrOx, the strong correlation between activity and local structure (corner-shared vs. edge-shared IrO6 octahedrons) has also been highlighted by Willinger et al.23. Besides, it is reported that certain lattice oxygen atom can be activated in a La2LiIrO6 perovskite. Such activated lattice oxygen atom can participate in the OER and is proposed critical for the observed high activity15. However, as surface reconstruction is observed in our catalysts, the activated lattice oxygen atom from perovskite is unlikely. We hypothesized that the high activity of IrOxHy evolved from SrCo0.9Ir0.1O3−δ due to the strong correlation between the IrOxHy and the initial/bulk lattice of SrCo0.9Ir0.1O3−δ.Till now, several facts have been put forward to explain the high activities measured from Ir-based complex oxides with perovskite or perovskite-related structures0.9Ir0.1O3−δ, first of all, its pseudo-cubic structure with initially corner-shared octahedrons can induce the formation of a local structure optimized IrOxHy phase, which still contains a high ratio of corner-shared IrO6 octahedrons. However, this is insufficient to explain the measured higher activity when compared with that from the reported PLD-SrIrO313. As compared with SrIrO3 and other reported Ir-based perovskites, the SrCo0.9Ir0.1O3−δ here is expected to possess a high amount of oxygen vacancies in the lattice since the SrCoO3−δ matrix is highly oxygen deficient37. Accordingly, the oxygen vacancy formation energies for m-SrIrO3, PLD-SrIrO3, and SrCo0.9Ir0.1O3 were calculated by density functional theory (DFT). Moreover, the O p-band centers of different materials were also calculated. This is because a perovskite with an O p-band center close to Fermi level was found easier to release oxygen, i.e., the formation of oxygen vacancies38  was performed to check the valence state and coordination environment of Co and Ir. As shown in Fig. 0.9Ir0.1O3−δ is approximately 3+ by comparing with the Co K-edge from the standard LaCoO3, in which the Co is strictly trivalent state40. Considering the Ir-LIII edge positions from m-SrIrO3 and IrO2 are similar to each other  in SrCo0.9Ir0.1O3−δ can reach a minimum value of 0.4 if assuming all Ir is pentavalent state. Therefore, a high amount of oxygen vacancies should exist in SrCo0.9Ir0.1O3, and thus both Co and Ir in SrCo0.9Ir0.1O3−δ are highly under-coordinated. Accordingly, the Fourier transformed (FT) EXAFS spectra in R-space for Co and Ir in SrCo0.9Ir0.1O3−δ are compared with the spectra from standard LaCoO3, m-SrIrO3, and IrO2, in which Co and Ir are fully or almost fully coordinated. In FT EXAFS spectra, the peaks are related to the coordination shells of Ir and Co. Specifically, the first peak represents the first Ir/Co–O shell caused by the interference between the electronic back scatterings from Ir/Co to neighbor O. Figure 0.9Ir0.1O3−δ. The first peak at a reduced distance of ~1.5 Å represents the first Co–O coordination shell in SrCo0.9Ir0.1O3−δ and it shows a much lower intensity as compared with the first peak from LaCoO3, indicating the highly under-coordinated Co. Additional evidence of such high oxygen-deficiency is obtained from the fitting of the first peaks, where the coordination number of Co in SrCo0.9Ir0.1O3−δ is approximately 4.1 O6 octahedrons. Due to the presence of oxygen vacancies, the Ir in the lattice is under-coordinated. During electrochemical cycling, fast leaching of Sr and Co leaves an Ir rich surface. The initial pseudo-cubic structure will collapse on the surface and the formed IrOxHy phase(s) is amorphous without long-range ordering. The formed IrOxHy phase(s) may contain a high amount of structural domains with corner-shared IrO6 octahedrons. Moreover, the Ir in the amorphous phase could be more under-coordinated as compared with those derived from SrIrO3 after Sr leaching. This deduction also explains why the activity of Ir from SrCo0.9Ir0.1O3−δ is higher than that of Ir from electrochemically cycled SrIrO3.Based on the results shown in Figs. 41. Thus, in this study, we also calculate the S-number of Ir in SrCo0.9Ir0.1O3−δ. The dissolution of cations, including Sr, Co, and Ir, from SrCo0.9Ir0.1O3−δ during electrochemical tests are studied. The amount of dissolved cations during the cycling are shown in Fig. 0.9Ir0.1O3−δ during the cycling are shown in Supplementary Figure 0.9Ir0.1O3−δ. The simultaneous dissolution of Sr and Co likely induces such high dissolution rate of Ir. In the following cycles, the dissolution rate of Ir from SrCo0.9Ir0.1O3−δ is found slowed down. Interestingly, the apparently reduced dissolution rates of Sr and Co suggests that the dissolution of Sr and Co from SrCo0.9Ir0.1O3−δ is kinetically hindered by the formed Ir-rich surface layer. Similarly, The Ir metal also showed a gradually decreased rate of Ir dissolution. That is the formation of active Ir hydrous oxide also causes a fast Ir dissolution at the early stage. Figure 0.9Ir0.1O3−δ, Ir metal, and IrO2 by chronopotentiometry method. Regardless of potential fluctuations caused by intermittent O2 bubbles released from the electrode surface, fairly stable performance is observed for all samples. The stability for different catalysts is then compared using their S-numbers, which are calculated by dividing the amount of oxygen molecules evolved by the amount of Ir dissolved in the electrolyte41. Moreover, as the surface reconstruction with fast cation dissolution  are observed from SrCo0.9Ir0.1O3−δ and Ir metal during the pre-treatment stage . Interestingly, from the following 180 min test, the S-number of SrCo0.9Ir0.1O3−δ steeply increased to approximately 105, which is approximately one order of magnitude higher than the S-numbers of reported perovskites. In addition, the S-numbers of SrCo0.9Ir0.1O3−δ and Ir metal are comparable with the S-number of IrO2 (by Sigma-Aldrich). The S-number of IrO2 we measured, however, is much lower than that of reported IrO2 (by Alfa-Aesar)41. Such higher stability of reported IrO2 can be related to the improved stoichiometry on the surface after additional annealing step. It is likely the production or condition of raw materials may also affect the S-numbers. From these results, we can conclude that the eventually formed Ir-rich layer over SrCo0.910Ir0.1O3−δ is rather stable if neglecting the initial instability caused by surface reconstruction process.Most recently, Geiger et al. proposed a stability number (S-number), considering the dissolution of active sites, is a proper metric to evaluate the stability of catalystsion Fig.  may grea0.9Ir0.1O3−δ perovskite. Such material can be easily prepared with a simple solid state method without high pressure condition. The Ir from pseudo-cubic SrCo0.9Ir0.1O3−δ showed an intrinsic activity (TOF) more than two orders of magnitude higher than that in IrO2, and approximately 10 times higher than that in the benchmark PLD-SrIrO3. The chemical state of surface Ir in SrCo0.9Ir0.1O3−δ is found changed after electrochemical cycling. The reconstructed surfaces are responsible for the observed activities. Although the initial bulk catalysts may not directly influence the OER, this study highlights that the chemical states of bulk catalysts, such as the crystal structure (pseudo-cubic structure with corner-shared IrO6 octahedrons) and the content of oxygen vacancy in the lattice (under-coordinated Ir), will affect the activity of the in-situ formed active sites (Ir-rich surface) for OER.In summary, we developed a pseudo-cubic SrCo3, IrO2, TiO2, and Co3O4 from Sigma-Aldrich Corporation were used as raw materials. Briefly, stoichiometry amount of raw materials were weighted and then mixed in mortar. The mixed precursors were finally calcined with box furnaces in ambient air. A sintering condition of 850 °C for 12 h, 1100 °C for 12° h, and 1200 °C for 12 h is applied for m-SrIrO3, SrCo0.9Ir0.1O3−δ, and SrCo0.9Ti0.1O3−δ respectively.All composite oxides were synthesized with solid state method. Powders of SrCO3 and SrCo0.9Ir0.1O3−δ were identified with X-ray Powder Diffractometer . The phase structures were analyzed by the Rietveld method with GSAS program and EXPGUI interface42. The morphologies of the materials were studied by FESEM (JEOL 6340F). TEM images were collected on JEM-2010F (JEOL). The XPS tests were performed using a PHI-5400 equipment with Al Ka beam source (250 W) and position-sensitive detector. An XPSpeak41 software is applied for peak fitting. Considering the spin–orbit splitting, a relative area ratio of 2:3 and 3:4 is considered for the doublets in Sr_3d and in Ir_4f, respectively. A spin–orbit splitting of 3 eV is considered for the doublets in Ir_4f. A Shirley background was applied during the fitting. All peaks are described as convolution of Gaussian and Lorentzian function.The phase structures of m-SrIrOXAS experiments were performed at Singapore Synchrotron Light Source, XAFCA beamline. Data analysis were performed with the Athena software package. The surface areas were characterized by nitrogen adsorption–desorption tests (ASAP Tri-star II 3020) with the BET method.−2 was applied. The catalyst ink with a concentration of 5 mg mL−1 was prepared by ultra-sonically dispersing 2.5 mg oxide and 1 mg acetylene black (Alfa Aesar) in an water (375 µL)–isopropanol (112.5 µL)–Nafion (12.5 µL) solution. 10 µL of well dispersed ink was dropped onto the glassy carbon and dried overnight. For ICP-MS tests, the amount of oxide increased to 5 mg for SrCo0.9Ir0.1O3−δ and Ir metal. As a higher stability is expected for IrO2, its amount further increase to 10 mg. An SP-150 workstation (Bio-Logic Science Instruments) was applied to perform cyclic voltammetry scanning and chronopotentiometric tests. The tests were performed in a 0.1 M HClO4 solution, which was purged with ultra-pure oxygen before each measurement for approximately 30 min. A rotation speed of 1600 rpm was used. A Pt wire was used as the counter electrode and a saturated calomel electrode (SCE) was used as the reference electrode.The electrochemical experiments were carried out on a rotating electrode configuration from Pine Instrument at room temperature. The working electrode of glassy carbon (rotating disk) with an area of 0.196 cmj is the BET surface area normalized current density at an overpotential of 270 mV. AOX is the total surface area of the catalyst deposited on the GC electrode. e is the electric charge carried by a single electron. NA is the number of Ir atoms. While calculating the NA, three cases are considered.The TOF is calculated from the equation:3, the calculation is performed by assuming PLD-SrIrO3 with an ideal P m-3m structure and a perfect (100) surface. A lattice parameter of 4.024 Å reported in the literature is used13. For m-SrIrO3, the lattice parameters from XRD refinement are used and a (001) surface, with the highest Ir surface density, is chosen (thus the activity will not be overestimated). For SrCo0.9Ir0.1O3−δ, the lattice parameters from XRD refinement are used and a (010) surface, with the highest Ir surface density, is chosen (thus the activity will not be overestimated). For IrO2, lattice parameters of a = 4.505 Å and c = 3.159 Å are used and a (110) surface is considered43. In this case, the active Ir atoms in reconstructed surface region are not counted and thus the activity is over-estimated. Thus, this way cannot be used for comparing TOF.In the first case, only the Ir atoms from the outermost surface are considered. For PLD-SrIrOIn the second case, all Ir atoms for each catalyst loaded on the electrode are considered active for OER. Please note this case over-estimates the active Ir atoms and thus the activity will be significantly under-estimated. Therefore, this way does not make sense, too.3 and SrCo0.9Ir0.1O3−δ is 5 and 10 nm, respectively. In PLD-SrIrO3 film, it was reported that the root mean square roughness is <4 nm before OER and <20 nm after cycling13. Thus, we assume a surface reconstruction with a depth of 8 and 40 nm to normalize the initial activity and final activity (after 30 h test) of Ir in PLD-SrIrO3, respectively. For IrO2, the surface reconstruction effect is negligible, and thus is not considered.In the third case, to include the effect of surface reconstruction, all Ir atoms from reconstructed surface regions are considered. Based on the TEM results Fig. , we assu2+ and Co3+ leached is compensated by electron lose from the catalyst, i.e., all leached Sr2+ and Co3+ cations will contribute to the current.The Sr2+ and Co3+ from the surface region  leached in 5 cycles at a constant speed. This is because we did not see steep current changes due to cation leaching.All SrHere, we estimate the maximum current contributed by Sr and Co leaching (possible leaching current). Several assumptions are made for the calculation/estimation:2+ and Co3+ from the surface region (10 nm depth). e is the electric charge of an electron. t is the time used for 5 cycles. Our cycling test for SrCo0.9Ir0.1O3−δ is performed in the potential range (vs. RHE) from 1 to 1.7 V. The scan rate is 10 mV s−1. Sox is the surface area of the loaded catalyst.Then, the current from cation leaching 0.9Ir0.1O3−δ. For example, at an overpotential of 270 and 320 mV, the measured OER current from SrCo0.9Ir0.1O3−δ can reach 2.8 and 16.3 Based on that, a maximum current of 0.0186 45 with the projector-augmented wave (PAW) approach46 and the Perdew–Burk–Ernzerhof (PBE) exchange-correlation functional47. To account for the strongly localized d-electrons of Co, a DFT+U approach was adopted and an effective Hubbard U parameter of 3.32 eV was used49. The electronic energy tolerance was set to 10−6 eV and the force tolerance for structural relaxation was 0.015 eV Å−1. The structural model for PLD-SrIrO3 is a 2 × 2 × 2 cubic supercell, for m-SrIrO3 is a conventional standard unit cell and for pc-SrCo0.875Ir0.125O3 is a 2 × 1 × 1 orthorhombic supercell. For all compositions, the lattice constants and ion positions were first fully relaxed. Then, the defect calculations were performed on the fully relaxed stoichiometric structures, and all the symmetry distinct oxygen sites in the corresponding model were explored to search for the most stable vacancy site. The oxygen vacancy concentration for PLD-SrIrO3 and pc-SrCo0.875Ir0.125O3 is 4.167% while for m-SrIrO3 is 3.125%. The total energy calculations of all structures were performed with the tetrahedron method with Blöchl corrections50 and an energy cutoff of 520 eV. And a 6 × 6 × 6 Monkhorst–Pack k-point mesh was employed for PLD-SrIrO3, a 4 × 6 × 8 mesh for pc-SrCo0.875Ir0.125O3 and a 10 × 6 × 4 mesh for m-SrIrO3. For all compositions, the bulk oxygen vacancy formation energy was calculated based on the most stable oxygen-deficient structure and with respect to H2O (g) and H2 (g) at standard condition. The oxygen vacancy formation enthalpy will be shifted around +2.33 eV larger if O2 (g) is used as the reference.Spin-polarized DFT calculations were performed using the Vienna ab initio simulation package (VASP)Supplementary Information


Silver nanoparticles (AgNPs) are widely used in many consumer products due to their anti-inflammatory properties. Therefore, the effect of exposure to AgNPs should be investigated in diseased states in addition to healthy ones. Tumor necrosis factor-α (TNFα) is a major cytokine that is highly expressed in many diseased conditions, such as inflammatory diseases, sepsis, and cancer. We investigated the effects of two different sizes of AgNPs on the TNFα-induced DNA damage response. Cells were exposed to 10 and 200 nm AgNPs separately and the results showed that the 200 nm AgNPs had a lower cytotoxic effect with a higher percent of cellular uptake compared to the 10 nm AgNPs. Moreover, analysis of reactive oxygen species (ROS) generation and DNA damage indicated that TNFα-induced ROS-mediated DNA damage was reduced by 200 nm AgNPs, but not by 10 nm AgNPs. Tumor necrosis factor receptor 1 (TNFR1) was localized on the cell surface after TNFα exposure with or without 10 nm AgNPs. In contrast, the expression of TNFR1 on the cell surface was reduced by the 200 nm AgNPs. These results suggested that exposure of cells to 200 nm AgNPs reduces the TNFα-induced DNA damage response via reducing the surface expression of TNFR1, thus reducing the signal transduction of TNFα. Nanotechnology is an advanced field that studies very small materials ranging from 0.1 to 100 nm . BecauseThe characteristics of AgNPs, including morphology, size, size distribution, surface area, surface charge, stability, and agglomeration, have a significant impact on their interaction with biological systems ,9,10. AlSeveral research groups have investigated the effects of AgNPs with sizes ranging from 5 to 100 nm on different cell lines; the cytotoxic effect of AgNPs on human cell lines  is size-dependent, with 5 nm being more toxic than 20 or 50 nm and inducing elevated reactive oxygen species (ROS) levels and S phase cell cycle arrest . In RAW To increase applications for AgNPs, we should consider their effects on diseased subjects as well as healthy ones. Inflammatory diseases, asthma, infections, and cancer are common diseases for which the effects of exposure to AgNPs should be investigated. Tumor necrosis factor-α (TNFα), a pro-inflammatory cytokine and a regulator of immunological reactions in many physiological and pathological conditions , is a coWe investigated the size-dependent effect of AgNPs, and our results revealed that the expression of TNFR1 on the cell surface was reduced by 200 nm AgNPs but not by 10 nm AgNPs, suggesting a reduction in TNFα-induced DNA damage by 200 nm AgNPs.The size of AgNPs is one of their most important characteristics and influences their uptake by cells and the cellular response. Our aim was to clarify the size-dependent cytotoxic effect of AgNPs. Many studies have investigated the effect of AgNPs in particle sizes ranging from 10 to 100 nm; however, nanoparticles larger than 100 nm might have different effects because they can induce different mechanisms of cellular uptake or have a different uptake ratio. We therefore conducted a cell viability assay to determine the differences between 10 nm and 200 nm AgNPs on the viability of NCI-H292 cells. As shown in Cellular uptake of nanoparticles plays an important role in cellular responses including proliferation, inflammation, DNA damage, and cell death. We therefore estimated the cellular uptake of 10 nm and 200 nm AgNPs, and the results are shown in In many disease states such as inflammatory disease, infections, and cancer, TNFα acts as a major cytokine. TNFα has been reported to be involved in ROS generation resulting in DNA damage and cell death . TherefoROS-mediated DNA damage is well known to be induced by TNFα. Since TNFα-induced ROS generation was affected by AgNPs, DNA damage was also evaluated as an important cellular response that is influenced by ROS generation and regulates cell fate.BTG2) promoter-reporter plasmid to detect the DNA damage response. As shown in BTG2 response in cells exposed to TNFα (20 ng/mL) only, 10 nm AgNPs (100 µg/mL) only, or both, were significantly increased compared to the control group. However, cells exposed to TNFα (20 ng/mL) + 200 nm AgNPs (100 µg/mL) showed a significant decrease in the fold induction of the response compared to the TNFα-exposed group. These results suggest that 200 nm AgNPs might regulate TNFα-induced DNA damage.For this purpose, we transfected a B-cell Translocation Gene 2 , the RAD21 homolog of S. pombe (RAD21), structural maintenance of chromosomes 1A (SMC1A), and tumor protein p53 (TP53) genes were increased by ≥ 1.9-fold in cells exposed to TNFα (20 ng/mL). Also, these same genes exhibited ≥ 1.2-fold induction in cells exposed to TNFα (20 ng/mL) + 10 nm AgNPs (100 µg/mL). However, in cells exposed to TNFα (20 ng/mL) + 200 nm AgNPs (100 µg/mL), the expression of these genes showed downregulation of ≤ 0.8-fold, indicating a reduction in TNFα-induced DNA damage by 200 nm AgNPs. To confirm the induction of the above five genes, we conducted real-time PCR analysis. As shown in SMC1A, which showed a significant decrease from 2.5- to 1.8-fold induction. In contrast, for cells exposed to TNFα (20 ng/mL) + 200 nm AgNPs (100 µg/mL), all five genes showed significant downregulated expression compared to the TNFα-exposed group, especially TP53, RAD21, and CHEK1, which were downregulated from 2.5 to 0.9, 1.6 to 0.4, and 2.3 to 0.5, respectively. The mRNA expressions of these genes involved in the DDR signaling demonstrated that most of the TNFα-induced upregulated genes were downregulated by 200 nm but not 10 nm AgNPs, suggesting that 200 nm AgNPs reduced the TNFα-induced DNA damage.DNA damage is regulated by the DNA damage response (DDR) signaling pathway, which uses signal sensors, transducers, and effectors; the ataxia-telangiectasia mutated (ATM) and ATM- and Rad3-Related (ATR) proteins are the most upstream DDR kinases that are activated by the sensors of the DDR pathway . TherefoTNFα has two receptors, TNFR1 and TNFR2. TNFR1 is the major receptor, exists in most cell types, and mediates the NF-KB activation signaling pathway, which is involved in ROS generation and DNA damage . Our aboAgNPs are considered to be a double-edged sword that can induce opposing effects. AgNPs have a well-known potential anti-inflammatory effect ,27, but Here, we investigated the size-dependent effects of polyvinylpyrrolidone (PVP)-coated AgNPs. We used 10 and 200 nm particles, hypothesizing that they would have different behaviors when interacting with lung epithelial cells. Interestingly, our results showed that the 200 nm particles were less cytotoxic , despiteBTG2 response  heat-inactivated fetal bovine serum , 100 μg/mL penicillin, and 10 μg/mL streptomycin  was used to culture the cells.Human pulmonary epithelial cell line NCI-H292 (ATCC CRL-1848TM) cells were cultured in an incubator with a humidified atmosphere containing 5% CO2 at 37°C. RPMI-1640 medium  supplemented with 10% (Polyvinylpyrrolidone (PVP)-coated AgNPs with two different sizes of 10 nm and 200 nm  were used in this study. Electronic light scattering  was used to analyze the particle sizes and zeta potentials. The average hydrodynamic diameter of 10 nm AgNPs in deionized water was 12.0 ± 1.8 nm, the polydispersity index was 0.191, and the zeta potential was −21.45 mV. For the 200 nm AgNPs, the average hydrodynamic diameter in deionized water was 221.9 ± 50.9 nm, the polydispersity index was 0.026, and the zeta potential was −27.59 mV. AgNP suspensions were concentrated and sterilized by autoclaving (121 °C for 20 min), then the working solutions were prepared by resuspension in RPMI 1640 medium for cell exposure.Recombinant human TNFα  was reconstituted in water to 100 µg/mL. The working dilutions were prepared using sterilized culture medium (RPMI-1640) containing a carrier protein.® luminescent cell viability assay  according to the manufacturer’s protocol. Cells were seeded at a density of 1 × 104 cells/well in an opaque 96-well plate and incubated at 37 °C and 5% CO2 overnight, then the cells were checked for 80–90% confluency. Cells were exposed to 10 nm and 200 nm AgNPs  separately for 24 h. CellTiter-Glo® reagent was added to each well, and the percent of viable cells was calculated based on quantification of adenosine triphosphate (ATP) using a luminometer .The viability of NCI-H292 cells was measured using a CellTiter-GloTo check the percentage of cells that incorporated AgNPs, NCI-H292 cells were seeded at a concentration of 8 × 104 cells/well in a 24-well plate . After overnight incubation, the cells were exposed to a final concentration of 100 µg/mL of both 10 nm and 200 nm AgNPs. After 24 h of exposure, the cells were washed twice with phosphate buffered saline (PBS) and collected by trypsinization using trypsin EDTA  and centrifugation. Then, the cells were resuspended in 1 mL PBS supplemented with 6% HFBS and kept on ice until analysis.The percentage of cells taking up AgNPs was analyzed using a flow cytometer . Forward scatter (FSC) is the laser light scattered at narrow angles to the axis of the laser beam and is proportional to the cell size. Side scatter (SSC) is the laser light scattered at a 90° angle to the axis of the laser and is proportional to the intracellular density, which is increased by the uptake of nanoparticles. The mean SSC for each group of cells was calculated depending on the peak intensities of treated cells compared to the control cells using the software supplied with the instrument.To quantify intracellular ROS, a ROS assay kit  was used. This assay is based on the cell permeable fluorogenic probe 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA), which diffuses into cells and is deacetylated by intracellular esterases to the nonfluorescent 2′,7′-dichlorodihydrofluorescein (DCFH). In the presence of ROS, DCFH is rapidly oxidized to the highly fluorescent 2′,7′-dichlorodihydrofluorescein (DCF). The fluorescence intensity is proportional to the intracellular ROS levels.2O2) and used as a positive control. The fluorescence of DCF was measured at regular intervals at an excitation/emission wavelength of 480 nm/530 nm using a fluorometric plate reader . The amounts of produced DCF were calculated based on a DCF standard curve.According to the manufacturer’s protocol, cells were cultured at a density of 1 × 104 cells/well in a black 96-well plate and incubated overnight. Subsequently, media were removed, and cells were washed twice gently with DPBS  and incubated with DCFH-DA/media solution for 30 min in the dark at 37 °C. Then, after removing the solution and washing the cells with DPBS, the DCFH-DA-loaded cells were exposed to TNFα (20 ng/mL) and 10 nm AgNPs (100 µg/mL) or 200 nm AgNPs (100 µg/mL) separately and together for 24 h. Parallel sets of wells containing DCFH-DA-loaded cells without any further exposure were used as a negative control. Another set of DCFH-DA-loaded cells were exposed to hydrogen peroxide (HBTG2 promoter-reporter plasmid (the region from nt −100 to −20 bp of the BTG2 gene containing a p53 binding site mutation) was used to detect DNA damage. Both reporter plasmids contain SV40 promoters and enhancer sequences that result in strong expression of the luciferase encoding gene (luc+) in different types of mammalian cells. Also, pRL-CMV vector , which is a Renilla luciferase-encoding control plasmid, was used as an internal control for variations in the transfection efficiency.pGL3-Control vector  was used as an empty control reporter plasmid. BTG2 promoter-reporter plasmid and pRL-CMV internal control plasmid were co-transfected into NCI-H292 cells. LipofectamineTM LTX reagent with a PlusTM reagent kit (Invitrogen) was used to perform the transfection according to the manufacturer’s protocol. Cells were cultured at a density of 2 × 105/mL in a 24-well plate and incubated overnight at 37 °C. Opti-MEM medium  was used to dilute the Lipofectamine LTX reagent and plasmids, then the Plus reagent was added to the diluted plasmids. Diluted plasmids with Plus reagent were added to the diluted Lipofectamine LTX reagent at 1:1 ratio and incubated for 5 min at room temperature. Finally, the plasmid–lipid complexes were added to the cells and incubated at 37 °C for at least 24 h before exposure to TNFα and AgNPs.pGL3 blank control reporter plasmid or After exposure of transfected cells to TNFα (20 ng/mL) and 10 nm AgNPs (100 µg/mL) or 200 nm AgNPs (100 µg/mL) separately and together for 24 h, the luciferase activities were assessed using a Dual-Luciferase Reporter Assay System  according to the manufacturer’s protocol. Cells were lysed using 1× passive lysis buffer and gentle shaking for 10 min, then cell lysates were transferred to tubes containing luciferase assay reagent II (LAR II). Firefly luciferase (F) signals were measured, then Stop & Glo reagent was added to the tubes and the Renilla luciferase (R) signals were also measured. The firefly and Renilla luciferase signals were recorded using a luminometer  according to the instrument manual. The changes in luciferase activities were calculated using the following equation:To analyze the expression of genes involved in DNA damage, PCR array analysis was conducted as follows. NCI-H292 cells were seeded at a concentration of 4 × 105 cells/60 mm cell culture dish. After overnight incubation, the cells were exposed to TNFα (20 ng/mL) only, or together with 10 nm AgNPs (100 µg/mL) or 200 nm AgNPs (100 µg/mL). After 8 h of exposure, the cells were detached by trypsinization and collected by centrifugation, and then the total cellular RNA was extracted using an RNeasy kit  according to the manufacturer’s protocol. An aliquot (1 µg) of the extracted total RNA was reverse transcribed into cDNA using a RT2 First Strand kit (SABiosciences/Qiagen), and the expression of 89 Human DNA Damage Signaling Pathway genes was measured using a RT2 profiler PCR array kit (SABiosciences/Qiagen) according to the manufacturer’s protocol. PCR array analysis was performed using an ABI PRISM 7000 sequence detection system .SMC1A, ATM, TP53, RAD21, and CHEK1 were purchased from SABiosciences/Qiagen. The reaction mixture was composed of 12.5 µL RT2 SYBR Green qPCR Master Mix (SABiosciences/Qiagen), 1 µL 10 µM gene-specific RT2 qPCR forward and reverse primers, 2 µL cDNA, and nuclease-free water to a final volume of 25 µL. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a house-keeping gene to normalize the data. RT-PCR analysis was performed using the same machine used for PCR array, and the thermocycling conditions were 95°C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min.For mRNA expression analysis, cells were seeded and exposed to TNFα and AgNPs, then total RNA and cDNA were synthetized as mentioned for the PCR array. The PCR primers for human To localize tumor necrosis factor receptor 1 (TNFR1), NCI-H292 cells were seeded in a CELLview cell culture dish  at a density of 1.5 × 104 cells/compartment and incubated for 24 h. The cells were exposed to TNFα (20 ng/mL) only, or together with 10 nm AgNPs (100 µg/mL) or 200 nm AgNPs (100 µg/mL). After 24 h of exposure, the cells were washed with 1× PBS fixed with 4% formaldehyde solution in PBS (Wako) at room temperature, permeabilized with 0.1% Triton X-100, and then blocked with 10% normal goat serum in PBS for 1 h. The cells were then incubated overnight at 4°C with rabbit polyclonal anti-TNF receptor 1 antibody  followed by incubation with labeled goat anti-rabbit IgG H&L (Alexa Fluor 488) (Abcam) for 1 h at room temperature. Nuclear DNA was stained with DAPI   for 5 min at room temperature. Microscopic observations and images were acquired using a confocal laser-scanning microscope  with a 63 × 1.4 Plan-Apochromat oil immersion lens.n ≥ 3).Statistical analysis was performed using Student’s t-test. Differences and significances between means of different groups were determined using one-way ANOVA with Duncan’s multiple comparison tests. P values less than 0.05 were considered statistically different. Data are presented as means ± standard deviation (SD) with at least three independent replicates (In this study, we found that 200 nm AgNPs, but not 10 nm AgNPs, reduced DNA damage in NCI-H292 cells and proposed a mechanism for this effect. This mechanism works by reducing membrane localization of TNFR1 and thus decreasing TNFα signal transduction, leading to a reduction in TNFα-induced DNA damage. Also, the mechanism explains why 10 nm AgNPs induced ROS-mediated DNA damage by their own action without affecting TNFR1 and TNFα signal transduction.


Sonchus oleraceus L. (Asteraceae) leaf extracts. We hypothesize that S. oleraceus extracts protect cells against H2O2-induced senescence by mediating oxidative stress. Premature senescence of young WI-38 cells was induced by application of H2O2. Cells were treated with S. oleraceus extracts before or after H2O2 stress. The senescence- associated β-galactosidase  activity was used to indicate cell senescence. S. oleraceus extracts showed higher cellular antioxidant activity than chlorogenic acid in WI-38 cells. S. oleraceus extracts suppressed H2O2 stress-induced premature senescence in a concentration-dependent manner. At 5 and 20 mg/mL, S. oleraceus extracts showed better or equivalent effects of reducing stress-induced premature senescence than the corresponding ascorbic acid treatments. These findings indicate the potential of S. oleraceus extracts to be formulated as an anti-ageing agent.Antioxidants protect against damage from free radicals and are believed to slow the ageing process. Previously, we have reported the high antioxidant activity of 70% methanolic  Ageing or senescence is a complex and inevitable biological process, which is not only attributed to individual genetic variation but also to external factors such as environmental conditions, nutrition, alcohol and diseases ,2,3,4. T2O2-stressed fibroblasts experienced an extended lifespan in the presence of cyanidin, the most prevalent anthocyanin in plants ethanesulfonic acid (HEPES) (Sigma®). Dried S. oleraceus extracts were re-dissolved and further diluted to required working concentrations in treatment medium.For the Cellular Antioxidant Activity (CAA) assay, a 20 mM stock solution of 2',7'-dichloro-fluorescin diacetate (DCFH-DA) (SigmaS. oleraceus extracts and ascorbic acid stock solution were further diluted in treatment media to working concentrations. 30% H2O2 was diluted to required working concentrations using treatment media. Bafilomycin A1 (>95%)  was dissolved in dimethyl sulfoxide (DMSO)  to a final concentration of 0.1 mM. For experimental application, the bafilomycin A1 stock was diluted to 100 nM in cBME  , 2 mM GlutaMAX plus 25 mM HEPES, pH 7.2). 5-Dodecanoylaminofluorescein Di-β-d-galacto-pyranoside (C12FDG)  stock solution was also prepared in DMSO to a final concentration of 20 mM. The 20 mM stock solution of C12FDG was diluted 1:10 with fresh cBME to make a 2 mM working solution immediately before its addition into the cells. The final concentration of C12FDG obtained in cells was 33 μM. Fluorescence-activated cell sorting (FACS) buffer was prepared by dissolved 1% (w/v) bovine serum albumin  and 0.01% (w/v) sodium azide (Sigma®) in PBS.For the ageing study, dried leaf extracts were re-dissolved in treatment media resulting in a concentration of 100 mg/mL (fresh weight). 1 mM stock solution of ascorbic acid was prepared in purified ionized water immediately prior to experiments. The filtered 2. The medium was changed every 3–4 days. Cells used for this study were at population doubling level (PDL) 19–27. The PDL of the cell culture was determined as follows: current PDL = last PDL + 3.32 × Log (harvested cell population/seeded cell population).WI-38 human lung diploid fibroblast cells  were cultured in cBME at 37 °C with 5% COet al. [4 cells per cm2 and cultured in cBME. After 24 h incubation, cells were treated with various concentrations of S. oleraceus extracts or ascorbic acid in treatment media for 3 h or 24 h, or treated with H2O2 only for 2 h. Cells were then washed with 100 µL HBSS and 50 µL methylene blue staining solution was added into each well. After another hour of incubation at 37 °C, the dye was washed off using fresh deionized water and the plate was air-dried following elution.Cell viability was measured using the method of Wolfe et al.  with modet al. [4 cells per cm2 on a 96-well plate in 100 µL of cBME, excluding the outside wells of the plate. After incubation for 24 h, the cBME was removed and the wells were washed with PBS. Triplicate wells were treated with 100 µL of S. oleraceus extracts and 25 µM DCFH-DA dissolved in treatment media and incubated for 1 h. Cells were then washed with 100 µL PBS, before 600 µM ABAP was applied to the cells in 100 µL of HBSS. Fluorescence was read with emission at 538 nm and excitation at 485 nm every 5 min for 1 h at 37 °C .The cellular antioxidant activity was measured using a modified method from Wolf et al. . WI-38 cversus time was integrated to calculate the CAA unit as:50) was determined for the S. oleraceus extracts and chlorogenic acid from the dose-response curve where the CAA unit is equal to 50.After blank subtraction from the fluorescence readings, the area under the curve of fluorescence ectively . The medS. oleraceus leaf extracts can protect human cells from exposure to oxidative stress, WI-38 cells were seeded at a density of 5 × 104 cells per cm2 in a 24-well plate. After 24 h incubation, cells were treated with 1, 5 or 20 mg/mL (fresh weight) S. oleraceus leaf extracts or 88 or 352 µM ascorbic acid in treatment media for 1 h. 88 or 352 µM ascorbic acid were chosen as the equivalent concentrations to the total concentrations of the three key antioxidants in 5 and 20 mg/mL extracts, respectively. Then cells were washed with HBSS before being stressed with 100 µM H2O2 in treatment media for another 1 h. Fresh cBME was added after the cells were washed with HBSS. Cells without any treatment were used as unstained controls. Cells with H2O2 treatment only were used as positive controls. After 48 h rest from stress, cells were treated for FACS measurement as detailed below.To investigate if S. oleraceus leaf extracts was conducted in the same way as the protective effect study described above with one modification: WI-38 cells were stressed with 100 µM H2O2 for 1 h before being treated with 1, 5 or 20 mg/mL (fresh weight) S. oleraceus leaf extracts or 88 or 352 µM ascorbic acid.The recovery ability of S. oleraceus leaf extracts or 80 or 352 µM ascorbic acid before chronic stress. Chronic stress was applied by adding 30 µM of H2O2 for 1 h per day 5 days after a single treatment of S. oleraceus leaf extracts or ascorbic acid. General procedures were otherwise the same as described above in the protective effect experiment.WI-38 cells were treated for 1 h with 1, 5 or 20 mg/mL (fresh weight) et al. [12FDG working solution  was added to the media to give a final concentration of approximately 33 µM and the incubation was continued for another 1 h. The solution was removed and the cell monolayers were washed twice for approximately 30 s with 0.5 mL of PBS at room temperature. Cells were then trypsinized (TrypLE™) (Gibco®) and harvested followed by centrifugation at 100 g for 10 min at 4 °C. Cells were re-suspended in ice-cold FACS buffer and analysed immediately in a FACScalibur flow cytometer . FlowJo software  was used for data analysis. Propidium iodide  was used as dead/living cell indicator for all the cell samples.The SA-β-gal activity was determined as described by Debacq-Chainiaux et al.  with modt-test using Microsoft Excel (Version 2007) or analysis of variance with post-hoc Tukey’s multiple comparisons using SPSS Statistics from IBM  were used to establish significant differences between the groups. p-values < 0.05 were considered to be significant.Either a Student’s S. oleraceus extracts possessed high cellular antioxidant activity, compared to chlorogenic acid or other plant samples reported in literature. H2O2 stress-induced premature senescence can be significantly suppressed by S. oleraceus extracts at 5 mg/mL or above, and this anti-ageing effect is concentration-dependent. When compared to the corresponding ascorbic acid treatments, 5 and 20 mg/mL S. oleraceus extracts showed better or equivalent effects. These data suggest the therapeutic potential of S. oleraceus extracts as an anti-ageing agent.In summary, our results demonstrate that


The electrochemical study of the modified electrode, as well as its efficiency for simultaneous voltammetric oxidation of acetaminophen and tryptophan is described. Compared with bare SPE the GO/Fe3O4@SiO2/SPE exhibited excellent electrocatalytic activity toward the oxidation of acetaminophen. The plot of catalytic current versus acetaminophen concentration showed a linear segment in the concentration range 0.5 to 100.0 µM. The detection limit of 0.1 µM was obtained using calibration plot. Also the anodic peaks of acetaminophen and tryptophan in their mixture can be well separated. The GO/Fe3O4@SiO2/SPE has been successfully applied and validated by analyzing acetaminophen and tryptophan in urine and pharmaceutical samples.A high sensitive electrochemical nanostructure sensor based on graphene oxide/Fe Acetaminophen (N-acetyl-p-aminophenol or paracetamol), is a long-established substance being one of the most extensively employed drugs in the world . AcetamiTryptophan (2-amino-3-(1H-indol-3-yl)-propionic acid, TRP), is one of the twenty essential amino acids in the human diet . TryptopOverdose consumption of acetaminophen can alter tryptophan metabolism by inhibiting tryptophan 2, 3-dioxygenase activity thus increasing the availability of tryptophan for the production of serotonin in brain . Thus, tThe screen-printed electrodes have been designed especially for miniaturization of electrochemical analytical systems , 23. SPE3O4 nanoparticles have been extensively used for the modification of electrodes because they increase the electrode surface and electrical conductivity as well as improving electron transfer kinetics between the electrode surface and wide range of electroactive species (2) nanoparticle coating on the surface of magnetite nanoparticles prevents their aggregation, improves their chemical stability, and provides better protection against toxicity. The silica coating stabilizes the magnetite nanoparticles in two different ways, one of which is by shielding the magnetic dipole interaction with the silica shell. On the other hand, the silica nanoparticles are negatively charged. Therefore, the silica coating enhances the coulomb repulsion of the magnetic nanoparticles  is one of the most important derivatives of graphene having large surface area, excellent conductivity, and strong mechanical strength and contains abundant C–O–C (epoxide), C–OH, and terminated COOH groups decoration of GO by magnetic iron oxide  nanoparticles, which are very important magnetic materials, can improve optical, magnetic and electrochemical properties of GO. These properties make GO a great candidate for electrode surface modification . The experimental conditions were controlled with General Purpose Electrochemical System (GPES) software. The screen-printed electrode  consists of three main parts which are a graphite counter electrode, a silver pseudo-reference electrode and, a graphite working electrode.All solutions were freshly prepared with double distilled water. Acetaminophen, tryptophan, and all other reagents were of analytical grade and were obtained from Merck chemical company . The buffer solutions were prepared from orthophosphoric acid and its salts in the pH range of 2.0-9.0. For real samples analysis acetaminophen tablets and acetaminophen oral solution were purchased from Aria Pharmaceutical Company, Iran, and the urine samples were collected from a healthy person. Synthesis of GO/Fe3O4@SiO2 nanocomposite −1, and then bath sonicated for 1 h to give a clear solution. NaOH (12 g) and chloroacetic acid (Cl–CH2–COOH) (10 g) were added to the GO suspension and bath sonicated for 2 h to convert the –OH groups to –COOH via conjugation of acetic acid moieties giving G-COOH. The resulting G-COOH solution was neutralized, and purified by repeated rinsing and filtration.For carboxylation of GO, an aqueous suspension (50 mL) of GO was diluted by a factor of 2 to give a concentration of 2 mg mL3·6H2O was added under stirring. After the mixture was stirred vigorously for 30 min under N2 atmosphere, 57 mg of FeSO4·7H2O was added and keeping it stirred under N2 atmosphere for 30 min. At last 18 mL of 6% NH4OH aqueous solution was added into the mixture drop by drop at 60 °C during 1 h and reacted for another 2 h. N2 atmosphere was used during the reaction to prevent critical oxidation. The reaction mixture was then centrifuged, washed with double distilled water, and dried. The obtained black precipitate was GO/Fe3O4 nanoparticle and was ready for use. Core–shell GO/Fe3O4@SiO2 nanocomposites were prepared by growing silica layers onto the surface of the GO/Fe3O4 as described by Lu et al. Fifteen milliliters of ethanol, 0.6 mL water, 0.6 mL ammonium hydroxide and 90 μL of TEOS were added in a 250 mL three neck flask in a 40 °C water bath. GO/Fe3O4 was added to the above solution under mechanical stirring. Aliquots of the mixture were taken out after 12 h by centrifugation and washed with water and vacuum-dried at 60 °C overnight. About 0.06 g of GO–COOH was dissolved in 42 mL of water by ultrasonic irradiation  for 20 min. The mixture was further stirred vigorously for 30 min at 60 °C. Then 106.2 mg of FeClPreparation of the electrode 3O4@SiO2 as follows. A stock solution of GO/Fe3O4@SiO2 in 1 mL aqueous solution was prepared by dispersing 1 mg GO/Fe3O4@SiO2 with ultrasonication for 1 h, and a 5 µL aliquot of the GO/Fe3O4@SiO2 /H2O suspension solution was casted on the carbon working electrodes, and waiting until the solvent was evaporated in room temperature. The bare screen-printed electrode was coated with GO/FePreparation of real samples Five acetaminophen tablets  were grinding. Then, the tablet solution was prepared by dissolving 325 mg of the powder in 25 mL water by ultrasonication. Then, different volume of the diluted solution was transferred into a 25 mL volumetric flask and diluted to the mark with PBS (pH 7.0). The acetaminophen content was analyzed by the proposed method using the standard addition method.The acetaminophen oral solution was diluted 100 times with water; then, different volume of the diluted solution was transferred into a 10 mL volumetric flask and diluted to the mark with PBS (pH 7.0). The acetaminophen content was analyzed by the proposed method using the standard addition method.Urine samples were stored in a refrigerator immediately after collection. 10 mL of the sample was centrifuged for 15 min at 2000 rpm. The supernatant was filtered out using a 0.45 μm filter. Then, different volume of the solution was transferred into a 25 mL volumetric flask and diluted to the mark with PBS (pH 7.0). The diluted urine sample was spiked with different amounts of acetaminophen and tryptophan.Electro-oxidation of acetaminophen at a GO/Fe3O4@SiO2/SPE Scheme 1 shows a cyclic voltammogram was recorded for acetaminophen in 0.1 M PBS (pH = 7.0). The voltammogram shows an anodic peak in the positive-going scan and a cathodic counterpart peak in the negative-going scan which corresponds to the transformation of acetaminophen to N-acetyl-p-benzoquinone-imine and vice-versa within a quasi-reversible two-electron process.3O4@SiO2/SPE by voltammetry. It was found that the electro-oxidation of acetaminophen at the surface of GO/Fe3O4@SiO2/SPE was more favored under neutral conditions than in acidic or basic medium. This appears as a gradual growth in the anodic peak for oxidation current of acetaminophen at the surface of GO/Fe3O4@SiO2/SPE. Thus, the pH 7.0 was chosen as the optimum pH for electro-oxidation of acetaminophen at the surface of GO/Fe3O4@SiO2/SPE.The electrochemical behavior of acetaminophen is dependent on the pH value of the aqueous solution. Therefore, pH optimization of the solution seems to be necessary in order to obtain the best results for electro-oxidation of acetaminophen. Thus, the electrochemical behavior of acetaminophen was studied in 0.1 M PBS in different pH values (2.0-9.0) at the surface of GO/Fe3O4@SiO2/SPE (curve a) and bare SPE (curve b). The anodic peak potential for the oxidation of acetaminophen at GO/Fe3O4@SiO2/SPE (curve a) is about 330 mV compared with 500 mV for that on the bare SPE (curve b). Similarly, when the oxidation of acetaminophen at the GO/Fe3O4@SiO2/SPE (curve a) and bare SPE (curve b) are compared, an extensive enhancement of the anodic peak current at GO/Fe3O4@SiO2/SPE relative to the value obtained at the bare SPE (curve b) is observed. In other words, the results clearly indicate that the combination of graphene and Fe3O4@SiO2 nanocomposites improve the acetaminophen oxidation signal.p) on the square root of the potential scan rate (ν1/2) over a wide range from 10 to 1000 mV s−1.The effect of potential scan rates on the oxidation current of acetaminophen has been studied . The resChronoamperometric measurements 3O4@SiO2/SPE were carried out by setting the working electrode potential at 0.4 V for the various concentration of acetaminophen in PBS (pH 7.0) (Chronoamperometric measurements of acetaminophen at GO/Fe(pH 7.0) . For an (pH 7.0) .1/2Cbπ-1/2t-1/2                                   Equ. 1I =nFADb are the diffusion coefficient (cm2 s-1) and the bulk concentration (mol cm−3), respectively. Experimental plots of I vs. t−1/2 were employed, with the best fits for different concentrations of acetaminophen  experiments were done for different concentrations of acetaminophen. The oxidation peak currents of acetaminophen at the surface of a modified electrode were proportional to the concentration of the acetaminophen within the ranges 0.5 to 100.0 μM. The detection limit (3σ) of acetaminophen was found to be 0.1 µM. These values are comparable with values obtained by other research groups .Simultaneous determination of acetaminophen and tryptophan3O4@SiO2/SPE.Determination of two compounds was performed by simultaneously changing the concentrations of acetaminophen and tryptophan, and recording the DPVs . The vol-1, which is very close to the value obtained in the absence of acetaminophen and tryptophan (0.0892 µA µM-1)  . These rReal sample analysis In order to evaluate the analytical applicability of the proposed method, also it was applied to the determination of acetaminophen and tryptophan in acetaminophen tablet, acetaminophen oral solution and urine samples. The results for determination of these species in real samples are given in 3O4@SiO2 nanocomposite modified screen printed electrode was successfully fabricated. Compared with bare SPE, the voltammetric responses of GO/Fe3O4@SiO2/SPE were significantly enhanced. The reason is the increase of the electrochemical active surface area and the conductivity of the electrode by introducing GO/Fe3O4@SiO2 nanocomposite. Under optimum condition, the peak current was proportional to the acetaminophen concentration in the range of 0.5–100.0 µM with the detection limit of 0.1 µM. Additionally, the GO/Fe3O4@SiO2/SPE exhibited an excellent electrocatalytic activity for acetaminophen and tryptophan in biological and pharmaceutical samples.A GO/Fe


Kinesin-1 transports numerous cellular cargoes along microtubules. The kinesin-1 light chain (KLC) mediates cargo binding and regulates kinesin-1 motility. To investigate the molecular basis for kinesin-1 recruitment and activation by cargoes, we solved the crystal structure of the KLC2 tetratricopeptide repeat (TPR) domain bound to the cargo JIP3. This, combined with biophysical and molecular evolutionary analyses, reveals a kinesin-1 cargo binding site, located on KLC TPR1, which is conserved in homologs from sponges to humans. In the complex, JIP3 crosslinks two KLC2 TPR domains via their TPR1s. We show that TPR1 forms a dimer interface that mimics JIP3 binding in all crystal structures of the unbound KLC TPR domain. We propose that cargo-induced dimerization of the KLC TPR domains via TPR1 is a general mechanism for activating kinesin-1. We relate this to activation by tryptophan-acidic cargoes, explaining how different cargoes activate kinesin-1 through related molecular mechanisms. •Crystal structure of the kinesin-1 KLC2 TPR domain bound to the cargo JIP3•JIP3 binding site is located on KLC TPR1 and is conserved from sponges to humans•Molecular mechanism by which ARF6 regulates kinesin-1 recruitment by JIP3/4 revealed•A unified framework explaining how unrelated cargoes activate kinesin-1 Cockburn et al. present the structure of the cargo binding TPR domain of kinesin light chain bound to the cargo molecule JIP3. From this they propose a mechanism by which multiple, unrelated cellular cargo molecules can “hotwire” the kinesin-1 molecule into motility. Kinesin-1 transports a wide variety of cellular cargoes toward the plus-ends of microtubules, including proteins, vesicles, mRNP complexes, and organelles, and is implicated in a number of diseases including neurodegeneration, viral and bacterial infections, and cancer . The simThe KHC motor domain hydrolyses ATP to power microtubule-based motility. At the other end of the molecule, the KHC tail domain and KLC harbor binding sites for cargoes . The ATPIn vitro binding studies using purified recombinant proteins also showed that KLC reduces the affinity for the KHC tail domains for both the motor domains and microtubules  locks the motor domains together, preventing their stable association with microtubules . Autoinhotubules . Recent otubules   is unusual in binding to multiple ligands using distinct sites  and prevents KLC binding  B and the binding . Kinesin binding . JIP3 ac binding . The JIPding (LZ B binds trmediate . Bindingrmediate . JIP3 thTPR) bound to the JIP3LZ. This reveals how ARF6 regulates kinesin-1 recruitment by JIP3/4. We show that JIP3 activates kinesin-1 by a different mechanism to tryptophan-acidic cargoes, and propose a unified framework explaining how unrelated cargoes activate kinesin-1 through related mechanisms.Here, we describe the crystal structure of the KLC2 TPR domain (KLC2TPR) in complex with the murine JIP3LZ to a resolution of 3.2 Å  binds to both JIP3LZ subunits (chains C and D). The KLC2 TPR1 α helices pack flat against the side of the JIP3LZ, lying across the JIP3LZ helices at a ∼90° angle, and oriented with helix 1B proximal to the JIP3LZ N terminus   to the ITC data, which gave a good fit across the entire dataset  spectroscopy  to facilitate protein concentration determination since JIP3LZ does not contain any Try or Trp residues. Analysis of the Trp-JIP3LZ CD spectra revealed a mean α-helical content of only 59% ± 3%, compared with 77% calculated for JIP3LZ from the KLC2TPR:JIP3LZ crystal structure  A–2D. The dataset  and 2. Ttroscopy E. For thtructure F. By comtructure G. The Trt 222 nm E: the rat 222 nm . Nonethe15.9 kDa H, showin KLC2TPR H.Figure TPR binding site spans heptad repeats 2–5  of the JIP3LZ , and Ile446 (chain C) form a hydrophobic patch, which is flanked by the hydrophilic residues Glu433, Thr434, and Asp450 (chain D) and Lys435, Asn439 Lys442, and Asp450 (chain C). The JIP3LZ hydrophobic patch is recognized by a number of hydrophobic side chains that line the inside of KLC2 TPR1  , and Gln223 and Asp227 (helix B), hydrogen bond to the side chains of JIP3 residues Asp450, Asp444, Lys435, and Asn439 (The KLC2e JIP3LZ A. The JIe JIP3LZ B. The fo Leu220) C. Aroundd Asn439 D. The ind Asn439 A and 3E,d Asn439 .Figure 3TPR:JIP3LZ complex is the same as in solution, we probed the KLC2TPR:JIP3LZ interface using ITC binding experiments with wild-type and mutant KLC2TPR-myc. -labeled LFP motif peptide : the amino acids involved in KLC2TPR binding are totally conserved in almost all Bilaterian JIP3/4 homologs, but not those from any species in the other animal phyla. However, the JIP3LZ binding site on TPR1 is more widely conserved, being totally conserved in essentially all Bilateria, but also in over half of the non-Bilaterian species in our datasets, across all phyla   values of 0.7–1.0 Å between 81 and 83 structurally equivalent Cα atoms   (Araki eSTN-WD2) . The TPRructures D, suggesructures . The dimCα atoms E. The amCα atoms  and S4. TPR:JIP3LZ interface with the crystallographic TPR1:TPR1 packing interfaces observed in other KLCTPR crystal structures   shows that the KLCTPR concentration inside the kinesin-1 molecule would be at least 100 μM. Nevertheless, we anticipate that the monomer-dimer equilibrium of KLCTPRs in the kinesin-1 molecule would favor the monomer in the autoinhibited state, since dimerization would mask the JIP3 binding site and lead to relief of KLC regulation. However, cargoes that tether the TPR domains together would promote dimerization via TPR1, leading to relief of KLC regulation. Indeed, KLC cargoes are usually dimeric or possess two KLC binding sites. This would allow cargoes binding to different regions of the KLCTPR to relieve KLC regulation by the same mechanism. For example, kinesin-1 activation by JIP1  follows a similar scheme to JIP3 despite engaging the KLCTPR in a completely different manner  F (NguyenB: 5OJF) . This sh binding A–6C, and binding  and S4. gulation , then so is weak . Considet manner . Interesner Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Joseph Cockburn (E.coli Rosetta (DE3) cells for production of all recombinant proteins used in this study. The cells were cultured using standard practices in LB media.We used Plasmids pMW his-SUMO and pMW GST3C  are bactTPR construct used in this work comprised murine KLC2 residues 191-480, which is similar to those used in previous studies  was amplified by PCR from a murine template  were generated by overlap PCR.The KLC2 studies  but withtemplate  and clonLZ domain construct used in this work (residues 417-487 of murine JIP3 isoform A) is equivalent to the JIP4LZ construct that was previously co-crystallised in complex with ARF6  and cloned into the NotI/EcoRI sites of pMW-GST3C. For CD and binding studies, we used a JIP3LZ construct N-terminally fused to a tryptophan residue (Trp-JIP3LZ) for quantitation of protein concentration by UV-VIS. The pMW-GST3C-Trp-JIP3LZ vector was constructed as above but using a forward primer encoding an in-frame, N-terminal tryptophan residue. All inserts were sequence-verified.The JIP3ith ARF6 . To conslac repressor gene or a lac operator sequence downstream from the T7 promoter. Proteins were produced by leaky expression in BL21(DE3) Rosetta cells. The relevant plasmid was transformed into BL21(DE3) Rosetta cells. 5 ml LB-ampicillin (100 μg/ml) cultures were inoculated from the resulting colonies and grown at 37°C for 8 hours. Each 5 ml culture was then used to inoculate a 1 L LB-ampicillin culture. The 1 L cultures were grown in 2 L baffled flasks overnight at 30°C and 185 rpm. Cultures were clarified by centrifugation and the pellets were re-suspended in cold binding buffer supplemented with cOmplete EDTA-free protease inhibitor cocktail (Roche). Re-suspended pellets were stored at -80°C until further use. Re-suspended bacteria were thawed and lysed by sonication on ice. Lysates were clarified by centrifugation.The pMW plasmids do not contain the TPR protein was purified by nickel-ion affinity chromatography. A 1 ml His-trap column  was equilibrated in binding buffer . The clarified lysate was loaded onto the column, and unbound material washed out back to baseline with binding buffer. Bound protein was then eluted in a linear gradient of elution buffer . Fractions were analysed by SDS-PAGE and pure fractions pooled. The his-SUMO tag was cleaved off overnight at 4°C with ULP1 protease. The KLC2TPR protein was separated from the his-SUMO tag by size-exclusion chromatography on a Superdex 75 16/600 column  equilibrated in 100 mM NaCl, 25 mM HEPES, 1 mM TCEP .The His-SUMO-KLC2LZ protein was batch-purified using glutathione sepharose 4B resin . Resin was pre-equilibrated in binding buffer  and incubated in suspension with the clarified lysate for at least 4 hours at 4°C. Unbound material was removed by washing the resin 5 times in wash buffer . The JIP3LZ was obtained by cleaving the resin-bound GST3C-JIP3LZ protein with 3C protease overnight at 4°C, and purified by size-exclusion chromatography as described above. The pooled fractions were further purified to remove contaminating GST by anion exchange chromatography as follows. The protein was diluted 4-fold in 25 mM HEPES pH 7.0 to a final NaCl concentration of 25 mM. The diluted protein was applied to a 1 ml HiTrap Q XL column  pre-equilibrated in 25 mM HEPES pH 7.0. The pure JIP3LZ was collected as the flow through. The NaCl concentration in the pure protein was increased to 100 mM using a 5 M NaCl solution.The GST3C-JIP3TPR and GST-JIP3LZ, the high KLC2TPR concentrations required to use this protein as titrant necessitated a higher salt concentration than that used for crystallographic studies. The KLC2TPR was prepared as above for crystallographic studies but the final size exclusion chromatography step was performed in 500 mM NaCl, 25 mM HEPES, 5mM MgCl2, 1 mM TCEP . The GST-JIP3LZ protein was bound to glutathione sepharose resin in batch as described above for the JIP3LZ. The GST-JIP3LZ protein was eluted in 0.25 M NaCl, 50 mM TRIS.HCl, 1 mM TCEP, 25 mM glutathione; final pH 7.5, and purified by size exclusion chromatography on a Superdex 200 16/600 column  equilibrated in 500 mM NaCl, 25 mM HEPES, 5mM MgCl2 and 1 mM TCEP .For ITC studies with the KLC2LZ protein used for CD and ITC experiments was prepared as described above for the JIP3LZ, with the following modifications. Following 3C cleavage, contaminating GST3C was removed by incubation of the eluate with fresh, pre-equilibrated glutathione sepharose 4B resin for 2 hours at room temperature. The Trp-JIP3LZ was then further purified by size exclusion chromatography using a Superdex 75 16/600 column equilibrated in 100 mM NaCl, 25 mM HEPES, 1 mM TCEP . Wild-type/mutant KLC2TPR-myc samples used for CD and ITC experiments were prepared as described above the KLC2TPR used in crystallographic studies. The Trp-JIP3LZ and KLC2TPR-myc samples used in the fluorescence anisotropy-based binding competition assays were prepared as for those used in the CD and ITC studies, but the final size-exclusion step was performed in 100 mM NaCl, 25 mM HEPES, 5mM MgCl2, 1 mM TCEP .The Trp-JIP3LZ protein was concentrated in a Vivaspin column  to a concentration of 0.63 mg/ml as quantified by a Bradford assay, and mixed with pure KLC2TPR (at 4.1 mg/ml) to give a final KLC2TPR:JIP3LZ stoichiometry of 3:1. The complex was concentrated in a spin concentrator  to a total protein concentration of 4.5 mg/ml, and incubated at 4°C overnight. Sitting drop crystallisation trials were set up the following day in SWISSCI MRC 3-well crystallisation plates (Jena Bioscience) using a Formulatrix NT8 crystallisation robot before storage and imaging in a Formulatrix Rock Imager at 19°C. Three-dimensional crystals grew in condition A10 of the Wizard Classic 3/4 HT96 screen (Molecular Dimensions) in a drop composed of 200 nl protein and 100 nl reservoir ; the drop ratio was further optimised to 200 nl protein plus 50 nl reservoir. Crystals were cryoprotected in 10% PEG 3350, 75 mM NaCSN, 150 mM NaCl, 37.5 mM HEPES pH 7.5 and 25% glycerol and flash frozen in liquid nitrogen. X-ray diffraction data were collected at beamline I24 of Diamond Light Source  at a wavelength of 0.96861 Å. Data were processed using XDS (TPR (PDB: 3CEQ) and the JIP4LZ (chains C and D from PDB: 2W83) (LZ (PDB: 2W83) and KLC1TPR (PDB: 3NF1) (http://molprobity.biochem.duke.edu) (th percentile), with 96.75/0% residues in the favoured/forbidden regions of the Ramachandran plot. Figures were prepared using PYMOL (Schrodinger).The purified JIP3sing XDS  and progsing XDS . Phases sing XDS , using tB: 2W83) . The strB: 2W83)  and refiB: 3NF1) . The moduke.edu) . The finTPR gel filtration buffer and mixed with purified KLC2TPR protein, at 2:1 (peptide:TPR) stoichiometry, giving a final KLC2TPR concentration of 5.5 mg/ml. The complex was incubated at room temperature for 1 hour prior to setting up robotic crystallisation trials at 19°C. Crystals were grown in condition D1 of the JCSG Core III screen  in a drop composed of 100 nl protein and 100 nl reservoir. This was further optimized to 0.893 M sodium potassium tartrate, 0.2 M NaCl, 0.1 M imidazole pH 8.0. Crystals were cryoprotected in reservoir plus 25% glycerol and flash frozen in liquid nitrogen. X-ray diffraction data were collected at beamline I04-1 of Diamond Light Source  at a wavelength of 0.9200 Å. Data were processed as described above. Phases were obtained by molecular replacement with PHASER (3ZFW (TPR (3NF1). The CSTN-WD2 peptide structure was rebuilt and the model refined in BUSTER 2.10.3  to 4.0 Å resolution with each TPR domain:peptide complex as a separate TLS group, LSSR NCS restraints, and LSSR restraints to the higher resolution structure of the KLC1 TPR domain (PDB: 3NF1) (CSTN-WD2 peptide was dissolved in KLC2h PHASER  using PDER (3ZFW  as the sZFW (TPR  (PDB: 3NB: 3NF1) . The strB: 3NF1)  with NCSTPR to GST3C-JIP3LZ, the proteins were prepared by size exclusion chromatography in 0.5 M NaCl, 25 mM HEPES, 5 mM MgCl2, 1 mM TCEP, final pH 7.5. ITC measurements were performed on a MicroCal iTC200 calorimeter  at 25°C, with a differential power of 5.0 μcal/s and stirring at 750 rpm. Experiments consisted of an initial sacrificial 0.5 μl injection, followed 120 s later by 19 injections of 2 μl spread over 4 s, spaced 120 s apart. Thermograms were integrated and corrected for heats of dilution using NITPIC (http://biophysics.swmed.edu/MBR/software.html). The resulting isotherms were analysed in SEDPHAT  (http://biophysics.swmed.edu/MBR/software.html).To study the binding of the KLC2edphat/) . Four exLZ and FITC-CSTN-WD2 peptide to wild-type and mutant KLC2TPR-myc, the recombinant proteins were prepared by size exclusion chromatography in 0.1 M NaCl, 25 mM HEPES, 1 mM TCEP, final pH 7.5. Wild-type or mutant KLC2TPR-myc at 44-50 μM were placed in the cell. The Trp-JIP3LZ was used as the titrant at dimer concentrations of 297-330 μM. The FITC-CSTN-WD2 peptide was used as the titrant at a concentration of 380 μM. The peptide concentration was determined by measuring the absorbance at 483 nm and using a molar extinction coefficient of 68,000 M-1cm-1. ITC experiments were performed as described above with injections spaced 120-150 s apart. Thermograms were integrated and corrected for heats of dilution using NITPIC (http://biophysics.swmed.edu/MBR/software.html). The resulting isotherms were analysed in SEDPHAT. Data for the Trp-JIP3LZ as titrant were fitted with the A + B + B → AB + B → ABB model, whilst binding of the FITC-CSTN-WD2 peptide was analysed using the A + B → AB model. Between 2 and 3 experiments were performed for each titrand/titrant pair. For each titrand/titrant pair, the isotherms from the experiments were globally fitted with the relevant model. During the fitting, the inactive fractions of A and B were globally fitted, whilst the cell and syringe concentrations and baselines of each experiment were fitted locally. The results are listed in Napp) for each titrand/titrant pair, defined asFKLC2,i and Flig,i are the locally-fitted correction factors for KLC2 and ligand concentrations for experiment i, and AKLC2 and Alig are the active fractions of KLC2 and ligand globally fitted over the n experiments. During fitting, the FITC-CSTN-WD2 peptide concentrations consistently refined to values around 30% greater than the measured value of 380 μM, with Napp values around 0.7  (All CD spectra were recorded on a Chirascan CD spectrometer (Applied Photophysics) in quartz cuvettes with a 0.1 cm path length. Data were recorded at 20°C between 260 - 180 nm in 1 nm increments and a 2 nm bandwidth over an average of two scans. The KLC2b server . KLC2TPRet set 4 . For eacecgi.py) .LZ was injected onto a WTC-030S5 column, pre-equilibrated with buffer containing 25 mM HEPES pH 7.5, 0.1 M NaCl and 1 mM TCEP at a flow rate of 1 ml/min. Data were recorded using a DAWN 8+ multi-angle light scattering (LS) detector, an Optilab T-rEX differential refractive index (dRI) detector and UV absorbance (UV) detector (Wyatt Technology) and analyzed with the Astra 6.2 software package provided by the manufacturer (Wyatt Technology).Fifty microliters of JIP3TPR-myc was measured essentially as described previously  in triplicate in 384-well plates, for 1 hour at room temperature. Fluorescence measurements were recorded on a TecanSpark plate reader at excitation and emission wavelengths of 488 nm and 535 nm respectively. Fluorescence anisotropy values were calculated at each KLC2TPR-myc concentration and the data were fitted with a fixed-slope dose response curve in Prism (GraphPad). This gave an affinity of 37 μM, similar to that found previously (LZ were incubated with the FITC-labelled LFP motif peptide (300 nM) and a fixed concentration of KLC2TPR-myc (21 μM). A KI value was obtained for the CSTN-WD1 peptide data by fitting the anisotropy values A with following equation using Prism (Graphpad):Amin and Amax are the minimum and maximum anisotropy values, [KLC] and [CSTN] are the total concentrations of KLC2TPR-myc and calsyntenin-1 peptide, respectively, and KLFP is the dissociation constant for the FITC-LFP motif peptide binding to the KLC2TPR-myc . Two independent experiments were performed with the Trp-JIP3LZ. One experiment was performed for the CSTN-WD1 peptide. Graphs in The binding affinity between the FITC-labelled LFP-motif peptide and the KLC2eviously . FITC-laeviously . For comChordata, 10 Arthropoda, 3 Nematoda, 2 Annelida, 1 Playthelminthes and 1 Mollucsa) and 3 non-Bilaterian genomes . Dataset 2 was assembled from Simion et al. , via residues 433-450 from JIP3LZ chains C and D and the equivalent residues in the JIP4LZ using LSQKAB from the CCP4 suite (α atoms). Full length ARF6 contains an N-terminal myristoylation group on residue 2 and an amphipathic helix (residues 2-11), both of which were absent from the ARF6:JIP4LZ crystal structure. The thickness of the membrane and positioning of the ARF6 amphipathic helix along the bilayer normal in The KLC2 complex   showed that the TPR1 polypeptide chain conformations were very similar . Taken together with the fact that the KLC1 and KLC2 TPR1s are 100% identical in amino acid sequence, we interpreted this discrepancy as being due to difficulties in modelling the PDB: 5FJY TPR1s into the original electron density map, rather than a difference in structure of the TPR1:TPR1 interfaces between these two crystals.For each copy of the KLC in COOT  using a UPERPOSE  from theUPERPOSE . This gaP4 suite . TPR1 wae-chains . Comparisolution ; PDB: 3NInformation on the number of experiments performed and definitions of errors and means can be found in the relevant sections of the methods and the table/figure legends.TPR:JIP3LZ and KLC2TPR:CSTN-WD2 crystal structures reported in this paper are PDB: 6EJN and PDB: 6F9I respectively.The accession numbers for the coordinates and structure factors for the KLC2