However, highly active, selective, and energy‐efficient CO2 conversion to multicarbon hydrocarbons, such as C2H4, remains challenging because of the lack of efficient catalysts. Herein, an ultrasonication‐assisted electrodeposition strategy to synthesize CuO nanosheets for low‐overpotential CO2 electroreduction to C2H4 is reported. A high C2H4 Faradaic efficiency of 62.5% is achieved over the CuO nanosheets at a small potential of −0.52 V versus a reversible hydrogen electrode, corresponding to a record high half‐cell cathodic energy efficiency of 41%. The selectivity toward C2H4 is maintained for over 60 h of continuous operation. The CuO nanosheets are prone to in situ restructuring during CO2 reduction, forming abundant grain boundaries (GBs). Stable Cu+/Cu0 interfaces are derived from the low‐coordinated Cu atoms in the reconstructed GB regions and act as highly active sites for CO2 reduction at low overpotentials. In situ Raman spectroscopic analysis and density functional theory computation reveal that the Cu+/Cu0 interfaces offer high *CO surface coverage and lower the activation energy barrier for *CO dimerization, which, in synergy, facilitates CO2 reduction to C2H4 at low overpotentials.Electrochemical CO +/Cu0 interfaces are in situ reconstructed in the CuO nanosheets during CO2 electroreduction. The reconstructed grain boundaries with Cu+/Cu0 interfaces increase the *CO surface coverage and lower the energy barrier for *CO dimerization, leading to a record maximal half‐cell cathodic energy efficiency of 41% for C2H4 formation.Abundant grain boundaries involving stable Cu According to recent technoeconomic assessment studies, the production cost of C2H4 in the eCO2RR process is estimated to be as high as US$2.48 per kg C2H4; thus, it is not economically beneficial, as the market price is US$0.55 per kg C2H4. Increasing the energy efficiency of a CO2 electrolyzer is a feasible approach to reducing production costs. Cu catalysts have achieved significant advances in the production rate and Faradaic efficiency (FE) of C2H4. However, they still suffer from low energy efficiency as the peak FE of C2H4 appears at considerably negative cathodic potentials from −0.7 to −1.0 V (vs reversible hydrogen electron (RHE hereafter)). Reducing the overpotential of the eCO2RR over Cu catalysts while maintaining the selectivity and productivity toward a target C2+ product can improve the market competitiveness of the eCO2RR; however, this remains a big challenge.Electrochemical COl energy.1, 22RR toward C2+ products than Cu metal catalysts. The two main features of the OD‐Cu catalyst, including defect sites with low‐coordinated Cu atoms and polarized Cuδ+ (0 < δ ≤ 1) induced by residual subsurface O, have been deemed responsible for promoting the C—C coupling step at low overpotentials. The defect sites combining the strain and low coordination number effects enhance *CO adsorption, increasing the CO coverage of the catalyst, which kinetically favors C—C coupling over hydrogenation. Similarly, both the experimental results and theoretical calculations provide solid evidence that *CO adsorption on the Cu+ site is stronger than that on the Cu0 site because of the unoccupied 3d orbital of Cu+. Moreover, Cu+/Cu0 interfaces promote CO2 activation and CO dimerization. The Cu+/Cu0 interface has more favorable energetics for C—C coupling than a single Cu+ or Cu0 site because attractive electrostatic interactions between the two Cs of CO assist the C—C bond formation. However, the existence of subsurface O during the eCO2RR is a long‐standing debate in literature because its presence depends on the history of the material. Further, the theoretical prediction of the stability of subsurface O differs because of the difference in the selection of the material model. Thermodynamically, the CuO will be fully reduced at neutral and alkaline pH for applied potentials lower than −0.1 V. However, the grand canonical potential kinetics density functional theory method has predicted that subsurface O is preserved in the disordered Cu region with low coordination. Notably, the electrochemical standard redox potentials of Cuδ+/Cu0 shift to a more negative value with the decrease in the bulk size to a few atoms . This suggests that the Cuδ+ species with the subsurface O can be thermodynamically trapped in the grain boundaries (GBs) containing abundant low‐coordinated Cu atoms. Therefore, it is reasonable to assume that stable Cuδ+ species can induce active Cuδ+/Cu0 interfaces across Cu GBs.Oxide‐derived Cu (OD‐Cu) catalysts exhibit a significantly lower overpotential of the eCOatalysts.11 The2RR. The electrochemical reduction of CuO or Cu2O to Cu can create more lattice distortions and defects, which decrease the structural stability by improving the surface energy of the Cu atoms. The undercoordinated Cu atoms around the lattice distortions and defect regions are conventionally unstable and inclined toward surface reconstruction to form small‐sized grains. A nanoclustering process is proposed to form relatively small Cu nanoclusters and nanoparticles with an increasing number of low‐coordinated sites during the eCO2RR. Resultantly, large CuO or Cu2O nanoparticles spontaneously evolve into small Cu nanograins with abundant GBs during dynamic reconstruction. The dislocated lattices with low‐coordinated Cu atoms in the reconstructed Cu GBs provide defect sites for oxide nucleation, affording Cuδ+ species along the Cu GBs.The GBs in the OD‐Cu are formed by the in situ reduction and reconstruction of CuO nanostructures during the eCO2RR.36 The2‐to‐C2H4 reduction over GBs involving Cu+/Cu0 interfaces. Rich‐density GBs are formed via in situ reduction of CuO nanosheets and subsequent reconstruction during the eCO2RR. An ultrasonication‐assisted electrodeposition method is developed for the facile synthesis of CuO nanosheets. In contrast to the electrodeposition onto a substrate in the conventional process, ultrasonication‐assisted electrodeposition attains freestanding CuO nanosheets formed by peeling Cu nanosheets off the substrate by ultrasonication, followed by the spontaneous oxidation of Cu in an alkaline solution. To the best of our knowledge, the prepared CuO nanosheets exhibit the lowest potential of −0.52 V to achieve an industrially relevant C2H4 partial current density (jC2H4) of 173 mA cm−2 in the eCO2RR. A high FE of C2H4 (FEC2H4) of 62.5% at such a low potential contributes to a maximal half‐cell cathodic energy efficiency (CEE) of 41%. The low‐coordinated Cu atoms in the GB regions shift the electrochemical redox potential to more negative values, thereby stabilizing the Cu+ species and creating the Cu+/Cu0 interfaces across the GB width. The combined results of in situ Raman spectra and density functional theory (DFT) computations suggest that the reconstructed GBs with the Cu+/Cu0 interfaces serve as primary active sites that steer the C—C coupling to produce C2H4 at low overpotentials along the pathway of the *CO dimerization.Here, we report a low overpotential yet highly active and selective CO22.1m KOH electrolyte solution, as shown in FigureE–t curves 4]2− complexes, followed by reduction to Cu on the carbon‐paper cathode (stage I). Subsequently, the gradually cumulative Cu‐nanosheet electrodeposits on the cathode were peeled off under ultrasonication and oxidized into CuO nanosheets in the KOH electrolyte (stage II). Owing to the accumulation of Cu(OH)2 on the surface of the Cu foam, the predominant reaction on the anode was switched from Cu anodization to the O evolution reaction (OER) on Cu(OH)2 during electrodeposition for 40 min. The applied potential abruptly increased because of the activation polarization caused by the sluggish kinetics of the OER. The real‐time captured photo images of the electrolyte revealed that this proposed two‐stage reaction process produced large quantities of CuO nanosheets , the Cu sample electrodeposited on the carbon paper showed a microsphere structure composed of nanoparticles at a deposition current density of 190 mA cm4]2− complexes near the carbon‐paper cathode and continuously grown into Cu nanoclusters upon the reduction of more [Cu(OH)4]2− complexes (step 1). The ultrasonication induced the cavitation effect, which provided vacuum bubble reaction conditions with extremely high temperatures and pressures. The bubbles were born and grown under ultrasonication and finally collapsed, facilitating the symmetrical breaking of the Cu nanoclusters (step 2). Resultantly, the cracked Cu nanoclusters were oriented toward the growth of the nanosheet structures attached to the cathode (step 3). Small nanosheets with adherent nanoparticles were observed on the electrodeposited Cu on the carbon paper  from cyclic voltammetry curves at various scan rates  and CuO‐0W (12%) electrodes, respectively , and jC2H4 for the peak FEC2H4, and the corresponding half‐cell CEE between the CuO‐160W (deposited at 190 mA cm−2) and previous Cu‐based electrodes. The CuO‐160W electrode possessed the lowest onset potential for C2H4 production. Moreover, the CuO‐160W electrode yielded the highest jC2H4 at the lowest corresponding potential of −0.52 V. Accordingly, the CuO‐160W electrode attained the highest half‐cell CEE of 41%. Additionally, the long‐term stability test demonstrated that the CuO‐160W electrode maintained a steady jtotal of 300 mA cm−2 and a stable FEC2H4 of ≈60% for at least 60 h when CO2 electrolysis was performed at a constant potential of −0.52 V  (outlined in green) and Cu(111) (outlined in blue), resulting in high‐density GBs  and Cu(111) grains afforded abundant Cu2O(111)/Cu(111) (Cu+/Cu0) interfaces (outlined in red) in the post‐CuO‐160W nanosheets. The high‐resolution HAADF‐STEM image shown in Figure +/Cu0 interfaces in the fragmented post‐CuO‐160W nanosheets. Electron energy loss spectroscopy (EELS) mapping showed the distinguished Cu2O and Cu grains  were more negative than the value of the bulk metal (n >> 20). In a limiting case, the electrochemical standard reduction potentials for a single metal atom were several volts more negative than for bulk metal . Lattice deformation and dislocation in reconstructed GBs created many Cu and/or Cu+ surface sites with a low coordination number. Thus, although the Cu2O(111) grains will likely be reduced to Cu0 during eCO2RR, the Cu2O surface sites in the region of the GBs could maintain a Cu+ state because they held more negative reduction potentials than the applied potential . We believe that the electrochemically stable Cu+/Cu0 interfaces across the reconstructed GBs were responsible for the low‐overpotential yet highly selective and productive CO2‐to‐C2H4 conversion over the CuO‐160W electrodes.Notably, the electrochemical standard potentials for small metal clusters containing n atoms (n >> 20).52 In Figure−1 were observed for the CuO‐160W electrode at an open‐circuit potential (OCP). The CuO Raman signals disappeared when a constant potential of −0.15 V was applied. Meanwhile, a peak at 523 cm−1 was observed, which could be assigned to the Cu2O species. This Raman signal intensity of Cu2O continued to increase as the applied potential increased to −0.30 V and remained almost constant from −0.35 to −0.55 V. When the applied potential exceeded −0.55 V, the peak intensity of Cu2O slightly decreased but was still retained at a high potential of −0.65 V. The decrease in the peak intensity was ascribed to the applied potential exceeding the standard reduction potential of −0.36 V for bulk Cu2O reduction to Cu in an alkaline solution. The Raman spectra results indicated that the Cu2O species remained in existence during the eCO2RR, which was largely attributed to abundant undercoordinated Cu atoms or clusters in the dense Cu GBs. Further, we investigated time‐dependent Raman spectra on the CuO‐160W electrode at −0.50 V to gain in‐depth insight into the evolution of reconstruction with time  were selected as the model planes based on the TEM results  for eCO2RR. Further, previous studies found that the synergistic effect between the surface Cu0 and Cu+ sites promoted *CO dimerization to form *OCCO. The positively charged *CO on the Cu+ site and the negatively charged *CO on the Cu0 site were dimerized with feasible thermodynamics and kinetics.We further studied the kinetics of the dimerization of *CO to *OCCO on the Cu GBs, Curm *OCCO.10 The2 reduction to C2H4 over the Cu+/Cu0 interfaces. The *CO dimerization rate had a reaction order of two with respect to the *CO surface coverage. Further, we compared the CO generation and dimerization rates as a function of the applied potential among three different CuO electrodes . XPS was performed on an ESCAL AB250Xi spectrometer with Al Kα X‐ray radiation. X‐ray absorption spectroscopy measurements at the Cu K‐edge were conducted at the National Synchrotron Light Source II of the Brookhaven National Laboratory. In situ Raman spectra were collected on a Horiba LabRAM HR evolution Raman spectrometer with a 633 nm laser equipped with a gas–solid–liquid three‐phase flow cell . During the in situ Raman testing, a 1.0 m KOH solution serving as both the catholyte and anolyte was pumped into the electrolyte compartment at a flow rate of 0.1 mL min−1, whereas CO2 (99.99%) was continuously supplied to the gas compartment at a flow rate of 5 sccm.SEM images were collected using an SU8020 microscope to analyze the morphology of the catalysts. STEM was performed using an FEI Talos 200F microscope with an acceleration voltage of 200 kV and an HAADF detector. XRD was performed using a PANalytical X‐Pert PRO MPD instrument with a Cu‐target X‐ray source , a carbon paper with a microporous carbon layer (Sigracet 34BC), to prepare gas diffusion electrodes (GDEs). The loading of the CuO catalysts was 0.5 mg cm−2 for all GDEs. The as‐prepared, relatively large CuO‐based GDEs were cut into 1 × 1 cm2 pieces, which served as the cathode for multiple independent tests, whereas Ni foam pressed onto a 2 × 2 cm2 GDL acted as the anode. The current densities were calculated based on the active geometric area of the cathode (1 cm2). Additionally, 1.0 m KOH solution was pumped into the cathodic and anodic compartments at a flow rate of 1.0 mL min−1 and separated using an anion exchange membrane (Fumasep FAA‐3‐PK‐75). High‐purity CO2 gas was fed into the serpentine flow channel on the cathode side at a flow rate of 50.0 sccm. A constant cell voltage was applied to the flow cell during CO2 electrolysis. An Ag/AgCl (saturated KCl) electrode was bridged to the cathodic compartment to measure the electrode potential. All potentials were converted to an RHE scale with manual iRs compensation: ERHE = EAg/AgCl + 0.0591 × pH + 0.197 V − iRs. The iRs was determined by potentiostatic electrochemical impedance spectroscopy measurements under an OCP at frequencies ranging from 100 kHz to 0.1 Hz.The eCO2, CO2, and CO; FID for CH4, C2H4, and C2H6). The liquid products were quantified by 1H nuclear magnetic resonance (NMR) spectroscopy (Bruker AV 400 MHz spectrometer). The concentration of the liquid products was calculated based on the NMR peak integral areas and calibration curves. To prepare the NMR samples, 500 µL of the collected electrolyte was mixed with 100 µL of D2O solution consisting of 5 × 10−3m 3‐(trimethylsilyl)propionic‐2,2,3,3‐d4 acid sodium salt (TSP). TSP was used as an internal standard.The gaseous products were analyzed by online gas chromatography  with a flame ionization detector (FID) and a thermal conductivity detector (TCD). The molar percentages of the gaseous products were calculated from the GC peak areas based on the calibration curves (TCD for HEC2H40denotes the standard potential of CO2 reduction to C2H4, 0.08 V, FEC2H4 is the FE of C2H4, and Ecathode is the applied potential of the cathode.The half‐cell cathodic energy efficiency was calculated by the following equation The projected augmented wave potentials were chosen to describe the ionic cores and considered valence electrons using a plane‐wave basis set with a kinetic energy cutoff of 400 eV. The symmetrical low‐angle tilt boundary model was chosen for the DFT simulation based on the HRTEM images. The partial occupancies of the Kohn–Sham orbitals were allowed using the Gaussian smearing method with a width of 0.05 eV. The electronic energy was considered to be self‐consistent when the energy change was less than 10−4 eV. Geometry optimization was deemed to be convergent when the energy change was smaller than 0.04 eV Å−1. The vacuum spacing in the direction perpendicular to the plane of the structure was 16 Å. Brillouin zone integration was performed using 2 × 2 × 1 Monkhorst–Pack k‐point sampling for the structure. Gibbs free energy was calculated using the following equationG, E, ZPE, and TS are the Gibbs free energy, total energy from DFT calculations, zero‐point energy, and entropic contributions, respectively. The U correction was set to 3.41 eV for the Cu atoms in the systems.First‐principles were employed to perform all spin‐polarization DFT calculations within the generalized gradient approximation using the Perdew–Burke–Ernzerhof formulation.61 TheThe authors declare no conflict of interest.Supporting InformationClick here for additional data file.


Correction to:EMBO Mol Med (2020) 12: e10605. DOI: 10.15252/emmm.201910605 ¦ Published online 24 April 2020The authors wish to correct electrophysiological and behavioral data shown in Figs 1E, and 5E and I, and the corresponding Materials and Methods.Figure 1E: The authors correct the labeling of the Y‐axis in the bottom panel of Fig 1E.Figure 1E. Original.Figure 1E. Corrected.Figure 5E: A scale bar has been added to Fig 5E.Figure 5E.Figure 5I: Some data points from the Veh and THC plots in 5D were incorrectly copied into 5I, which slightly affected the mean without affecting the statistics. We are herewith retracting 5I and replacing it with the corrected figure. The authors note that the interpretation of the data remains the same.Figure 5I. Original.Figure 5I. Corrected.In addition, two sentences in the Electrophysiological recordings section of Materials and Methods are updated:The sentence,“Action potentials (APs) were evoked by a ramp current injection with 10‐pA increments for 2 ms, and only the first AP was used to determine the AP threshold and the minimal current necessary to induce firing (rheobase).”Is updated to“Action potentials (APs) were evoked by a ramp current injection with 10‐pA increments for 2 ms to determine the AP threshold and the minimal current necessary to induce firing (rheobase).”The sentence,“Hyperpolarization‐activated cation current (Ih) was measured as the voltage sag to a 500 ms hyperpolarizing current injection (50‐pA increment from −400 pA to 0).”Is updated to“Hyperpolarization‐activated cation current (Ih) was measured as the voltage sag to a 2,000 ms hyperpolarizing current injection (50‐pA increment from −400 pA to 0).”The authors acknowledge the constructive input from Sodikdjon Kodirov and apologize for these errors and any confusion they may have caused.


PKHD1) gene, which usually leads to end‐stage renal disease. Previous studies suggested that the P2X purinoreceptor 4 (P2X4R) may play an important role in the progression of ARPKD. To test this hypothesis, we assessed the chronic effects of ivermectin  and 5‐BDBD (P2X4R antagonist) on the development of ARPKD in PCK/CrljCrl‐Pkhd1pck/CRL (PCK) rats. Our data indicated that activation of ATP‐mediated P2X4R signaling with ivermectin for 6 weeks in high dose  decreased the total body weight of PCK rats while the heart and kidney weight remained unaffected. Smaller doses of ivermectin  or the inhibition of P2X4R signaling with 5‐BDBD  showed no effect on electrolyte balance or the basic physiological parameters. Furthermore, cystic index analysis for kidneys and liver revealed no effect of smaller doses of ivermectin (0.5 or 5 mg/L) and 5‐BDBD on the cyst development of PCK rats. We observed a slight increase in the cystic liver index on high ivermectin dose, possibly due to the cytotoxicity of the drug. In conclusion, this study revealed that pharmacological modulation of P2X4R by ivermectin or 5‐BDBD does not affect the development of ARPKD in PCK rats, which may provide insights for future studies on investigating the therapeutic potential of adenosine triphosphate (ATP)‐P2 signaling in PKD diseases.Autosomal recessive polycystic kidney disease (ARPKD) is an inherited pathology caused mainly by mutations of the polycystic kidney and hepatic disease 1 ( Recent studies demonstrated the significant involvement of P2X4 and P2X7 receptors in cyst cell calcium signaling. However, neither allosteric modulation nor an antagonism of P2X4 receptors by chronic administration of ivermectin or 5‐BDBD, respectively, affect the progression of ARPKD in PCK rats. For example, both P2X7R agonists and antagonists have been shown to be capable of modulating the development of renal cysts  has been linked to epithelial transport in the nephron  actions converge at receptor or post‐receptor signaling pathways in ANG II‐dependent hypertension    were delivered to PCK rats in drinking water starting at 6 weeks of age for 42 days, respectively. DMSO was used to dissolve ivermectin to stock concentrations of 10 mM. For oral administration, we used the maximum approved dose of 50 mg/L in our protocol based on the recommendation for rats as described previously  starting at 4 weeks of age for 56 days. Based on the previous reports . Blood and organ collections were performed at the end of the treatment as previously described  through the abdominal aorta. Kidneys were extracted, decapsulated, and placed into ice‐cold PBS /medical grade O2.4t‐test or ANOVA with Tukey post hoc test. *p < 0.05.Data are expressed as mean ± SEM and analyzed using GraphPad Prism 9 (GraphPad Software). Different groups were compared using the student's 33.14R and significantly increases purinergic response effectiveness, reducing ATP EC50 values up to ten times , 0.5 (N = 17), 5 (N = 10), and 50 mg/L (N = 8) ivermectin delivered in drinking water, respectively, starting from 6 weeks of age for 42 days  or 18 mg/kg/day 5‐BDBD (N = 8), respectively, starting from 4 weeks of age for 56 days  had no significant effects on the body, kidney, and heart weights of PCK rats Figures . Higher s Tables  and 2.3.3Histological examination and cystic analysis of kidney and liver demonstrated similar cystic areas for small doses of ivermectin (0.5 and 5 mg/L) compare to corresponding control groups Figure . Althoug44R in this pathway . The contradictory results of mentioned studies indicate that P2X receptor pathway activation and consecutive intracellular Ca2+ release may vary in the different models depending on the treatment protocol timeline or possible compensatory expression mechanisms  or bx‐430 , in combination therapy with corresponding P2X7R pharmacology  or 5‐BDBD (P2X4R antagonist) does not affect the development of ARPKD in PCK rats, which may provide insights for future studies on investigating the therapeutic potential of adenosine triphosphate (ATP)‐P2 signaling in PKD diseases.In conclusion, this study revealed that modulation of P2XOP and AS conceived the study. ON, VL, EI, and OP provided investigation. BX, ON, ASC, and OL analyzed the data. BX wrote the original draft of the manuscript. BX, ON, ASC, EI, VL, AS, and OP reviewed and edited the manuscript. All authors approved the final version of the manuscript.This research was supported by the National Institutes of Health grants R01 DK126720 (to OP), R35 HL135749 (to AS), Department of Veteran Affairs grant I01 BX004024 (to AS), endowed funds from the SC SmartState Centers of Excellence (to OP).None declared.The Institutional Animal Care and Use Committee (IACUC) of the Medical College of Wisconsin gave their approval to perform this study on 5/23/2017, with the reference number AUA1061. The Medical College of Wisconsin IACUC guarantees that institutional guidelines and regulations are followed and that all adverse events are reported to the IACUC as soon as possible.


Compared with C3 species, there is less information on gm responses to short‐term changes in environmental conditions such as partial pressure of CO2 (pCO2) across diverse C4 species and the potential determinants of these responses.Mesophyll CO4 grasses we investigated the response of gm to short‐term changes in pCO2 and its relationship with leaf anatomy and biochemistry.Using 16 Cgm increased as pCO2 decreased , with percentage increases in gm ranging from +13% to +250%. Greater increase in gm at low pCO2 was observed in species exhibiting relatively thinner mesophyll cell walls along with greater mesophyll surface area exposed to intercellular air spaces, leaf N, photosynthetic capacity and activities of phosphoenolpyruvate carboxylase and Rubisco. Species with greater CO2 responses of gm were also able to maintain their leaf water‐use efficiencies (TEi) under low CO2.In general, 2 response of gm in diverse C4 species, identifies the key leaf traits related to this response and has implications for improving C4 photosynthetic models and TEi through modification of gm.Our study advances understanding of CO This carboxylation occurs in the mesophyll chloroplast in species with the C3 photosynthetic pathway and mesophyll cytosol in species with the C4 photosynthetic pathway  across diverse plant groups     have been observed in response to short‐term changes in leaf temperature, quantity and quality of light, relative humidity and CO2 concentrations  and composition are considered invariable in the short term (minutes) and may not explain the rapid responses of gm , which catalyses the conversion of CO2 to HCO3−  used to calculate it in fluorescence and Δ13C methods, neglecting the contribution of respiratory and photorespiratory CO2 release to the total CO2 pool in the leaf and inaccurate measurement of day respiration or estimates of the Rubisco fractionation factor in the Δ13C method .Some studies have suggested that rapid changes in gm in diverse C4 species and in the leaf traits that could explain this variability , adaxial stomatal densities and Smes. We also demonstrated that C4 grasses adapted to low precipitation habitats exhibited traits related to greater gm but lower leaf hydraulic conductance compared with grasses from habitats with relatively high precipitation  identify the anatomical and biochemical traits that may explain the variable CO2 response of C4–gm, and (3) evaluate the impact of varying CO2 responses of gm on changes in photosynthesis and leaf water‐use efficiency. To address the above objectives, we estimated gm under changing pCO2 in 16 diverse C4 grasses , leaf nitrogen content (Narea), activities of key enzymes of C4 photosynthetic pathway such as CA, phosphoenolpyruvate carboxylase (PEPC) and ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco) and PEPC affinity for its substrate HCO3− (Km). Narea is integral to the proteins of photosynthetic machinery  that, along with the leaf structure, are responsible for the drawdown of CO2 inside the leaf  to investigate the response of , et al. . We also4 grasses  with five or six replicate pots per species. The plants were irrigated daily to pot saturation and fertilised twice a week with Peters 20–20–20 (2.5 g l−1). Pots were randomised daily within the growth chamber.Sixteen Ces Table  were selAnet, μmol CO2 m−2 s−1), stomatal conductance to water vapour , intercellular CO2 concentrations  and mesophyll conductance to CO2  were performed at four different CO2 levels inside the chamber or Ca  using an LI‐6400XT infrared gas analyser . Intrinsic water‐use efficiency  was calculated as Anet/gsw for each CO2 level. Maximum photosynthetic rates  were measured at saturating light of c. 1200 μmol photons m−2 s−1 and pCO2 of c. 1500 μmol mol−1.The measurements of net photosynthetic rates  of transpired H2O and oxygen isotope discrimination (Δ18O) in CO2 to calculate the CO2 concentration at the site of isotope exchange by CA. The Δ18O method assumes full isotopic equilibrium between CO2 and H2O at the site of CA (θ = 1), which may not always be true and therefore gm could be underestimated. In the current study we used the method described by Ogee et al.  coupled to a tunable diode laser absorption spectroscope  and a cavity‐ring down absorption spectroscope , as described previously  was set to c. 34, 27, 20 and 14 Pa. At each CO2 level, the leaves were allowed to adjust for at least 30 min or until stable values of Anet and gsw were achieved. Data for isotopologs of CO2 and H2O and physiological parameters  were collected and averaged over the next 20–30 min for each CO2 level (c. 10–15 cycles of TDLAS) with the Li6400XT set to log data only when the TDLAS analysed the sample line. At each CO2 level, three biological replicates were measured for the species eari and five biological replicates were measured for species' pvir and eser. For the remaining 13 species, measurements were conducted on four biological replicates. After the completion of above measurements, lights were tuned off and the leaves were allowed to stabilise for 15 min before logging the rates of dark respiration . Mesophyll conductance was estimated for each species at each of the four pCO2 levels using the Ogee et al.  , it was assumed that the pH of the mesophyll cytosol was constant due to the small micromolar shifts in dissolved CO2 at different pCO2  was equal to the dark respiration (Rn) for the C4 species.Several methods have been used to estimate C Peisker  calculate et al.  that buie et al.  method. e et al. ; Ubierna  Fig. . For C4 ada, number mm−2), abaxial stomatal density , SR (unitless) expressed as ratio of SDada : SDaba, leaf thickness (μm), mesophyll surface area exposed to intercellular air spaces  and TCW (μm). The details of sample preparation for microscopy and measurements are presented in Pathare et al.  and ribulose‐1,5‐bisphosphate carboxylase/oxygenase  activities were performed at 25°C, as described previously . In addition to enzyme activities, PEPC affinity for HCO−3  values were derived using a membrane‐inlet mass spectrometer. Km values have been published in DiMario et al. .Immediately following gas exchange measurements, leaf samples were taken from the same leaf and frozen in liquid nitrogen for enzyme assays. Measurements of CA, phosphoenolpyruvate carboxylase  were analysed using two different methods. First, the percentage change in gm in response to a decrease in pCO2 (from 34 to 14 Pa) was calculated for each biological replicate for the 16 species as follows:Changes in gm, we also calculated percentage change in Anet, gsw, Ci and TEi in response to the decrease in pCO2 (from 34 to 14 Pa). pCO2 levels of 34 and 14 Pa were chosen, as these two pCO2 levels were the highest and lowest levels, respectively, used in current study. Relationships of percentage change in physiological traits with key leaf anatomical and biochemical traits are given in the Figs Similar to y = a × (34/CO2)b) to model the changes in gm in response to changes in CO2 (here Ca). In the equation, y indicates the gm, coefficient a (μmol m−2 s−1 Pa−1) is the value of gm at 34 Pa pCO2, CO2 indicates the pCO2 inside the leaf chamber (Ca) and coefficient b (unitless) indicates the sensitivity of gm to changes in Ca. For further analysis  to m4/COb tpost‐hoc Tukey's test was used to examine differences in leaf‐level anatomical and biochemical traits among the 16 diverse C4 grasses. Results for post‐hoc Tukey's test are given in Table gm, ∆18O, Anet, gsw, Ci and TEi, two‐way ANOVA was performed with species and pCO2 as the main effects using the aov function in R . Normality and equal variances were tested and, when necessary, square root or log transformations were used to improve the data homoscedasticity Zar, . One‐wayes Table ; Fig. S9b with the key anatomical and biochemical traits such as TCW, Smes, ratio of TCW : Smes, SR, SDada, leaf thickness, PEPC activity, Rubisco activity, kCA, Km, Amax and Narea. To account for the combined effect of anatomy and biochemistry on the CO2 responses of gm, we derived ratios of TCW to biochemical traits . We examined the relationship between coefficient b and the ratio of TCW to biochemical traits. Similarly, we also examined the relationships between percentage change in gm and key anatomical and biochemical traits and the ratio of TCW to the biochemical traits mentioned above. Both, coefficient b and percentage change in gm showed similar relationships with anatomical and biochemical traits. In the main text we used coefficient b as a proxy for CO2 response of gm  were removed while deriving regression statistics if required  to changes in Ca are given in Figs 2 response of gm  also varied significantly among the 16 C4 grasses included in the current study  showed a 1.6‐fold variation across the 16 C4 grasses with values ranging from c. 29 to 46 μM HCO3−  with values ranging from c. 25 to 42.5 μmol CO2 m−2 s−1   Figs  and secoa from 3 to 14 PaKm Fig. . CoefficTCW that acts as a resistance in series to CO2 diffusion) and biochemistry (that can have a facilitating effect) on CO2 response of C4–gm, we derived the ratio of TCW with PEPC activity, Rubisco activity, kCA, Km, Amax and Narea and investigated the relationship between coefficient b and these ratios  and PCK (coefficient b = 0.0.73) subtypes showed 20% and 43% greater sensitivity of gm to Ca respectively compared with NAD‐ME (coefficient b = 0.512). However, there were no statistically significant differences in coefficient b among the subtypes  to the total CO2 pool in the leaf, particularly under low pCO2. Ignoring these CO2 pools while estimating C3–gm also overlooks the effects of spatial distribution of mitochondria and chloroplast on pathlength of CO2 movement. Inaccurate measurements of day respiration or estimates of the Rubisco fractionation factor in the ∆13C method and measurements conducted at low [O2] have also been suggested as potential sources of artefacts in determining the C3–gm response to pCO2   are greater compared with the gaseous phase  can enhance C4–gm . C4 species with a greater CO2 response of C4–gm tended to show greater activities of PEPC and Rubisco and greater Amax and Narea. Also, the CO2 response of C4–gm was strongly related to the ratio of TCW/photosynthetic capacity traits  are expected to provide a selective advantage by maintaining high rates of C4 photosynthesis, particularly under conditions such as drought when CO2 availability is low due to restricted stomatal conductance. Here, we observed that Km alone did not show a strong relationship with CO2 response of C4–gm  exhibited a greater CO2 response of gm. This contrasts with the general expectation and could be explained by the lower TCW leading to higher gm and with the corresponding greater CA activities leading to higher HCO3− in mesophyll cells under low Ci conditions. The higher HCO3− concentration in the mesophyll cells of species with lower TCW and greater CA can reduce the selective pressure on PEPC for lower Km values.We also investigated the relationship between the COgm Fig. . Howevergm Fig. , in whic4–gm is positively related to TEi under ambient pCO2  compared with the increase in gsw . Also, C4 grasses showing greatest increase in gm at low pCO2 also showed the lowest increase in gsw .Fig. S2 Sensitivity of mesophyll conductance .Fig. S3 Relationship of model coefficient a  with coefficient b  and relationship between coefficient b and percentage change in gm.Fig. S4 Relationship of percent increase in gm with mesophyll cell wall thickness (TCW) mesophyll surface area exposed to intercellular air spaces (Smes) ratio of TCW : Smes, stomatal ratio (SR), stomatal density adaxial (SDada) and leaf thickness among the 16 C4 grasses measured in current study.Fig. S5 Relationship of percent increase in gm with PEPC activity Rubisco activity CA activity expressed as kCA, PEPC affinity for HCO3− (Km), maximum photosynthetic capacity (Amax) and leaf N content (Narea) among the 16 C4 grasses measured in current study.Fig. S6 Relationship of percent increase in gm with ratio of mesophyll cell wall thickness (TCW) to PEPC activity Rubisco activity PEPC affinity for HCO3− (Km), CA activity expressed as kCA, maximum photosynthetic rates (Amax) and leaf N content (Narea) among the 16 C4 grasses measured in current study.Fig. S7 Relationship of percent increase in gm with percent decrease in leaf‐level water‐use efficiency (TEi) expressed as Anet/gsw , percent increase in stomatal conductance to water (gsw) and percent decrease in net photosynthetic rates (Anet)  for the 16 C4 grasses measured in current study.Fig. S8 PCA biplot showing major axes of variation in important leaf‐level anatomical and biochemical traits and percent change (increase or decrease) in response to CO2 in physiological traits such as gm, Anet, gsw and TEi for the 16 diverse C4 grasses measured in current study.Fig. S9 Response of mesophyll conductance (gm) to changes in pCO2 inside leaf chamber (Ca) in three C4 biochemical subtypes .Fig. S10 Response of mesophyll conductance to CO2 (gm) to changes in intercellular CO2 (Ci) in 16 diverse C4 grasses measured in current study.Fig. S11 Response of leaf net C18O16O discrimination (∆18O) to changes in pCO2 inside leaf chamber (Ca) in 16 diverse C4 grasses measured in current study.Fig. S12 Response of stomatal conductance to water (gsw) to changes in pCO2 inside leaf chamber (Ca) 16 diverse C4 grasses measured in current study.Fig. S13 Response of net CO2 assimilation rates (Anet) to changes in pCO2 inside leaf chamber (Ca) in 16 diverse C4 grasses measured in current study.Fig. S14 Response of leaf intercellular CO2 concentration (Ci) to changes in pCO2 inside leaf chamber (Ca) in 16 diverse C4 grasses measured in current study.Fig. S15 Response of leaf‐level water‐use efficiency (TEi = Anet/gsw) to changes in pCO2 inside leaf chamber (Ca) in 16 diverse C4 grasses measured in current study.Methods S1 Measurement of anatomical traits.Methods S2 Principal component analysis.Table S1 Key input parameters used in calculation of isotope parameters and estimation of mesophyll conductance (gm) for the 16 C4 grasses at four pCO2 levels  and a temperature of 25°C.Table S2 Results of one‐way ANOVA with species as main effects for all the leaf‐level anatomical and biochemical traits measured for 16 C4 grasses in current study.Table S3 C4 grasses used in current study along with their biochemical subtype and mean ± SE values for the equation (gm = a × (34/Ca)b) constants derived for the CO2 response of gm in the 16 C4 grasses.Table S4 Component loadings for important leaf‐level traits determined on 16 C4 grasses.New Phytologist Central Office.Please note: Wiley Blackwell are not responsible for the content or functionality of any Supporting Information supplied by the authors. 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Resulting from a strong directional dependence of adhesion and strain, a wetting tendency is only obtained for some low‐index facets under typical gas‐phase synthesis conditions. Thermodynamic stability in particular of lattice‐matched RuO2 films is instead indicated for more oxidizing conditions. Intriguingly, the calculations also predict an enhanced activity and stability of such epitaxial RuO2/TiO2 core‐shell particles under OER operation.Due to their high activity and favorable stability in acidic electrolytes, Ir and Ru oxides are primary catalysts for the oxygen evolution reaction (OER) in proton‐exchange membrane (PEM) electrolyzers. For a future large‐scale application, core‐shell nanoparticles are an appealing route to minimize the demand for these precious oxides. Here, we employ first‐principles density‐functional theory (DFT) and ab initio thermodynamics to assess the feasibility of encapsulating a cheap rutile‐structured TiO Using first‐principles density‐functional theory calculations combined with ab initio thermodynamics, we introduce a design protocol for RuO2‐based core‐shell catalysts which exhibit enhanced stability and activity under oxygen evolution reaction (OER) operating conditions. Proton‐exchange membrane (PEM) water electrolysis on TiO2,12, 132 or RuO2 enclosing the cheap core material. In this study we explore this idea with detailed first‐principles calculations. Analyzing adhesion, strain and surface energies, we show that prevailing gas‐phase synthesis protocols will only be able to stabilize thin films in the few‐monolayer regime at some low‐index facets of TiO2 for both IrO2 and RuO2. This rationalizes in particular the experimentally observed poor wetting behavior of IrO2 at the prevalent (110) facet of rutile TiO2 nanoparticles.[2/TiO2 or RuO2/TiO2 core‐shell nanoparticles as a promising target for future synthesis or advanced deposition endeavors.Titanium dioxide exhibits a stable rutile modification. This motivates the idea to instead pursue epitaxial core‐shell nanoparticles with thin coherent films of the equally rutile‐structured IrOarticles.18, 192/TiO2 and RuO2/TiO2 interfaces for all five symmetry inequivalent low‐index orientations of rutile, namely (001), (010)/(100), (011)/(101), (110) and (111). In the case of the (111) facet, there are two possible stoichiometric interfaces denoted as t1 and t2, see Figure S1 in the Supporting Information for a description of all interfacial geometries. In the calculation of γinterfhklaccording to equation (6) we consistently use the optimized TiO2 bulk lattice constants in the directions parallel to the interface for all three solid‐state terms which fixes the value of Ahkl. This effectively filters out the increasing strain in a coherent MO2 film of increasing thickness and allows to arrive at a purely interface specific quantity that reflects the intrinsic cost of creating the interface. Compared to other heterostructures[−2. This shows the expected propensity to form such interfaces between the lattice‐matched oxides.2 in TiO2 only at temperatures above 900 °C.Wadhhklin Table 2 bulk lattice constants in the directions parallel to the interface for all terms entering Equation (7) yields an interface specific quantity that is independent of the thickness of the shell layer. The large negative values obtained for both IrO2/TiO2 and RuO2/TiO2 designate a strong intrinsic adhesion, with the same trend over the five low‐index orientations found for both materials: (111) toughest to break and (010)/(100) and (110) offering weakest adhesion. This trend can be rationalized with the number of bonds formed per surface area, which is the number of broken bonds in the unit cell to divide the interface model into two separate TiO2 and MO2 slabs normalized by the interface area. This trend goes as 0.211, 0.191, 0.160, 0.148 and 0.105 Å−2 for (111), (001), (011)/(101), (010)/(100) and (110), respectively. Reformulating Wadhhklfrom Equation (7) in terms of the interface formation energy and the surface free energies of the stoichiometric TiO2 and MO2 terminations forming the interface,As starting point of our investigation we report in Table namely 00, (010)/(2/TiO2 interface to the higher respective surface free energies of IrO2 as compared to RuO2 (see below). Note that at an almost equal cohesive energy of both oxides (computed as −16.95 and −17.16 eV per MO2 unit at the present DFT Perdew‐Burke‐Ernzerhof (PBE) functionalalso allows to trace the consistently stronger adhesion found for the IrO2 as preferred shell material. However, the focus on the interface specific quantities γinterfhkland Wadhhkldisregards the increasing strain that will build up in pseudomorphic shell layers of increasing thickness. To this end, it is important to realize that the lattice mismatch of RuO2−TiO2 and IrO2−TiO2 is quite different for the two main rutile bulk lattice constants, a and c. Along the longer a axis all three materials exhibit almost identical values. With the employed PBE functional this is aTiO2=4.578 Å and shorter lattice constants by only −1.1 % for both RuO2 and IrO2. In contrast, along the shorter c axis, this mismatch is larger. Specifically, at PBE level cTiO2=2.955 Å, while RuO2 and IrO2 prefer a longer lattice constant by 5.6 % and 7.6 %, respectively. As a consequence of this anisotropy, hardly any strain will build up at the three interface orientations (001), (011)/(101) and (111), whereas much higher strain will build up at the two other orientations (110) and (010)/(100), see the Supporting Information for a detailed account of all values. While the larger strain for IrO2 might  scotch the intrinsic advantage of this material in terms of the afore discussed interface specific quantities, we note that much more problematic is the fact that precisely those two orientations that predominantly suffer from strain are those two that offer the weakest intrinsic adhesion anyway, cf. Table Consistently offering smaller interface formation energies and stronger adhesion, the analysis up to now would suggest IrO2, RuO2 and TiO2, in an oxygen environment. Equivalent behavior and trends are obtained for the surface free energies in an aqueous environment,2 and RuO2 are generally not smaller than those of TiO2, and in particular for the two orientations (010)/(100) and (110) that were already identified as problematic in terms of their interface stability and strain, TiO2 exhibits even significantly lower respective γsurfhkl,σthan the intended shell oxides.For the actual realization of epitaxial core‐shell nanoparticles, not only the interface matters. While growth itself is kinetics, there will always be a thermodynamic driving force to expose the material with the lower surface free energy at the shell. To this end, Figure γsurfhkl,σ1MLMO2/TiO2,100%, cf. Equation (9), of a model where a pseudomorphic one monolayer (ML) film of the shell material completely covers the TiO2(hkl) surface, and compares it to the surface free energy estimatesAll aspects, surface, strain and interface, can be combined in a rough estimate of the wetting tendency. As illustrated in Figure γsurfhkl,σxMLMO2/TiO2,100%is the surface free energy of a model where xMLs of the shell material pseudomorphically cover the entire TiO2(hkl) surface and γsurfhkl,σTiO2,100%is the surface free energy of the bare TiO2(hkl) surface. What Equations (2) and (3) thus evaluate is the cost, when the same total amount of shell material is not homogeneously dispersed on the entire surface as in γsurfhkl,σ1MLMO2/TiO2,100%, but instead forms 2 or 3 ML high islands, respectively, with the remainder of the TiO2 surface area uncovered. The simple linear superposition in Equations (2) and (3) thereby disregards any additional costs from the edges of the islands. Hence, γsurfhkl,σ2MLMO2/TiO2,50%and γsurfhkl,σ3MLMO2/TiO2,33%represent lower boundaries to the true surface free energies. According to these equations, wetting would require the γsurfhkl,σ1MLMO2/TiO2,100%of the fully dispersed monolayer to be lower than the surface energies of the two competing island models.Here, 2 and RuO2 as shell materials and using the most stable surface terminations under the synthesis conditions by Lee et al.,2 surface energies than TiO2. The latter lower surface energies result in fact from a qualitatively different behavior of the intended shell oxides that provides an important lead to future synthesis endeavors. Over the entire range of oxygen chemical potential shown in Figure 2 facets and their surface free energies correspondingly remain constant. In contrast, both shell MO2 are able to stabilize O‐rich terminations so that their surface free energies decrease with increasing ΔμO. The (001) and (111) facets are able to stabilize such terminations already at lowest oxygen chemical potentials, which is why their surface free energies are already quite low for the synthesis conditions of Lee et al., cf. Figure 2 surface free energies will continuously decrease.Figure O. The 00 and  thin films at TiO2(110) not to be stable under such conditions. Elevated pressures might instead be a route to achieve more favorable O‐rich conditions in gas‐phase synthesis. However, in light of our results we believe electrodeposition or advanced atomic layer deposition to be more promising routes with easier access to oxidizing conditions.[2/TiO2, all but the (110) orientation now exhibit a clear preference for wetting. While the (110) orientation is thus certainly the most difficult, we stress that the simple estimates for the 2 or 3 ML island models in Figure Unfortunately, for gas‐phase synthesis such conditions are harder to obtain. Most straightforwardly, they would be realized by lowering the temperature, cf. the temperature scales in Figure nditions.30, 312,2 and summarize these findings in Figure σ for all five low‐index facets have been computed to systematically consider the different possibilities to adsorb O, H, OH, OH2, OOH, and OO species in (1×1) and (1×2) surface unit‐cells. Figure U>1.3 V. The corresponding values at the OER equilibrium potential U=1.23 V were also used in the wetting model in Figure γsurfhkl,σline. Generally, we find the expected sequence from fully hydrated or hydroxylated surfaces at open‐circuit conditions to gradually deprotonated terminations at increasing potentials until pure O‐terminations and eventually terminations with higher oxidized superoxo species become most stable at OER‐relevant potentials. A detailed account of these findings and their very good consistency with existing theoretical and experimental data in particular for the best characterized (110) facet is provided in Figure S3 in the Supporting Information.Ir and Ru oxides are the primary current OER electrocatalysts. However, even they are known to degrade in the harsh acidic PEM operation conditions, involving a hitherto only incompletely characterized transformation to some amorphous hydrous state.38, 39ecies in ×1 and  facet that first turns negative already at a critical potential UcriticalRuO2111=1.27 V, that is, just at the OER onset. This reflects a potential thermodynamic instability, as the particle could gain energy by decaying and generating more such surfaces. With the current computational settings and pursuing the exact same approach, for IrO2 this critical potential where γsurf111,σturns negative is computed as UcriticalIrO2111=1.39 V. The concomitantly indicated thermodynamic stability up to higher applied potentials is fully consistent with the well‐established better corrosion resistance of this material[Ucritical as one of the useful thermodynamic descriptors for evaluating the catalyst stability. Intriguingly, when we compute exactly the same set of surface free energies for a pseudomorphic 2 ML film of RuO2 on TiO2, we find the decreasing surface free energies of all facets to be shifted toward higher potentials, and thereby obtain higher Ucritical where the surface energies turn negative, cf. Figure Ucritical2MLRuO2/TiO2111=1.36 V, that is, 0.1 V higher than for the pure RuO2 particle and close to the corresponding facet of pure IrO2. For other facets, this relative increase is up to 0.2 V as detailed in Table S4 in the Supporting Information. According to this thermodynamic descriptor the core‐shell RuO2/TiO2 particle thus exhibits trends of an increased stability, which is for all facets now essentially the same or better than for a pure IrO2 particle.More relevant for the present context is the pronounced decrease of the surface free energies with increasing potentials. As such, it is in particular Ucritical values result from increased surface free energies of the core‐shell particle compared to those of the native shell oxide, cf. Figure 2 particle. However, for the entire system a corresponding dewetting or other strain relieving alternatives like extended defects are not competitive as they would necessarily involve the formation of more surface area or expose TiO2 with its higher surface free energy at these OER operation conditions. For thicker films this will be changed by the accumulating strain. However, for few ML epitaxial films the increased stability of once formed core‐shell particles results simply out of lack of alternatives and this is how the thermodynamic descriptor should be read.This striking finding begs the question for its physical origin. Numerically, the shifted γsurf111,σwith increasing potential as compared to the surface free energies of the other facets shows that it becomes increasingly favorable to form (111) facets. Indeed, combining the fully analogous results for native IrO2 within a Wulff construction, we had shown in previous work that the thermodynamically preferred particle shape at OER operation conditions would be one that exclusively exhibits (111) facets.2 particles and epitaxial RuO2/TiO2 particles away from the familiar rutile form with its predominant (110) facets.2 core may lead to a very slow kinetics. Additionally, as long as the shell is intact, the TiO2 core does not contact the electrolyte and will at least be partially screened from the applied potential. It could thus well be that epitaxial core‐shell particles show an increased resistance to the driving force to reshape – and concomitantly against degradation, either in terms of general mass loss or the fraction of exposed (111) facets with their particularly low Ucritical, cf. Figure This cautious reading extends also to another interesting aspect reflected by the calculated surface free energies in Figure We note in passing that CHE is a simplified theoretical approach and that a negative surface energy resulting from such a treatment is not a sound indicator of what detailed physiochemical processes ensue, but merely one readily available stability descriptor which can be used to gauge trends between similar systems. This descriptor neither accounts for the chemical properties of the decomposition products nor for mass transport effects as has been pointed out recently.2−TiO2 core‐shell particles would not only exhibit an improved stability, but also catalytic activity. Figure 2 and for 2 ML RuO2−TiO2, as well as for RuO2 that is equally strained as in the epitaxial core‐shell system. Specifically, this is the reaction energetics along the classic OER peroxide pathway suggested by Rossmeisl et al.:According to our calculations, epitaxial RuO2(110),[ηTD=0.44 V to make all reaction steps exergonic. In contrast, we compute a much lower overpotential of only ηTD=0.26 V for the coherent 2 ML film of RuO2 on TiO2. This reduction results exclusively from the strain imposed on RuO2 in the core‐shell system, as demonstrated by the almost identical overpotential obtained for an equally strained RuO2(110) surface, cf. Figure 2(110) and that is correspondingly brought closer to the optimum value at the top of the volcano in the epitaxial core‐shell system.2 evolution, which would then also affect the Tafel slope for such particles. To demonstrate this, we compute the Tafel slope b for pristine RuO2(110) and the related core‐shell system. It is governed by the apparent transfer coefficient β, which specifies the number of electrons transferred counting from the catalyst resting state to the transition state in the free energy diagram[Here, we note that alternative mechanistic pathways, such as the lattice oxygen mechanism, have recently been investigated.46, 47b of RuO2(110) is 39.3 mV dec−1 at U=1.23 V. However, by increasing U beyond 1.57 V, the resting state changes from the surface oxo (*O) to the hydroperoxo (*OOH) intermediate, corresponding to a shift of the transition state from the second to the nearest neighbor position in the free energy profile, resulting in a Tafel slope of 118 mV dec−1 . This fully coincides with experimental data under OER conditions.η=0). The transition state is the third electron transfer step following the (*OH) state. The Tafel slope thus changes twice with increasing U, from b=23.6 to 39.3 and finally to 118 mV dec−1 by consecutively moving the transition and resting states closer to each other . Intriguingly, the transition bias between the two Tafel regimes (39.3 and 118 mV dec−1) has been lowered for the core‐shell system by more than 0.2 V from U=1.57 to 1.35 V. This phenomenon of lowering the transition bias may provide a validation and a guiding principle for the successful design of such core‐shell systems: the lower the transition bias, the better one achieves the desired core‐shell structure. As a further activity descriptor, we adopt the electrochemical‐step symmetry index (ESSI), which was proposed as a measure to quantitatively assess how close a catalyst approaches the catalytically ideal free energy profile:[As a result, the computed Tafel slope  profile:53, 54ΔGi+are the reaction energies of those steps in the OER peroxide pathway that are larger than 1.23 eV and E0=1.23 V is the OER equilibrium potential. For pristine RuO2 the relevant ΔGi+are the third and fourth steps in Figure 2 evolution, and the corresponding ESSI descriptor becomes 0.391 V. For the core‐shell system, the ESSI descriptor yields 0.153 V which is much smaller and indicates that it is significantly closer to the ideal catalyst than pristine RuO2. The reduction is, as discussed above, due to the strain induced destabilization of the O binding in the *O state. The equally strained RuO2 has an intermediate ESSI value of 0.226 V. In spite of the nearly identical overpotentials ηTD of the core‐shell and the strained RuO2 system, this large ESSI change can simply be attributed to a change in the number of terms n contributing to the average in Equation (5). The second reaction step at the strained RuO2 is slightly below (by −0.02 eV) the threshold of 1.23 eV while it is slightly above (+0.08 eV) for the core‐shell system. Such large deviations in the ESSI descriptor by a threshold induced discretization error for the average should be taken with a grain of salt based on typical DFT errors. The simple ηTD estimate is more robust for such cases. As shown in Figure 2/TiO2 core‐shell system, especially for the most relevant (011)/(101), (110) and (111) surfaces, could provide a promising RuO2‐based OER electrode as the free‐energy change of the second reaction step ranges between 1.03 and 1.43 eV and it exhibits ESSI values below 0.45 V.Here, 2 coherently encapsulates a rutile‐structured TiO2 core. A high directional dependence of adhesion and lattice mismatch disfavors in particular the (110) orientation, which suffers from maximum strain and offers the smallest density of interfacial bonds. While the same trend with regards to direction is obtained for IrO2, its intrinsically higher surface energies render this oxide a less suitable material for such core‐shell concepts.Systematically analyzing the interfacial stability, strain and surface free energies, our first‐principles calculations predict a general thermodynamic feasibility of epitaxial core‐shell particles, in which an ultrathin film of RuO2 to stabilize O‐rich surface terminations at more oxidizing conditions. This increasingly lowers their surface free energies relative to TiO2, for which only stoichiometric terminations are found as most stable up to high oxygen chemical potentials. Generally favoring wetting, this points towards growth protocols operating at oxidizing conditions, as for example achievable by electrodeposition. Apart from reducing the precious oxide content, our calculations further indicate two additional benefits of corresponding pseudomorphic core‐shell particles. On the one end, an increased stability at OER operation conditions results as the implied higher surface area and/or exposure of TiO2 penalizes a dewetting or strain‐relieving formation of extended defects. On the other end, the additional strain in particular of the prevalent (110) facet leads to a lowered thermodynamic overpotential for few monolayer, coherent RuO2 films at rutile TiO2. We hope that these insights will stimulate advanced growth endeavors to overcome the presently realized massively loaded composites with their incoherent thick, precious oxide films towards tailored epitaxial core‐shell particles with a predicted increased stability and activity at simultaneously minimized mass loading.A key aspect for the practical realization is the ability of both studied MOγinterfhkland the work of adhesion Wadhhklof a stoichiometric interface with crystallographic orientation (hkl). The prior indicates the cost of creating the interface from the respective bulk materials and is defined asIn order to assess the interfacial stability we use ab initio thermodynamicsAhkl) facet and the same stoichiometric surface termination as the interface model.where γinterfhkl, the central quantity determining the thermodynamic stability of a specific facet of a core‐shell particle with termination σ containing νMhkl,σ, νOhkl,σ, and νHhkl,σwith M=(Ir or Ru), O, and H atoms, respectively, in an aqueous environment and under an applied potential U is the surface free energyIn analogy to μH2Oat normal conditions. As a byproduct, the surface may be (de)protonated. At the metallic surface, we assume this to proceed in form of a proton coupled electron transfer, thus introducing as relevant reservoir the sum μH+aq.+μe-of the electrochemical potential of a solvated proton and the electron electrochemical potential in the system. Within the computational hydrogen electrode (CHE) concept of Nørskov and coworkersμH+aq.+μe-=12μH2+eU, where μH2is the chemical potential of hydrogen gas at normal conditions and the applied potential U is referenced to the reversible hydrogen electrode (RHE). Here, any treatment of aqueous surrounding is not included in our model. We note that both the solvent dielectric environment and specific interactions with surrounding water molecules can lead to different absolute energetics.[Here, the second line accounts for any off‐stoichiometries of the surface termination σ by releasing or taking water molecules from the water environment represented by the chemical potential of water When instead focusing on the stability of a facet in an oxygen gas environment, the surface free energy is analogously given asμO=ΔμO, now summarizes the dependence of the surface free energy on temperature T and oxygen pressure p.2 environment forms an ideal‐gas‐like reservoir, the chemical potential of oxygen, μO, is expressed aswhere the relative chemical potential of oxygen, ΔνTihkl=0and using the Gsurfhkl,σfrom a corresponding oxide slab model), as well as for the calculation of the Gibbs free energy change of a reaction step ΔG=Ahklγsurf1hkl,σΔμO-γsurf2hkl,σΔμOwhere the two surface terminations surf1 and surf2 differ in their composition according to the reaction step studied.The chemical potentials of oxygen at the standard state pressure are obtained from reported experiments.2 with experimental XPS spectra compared to hybrid functionals,2 and RuO2. ZPE and entropic contributions to the molecular chemical potentials μH2Oand μH2were obtained from experimental data and reference tables.[U approach has been treated in the fully localized limit.For the differences of solid‐state Gibbs free energies entering Equations (6)–(9) we follow the approach of Reuter and SchefflerThe authors declare no conflict of interest.1As 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.Supporting InformationClick here for additional data file.


Z)‐γ‐bisabolene synthase (BbS) from Cryptosporangium arvum, and germacrene A synthase (SmTS6) from Streptomyces mobaraensis. These enzyme reactions not only yielded several previously unknown compounds, showing that this approach opened the door to a new chemical space, but substrates with blocked or altered reactivities also gave interesting insights into the cyclisation mechanisms and the potential to catalyse reactions with different initial cyclisation modes.Four synthetic farnesyl diphosphate analogues were enzymatically converted with three bacterial sesquiterpene synthases, including β‐himachalene synthase (HcS) and ( Four synthetic FPP analogues were converted by three robust bacterial sesquiterpene synthases, yielding previously unknown compounds with high stereoselectivity, which demonstrates that this approach can open the doors to chemical terra incognita. The altered or partially blocked reactivity in the substrates also gave interesting insights into the enzyme mechanisms which adds to our understanding of terpene biosynthesis. The 1H NMR spectrum of 19 showed line broadening for all CH2 groups, pointing to slowly interconverting conformers, but sharp signals in the 13C NMR spectrum. As a result of line broadening, signals for the hydrogens attached to C12 were missing in all 1H based spectra. Therefore, to secure the structure of 19 a catalytic hydrogenation was performed that yielded an inseparable mixture of the two possible diastereomers, C3v symmetric 20 and Cs symmetric 21 . Compound 19 can be formed from 11 through a newly opened reaction path that is not possible for FPP, i. e. by 1,12‐cyclisation, leading to a cationic intermediate with a tertiary cation at C11 in A5, followed by deprotonation to 19 , the enantiomer of ent‐22  that we had previously obtained from 12 with dauc‐8‐en‐11‐ol synthase (DcS) from Streptomyces venezuelae (Scheme 22 requires isomerisation to (R)‐A6 and subsequent 1,6‐cyclisation by anti‐SN2’ reaction, resulting in (S)‐B 6. Final attack of water yields 22. These results show that HcS can also catalyse a 1,6‐cyclisation with substrate 12.Substrate 13 yielded with HcS compounds 23 and 24 ‐ and (R)‐25 were converted by catalytic hydrogenation, yielding silica gel chromatographically inseparable diastereoisomeric mixtures of ‐26 a and ‐26 b that were also inseparable by GC on a chiral stationary phase . However, the retention times for 26 a and 26 b were clearly different, allowing to conclude on the configuration at C3. Compound 23 was converted by Wittig reaction into 27  followed by catalytic hydrogenation that yielded a sample identical to 26 b with minor formation of 26 a (3 %). Thus, the enzyme product is (R)‐23 . Its formation can be explained from 13 either directly or by isomerisation to (S)‐A 7 and hydrolysis with inversion of configuration at C3.FPP analogue 4 Scheme A. Both c24 can be explained by 1,11‐cyclisation of A 7 to B 7 with anti‐SN2’ attack at C1. A reprotonation induced cyclisation of C 7 to D 7 and deprotonation yields 24. If (S)‐A 7 is a common intermediate for both products 23 and 24, the shown absolute configuration of 24 may result  was followed by trials to convert this compound into the bis‐dinitrophenylhydrazone for crystallisations. Unfortunately, this approach was not successful and clarification of the absolute configuration of 28 is open.The formation of t Scheme A. Its oz10–13 were enzymatically converted with BbS , while 13 yielded the acyclic product 23. These reactions proceed with similar efficiency as observed for the native substrate FPP, showing that the structural modifications did not influence acceptance by BbS. Based on the cyclisation mechanism of BbS with FPP, (R)‐A 7 in analogy to (R)‐NPP may be the intermediate which should further react by abstraction of diphosphate and SN2 attack of water with inversion of configuration to yield (S)‐23. The absolute configuration of 23 was confirmed by analysis on a chiral GC column, compared to (R)‐23 obtained from 13 with HcS .In a second series of experiments, the substrate analogues 10–13 were finally tested with SmTS6 . With substrate 10, only two acyclic products (6E)‐10,11‐dihydro‐β‐farnesene (32) and (6E)‐10,11‐dihydro‐nerolidol (33) were obtained ‐ and (S)‐25. Catalytic hydrogenation of 33 and comparison to the 25 hydrogenation products 26 a and 26 b by GC using a chiral stationary phase confirmed the structure of (R)‐33 . Taking analogue 11 as substrate, two products ‐1,5,9‐trimethylcyclododeca‐1,5,9‐triene  and ‐1,5,9‐trimethyl‐cyclododeca‐4,8‐dien‐1‐ol  were isolated. Their formation can be rationalised through a 1,12‐cyclisation of 11 to cation A5. Its deprotonation leads to 34, while attack by water gives raise to alcohol 35. Compound 34 is C3h symmetric and shows only five signals in the 13C NMR. Its structure was confirmed by catalytic hydrogenation that resulted in the same products as obtained with 19 ‐ and (S)‐IPP36R)‐ and (S)‐‐11 with FPP synthase (FPPS) from Streptomyces coelicolor35 can be followed by HSQC spectroscopy into one of the diastereotopic positions assigned by NOESY spectroscopy with the unlabelled compound. Assuming inversion of configuration in the cyclisation to 35, the absolute configuration of (S)‐35 was concluded . With substrate 12, SmTS6 showed only poor conversion that was insufficient for product isolation, again in line with the inability of SmTS6 to perform 1,6‐cyclisations. For analogue 13, (S)‐23 was obtained, as confirmed by GC analysis on a chiral stationary phase .The absolute configuration of Z)‐γ‐bisabolene synthase (BbS), and germacrene A synthase (SmTS6), yielding interesting products in most cases. Most of these compounds are unknown or only poorly described. One exception is 34 that has been obtained before from isoprene in a metathesis reaction using a tungsten‐carbene complex,19 was only obtained as a mixture with 34 and other cyclotrimers of isoprene.22 for which the enzymatic formation of its enantiomer was previously reported from the same FPP analogue.33 a synthesis from 6‐methylheptan‐2‐one has been reported.E)‐iso‐γ‐Bisabolene, a stereoisomer of 30, has been tentatively identified by GC/MS in headspace extracts from the fungus Fusarium where it could be a side product of trichodiene synthase,[E/Z mixture of 30 has been obtained by synthesis,Z isomer has not been made available before. Also the enzyme products (R)‐ and (S)‐23, 24, 29, 31, 32 and 35 and their synthetic derivatives 27 and 28 were isolated in this study for the first time and obtained in high stereoisomeric purity, showing that the enzymatic conversion of FPP analogues can open the doors towards a new chemical space.Four synthetic FPP analogues were converted with the bacterial β‐himachalene synthase (HcS), ‐23 was obtained. The 1,10‐cyclising SmTS6 gave acyclic products with 10 for which such a cyclisation is not possible, however, substrate 13 that could in principle undergo 1,10‐cyclisation also only gave an acyclic product, likely because the conformational fold of this substrate in the active site is disturbed. With FPP analogue 11 a 1,12‐cyclisation was observed, demonstrating that new cyclisation paths can be opened that follow the intrinsic reactivity of the substrate. The results with HcS are less clear. With 10 no product was obtained, while with 11 a 1,12‐cyclisation and with 13 a 1,10‐cyclisation was manifested. However, these observations were contrasted by the conversion of 12 through initial 1,6‐cyclisation. We have discussed in our previous work that the HcS mechanism can be understood either by initial 1,6‐ or 1,11‐cyclisation.BbS catalyses an initial 1,6‐cyclisation of FPP and yielded corresponding products from all substrate analogues, only with substrate 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.Supporting InformationClick here for additional data file.


The calculated rate constants of the two processes are in very good agreement with the experimental Photoelectron‐Photoion Coincidence (PEPICO) branching ratio. This study solves the intriguing and theoretically unexplained experimental observation that 2‐Nitroimidazole, at variance with the other two regio‐isomers is a source for only NO at low energies (<12.76 eV). This is a key point for biomedical application of the nitroimidazoles, because NO is the vasodilator that favors the reoxigenation of hypoxic tumor tissues.Nitroimidazoles are a class of chemicals with a remarkable broad spectrum of applications from the production of explosives to the use as radiosensitizers in radiotherapy. The understanding of thedynamics of their fragmentation induced by ionizing sources is of fundamental interest. The goal of this work is to theoretically investigate the  2The kinetic competition between the NO and NO loss channels in 2‐Nitroimidazole ions favours the former channel at low energies (<12.76 eV). Thus, at these energies, 2‐Nitroimidazole is an exclusive NO source, unlike 4 and 5‐Nitroimidazole isomers. This is important for biomedical applications since NO is a vasodilator that favors oxygenation of tissues. These molecules, which share with oxygen the radical nature, may act via a twofold mechanism.2 in their fragmentation, indirectly can produce NO through the reduction of nitrite NO2− into nitric oxide via reactions with several heme‐containing proteins, in particular in hypoxic states.2 losses after ionization of nitroimidazoles have been extensively investigated. and k67+/[k(83++k67+)] for the two channels are calculated and reported versus energy for the three isomers NIM mixture), where the theoretical analysis =2.05×108 s−1 and k(11.75 eV)=32.2 s−1 for the NO and NO2 losses, respectively.As a matter the fact, the s Figure  finds tw+, and of the sum of all ions that originate from fragments 83+, the so‐called ‘83+ family’:+ (30+), HCNH+ (28+), HNC(H)NCH+ (55+ only from 5NIM), HNC(H)CO+ (56+ only from 2NIM), and 67+ are shown in Figure + takes off at about 11.6 eV, in the case of 2NIM the BR of 67+ is zero.The branching ratios have been also experimentally estimated from the Photoelectron‐Photoion Coincidence (PEPICO) spectra of 2NIM and of the mixture 45)NIM.NIM.+, an+, however the parent ion does not contribute to the PEPICO experimental BR at energies higher than 11.4 eV, thus a comparison between experiments and theory can be attempted for the 4(5)NIM mixture NIM are to be searched in the structure of TS and VTS of the dissociation process at energies close to the AEs of ion 67+ of 4(5)NIM, i. e., 11.75 eV, as shown in Figure In summary, the puzzling discrepancy of about 1.0 eV between the theoretical and experimental AEs of fragment 672 group still substantially interacts with the imidazole ring, being the C2−N distance equal to 2.49 Å, with the positive Mulliken charge equally distributed on the two moieties. This implies that the vibrational frequencies in the low‐energy range (those associated with the inter‐fragments modes) of the TS of 2NIM are higher than those of 4 and 5NIM. Hence, because N#(E) increases more rapidly when the systems have frequencies with lower wavenumbers, the N#(E) of 4 and 5NIM increases more rapidly than for 2NIM. This makes the dynamics of the fragmentation in 2NIM slower than in the 4 and 5NIM isomers and the NO2 loss channel not competitive with the NO loss in the low energy region.The comparison of these TS and VTS structures (Tables S5 to S7) in the three isomers shows that only in 2NIM the NO2 loss from 2NIM is observed experimentally only 1.1 eV above its theoretically predicted AE; ii) highlights the importance of the kinetic studies in the fragmentation processes of nitroimidazoles and the competition among the different channels, even when no reaction barrier exists;In conclusion this work i) demonstrates why the NOThe 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.Supporting InformationClick here for additional data file.


TGFB‐induced factor homeobox 2 (TGIF2) has been reported to exert essential functions in brain development. This study aimed to elucidate the correlation of TGIF2 with autism, a neurodevelopmental condition which presents with severe communication problems.An autism‐related gene expression dataset GSE36315 was used to analyze aberrantly expressed genes in autistic brain tissues. Maternal mice were treated with valproate (VPA), and their offspring were selected as model mice with autism. The functions of TGIF2 in autism‐like symptoms in mice were examined by behavioral tests and histological examination of their hippocampal tissues. Mouse hippocampal neurons were extracted for in vitro studies. A gene set enrichment analysis was performed to analyze the signaling pathways involved, and the upstream factors influencing TGIF2 expression were explored in the ENCODE database and validated by ChIP‐qPCR assays.TGIF2 was poorly expressed in autistic patients in the GSE36315 dataset as well as in the temporal cortex tissues of autistic mice. Adenovirus‐mediated overexpression of TGIF2 suppressed autism‐like symptoms and neuronal apoptosis in autistic mice. TGIF2 activated the Wnt/β‐catenin signaling pathway. TGIF2 could be regulated by monomethylation of histone H3 Lys4 (H3K4me1). The histone demethylase LSD1 was highly expressed in the tissues of autistic mice and bound to TGIF2 promoter, which was possibly responsible for TGIF2 downregulation.This research suggests that the downregulation of TGIF2, possibly regulated by LSD1/H3K4me1, is correlated with neuronal apoptosis and development of autism in mice through the inactivation of the Wnt/β‐catenin pathway. This study suggests that TGIF2 is downregulated in autism, which is possibly regulated by LSD1/H3K4me1. Restoration of TGIF2 alleviates neuronal apoptosis and autism‐like symptoms in mouse by activating the Wnt/β‐catenin pathway. The findings suggest that TGIF2 may serve as a candidate tool for the management of autism. The neuA recent bio‐collections research by J Reilly et al. documented that hundreds of genes, which are distributed on over 10 chromosomes and are mainly cell adhesion molecules linked to synaptic formation and functions, are correlated with the development of autism  and 15 females (25–27 g), were procured from Hunan SJA Laboratory Animal Co., Ltd . The animals were housed in 25℃ specific animal rooms with free access to rodent feed and water and given 12 h of light exposure (7:00–19:00) every day. The animal procedures were ratified by the Ethical Committee of Hunan Provincial People's Hospital and abided by the Guide for the Care and Use of Laboratory Animals .n = 8 in one group and n = 7 in the other. On gestational day 12.5, the female mice (n = 8) were intraperitoneally injected with valproate . The newborn mice were allocated into the VPA group. In the other group, the female mice (n = 7) were treated with normal saline in a similar manner, and the newborn mice labored by these female mice were allocated into the negative control (NC) group. All female mice were successfully fertilized, and each of them labored 5–12 mice. Mice were raised until young adulthood, and 40 mice were selected from the VPA (n = 70) and the NC (n = 65) groups for behavioral tests . On the second day, the vaginal smears from female mice were collected for evaluation. The presence of sperms was considered as an indicator for pregnancy, and the date was recorded as gestational day 0.5. The female mice were then separately housed. The pregnant mice were randomly allocated into two groups, 2.212 PFU/ml. The glass pipette was gently retracted from the skull. The tails of injected mice were marked to distinguish them from the uninjected ones. All newborns were placed back to the nest for recovery (10–20 min) as measured by spontaneous limb mobility. Thereafter, the nest was returned to the parent cage.Adenovirus 9 (AAV9)‐based overexpression vector of TGIF2 (AAV9‐TGIF2) and the empty vector (AAV9‐NC) were procured from Genechem Co., Ltd . The AAV9‐based vectors were injected into the newborn mice (P1) in the VPA group intracerebroventricularly according to a previous report  with ad libitum access to feed and water. Considering that male autistic children account for most of the reported cases, and young adult/adult mice may behave differently due to the matter of oestrous cycle, only young adult male mice were included for the behavioral tests. At least 3 days before the tests, the mice were housed in the test rooms. Most of the tests were performed as previously reported  tests were performed to examine the learning and memory abilities of mice. The testing system included a movable transparent platform, an automatic recording system, and a round pool. The diameter of the pool was 120 cm, the height was 50 cm, and the depth of water was 30 cm (2 cm over the platform). The temperature of water was adjusted to room temperature. The pool was divided into four quadrants, and the hidden platform was placed in quadrant 1. The mice were acclimated 1 day before the tests, and they were allowed to practice for 3 days. In short, each mouse was placed into the water facing the pool wall in three nonplatform quadrants, respectively. The duration of each test was 60 s. The time for mice spent to find the hidden platform was recorded as the latency. If the mice failed to get to the platform within 60 s, the latency was defined as 60 s. The mean value of the escape latency from three tests was calculated. After 3 days of practice, the latency of each mouse was recorded twice a day for continuous 5 days.2.5The three‐chamber social test was performed as previously reported  as previously described /(n + f) where “n” refers to the time spent in the exploration of the novel object and “f” refers to the time spent in exploring the familiar object.A 40 cm × 40 cm × 40 cm instrument was used for the novel objective recognition assay. On the first day, the mice were allowed to explore freely for 10 min. During the sampling phase on day 2, the male mice were allowed to explore two identical objects for 10 min. During the test phase (24 h later), one of the two objects was replaced with a novel object. The time the mice spent in exploring each of the two objects within a period of 10 min was examined. The cases of mouse nose touching the object or they facing the object within 2 cm were regarded as object recognition. The discrimination index, as a measure of ability to distinguish unfamiliar and familiar objects, was calculated as  and allowed free movement during the first 2 min. Then, a 28‐s tone (85 db) and a 2‐s foot shock (0.75 mA) were applied for three times. After the stimulations, the mice were maintained in the chamber for 30 min. The rigidly standing time of each mouse at each stimulus interval was calculated as a percentage of the freezing response. On day 2, the mice were housed in the same chamber and allowed free movement during the first 2 min to assess environmental memory. On day 3, the chamber was remolded to have the mice placed in a novel condition. The mice were allowed free movement for 2 min and then subjected to 120 s of tone stimulation. The rigidly standing time in environmental trials and tone stimulation trials was recorded, respectively.2.9An in situ cell death detection kit  was used to examine cell apoptosis in hippocampal tissues. The paraffin‐embedded sections were dewaxed, rehydrated, added with 50 μl proteinase K  and hydrolyzed at room temperature for 20 min to remove the tissue protein. After PBS washes and antigen retrieval using citric acid buffer, the sections were reacted with 50 μl TUNEL reaction solution in a wet box avoiding light exposure for 50 min and then washed. The sections were dried, incubated with 50 μl DAPI (Beyotime) at 37℃ in the dark for 30 min sealed with neutral balsam, and observed under the microscope. The apoptotic neurons were observed under the microscope with five random fields selected. The apoptosis index was calculated as apoptotic cells/total cells 100%.2.10The paraffin‐embedded hippocampal tissue sections were dewaxed, rehydrated, and stained with hematoxylin (Sigma–Aldrich) at room temperature for 5 min. After differentiation in HCl‐ethanol for 30 s, the sections were stained in eosin solution (Sigma–Aldrich) for 2 min. For Nissl staining, the sections were incubated at 37˚C overnight, rehydrated, and stained with Nissl for 10 min. After staining, the tissue sections were routinely dehydrated and sealed for microscope observation.2.11® reagent  and reverse‐transcribed to cDNA using a PrimeScript RT kit . Thereafter, relative mRNA expression was determined using the 7500 Fast™ System  and a Sensi Mix SYBR kit  according to the manufacturer's instruction manual. The primers are presented in Table Total RNA from temporal cortex tissues or neurons were extracted using the TRIzol2.12The tissues and cells were lysed in the radio‐immunoprecipitation assay lysis buffer (Sigma–Aldrich) to collect total protein. The concentration of the isolated protein was examined using the bicinchoninic acid method. Next, an equal amount of protein sample (40 μg each lane) was separated by 10% SDS‐PAGE and loaded onto polyvinylidene difluoride membranes. The membranes were blocked with 5% nonfat milk at 4˚C overnight, and incubated with the primary antibodies against TGIF2 , β‐catenin , Wnt1 , c‐Myc , c‐FOS , Survivin , and GAPDH  at 4℃ overnight. Thereafter, the membranes were incubated with HRP‐labeled goat anti‐rabbit IgG  or goat anti‐mouse IgG  at room temperature for 1.5 h. The blot bands were developed using the enhanced chemiluminescence system (Thermo Fisher Scientific) and analyzed using Image J software .2.13g for 5 min, and the cells were cultured and resuspended. The cell suspension (2 × 106 cells/ml) was loaded into a sterile culture flask. The medium was replaced by complete medium after 24 h. After 72 h, the medium was added with cytarabine solution  till a final concentration of 2.5 mg/L. The culture solution was refreshed every 3 days. Hippocampal neurons cultured for 6 d were transferred to 96‐well plates at a 1 × 104 cells per well. Afterward, the expression of neuronal markers NeuN and MAP‐2 was examined by immunofluorescence to examine the successful extraction of neurons.The hippocampal tissues of the mice in the NC and VPA groups were collected and resuspended in 20% fetal bovine serum at 37℃ using a long glass pipette. The mixture was filtered using a sterile 200‐mesh cytoscreener, centrifuged at 178× 2.143 cells per well. Each well was added with 10 μl CCK‐8 solution, followed by 2 h of incubation at 37℃. The optical density at 490 nm was examined using a microplate reader.Viability of neurons was examined using the CCK‐8 method . In short, the cells were seeded in 96‐well plates at 6 × 102.155 cells/well) for 24 h. Annexin V‐fluorescein isothiocyanate (FITC) and propidium iodide  were added for 10 min of incubation at room temperature in the dark. Apoptosis rate of cells was then examined using a FACS Canto flow cytometer . The Annexin V‐FITC+/PI– cells were regarded as apoptotic cells.The hippocampal neurons were cultured in 24‐well plates  and treated using a high‐intensity ultrasonic processor on ice at 150 Hz. After centrifugation and collection of the supernatant, an equal amount of chromatin was used for immunoprecipitation at 4℃ overnight. The specific antibodies against LSD1  or H3K4me1  were used. Anti‐rabbit IgG was used as control, and total chromatin was used as input. After incubation with magnetic beads, the immunoprecipitates were collected. The magnetic beads were washed, and the chromatin was eluted by the proteinase K mixture in ChIP elusion buffer. The TGIF2 expression in the DNA fragments immunoprecipitated by LSD1 or H3K4me1 was examined by qPCR.A Magna ChIP™A/G One‐Day ChIP kit  was used according to the manufacturer's instructions. In short, fresh frozen cortex tissues were cut into 1–3 mm2.17The hippocampal neurons were fixed with 4% PFA for 15 min, penetrated with 0.5% Triton X 100 at room temperature for 20 min, and blocked with 5% normal goat serum for 30 min. For tissue staining, the paraffin‐embedded cortex tissue sections were dewaxed, rehydrated, boiled in 3% citrate sodium at 100℃ for 20 min, and blocked in the QuickBlock™ Blocking Buffer  at room temperature for 1 h. The sections were incubated with anti‐NeuN , anti‐MAP2 , and anti‐β‐catenin  at 4℃ overnight and then with FITC‐conjugated goat anti‐rabbit IgG  or goat anti‐mouse IgG  at room temperature for 1 h. The nuclei were stained with DAPI (Beyotime) at room temperature for 5 min. The staining was observed under a fluorescence microscope, and the mean signal intensity was examined using the Image‐pro plus 6.0 software .2.18At 24 h before transfection, the 293T cells  were cultured in six‐well plates for 24 h till a 70% cell confluence. The cells were transfected with 1.0 μg reporter vector BAT‐LUX TCF/LEF  and 50 ng pRL‐TK vector  using Lipofectamine 3000 (Thermo Fisher Scientific). BAT‐LUX induces the expression of a firefly luciferase gene via the seven TCF/LEF binding sites. After 24 h, the medium was refreshed, and the cells were treated with AAV9‐NC or AAV9‐TGIF2. After 48 h, the intensity of firefly and renilla fluorescence in cell lysates was examined using the dual‐luciferase reporter assay system  according to the manufacturer's instructions.2.19t test (two groups) or one‐ or two‐way analysis of variance (ANOVA) followed by Tukey's post hoc test. p < .05 was considered to show statistical significance.The GraphPad Prism 7 software  was used for statistical analysis. Measurement data collected from three independent experiments were expressed as the mean ± SD. Differences were analyzed by the Student's 33.1https://www.ncbi.nlm.nih.gov/geo/) for analysis. The GSE36315 dataset contained four autistic tissue samples and four normal brain tissue samples from healthy individuals. A total of 398 differentially expressed genes (DEGs) were identified  was significantly delayed compared to the mice in the NC group (hereafter termed NC mice) Figure . Moreove3.2To examine the function of TGIF2 in autistic mice, AAV9‐based overexpression vector containing the TGIF2 cDNA sequence was injected into the VPA mice through intracerebroventricular injection Figure . After A3.3The pathological changes in mouse hippocampal tissues were detected by the hematoxylin and eosin (HE) staining and Nissl staining. It was observed that AAV9‐mediated TGIF2 overexpression significantly alleviated the neuronal injury in the temporal cortex tissues of VPA mice Figure . The num3.4The hippocampal neurons from NC and VPA mice were extracted and identified by the positive immunofluorescence staining of neuronal markers MAP2 and NeuN Figure . Moreove3.5To explore the downstream signaling pathways regulated by TGIF2, a gene set enrichment analysis (GSEA) was performed to analyze the signaling pathways enriched by the DEGs in the GSE36315 dataset. It was observed that the Wnt/β‐catenin pathway had the highest correlation with TGIF2 Figure . Thereaf3.6https://www.encodeproject.org/). It was observed that there was significant H3K4me1 modification near the TGIF2 promoter (Figure To examine why TGIF2 was poorly expressed in autism, the ChIP‐seq data including the expression of H3K4me1, H3K4me3, H3K9me3, H3K27ac, and H3K9ac in mouse forebrain tissues were downloaded from the ENCODE database (r Figure . Thereafr Figure . The H3Kr Figure . Moreover Figure . Therefor Figure . Moreover Figure . Therefo4Considering the increasing prevalence of autism in children and the accompanying lifelong neuronal disorders (Christensen et al., Integrated bioinformatics analyzing tools including the GEO database have been largely used for the identification of key molecules implicated in the development of diseases, including autism (Li et al., When exploring the downstream signaling pathways using the GSEA, we found that the Wnt/β‐catenin pathway was highly enriched by the DEGs and it had the highest correlation with TGIF2. The levels of Wnt/β‐catenin‐related proteins as well as the nuclear translocation of β‐catenin in the tissues of autistic mice were restored after TGIF2 upregulation. The Wnt/β‐catenin signaling has been widely accepted to be essential for brain development and function and the cascade has a central role in neurodevelopmental pathology in autism (Medina et al., Like genetic mutations, epigenetic modifications such as histone modifications, DNA methylation, and RNA interference can affect gene expression and possibly lead to behavioral and neuronal changes in mental disorders, including autism (Alam et al., 5In conclusion, this study demonstrates that TGIF2 is downregulated in autism, which is possibly regulated by LSD1/H3K4me1. Restoration of TGIF2 alleviates neuronal apoptosis and autism‐like symptoms in mouse through activating the Wnt/β‐catenin pathway. The findings suggest that TGIF2 may serve as a candidate tool for the management of autism. We hope more studies will be carried out to validate our findings and offer more insights in the pathogenesis of autism for the development of new therapeutic options.The authors declare no conflict of interest.