However, in our current understanding, there are missing sources associated with these two species. Here we investigate the effect of copper(II) on CH3Br and CH3Cl production from soil, seawater and model organic compounds: catechol  and guaiacol (2-methoxyphenol). We show that copper sulfate (CuSO4) enhances CH3Br and CH3Cl production from soil and seawater, and it may be further amplified in conjunction with hydrogen peroxide (H2O2) or solar radiation. This represents an abiotic production pathway of CH3Br and CH3Cl perturbed by anthropogenic application of copper(II)-based chemicals. Hence, we suggest that the widespread application of copper(II) pesticides in agriculture and the discharge of anthropogenic copper(II) to the oceans may account for part of the missing sources of CH3Br and CH3Cl, and thereby contribute to stratospheric halogen load.Methyl bromide  chemicals induces abiotic production of methyl bromide and methyl chloride from soil and seawater, contributing to the atmospheric halogen load. They have average tropospheric lifetimes of 0.8 and 0.9 years1, respectively, which are long enough for them to reach the lower stratosphere and disassociate, releasing bromine and chlorine radicals that catalyze stratospheric ozone depletion. However, the sizes of identified sources of CH3Br and CH3Cl are smaller than those of the sinks with a −39 Gg yr−1 imbalance for CH3Br and a −748 Gg yr−1 imbalance for CH3Cl, respectively1. A recent reevaluation on CH3Cl emissions from tropical plants2, which was seen as the largest CH3Cl source, showed it may be overestimated by about 1300 Gg yr−1, suggesting the existence of even larger missing CH3Cl sources than previously thought. These discrepancies need to be resolved to better understand stratospheric halogen chemistry and to predict future ozone recovery. The present atmospheric CH3Br and CH3Cl originate from predominantly natural sources. These natural sources tend to be amplified under anthropogenic-induced influences, such as biomass burning3, global warming5, sea level rise7, agricultural cultivation8, land use change9, etc., posing persistent and growing challenges for the recovery of stratospheric ozone11.Methyl bromide  oxidizing organic matter in the presence of halide ions are responsible for the production of methyl halides12. This pathway is ubiquitous and makes a significant contribution to the global methyl halide budgets. It is reasonable to postulate that the Fenton-like mechanism may be catalyzed by other transition metal ions, such as copper(II).Among the known sources and sinks of CH4), are widely used, especially in the fields of rice, oranges, walnuts, and grapes16. For example, in 2008 in the U.S., copper compounds accounted for 17% (the second most) of total fungicides applied in agriculture17. Roughly 2.0 × 1011 kg CuSO4 per year are used in global agriculture -based pesticides, herbicides, and fungicides, such as copper(II) sulfate  chemicals on CH3Br and CH3Cl production. The results provide insight on copper-catalyzed production mechanisms of CH3Br and CH3Cl, as well as the potential impact of copper chemical application on atmospheric CH3Br and CH3Cl budgets.Here, we incubate agricultural soil, seawater, and model compounds of soil organic carbon with different copper treatments, and quantify CH3Br) and methyl chloride (CH3Cl) with average fluxes at −0.06 ng kg−1 hr−1 and −3.7 ng kg−1 hr−1, respectively  in the soil12. CH3Br and CH3Cl fluxes declined to near zero after organic matter depletion, demonstrating that soil organic matter provides the carbon substrate for CH3Br and CH3Cl formation rather than atmospheric CH4 or CO2.Live, unamended agricultural soil samples were net sinks of methyl bromide (CH4) at different dosages yielded enhancements in CH3Br and CH3Cl productions from both live and autoclaved soil samples, and the increments were positively correlated to the amount of CuSO4 added , as an electron acceptor, may oxidize organic matter in a similar manner as Fe(III) during the halogenation process to produce volatile CH3Br and CH3Cl.Addition of copper(II) sulfate , despite Cl− being ~135 times more abundant than Br− in the soils . These results suggested that Cu(II)-catalyzed methylation reaction favors bromide over chloride when both ions are present, possibly due to relative electronegativities and polarizabilities of the halogens20. This is a similar trend as found in the Fe(III)-catalyzed production of methyl halides21.Notably, live soils produced 4–6 times more CH3Br and CH3Cl production rates induced by copper(II)-addition were much larger in autoclaved soil samples than in live soil samples. For example, as the Cu(II) addition increased from 0 to 10 mM, average CH3Br fluxes from live soil samples gradually switched from −0.06 to 0.69 ng kg−1 hr−1, while emissions from autoclaved soils increased from 0.45 to 578 ng kg−1 hr−1. For CH3Cl fluxes, they shifted from −3.7 to 2.1 ng kg−1 hr−1, and from 6.9 to 25.2 ng kg−1 hr−1, respectively. It is possible that thermal treatment promoted the fracture of labile humic substances in soil24, making the organic carbon more readily available for the formation of volatile halogenated compounds.The increases of CH2O2 also enhanced CH3Br and CH3Cl production in both live and autoclaved soils  on CH3Cl and CH3Br production in both live and autoclaved soil samples  increased the production rates of CH3Br and CH3Cl by about 93-fold and 7-fold, respectively, in live soils; and by about 1.2-fold and 5-fold, respectively, in autoclaved soils. It is postulated that the combination of Cu(II) and H2O2 produced hydroxyl radicals (•OH)25, which provides a powerful, nonspecific oxidant agent. Subsequently, the •OH radicals react with organic compounds, halide ions, and/or copper ions to enhance the copper-mediated reaction to form volatile organochlorine compounds, such as CH3Br and CH3Cl.The addition of H26. To elucidate abiotic chemical reactions, in the absence of soil microbes or complex soil organic matter in soils that may be chemically altered during the autoclaving process, an experiment was designed with catechol to further test the essentiality of Cu(II) and H2O2 in CH3Br and CH3Cl production.Catechol  is regarded as a precursor of soil humic substances, and it is often used as a model substance of monomeric phenols that are commonly formed during the microbial degradation of many naturally occurring and anthropogenic aromatic substances4 (50 mM), and H2O2 (50 mM) produced large amounts of CH3Br and CH3Cl, respectively  and H2O2.Water solutions containing catechol 10 mM), KBr or KCl 20 mM), CuSO0 mM, KBr0 mM, CuS4 induces production of CH3Br and CH3Cl from the reaction with soil organic carbon. In addition, Cu(II) works in conjunction with H2O2, an oxidizing agent, to further increase CH3Br and CH3Cl production. In natural ecosystems, the formation of H2O2 results principally from exciting humic substances by solar radiation28. Therefore, time-series light vs. dark experiments were conducted with soil samples to explore how long the Cu(II) effect can persist, and to find out if a potential photochemical pathway involving ambient sunlight exists.The experiments described so far showed that the addition of CuSO3Br and CH3Cl were highest immediately following the addition of CuSO4, and then declined to negligible levels over the following week. The depletion of Cu(II) may contribute to the persistent decline of CH3Br and CH3Cl production. Therefore, under natural conditions where copper can undergo aerial oxidation20, it may apply a longer enhancing effect on CH3Br and CH3Cl production. It is noted that CH3Cl fluxes started to turn negative on day 8, indicating net consumption of CH3Cl from the ambient air, which might be attributable to the re-habitation of methyl halide-consuming microbial communities27 after the addition of Cu(II) chemical.The time-series experiments showed that, in the dark experiments  for them to resume near-background levels. These results suggested that solar radiation may amplify the effect of Cu(II) on CH3Br and CH3Cl production, and Cu(II) might be recycled under the light conditions to exert the longer enhancement effect.For sunlight experiments Fig. , the ave3Cl production. Results showed rapid production rates occurred when the chemical solutions with Cu(II) were exposed to sunlight, while the omission of sunlight yielded slow production of CH3Cl  can catalyze the reaction between dissolved organic carbon and chlorine in seawater to generate CH3Cl32. However, in natural environments, UV radiation with such a short wavelength would have been absorbed by stratospheric ozone before it can reach earth’s surface, which makes this pathway only plausible in sewage treatment industries. Similar to some other studies31, the light involved in our experiments was ambient sunlight. It has been proposed that the UVB region of sunlight may bring the complex of Cu(II) and halide ions to a redox excited state20, promoting methyl halide production. Therefore, the larger CH3Br and CH3Cl fluxes in samples with copper sulfate and sunlight imply that sunlight is an important driver for the reaction.A few studies have proven the importance of irradiation in the production of methyl halidesλ = 265–322 nm), UVA (λ = ~320–390 nm), and photosynthetic active radiation  by about 75%, <1%, and 23%, respectively  addition treatments. The treatment with both Cu(II) amendment and ambient sunlight exposure showed the greatest positive average fluxes of both CH3Br and CH3Cl  and sunlight. Similarly, a cruise study in the Atlantic Ocean also identified a photochemical production pathway for methyl iodide (CH3I) production under ambient sunlight conditions30.Natural seawater from San Francisco Bay produced relatively small amounts of CH3Cl Fig. , while s34. Therefore, this study also suggests a previously unaccounted for, primarily anthropogenically driven CH3Br and CH3Cl source from seawater through the interaction of copper, sunlight, and dissolved organic matter.Many discharge routes of anthropogenic copper to the oceans exist, such as copper leaching from boat antifouling paint and aquaculture nets, copper residue from automobile brake pads in urban runoff, and atmospheric aerosol deposition3Cl12; during this process, the only byproduct of this reaction was 1,2-benzoquinone and the aromatic ring remained intact  can foster the reaction between guaiacol and chloride ions to produce CHact Fig. . To test−) or cleavage of ·CH3 radical. The bond dissociation energy (BDE) required to cleave the ·CH3 radical from the methoxy group of guaiacol is approximately 240 kJ/mol36, the least within the structure. In comparison, the carbon bonds within the aromatic ring could possess up to 600 kJ/mol of BDE. Production of CH3Br and CH3Cl from catechol, which has no such methoxy group, must follow a different pathway, whereby the aromatic ring is fractured to provide ·CH3 radicals.This may be attributed to the more readily liberated methyl group from the methoxy group of guaiacol, either through an SN2 reaction  addition would trigger a higher production of CH3Br and CH3Cl from the autoclaved soil than from the live soil. Hence, it is speculated that autoclaved soil produced non-aromatic carbon fragments, which reacted more readily in the production of halogenated volatile compounds.Other studies showed that thermal decomposition of catechol and other aromatic compounds produces a range of C1–C6 productions, such as furans38, oxalic acid39, carbon suboxide40, chloroethane, iodoethane, and other chloroalkanes42, chlorinated acetic acid44 and other organic acids47  as byproducts of the Fenton-like reactions of catechol48. We find this reaction also produces chloroform (CHCl3) and CO2 -based chemicals are applied, as well as in coastal marine environments where Cu(II)-containing runoff is discharged51.The mechanism as noted in this study, in which Cu(II) and radiation enhance the methylation of bromide and chloride to produce CH3Br and CH3Cl production induced by Cu(II)-based chemical usage may depend on several environmental variables, such as sunlight intensity, soil organic matter , halide concentrations, etc. It has also been shown that pH of soil and water has an important influence on the occurrence of volatile halogenated compounds49. However, a simplified estimation without considering these factors suggests this process may be responsible for 4.1 ± 1.9 Gg CH3Br yr−1 and 2.5 ± 0.7 Gg CH3Cl yr−1, respectively -based chemicals may increase atmospheric concentrations of methyl halides, especially for CH3Br (~10% of the missing sources), contribute to stratospheric halogen load, and thereby affect ozone levels.CH4) or hydrogen peroxide (H2O2) or both, and different light conditions, light vs. dark, to investigate their impact on CH3Br and CH3Cl production in soils. The second set of experiments involved incubations of solutions with catechol  and guaiacol (2-methoxyphenol), compounds with (methoxy-) phenolic structures, which are often used as chemical models for natural organic matter in soil37. The third set involved incubation of seawater samples under different conditions: Cu(II) vs. no Cu(II) amendment, and light vs. dark. All three sets of incubations followed the same experimental set-up, as described in the section “Incubation and sampling”, unless otherwise stated.Three sets of experiments were conducted. The first set involved the incubation of soil samples with either unamended conditions or with chemical reagent supplements, such as copper(II) sulfate (CuSOSurface soil was collected from Oxford Tract (37°52′34″N 122°16′02″W), an agricultural research facility of the University of California, Berkeley. The soil type was a Tierra —Urban land complex with a clay loam texture  were mixed with 10 mL of solution containing CuSO4 at 0 mM (deionized water), 1.25 mM, 2.5 mM, 5 mM or 10 mM. The glass jars containing all these mixtures were sealed and situated in a thermostatted bath  at 20 °C during the experiments. Incubations to determine CH3Br and CH3Cl fluxes started within 30 min after the mixture preparation, and followed the method as described in the section “Incubation and sampling” below. Each treatment was conducted twice (n = 2).Experiment I  were incubated in glass jars under four different treatments. (i) soil samples were mixed with 20 ml of deionized water; (ii) soil sample was mixed with 10 ml of deionized water and 10 ml of H2O2 at 50 mM; (iii) soil sample was mixed with 10 ml of deionized water and 10 ml of CuSO4 at 50 mM; (iv) 10 ml each of H2O2 at 50 mM and CuSO4 at 50 mM were added into the soil samples. Incubations to determine CH3Br and CH3Cl fluxes started within 30 min after the mixture was completed, and followed the method described in the section “Incubation and sampling”. Each treatment was conducted twice (n = 2).Experiment II . To account for the potential effect of temperature associated with sunlight exposure, the temperature within the light glass jars was measured in the beginning and the end of the experiment; then the dark glass jars were incubated at the same temperature as the immediate prior light experiments by situating in a temperature-controlled thermostatted bath .Two groups of soil samples (50 g each) were mixed with 10 ml 50 mM CuSO52. Sample storage time in the syringe was less than 5 min. The air samples were cryotrapped and then cryo-focused prior to injection into a gas chromatograph-mass spectrometer  for CH3Br and CH3Cl analysis53.Prior to headspace air sampling, the glass jars were vented and flushed with ambient air for 1 min. After sealing the jars, 20 ml of headspace air was drawn into a glass syringe with Teflon plunger  and stainless steel needle through a septum on the cover of the glass jars at 30 min intervals for three samplingsn = 3). The incubations for the dark experiment lasted for 8 days while the light experiment lasted for 17 days, until negative CH3Cl fluxes were finally observed. The solar radiation strength, such as, UVA (λ = ~320–390 nm), UVB (λ = 265–322 nm), and photosynthetic active radiation , was also measured by UVA/UVB/PAR-BTA sensors  inside the glass jar in the sunlit experiments 2 ; guaiacol, C6H4(OH)(OCH3) ; potassium chloride, KCl , potassium bromide, KBr ; hydrogen peroxide, H2O2 ; copper(II) sulfate, CuSO4 .The following chemicals were used in the model substrate experiments: catechol, C4 and H2O2 were configured at 50 mM.All the chemicals above were prepared as solutions with deionized water  and stored in volumetric flasks. Catechol and guaiacol were configured at 10 mM ; KBr and KCl were configured at 20 mM; CuSO3Br studies or 20 mM KCl (Cl-control) for CH3Cl studies.Experiment IV , after which, 10 ml of H2O2 solution at 0 mM (deionized water), 10 mM, 20 mM, 30 mM, 40 mM or 50 mM were added into the mixture for incubation.In order to investigate the response of CH3Br and CH3Cl formation to varied amount of Cu(II) addition, 10 ml of 50 mM H2O2 was first added to a set of parallel control mixtures (both Br-control and Cl-control), after which, 10 ml of CuSO4 at 0 mM, 10 mM, 20 mM, 30 mM, 40 mM or 50 mM were added into the mixture for incubation.In order to investigate the response of CH3Br and CH3Cl fluxes following the method as described in the section “Incubation and sampling” below. Each of the incubation treatments was conducted twice (n = 2).All the aforementioned mixtures were incubated to determine CHExperiment V , the methoxy group of guaiacol can undergo nucleophilic attack by chloride to produce CH3Cl, without needing sunlight or microbial mediation12. To determine if Cu(II) behaved similarly to Fe(III) in this known pathway, two additional sets of experiments were conducted with 10 ml each of either 10 mM guaiacol or 10 mM catechol, 20 mM KCl, and 50 mM CuSO4.3Cl fluxes were determined. Each treatment was conducted twice (n = 2).All the aforementioned mixtures were incubated following the method as described in the section “Incubation and sampling” below, from which CHV = 1.9 L, Ball Corporation, California, USA). The jars were previously acid washed and then rinsed with deionized water, ethanol, and acetone to ensure purity. Samples were stored at 5 °C after collection, prior to the testing.Unfiltered seawater samples were collected from the San Francisco Bay (37°51′45″N 122°18′50″W) using glass jars (n = 2), sunlight control (n = 5), dark Cu(II) addition (n = 4), and sunlight Cu(II) addition (n = 7). The dark control experiment entailed storing the sample unsealed for 1.5 h in a dark environment. The sunlight control experiment entailed storing the sample unsealed for 1.5 h under ambient light . The Cu(II) experiments entailed the addition of 0.3 mg CuSO4 to the seawater, which were mixed and dissolved in the solution, before situating under dark or light conditions. Immediately following the above treatments, all the seawater samples were sealed for a closed headspace and incubated following the method described in the section “Incubation and sampling”.Experiment VI , the soils, seawater, or chemical mixtures were first mixed well in glass jars and then situated in a thermostatted bath  at 20 °C. The jars were sealed with a stainless steel lid and Viton gasket and connected to a previously-evacuated stainless-steel loop (14.5 mL), which served as a sampling volume. The sample valve is at the top of the jar while the samples are at the bottom. The initial headspace was filled with ambient air, either outdoor air or laboratory air. Headspace air samples from the jars were drawn into the sampling loop at 30 min intervals, and then drawn through a cryotrap, followed by desorption, cryofocusing, and injection into a gas chromatograph-mass spectrometer  for CH3Br and CH3Cl fluxes from the samples were calculated based on the hourly averaged change of headspace concentration multiplied by moles of headspace air5, and normalized to the dry mass of the soil (ng kg−1 hr−1) or the volume of the seawater (ng L−1 hr−1); or were reported as the total mass of CH3Br and CH3Cl produced in pure chemical experiments (ng). Positive fluxes represented production of CH3Br and CH3Cl while negative fluxes represented consumption of CH3Br and CH3Cl from the headspace air. At least two (n ≥ 2) incubations were conducted for each treatment of the soils, seawater, and chemical mixtures.CH8. The averaged instrumental precisions for CH3Br and CH3Cl after applying drift corrections were 1.9% and 1.0%, respectively8. Using the precision estimates, ambient concentrations, and sample size, the minimum detectable fluxes from soils were about ±0.02 ng kg−1 hr−1 for CH3Br and ± 0.58 ng kg−1 hr−1 for CH3Cl.Weekly calibration curves were constructed using whole ambient air standards collected at Niwot Ridge, Colorado, and calibrated at the Global Monitoring Division Laboratory of the National Oceanographic and Atmospheric Administration. Each calibration curve was made up of 10–18 data points by trapping various volumes of standard gas to capture the full range of chromatogram peak areas observed in the samples. The daily drift of instrumental signals was also corrected using multiple daily runs of the same standardSupplementary InformationPeer Review File


In this study, the activation processes of an in situ–prepared bulk La2O2CO3 sample and an ex situ as-prepared La(OH)3 sample are in situ investigated by X-ray photoelectron spectroscopy (XPS), X-ray diffraction (XRD), and online mass spectroscopy (MS). Results indicate that the La2O2CO3 sample, during linear heating to 800°C, always contains some carbonates near the surface region, which supports a two-step model of bulk carbonate decomposition through surface sites. The La(OH)3 sample structure evolution is more complex due to contaminations from air exposure. Together with TGA results, online mass analysis of water and CO2 signal loss showed that three major catalyst structure phase change steps and a preheating up to 800°C are required for the as-prepared material to be transferred to La2O3. This process is carefully investigated combining the three in situ methodologies. XPS and XRD data further reveal transformations of variety of in situ surface structures and forms including hybrid phases with hydroxyl, carbonates, and oxide as the sample heated to different temperatures within the range from 200 to 800°C. The results provide useful insights on the activation and deactivation of La-contained materials.Lanthanum-containing materials are widely used in oxidative catalytic and electrocatalytic reactions such as oxidative coupling of methane (OCM) and solid oxide fuel cells (SOFCs). However, many of these materials are highly susceptible to air contamination which means  Results also showed that the binding energy of La 4d7/2 is the easiest benchmark for XPS spectra calibration as its BE is always uniform (102.2 ev) after above pretreatments including air exposure. A recent DFT simulation further corroborated the experimental results revealing the catalyst surface structure under realistic process conditions  oxygen site in the (La2O22+)n layer of bulk La2O2CO3 overlaps with the lattice oxygen in La2O3.Our previous eratures . The resnditions . Using t2O2CO3 (in situ–prepared) and La(OH)3 (ex situ–prepared), are investigated by in situ XPS, XRD, and online MS for their surface and bulk structures. As these two samples represent two most popular La-related contamination species on many complex oxide surfaces, such as perovskites and MOF, their temperature stability and evolution of structure and phases are observed in detail. The in situ measurements are continued until the complete regeneration of La2O3 at about 800°C. All observed species are further interpreted by the characteristic species assignments and DFT surface structure model mentioned above. This study hopefully provides useful insight for understanding the related behavior of La-containing materials and the reaction performance of La-based catalysts.In this study, two samples, that is, La2O3 catalyst used in this work is described in detail elsewhere. The synthesis of the La2O3 nanorod catalysts follows the process proposed by Zhu and coworkers (3. The SEM result indicates the sample is in the shape of nanorods with 10 nm diameter in average. BET measurement (N2) yields specific surface area of 3.4 m2/g  and a 180° double-focusing hemispherical analyzer with a six-channel detector (30 eV pass energy). For in situ heating experiments simulating industrial high-temperature pretreatments, the sample analysis chamber (SAC) was specially equipped with a homemade heating stage. This sample stage is designed to be compatible with the original spectrometer manipulator which allows a sample to be heated up to 800°C with simultaneous XPS analysis. The in situ heating stage is also adapted to the regular flag-sample holder which allows the sample to be transferred to all the other UHV-connected functional chambers within the vacuum system. All heating treatments, if not specified additionally, are performed in the analysis chamber.The XPS surface analysis in this work is performed by the ThermoFischer ESCALAB 250Xi XPS spectrometer. All spectra were acquired using a monochromated X-ray irradiation Al−10mbar). The in situ XPS analysis for every step is conducted at the fixed temperature. The analysis location (Φ=500 μm ) on the sample surface is chosen randomly. A set of primary sample spectra  were acquired at 25°C before heating starts. Similarly, spectra were collected between 200 and 800°C with an increment of 100°C. Finally, the spectra after subsequent cooling to the room temperature were also collected. The monitoring time for spectra collected at fixed temperature was around 10°min; the heating rate in the XPS experiments was 10°C/min. During the whole process, the maximum pressure in the analysis chamber monitored by a wide-range gauge was around 2·10−8 mbar. All spectra processing analysis including O 1s and C 1s peak fitting are performed in Thermo Avantage software. The spectra are deconvoluted using the Lorentz–Gauss mix function after subtraction of modified Shirley (Smart) background. As proposed in our previous work .For XPS analysis, the fresh powder sample was pressed in a tiny 1.5 mm hole of a flag-shaped sample holder and then preheated in vacuum  was used. In situ XRD analysis was conducted in the Bruker D8 Advance X-ray diffraction system using in situ Anton Paar XRK 900 cell reactor. The system is equipped with Cu Kα X-ray source . The diffraction patterns are acquired with a step width of 0.02° in the (2θ) range from 20 to 50°. The sample temperature was ramped from room temperature to 800°C at a rate of 10°C/min. All presented survey scans are obtained at specified temperature.To confirm the phase change during the first heating of a fresh LaTemperature program desorption (TPD) in vacuum for a fresh sample was carried out using a quadrupole mass spectrometer  connected to HPGC .in situ–prepared La2O2CO3 is linearly heated directly in front of analyzer during the XPS analysis. The presented C 1s and O 1s spectra are collected at the actual temperature. The 529.8 eV peak in O 1s spectra is related to lattice oxygen in La2O3 or 4-coordinated oxygen atoms in La2O2CO3. The C 1s peak with BE around 285 eV is associated with adventitious carbon accumulated on the sample surface from background during the heating in HPGC. Both C 1s and O 1s spectra also show characteristic peaks of CO32− groups (BEs 532.4 eV for O 1s and 290.5 eV for C 1s). Our previous TPD studies showed that the bulk La2O2CO3 demonstrates two major desorption features: decomposition of the surface carbonate below 550°C and decomposition of the bulk La2O2CO3 from 600 to 800°C  desorbing from the freshly prepared sample during the heating process. The inset of the figure plots our previously obtained TGA result including the raw data (black curve) and its derivative curve (red) for a comparison  is also detected at this step. No more water signal is observed in later TPD process, and this step is considered as sample dehydration. In the 2nd step at about 530°C, around 2.5% loss of total mass is observed which is identified as partial carbonate decomposition since it is accompanied by CO2 desorption. Finally, within 3rd step at around 700°C, about 3.0% mass loss is observed and identified as the complete carbonate decomposition accompanied by CO2 desorption 4CO3 and La(OH)3 phases are decomposed forming La2O2CO3 and LaOOH, respectively, both yielding H2O as desorbed product. During the 2nd and 3rd steps, La2O2CO3 is decomposed to La2O3 yielding CO2 in two stages at ∼ 520 and 700°C.To arbonate . Figure mparison . The comsorption . The MS terature , similarin situ precleaning procedure by heating in vacuum at 800°C for 1 h. The C 1s spectrum of the as-prepared sample shows two main peaks with similar intensities: the adventitious carbon peak at 284.6 eV and a higher binding energy peak at 289.5 eV associated with carbonate. The O 1s spectrum of the as-prepared fresh sample has three features: a main peak at 531.0 eV and two relatively small shoulders at 532.3 and 528.7 eV. The shape of C 1s and O 1s spectra correlate very well with our previously published data 3. After the sample is heated to 300°C, the XRD pattern is significantly changed. Comparing with known PDF database, the main peaks in La(OH)3 diffraction pattern are not observed in the new pattern, suggesting that the La(OH)3 is completely decomposed. TGA and MS results above also support that heating to 300°C is accompanied by simultaneously removing most of hydroxide species from the bulk. Therefore, the main contamination in the sample bulk should be carbonates. However, the peaks in the new XRD patterns obtained at 300 and 400°C [attributed mainly to tetragonal La2O2CO3 (011)] are not found in good consistency with any single XRD PDF of the known La-based compounds. In addition, all the peaks obtained, such as the one with the highest intensity at 30.3°, are very broad, which indicates that after heating to 300°C, the new crystallization is only started forming very small grains. Most of the peaks in the obtained experimental XRD patterns can be covered by superposition of the tetragonal La2O2CO3 and hexagonal La2O3 PDF data. The behavior of XRD pattern suggests that the carbonate is highly dispersed in the sample below 300°C. However, there are still a few broad peaks in the experimental curves which are different from the carbonate and oxide references. This indicates that the sample, after being exposed to air and contaminated, is still not only a mixture of La(OH)3 and La2O2CO3 but also contains other complex compounds, such as La2(OH)4CO3 or LaOOH.in situ XP spectra obtained for the same batch of sample are shown in in situ heated up to 200°C, the C 1s peak shape is not changed significantly. The O 1s spectrum taken at 200°C shows the 529.1 eV peak which can be assigned to La2O3 lattice oxygen  in (La2O22+)n layer of bulk La2O2CO3 3 and La2O2CO3 at 200°C to a mixture of La2O2CO3 and La2O3 at 400°C during a gradual dehydration. For the O 1s peak with binding energy at ∼ 529.5 eV observed at 400°C, our previous publication assigned it to either the characteristic lattice oxygen species [(O4c) and (O6c)] in La2O3 or the (O4c) in the (La2O22+)n layer of bulk La2O2CO3 3 phase. The strong interaction between carbonate and hydroxide or formed complex compounds does have an effect on the electronic structure.Comparison of the h sample . An earlple  and 285.5 eV (300 and 400°C). The atomic percentage of this peak carbon increases from 14 to 20% as the sample heated from 200 to 400°C . The ass3, is rather easy to absorb CO2 from air (during synthesis and preparation on XPS sample holder) forming highly dispersed carbonate compounds in the bulk. If the sample is kept at temperature lower than 200°C, La(OH)3 still dominates in the bulk structure, and especially in the near surface region.Combining the XPS and XRD results, it clearly suggests that the as-prepared fresh sample, although to be synthesized as La(OH)in situ XRD obtained at this step demonstrates the patterns similar to that obtained at 300 and 400°C. The main difference is that the peaks become sharper than in the patterns collected at lower temperatures indicating the polycrystalline grain size is increasing. Following the 1st step (25–400°C) data presented in nd step (400–600°C) is investigated. Increasing temperature to 500°C gives rise to further decrease of carbonate feature at 290.4 eV in the C 1s spectrum. Meanwhile, in the O 1s spectrum, the intensity of carbonate component at 532.5 eV changes from slightly higher than the lattice oxygen peak to significantly lower.Taking into account previous literature studies , TGA and2O2CO3 through online MS-measured TPD has been studied in our recent publication  and then surface carbonate can be easily correlated with in situ La2O2CO3 decomposition considered in 3 sample can be fully recovered to La2O3. This is also supported by the La 3d 5/2 spectra taken from room temperature to 800°C . The significant volume change can result in a loss of electrical contact between surface carbon species and the bulk of the sample which explains the charging built up during XP spectra collection.The evolution of both oxygen and carbon components depending on heating temperature is summarized in in situ–prepared La2O2CO3 and ex situ–prepared La(OH)3, are investigated by in situ XPS, in situ XRD, and online MS. All observed species are further interpreted by previous obtained characteristic assignments and DFT surface structure model. After heated up to 800°C, both samples are completely regenerated to La2O3. For La2O2CO3 sample, during linear heating to 800°C, a partial carbonate coverage near the surface region is always observed, which confirms the previously suggested two-step model of bulk carbonate decomposition. The ex situ–prepared La(OH)3 sample is a hybrid of carbonates and hydroxyl due to air exposure–induced contamination. Three major steps of catalyst structure phase changes are investigated by the three in situ methods. Evolution of surface structures showing the changes of hybrid phases with hydroxyl and carbonates is observed by XPS and XRD from 200 to 800°C. Results provide surface and bulk structure details of the two samples during the heating treatment which bring insight into the activation and deactivation of La-containing materials.The temperature-correlated structure changes of two most popular La-related contamination species,


Here, by means of first-principles calculations, we identify six ferroelectric catalysts comprising transition-metal atoms anchored on In2Se3 monolayer, whose catalytic performance can be controlled by ferroelectric switching based on adjusted d-band center and occupation of supported metal atoms. The polarization dependent activation allows effective control of the limiting potential of CO2 reduction on TM@In2Se3  as well as the reaction paths and final products on Nb@In2Se3 and Re@In2Se3. Interestingly, the ferroelectric switching can even reactivate the stuck catalytic CO2 reduction on Zr@In2Se3. The fairly low limiting potential and the unique ferroelectric controllable CO2 catalytic performance on atomically dispersed transition-metals on In2Se3 clearly distinguish them from traditional single atom catalysts, and open an avenue toward improving catalytic activity and selectivity for efficient and controllable electrochemical CO2 reduction reaction.Efficient and selective CO 2 into chemical fuels holds promise for mitigating environmental pollution and energy crisis. This work presents a distinct design of ferroelectric catalysts with high catalytic activity and selectivity for efficient and controllable electrochemical CO2 reduction reaction.Electroreduction of CO Public health is severely threatened by the greenhouse effect due to the excessive fossil fuel usage. It is highly desirable to develop technologies that can convert the greenhouse gas  into sustainable and clean energy sources. Among various possibilities, catalytic reduction of CO2 into hydrocarbon fuels, which features environmental friendliness, high efficiency, and low cost, has been recognized as one of the most promising approaches. Single atom catalysts (SACs), first proposed for CO oxidation in 20118, offer us excellent candidates to activate and convert CO2 efficiently when trasition metal (TM) atoms are used13, because the coexistence of empty and occupied TM d-orbitals can accept lone-pair electrons, and then back-donate these electrons to the antibonding orbitals to weaken the C=O bonds. Notably, two-dimensional (2D) materials serve as promising substrates for atomically dispersed transition metal atoms for CO2 reduction due to their large surface-to-volume ratios, short carrier diffusion distances, unique electronic properties, and abundant active sites. The catalytic performance of these 2D SACs stems from versatile advantageous characteristics. For example, catalysts with singly dispersed Ni, Co, Cu, Pt, and Pd atoms on graphene14, Pt or Pd atom anchored on C3N415, V-β12 boron monolayer16 and Co decorated metal–organic frameworks18 all have shown great potential for CO2 reduction.The ever-rising global energy consumption and its negative impact on the environment are major challenges in front of humanbeing in the 212 reduction in order to satisfy large-scale industrial applications. Recent studies have shown that electric polarization plays an important role in determining catalytic activities and selectivities, and can greatly improve the catalytic performance compared with the unpolarized counterparts21. For instance, Janus TM dichalcogenides possess better catalytic abilities for water splitting and N2 fixation21, since the catalysis-related properties, including surface stoichiometry, electronic structure26, adsorption strengths, and reaction activation energies, can be tuned by the polarization27; Moreover, by utilizing the polarization from the substrate, the overpotential of oxygen evolution reaction on TiO2 surface can be strongly reduced28.Despite latest advances, there is still plenty of room to improve the catalytic activity and selectivity of COviz., materials with switchable ferroelectric polarization, hold promise as efficient catalysts since their unique reversible polarization provides an additional mechanism to adjust empty and occupied d-orbitals of adsorbed metal atoms. The activation of reactants can thus be controlled, while the catalytic properties of the supported metal, such as activity32 and selectivity, can be tuned33. These expectations have been partially confirmed in ABO3 perovskite ferroelectrics. For instance, reversing the polarization of ferroelectric PbTiO3 substrate makes CrO2 monolayer overcome the limiting factor stemming from the Sabatier principle, and thus display excellent catalytic behaviours for both NOx decomposition and CO oxidation34. Reorienting the polarization direction of the ferroelectric PbTiO3 substrate can dramatically change the chemisorption energies of CO, O, C, and N on the PbTiO3-supported Pt films, and alter the reaction paths of the dissociations29. The oxygen evolution reaction activity of TiO2 film on a ferroelectric SrTiO3 substrate can be strongly enhanced relative to unsupported TiO2 due to the presence of dynamic dipoles in response to the charge on the adsorbed species, while the reaction path can be modulated via ferroelectric switching28. However, these predictions have not been experimentally realized so far, mainly due to the instability and depolarization in traditional ABO3 perovskite ferroelectrics when the thickness is smaller than a critical value. Encouragingly, the emergence of 2D ferroelectric materials, exemplified by In2Se335, CuInP2S636, and SnTe37, provides an excellent opportunity to verify controllable catalysis in ferroelectrics. The realization of ferroelectricity at room temperature makes it feasible for 2D ferroelectrics to be used for controllable catalytic CO2 reduction.Inspired by these recent achivements, we expect that ferroelectrics, 2Se3 monolayers as the ferroelectric SACs toward electrochemical CO2 reduction. Our efforts identify six catalysts with singly dispersed TM atoms anchored on the 2D ferroelectric substrate, namely α-In2Se3 monolayer, with polarization downwards or upwards (denoted as TM@P ↓ -In2Se3 or TM@P ↑ -In2Se3). The switchable polarization can not only alter the reaction barrier and paths of CO2 reduction, but also lead to different final products. It can even reactivate the stuck CO2 reduction. These performance improvements stem from the synergistic effects of adjusted empty and occupied d-orbitals  of adsorbed metal atom, polarization dependent electron transfer, and CO2 adsorption energies under ferroelectric switching. These ferroelectric SACs, catalytic mechanism, and the polarization dependent catalytic CO2 reduction open an avenue for controllable CO2 reduction reaction (CO2RR), and introduce a feasible approach to significantly improve the efficiency of such reactions.In this work, based on the experimentally available 2D ferroelectrics, we theoretically investigate the potential of using transition metal decorated α-In2Se3 monolayer was chosen as the substrate of potential catalysts due to the following reasons: (1) it is the ground-state phase among possible phases  phases have been predicted to share similar atomic structures and are almost energetically degenerate, and they have the same CO2RR performance  it is stable at room temperature and has been experimentally synthesized39, and its ferroelectricity with switchable polarization under room temperature has been explicitly demonstrated40. With the polarization locking from the asymmetric arrangement of its quintuple layers 38, α-In2Se3 monolayer does not have enough electrons to be injected into the antibonding 2πu orbitals of CO2 so that the strong sp-hybridization symmetry of the carbon atom can not be disrupted41. Thus, the material itself is not suitable as a catalyst for CO2 reduction42, which is corroborated by our theoretical study: upon adsorption on In2Se3, the inherent linear O=C=O structure of CO2 molecule is well maintained .After a comprehensive investigation of the energetically most favorable adsorption configurations Fig. a, b and 2Se3 are stable  and able to activate CO2 molecules, transition metal atoms need to uniformly disperse on the substrate to prevent any aggregation of the deposited atoms and maximize the catalytic efficiency47.Although the screened TM@In54. At the energetically most favorable site, all the six TM atoms have rather strong binding energies  simulations with a Nose–Hoover thermostat at 300 K on Pd@In2Se3 as a representative example. The neagive clustering energies  indicate that it is not energetically favorable to form clusters on the catalyst surface. In the AIMD simulations, the out-of-plane polarization of ferroelectric In2Se3 monolayer can be well retained, and the metal atom stays anchored at the energentically favorable site even at room-temperature (300 K) for at least 15 ps . The two Pd atoms can maintain dispersedly adsorbed features without metal clustering and structural phase transition for 15 ps  is higher than that of Pd@P ↑ -In2Se3 (−4.13 eV), indicating that Pd@P ↓ -In2Se3 possesses better catalytic ability46. Similar phenomena occur in the other five TM@In2Se3 catalysts  are rather pronounced, while such differences for TM@In2Se3  are only moderate. These contrasting results imply that both reaction paths and barriers are affected by the polarization . Considering that the localized electronic states near the Fermi level lead to high N2 catalytic activity in the catalyst Co2@GDY 49, we expect that the modulation of the electronic structure of TM@In2Se3 caused by the polarization switching has a major influence on the subsequent CO2 hydrogenation process.Different from traditional SACs like Pd@Cnt Table . Comparesee Fig. , and thesee Fig.  under th2Se3 systems is polarization dependent, the effective ferroelectric switching of TM@In2Se3 is essential to achieve controllable catalytic reduction. We reverse the polarization orientation of Pd@In2Se3  for P ↑ to P ↓ (P ↓ to P ↑ ), which is slightly larger than the value for the bare ferroelectric layer (0.13 eV/UC), but it is still accessible to experimental manipulation38. It is noted that the ferroelecitrc switching behaviors are not significantly affected by the metal adsorption. Although the local area of metal anoching site is metallized due to the hybridization between the metal and surrounding Se atoms, the semiconduting feature of the whole substrate and the associated ferroelectricity can be well preserved  because of the monodentate TM-O configuration. Moreover, the binding energies of the catalysts with the activated CO2 molecule  is significantly higher than that on TM@In2Se3 , as indicated by the larger binding energies (−3.39~−2.02 eV vs −1.39~−0.76 eV), more electron transfer (0.61~0.92e vs 0.26~−0.45e), and smaller angles of ∠OCO (131.1°~136.8° vs 146.4°~151.2°).Effective activation of COsee Fig.  by formisee Fig. . Note th2 catalytic activation, namely coexistence of charge depletion and accumulation between the TM atoms and CO2 molecules. The empty TM d orbital could accept electrons from the highest occupied molecular orbitals  of the CO2 molecule, while the occupied TM d orbital could back-donate electrons to the lowest unoccupied molecular orbitals  of the CO2 molecule , upon adsorption on the In2Se3 substrate, the Pd atom loses partial d electrons to form the Pd-Se bond, and the resulting empty Pd d orbitals provide channels for electron acceptance and back donations to activate CO2 molecules. Consequently, compared with other TM@In2Se3 catalysts, where the TM atoms have intrinsic partially occupied d orbitals, Pd@In2Se3 catalyst has a lower degree of CO2 activation as indicated by the relatively larger ∠OCO and smaller charge transfer  variations are more evident than the CO2 binding energy and electron transfer. This is due to the fact that the DOS variation is directly related with the orbital shift under the polarization flip, such as the d band center, as seen in Fig. 2 binding energy are mainly related to the itinerant electrons induced by the polarization.The distinct behaviours on different ferroelectric surfaces are attributed to the mechanism of COule Fig. . As indi2RR by consuming proton–electron pairs from the electrolyte solution64. To check if CO2RR is more favourable, we first calculated Gibbs free energy changes (ΔG) at the first hydrogenation step of CO2RR (* +CO2 + H + +e− → OCOH* or OCHO*) and HER (* +H + +e− → H*). According to the Brønsted–Evans–Polanyi relation66, reactions with lower ΔG have smaller reaction barriers and consequently are kinetically more favoured.As an important competing side reaction, hydrogen evolution reaction (HER) may significantly restrain the Faradaic efficiency of CO2RR, Rh@In2Se3, Pd@In2Se3, and Re@P ↑ -In2Se3 prefer to form carboxyl (OCOH*), while Ni@In2Se3, Nb@In2Se3, Zr@P ↑ -In2Se3, and Re@P ↓ -In2Se3 tend to produce formate (*OCHO). Since CO2 molecule cannot be activated on Zr@P ↓ -In2Se3, it is not considered here. The Gibbs free energy changes  catalyst , and final product.Considering the complexity of CO2RR can be classified into three categories: (i) TM@In2Se3 , for which the polarization switching changes the reaction barriers, but not the reaction paths and the intermediate/final products; (ii) Zr@In2Se3, for which the ferroelectric switching can reactivate the stuck CO2 reduction; (iii) TM@In2Se3 , for which all the reaction paths, reaction barriers, and final products are polarization dependent. This classification is well consistent with d band center analysis , the reaction barrier of the CO2 hydrogenation process is also affected. Taking the Rh@In2Se3 catalyst as an example  under different polarization directions are the same, the energy barriers are obviously different: the reaction of CO2* →OCOH* is downhill on Rh@P ↑ -In2Se3, but uphill on Rh@P ↓ -In2Se3. In the subsequent hydrogenation steps, the polarization reversal does not change the most favourable path of CO2RR, both following the paths of CO2* →OCOH* →CO* →CHO* →CH2O* →CH2OH* →CH3OH* →OH* →H2O*, with the final product of CH4. However, the reaction barrier for each step, the potential-determining step (PDS), and the limiting potential are all different and polarization dependent. On Rh@P ↓ -In2Se3, the PDS of CO2 reduction is CO* →CHO* with a limiting potential of −0.43 V vs. RHE; in contrast, the corresponding PDS on Rh@P ↑ -In2Se3 is the protonation of CHO* to CH2O* with the estimated Ul of −0.26 V vs. RHE. Obviously, the reaction barrier of CO2 reduction is polarization tunable via ferroelectric switching. Notably, the limiting potential (−0.26 eV vs. RHE) is better than most reported CO2RR catalysts  and mild charge gained by the adsorbed CO2 molecule  . More importantly, HOCHO* on the Zr@P ↓ -In2Se3 is merely 40 meV higher than that on Zr@P ↑ -In2Se3, which indicates that the polarization switching is relatively easy to achieve. The conclusion holds true under dilute Zr concentrations, while the local ferroelectric switching around HOCHO* is prohibited due to the higher energy barrier . Moreover, the H2O* at Zr@P ↓ -In2Se3 could be removed spontaneously, in contrast to the 0.36 eV energy barrier for the dehydration process on Zr@P ↑ -In2Se3.Since polarization can modulate interaction strength as indicated above, it is expected that the binding strength of the intermediate HOCHO* can be weakened when the polarization direction of In2 molecule cannot be activated on Zr@P ↓ -In2Se3 for reduction, the polarization reversal can reactivate the catalytic reduction on Zr@P ↓ -In2Se3 with a reasonable limiting potential. During the hydrogenation process, appropriate polarization switching could either reactivate the CO2 reduction or accelerate the reaction rate by reducing the reaction barriers.Therefore, though CO2Se3 , the activations of CO2 molecule are much deeper on Nb@In2Se3 and Re@In2Se3, as evidenced by the smaller bond angle ∠OCO , and more pronounced charge transfer between CO2 and the catalyst . The huge differences of d band center  as effective ferroelectric catalysts for electrochemical CO2RR. We found that the polarization reversal of ferroelectric In2Se3 monolayer can adjust the empty/occupied d orbitals of adsorbed TM atom and the energy of the d electrons, thereby tuning the catalytic performance for CO2RR, including the degree of CO2 activation, limiting potential, reaction path, and final product, even reactivating stuck catalytic reduction. Especially, the Rh@P ↑ -In2Se3 catalyst is indentified as a highly efficient electrochemical catalyst for CO2RR due to the fairly low limiting potential (<0.5 V)71. Moreover, the Re@In2Se3 and Nb@In2Se3 catalysts can realize selective generation of different desired products with the same catalyst via polarization switching, which opens a path toward ferroelectric controllable catalysis. These SACs based on 2D ferroelectric materials hold great promise for improving catalytic activity and selectivity for electrochemical CO2RR.In conclusion, by comprehensive DFT computations, we have screened out six TM@In73. The spin-polarized generalized gradient approximation (GGA) in the form of Perdew − Burke − Ernzerhof (PBE) treats the exchange − correlation interactions, while the frozen-core projector augmented wave (PAW) approximation describes the interaction between the ion and electron76. The van der Waals (vdW) interactions are described with the DFT-D3 method in Grimme’s scheme77. The models with one TM atom anchored 2 × 2 In2Se3 are built as the potential catalysts for the simulations, since it corresponds to the coverage of 25% with the optimal adsorption energies, as shown in Supplementary Fig. 2Se3 supercell act as the catalytic active sites. More than 20 Å vacuum space perpendicular to the surface is added to avoid interactions between periodic slabs. The dipole correction is taken into account for all the asymmetric structures78. The 2D Brillouin sampling is presented with a 4 × 4 × 1 gamma-centered Monkhorst-pack k-mesh. The cutoff energy for plane-wave basis sets is 500 eV. The convergence thresholds for force and total energy are 10−2 eV/Å and 10−5 eV, respectively. Moreover, the Gibbs free energy calculation is operated with computational hydrogen electrode (CHE) model79, and the solvent effect is considered with the implicit solvent model implemented in VASPsol61. The site-specific charge differences are obtained using Bader analysis. Besides the standard PBE calculations and the models of TM@2 × 2 In2Se3, we also reexamined the chemical reactions by using larger supercells  computations are performed with the Vienna ab initio simulation package (VASP) codesupporting informationPeer Review File


Non–coding RNAs including microRNAs (miRNAs), long non–coding RNAs (lncRNAs) and circular RNAs (circRNAs) have been found to be closely involved in the development and progression of HNSCC and partially associated with clinical significance of HNSCC patients.Head and Neck Squamous Cell Carcinoma (HNSCC) is one of the most commonly reported malignant tumors in human beings, representing 5.3% of all cancers . In the MicroRNAs are non–coding RNAs with about 22 nucleotides long that participate in the post–transcriptional regulation of gene expression involved in the proliferation, migration, metastasis and angiogenesis of cancer . For insin vitro and fewer metastases in vivo in HNSCC cells (homeobox C cluster (HOXC) and lncRNA HOTAIR transcriptionally activated by STAT3 interact with pEZH2–S21 resulting in proliferation and the growth of HNSCC xenograft tumors in vivo , long non–coding RNA Fer–1–like protein 4 (FER1L4) .LncRNAs also belong to a subgroup of non–coding RNAs that are at least 200 nucleotides long and the majority of them do not have proteincoding ability . lncRNAsCC cells . MALAT–1CC cells . HOX tra in vivo . Besides in vivo  or LINC0(FER1L4) , and gro(FER1L4) , but oth(FER1L4) , FOXD2 a(FER1L4) , KTN1 an(FER1L4) , LINC004(FER1L4) , H19 imp(FER1L4) , small n(FER1L4) , urothel(FER1L4) , or ZNFX(FER1L4) , and can(FER1L4) . A three(FER1L4) . Immune–circRNAs are a novel subclass of non–coding RNAs, which are produced by reverse splicing and are characterized by a closed single–stranded structure and lack of 5′cap and 3′polyadenylation [poly(A)] tail . Current“Non–coding RNAs in Head and Neck Squamous Cell Carcinoma: Functional and Clinical Implications” includes 21 original articles and 3 review articles, which highlight the regulatory mechanism and clinical relevance of non–coding RNAs in HNSCC. For example, Wan et al. identifies a super–enhancer regulatory model of miR–21–5p by FOS like 1 (FOSL1), promoting malignant progression of HNSCC. The article by Li et al. discuss LINC02195 as a regulator of MHC I and the number of CD8+ and CD4+ T cells in the tumor microenvironment. Moreover, Wang et al. summarizes the crosstalk between lncRNAs and microRNAs as well as the detailed regulatory mechanism of the interaction. Additionally, Luo et al. identifies miRNAs signatures respectively for HPV+ and HPV− HNSCC, which are of great significance in evaluating patient survival. All of other articles mainly discuss the potential function of non–coding RNA as a ceRNA in HNSCC.This Research Topic All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.WC was supported by grants from the National Natural Science Foundation of China (81972589).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


In this study, IGF2BP3 mRNA and protein levels were elevated in NPC tissues. In addition, IGF2BP3 exerted an oncogenic role by promoting epithelial‐mesenchymal transition (EMT), thereby inducing NPC cell migration and invasion. Further studies revealed that IGF2BP3 regulated the expression of key regulators of EMT by activating AKT/mTOR signalling, thus stimulating NPC cell migration and invasion. Remarkably, targeting IGF2BP3 delayed NPC metastasis through attenuating p‐AKT and vimentin expression and inducing E‐cadherin expression in vivo. Moreover, IGF2BP3 protein levels positively correlated with distant metastasis after initial treatment. Importantly, IGF2BP3 expression served as an independent prognostic factor in predicting the overall survival and distant metastasis‐free survival of NPC patients. This work identifies IGF2BP3 as a novel prognostic marker and a new target for NPC treatment.Metastasis contributes to treatment failure in nasopharyngeal carcinoma (NPC) patients. Our study aimed at elucidating the role of insulin‐like growth factor 2 mRNA binding protein 3 (IGF2BP3) in NPC metastasis and the underlying mechanism involved. IGF2BP3 expression in NPC was determined by bioinformatics, quantitative polymerase chain reaction and immunohistochemistry analyses. The biological function of IGF2BP3 was investigated by using  IGF2BP3 functions as a post‐transcriptional regulator that recruits target coding and non‐coding transcripts to protein‐RNA complexes and affect the expression and translation of target RNAs.IGF2 mRNA, thus facilitating IGF2 expression.The PI3K/AKT signalling pathway plays a critical role in regulating various cell functions, including cell survival, cell differentiation, cell growth and cell motility.Here, we not only analysed the mRNA levels of IGF2BP3 in Gene Expression Omnibus (GEO) datasets but also determined the mRNA and protein levels of IGF2BP3 in clinical samples. Both mRNA and protein levels of IGF2BP3 were found to be higher in NPC than those in adjacent normal tissues. Moreover, IGF2BP3 protein expression was identified as an independent factor in predicting OS and distant metastasis‐free survival of NPC patients. Therefore, IGF2BP3 might be used to optimize therapeutic and follow‐up decisions in NPC.22.1The NPC mRNA‐Seq datasets  were downloaded from the GEO database and used to perform differential expression analysis. The differential analysis of IGF2BP3 expression was performed in NPC and adjacent normal tissue. The involvement of IGF2BP3 in specific signalling pathways was elucidated by gene set enrichment analysis (GSEA).2.22 at 37°C. As for the use and management of SC79, after cell transfection, cells were cultured in a complete medium with 8μg/mL SC79 for 30 mins in the incubator and then collected cells for follow‐up experiments.Three NPC cell lines  and a nasopharyngeal epithelial cell line (NP69) were obtained from the Cell Bank of Central South University  and used in this study. Cells were cultured in RPMI‐1640 medium containing 10% foetal bovine serum  and supplemented with 1% streptomycin/penicillin  in humidified air with 5% CO2.3Two small hairpin RNAs (shRNAs) targeting IGF2BP3 (shIGF2BP3‐1 and shIGF2BP3‐2) were designed. A scramble shRNA, shIGF2BP3‐1 and shIGF2BP3‐2 were cloned into pLVX‐shRNA2‐puro. To construct the IGF2BP3 overexpression vector, IGF2BP3 cDNA was synthesized and cloned into pLVX‐IRES‐ZsGreen1. For lentiviral particle preparation, targeted viral plasmids were used to transfect cells with Lipofectamine 2000 . The cell lines were infected with shRNAs or overexpressed viral supernatants, and qRT‐PCR analysis and Western blot analysis were used to determine IGF2BP3 mRNA and protein expression levels, respectively.2.42O. The All‐in‐One™ First‐Strand cDNA Synthesis Kit  was used for reverse transcription to generate high‐quality cDNA, according to the manufacturer's protocols. A mixture containing SYBR® Green Master Mix, cDNA, forward and reverse primers, RNase‐free H2O was prepared and subjected to qRT‐PCR. The primers used in qRT‐PCR are listed in Table S1. All experiments were performed in triplicate using glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) as an internal reference control. The 2−ΔΔCT method was used to calculate relative mRNA levels.Total RNA was extracted using TRIzol reagent  from cells or tissue samples and stored at −80 °C in RNAse‐free H2.5Cell lysates from cell lines were prepared with a RIPA lysis buffer kit and were used for the quantification of protein concentrations using the bicinchoninic acid assay . After heating and denaturation, proteins were subjected to sodium dodecyl sulphate‐polyacrylamide gel electrophoresis followed by transfer to polyvinylidene fluoride membranes. After blocking with 5% skimmed milk, the membranes were individually incubated with the primary antibodies, including anti‐IGF2BP3 , anti‐E‐cadherin , anti‐vimentin , anti‐p‐mTOR , anti‐mTOR , anti‐p‐AKT , anti‐AKT , anti‐GAPDH  and corresponding secondary antibodies. Eventually, the protein bands were detected using the enhanced chemiluminescence reagent and then scanned using the electrophoresis gel imaging analysis system. The ImageJ2 software  was used to analyse all imaging results. The density ratios of target bands to GAPDH bands were calculated and used to determine the relative protein expression.2.65 cells/well NPC cells were seeded into 24‐well plates and cultured overnight at 37°C to obtain a confluent monolayer. A 10‐µL plastic pipette tip was used to generate wounds by scratching the cell monolayer. The scathing cells were then washed with PBS and cultured in serum‐free RPMI‐1640. An inverted microscope was used for recording the wounds at time points of 0 and 24 h. Ultimately, the ImageJ (version 1.53a) was used to measure the width of wound‐healing assays. After measurement of the grey value of wounds, the formula /255) was used to calculate the width.Briefly, 5 × 102.75 cells/well in 100 μL serum‐free RPMI‐1640 was added to the upper wells of the chambers, and RPMI‐1640 with 10% FBS was added to the lower wells of the chambers. After 30 h for the migration assays or 48 h for the invasion assays, the cells that passed through the chambers were fixed with 4% paraformaldehyde and were then stained with 0.5% crystal violet followed by counting under a microscope.Transwell chambers  with or without Matrigel  were used for Transwell invasion and migration assays. Briefly, cell suspension was prepared in serum‐free RPMI‐1640, and 1 × 1036 cells per mouse). After 3–4 weeks of cell inoculation, the mice were euthanized and the number of lung surface metastatic foci was counted. The lung tissues were also harvested for immunohistochemistry (IHC) staining and Western blot analysis.The 4‐ to 6‐week‐old male Balb/c athymic nude mice used in this study were obtained from the Shanghai Laboratory Animal Company (SLAC), Ltd. . All mice were housed in a specific‐pathogen‐free animal facility. All animal experiment protocols used in this work were approved by the Ethics Committee for Animal Experiments of the Fujian Medical University Cancer Hospital, and experiments were conducted according to the Guide for the Care and Use of Laboratory Animals. For the lung metastasis model, cell lines assigned to the shNC, shIGF2BP3‐1, and shIGF2BP3‐2 groups were inoculated into nude mice through the tail vein  histologically confirmed NPC; (2) with complete follow‐up data; (3) with enough tissue section. The exclusion criteria were as follows: NPC patients who received treatments before a biopsy, such as chemotherapy, radiotherapy, molecular targeted therapy or immunotherapy. All patients underwent a biopsy and had a definitive pathological diagnosis. The medical records of the patients were used for clinical data extraction. All the patients provided written informed consent before sample collection, and the Ethics Committee of the hospital approved the protocols, the ethics approval code was K2021‐092‐01.3.2The immunohistochemistry (IHC) analysis was performed on 4‐µm paraffin‐embedded NPC tissue sections. After deparaffinized in xylene and rehydrating in alcohol, the sections were boiled in antigen retrieval buffers and immersed with 3% hydrogen peroxide to block endogenous peroxidase activity. The sections were treated with normal goat serum followed by individual incubation with the appropriate primary antibody against IGF2BP3 , E‐cadherin , vimentin , p‐AKT antibody  and AKT . After incubation with corresponding secondary antibodies, the sections were counterstained with Mayer's haematoxylin to stain the nucleus and were then dehydrated and mounted for imaging. The images of the slides were captured under a microscope. Two independent pathologists blindly evaluated the IHC scoring of these slides with software assistance. For IGF2BP3 evaluation, the intensity scores were designated as 0 (no), 1 (weak), 2 (moderate) and 3 (strong) and the percentages of the stained cells were divided into five levels as follows: 0 (<5%), 1 (5–25%), 2 (26–50%), 3 (51–75%) and 4 (76–100%) and used to record the percentage score. The final score was calculated by multiplying the intensity score and percentage score. A score >6 was defined as high expression, while a score ≤6 was defined as low expression.3.32 test was performed to elucidate the association between IGF2BP3 expression and clinical parameters of NPC. Survival analyses were performed by plotting Kaplan‐Meier survival curves, and log‐rank tests were performed to evaluate the difference. The univariate and multivariate Cox proportional hazards models were used to evaluate the effects of various variables on NPC patient survival, and only the significant factors identified in univariate analyses were further used for multivariate analyses. As for the in vitro and in vivo experimental results, the differences between groups were assessed by one‐way analysis of variance (ANOVA) and two‐tailed t tests. All statistical tests were two‐sided and a P value of <0.05 represented statistical significance.Statistical analyses were conducted using the SPSS (version 21.0) software  and GraphPad Prism (version 9.0.0) software . The experimental results were expressed as the mean ±standard deviation (SD). Wilcoxon rank‐sum tests were adopted for evaluating the differential IGF2BP3 expression between NPC and normal tissues. The χ44.1http://gepia.cancer‐pku.cn/). To further characterize the role of IGF2BP3 in NPC, we performed bioinformatic analysis of the GEO datasets and determined that the IGF2BP3 mRNA levels were differentially expressed between NPC and adjacent normal tissues (p < 0.001)  database and suggested that the IGF2BP3 mRNA levels were elevated in most human cancers compared to the corresponding normal tissues based on the pan‐cancer view of IGF2BP3 in human cancers, including head and neck cancer  Figure . Also, t) Figure .In addition, the mRNA and protein levels of IGF2BP3 were measured in 5‐8F, C666‐1, HNE‐2 and NP69 to ascertain whether IGF2BP3 overexpression is a characteristic of NPC cell lines. As shown in Figure 5The evaluation of the role of IGF2BP3 in the migration and invasion of NPC cells by the wound‐healing and transwell assays revealed that IGF2BP3 knockdown could impair the wound‐healing rate of 5‐8F cells. In contrast, the ectopic overexpression of IGF2BP3 in C666‐1 cells had the opposite effect Figure . Moreove5.1The GSEA analysis revealed that IGF2BP3 expression was associated with the mTORC1 signalling pathway, which is well known to be involved in modulating cell migration and invasion. The evaluation of the effect of IGF2BP3 on AKT/mTOR signalling in NPC cells by Western blot analysis indicated that silencing of IGF2BP3 reduced the phosphorylation of AKT and mTOR Figure A and B. 5.2Consistent with the results obtained by bioinformatics analysis, the results of the determination of the IGF2BP3 expression levels by qRT‐PCR and IHC analyses revealed upregulated mRNA and protein expression levels of IGF2BP3 in NPC tissues Figure  A and B Survival analyses to determine the association between IGF2BP3 protein levels and the OS of NPC patients suggested a positive association between high IGF2BP3 protein levels and poor OS of NPC patients Figure . Given t6IGF2BP3 acts as an oncogene in various human cancers.Both IGF2BP3 mRNA and protein levels were found to be upregulated in cutaneous squamous cell carcinoma.IGF2BP3 was previously demonstrated to be an upstream regulator of AKT/mTOR signalling in hepatocellular carcinoma.IGF2BP3 expression was also shown to be correlated with the clinical stage in urachal carcinoma and ovarian clear cell carcinomaOverall, we proposed that IGF2BP3 functions as a promoter of metastasis in NPC. IGF2BP3 protein levels are positively correlated with distant metastasis after initial treatment and negatively related with OS and DMFS in patients with NPC. IGF2BP3 may be used as a new target for the treatment of NPC.The authors confirm that there are no conflicts of interest.Yun Xu: Conceptualization ; Formal analysis ; Funding acquisition ; Investigation ; Writing – original draft . Zhoubo Guo: Formal analysis ; Investigation ; Writing – original draft . Hewei Peng: Investigation ; Resources ; Supervision . Lanyan Guo: Formal analysis ; Resources ; Supervision . Ping Wang: Conceptualization ; Funding acquisition ; Project administration (lead).Table S1‐S5Click here for additional data file.


The method yields, in addition, the intraerythrocytic carbonic anhydrase activity and the membrane HCO3− permeability. For normal rat erythrocytes, we find at 37 °C a CO2 permeability of 0.078 ± 0.015 cm/s, an intracellular carbonic anhydrase activity of 64,100, and a bicarbonate permeability of 2.1 × 10−3 cm/s. We studied whether the rat erythrocyte membrane possesses protein CO2 channels similar to the human red cell membrane by applying the potential CO2 channel inhibitors pCMBS, Dibac, phloretin, and DIDS. Phloretin and DIDS were able to reduce the CO2 permeability by up to 50%. Since these effects cannot be attributed to the lipid part of the membrane, we conclude that the rat erythrocyte membrane is equipped with protein CO2 channels that are responsible for at least 50% of its CO2 permeability.We have studied the CO Thereafter, Endeward and co-workers used human red cells deficient in AQP1 or in the Rhesus-associated glycoprotein (RhAG), to show that about 90% of the CO2 permeability of the human red cell membrane is due to these two proteins acting as CO2 channels2 pathway. Analogous studies have not been reported so far for any other mammalian species.In 1998, Nakhoul et al. and Cooper and Boron2 pathway. We chose the rat because this animal is relatively easily accessible to physiological studies assessing the exchange of CO2in vivo. We used the previously described mass spectrometric method to determine the CO2 permeability of rat red cells in suspension,2 pathway across the red cell membrane of the rat. In addition, we report here the first estimates of the intraerythrocytic carbonic anhydrase activity under physiological conditions of 37 °C, pH 7.2, and a CO2 partial pressure of 40 mmHg.The aim of the present study was to investigate whether red blood cells of the rat are similarly equipped with a gas channel-mediated COg for 20 min, plasma removed and cells washed three times in 0.9% NaCl. Haematocrit, cell count, and haemoglobin concentration were determined by standard techniques. Mean corpuscular volume (MCV) was ∼63 fl, which is in agreement with previous reports,CO2 and PHCO3−, was estimated from an established relation between red cell area and volume2. This may be compared to the published red cell surface areas published for mice and humans -pyridinium perchlorate salt)−5 M. Thus, it was ensured that no extracellular carbonic activity was present during the mass spectrometric experiment with dilute red cell suspensions. Inhibition of channel-mediated membrane CO2 permeability was attempted by the following chemicals: DIDS , which has previously been shown by us to be an efficient inhibitor of human red cell PCO2 as well as PHCO3,,3−–Cl− exchangerCO2 in human red cells2,Any potential extracellular carbonic anhydrase activity resulting from red cell lysis that may occur during the mass spectrometric determination of P2 permeability of plasma membranes can be determined for red cells or other cells in suspension using a mass spectrometric method,,,18O16O/HC18O16O2− that is labelled with 18 O to a degree of 1%. In this solution, C18O16O and HC18O16O2− react with water or H+, thereby transferring by a defined probability the label 18 O from the CO2–HCO3− pool into the much larger pool of water. This reaction is slow, but inside red cells due to their high carbonic anhydrase activity becomes much faster. The exchange of 18 O from CO2–HCO3− into the water pool causes a decay of the species C18O16O (mass 46), and we observe this decay vs. time after the start of the exposure of the cells to the solution. In a first rapid phase, the carbonic anhydrase-containing cells rapidly take up C18O16O. The kinetics of this process depends on the permeability of the membrane to CO2 and on the speed of the intracellular conversion of CO2 to HCO3−, that is, on intracellular carbonic anhydrase activity. The rate of disappearance of C18O16O from the extracellular fluid is followed by a mass spectrometer equipped with a special inlet system for fluids as first described by Itada and ForsterWe have previously reported how the CO18O16O  × dilution factorTo perform mass spectrometric measurements, a chamber with a volume of 2.2 ml was used that was attached to the high vacuum of the mass spectrometer via the previously published inlet systemCO2 and PHCO3− from the data for this same blood’s red cells using the system of equations that describe the entire 18 O-exchange processn = 31). Ai was defined as (acceleration factor − 1), where the acceleration factor is the factor by which the speed of CO2 hydration in the undiluted red cell interior was accelerated over the uncatalysed rate.Intraerythrocytic carbonic anhydrase activity (Ai) was determined at pH 7.2 from mass spectrometry of lysed red cells. Activity was determined for the blood sample from each animal studied, and used to calculate PCO2 of the rat erythrocyte membrane at 37 °C. The control value in the absence of potential gas channel inhibitors is 0.078 ± 0.015 cm/s . The other columns of CO2. 300 µM phloretin and DIDS at concentrations both of 10−5 and 10−4 M significantly reduce CO2 permeability. In the presence of 10−4 M DIDS, PCO2 amounts to 0.037 ± 0.006 cm/s (n = 10), that is, to about 1/2 of the control value. 1 mM pCMBS has no effect on PCO2, and 5·× 10−7 M Dibac, a rather specific inhibitor of the erythrocytic Cl−–HCO3− exchanger AE1CO2 . In conclusion, two of the four inhibitors used reduce PCO2 to a major extent.HCO3−. The control value of PHCO3− amounts to 0.0021 ± 0.0002 cm/s . 1 mM pCMBS has again no significant effect, but all other inhibitors including Dibac have major and statistically significant effects. With 5·× 10−7 M Dibac, PHCO3− is reduced to 0.00091 ± 0.00011 cm/s (n = 8), that is, to less than 1/2 of the control value.+ from CO2, thus accelerating the contribution of the Bohr effect to O2 releaseWe report here an intraerythrocytic carbonic anhydrase activity of 64,100, which may be compared with the value of 21,000 we obtained for human red cells with the same method under identical conditionsCO2 of red cells of the rat of 0.078 cm/s at 37 °C. This value is half of the value reported previously for human red cells, 0.15 cm/s,,We report here a P2 permeability of the pure phospholipid bilayer. It has been shown that DIDS has no effect on PCO2 of pure phospholipid vesicles and cell membranes without gas channels,CO2 in both types of membranes when gas channels are present,,,CO2 of membranes in the absence of gas channels, but it does reduce PCO2 when the gas channel aquaporin 1 is present,CO2 by acting on a protein CO2 channel present in the membrane of rat erythrocytes. The maximal inhibition of PCO2 achieved here amounts to 50% of the control PCO2. This suggests that at least 50% of the PCO2 of rat red cells is due to a gas channel. This is a little less than has been reported for normal human red cells, where PCO2 fell by DIDS to about 1/3 of the control value,None of the potential gas channel inhibitors studied here is expected to have an effect on the COCO2 in human erythrocytes, although it drastically reduces PHCO3CO2 in rat erythrocytes by Dibac. However, we find here also no effect of pCMBS on the PCO2 of rat red cells, while previously we found a moderate suppressive effect of 1 mM pCMBS on PCO2 of human red cells. Importantly, this effect was substantially less pronounced than the effect of DIDS,Our previous work has shown that Dibac does not affect P2 channels, AQP1 and RhAG, that contribute about equally to the CO2 permeability of the membrane and together are responsible for about 90% of the total CO2 permeability,In human red cells, there are two major CO2 channels, perhaps RhAG even a little more markedly than AQP1It is clear that DIDS inhibits quite effectively both AQP1 and RhAG COCO2 of human red cells clearly less than does DIDS, and this inhibitory effect seems to be exclusively due to AQP1, because in AQP1-deficient human red cells, whose only CO2 channel is RhAG, an effect of pCMBS is not detectablepCMBS inhibits P2 across aquaporin 1, and the water channel of the aquaporin 1 monomer is a minor CO2 pathway,According to molecular dynamics studies, the central pore of the aquaporin 1 tetramer is the major pathway of COCO2 of human red cells is mainly due to inhibition of the water (and CO2) channel of monomeric AQP1. It is well known that pCMBS, by acting on this pathway, largely blocks its water as well as its CO2 permeability via binding to a cysteine in position 189 in the water pore,,2 pathway and likely does not act on the central pore of the aquaporin tetramer. It is probable that the properties of human AQP1 discussed under C) to E) apply analogously to rat AQP1.Thus, it seems likely that the inhibitory effect of pCMBS we see on PIn rat erythrocytes it is well established that AQP1 as well as RhAG are present in the membraneA reasonable hypothesis to explain these observations comes from the following considerations:2 channels in their membrane, which constitute a sizable part of the membrane CO2 permeability. These channels can very effectively be inhibited by DIDS and also, to a lesser extent, by phloretin. The effect of DIDS is qualitatively compatible with results obtained for human red cells, suggesting that AQP1 and RhAG represent the CO2 channels of rat erythrocytes. Likewise, the effect of phloretin reported here is similar to that observed in human red cells (own unpublished observations).Rat, as well as human red cells, express COCO2 is smaller in comparison to human red cells, suggests that the role of CO2 channels in the rat may be somewhat less pronounced than it is in human red cells.However, the fact that the effect of DIDS on PCO2, which might indicate that it is AQP1, which is expressed to a lesser degree in rat erythrocytes. However, conclusive evidence on this last point is lacking. An alternative explanation might a lower cholesterol content of the rat erythrocyte membrane, which would increase the membrane’s intrinsic permeability to CO22 channels less visible. However, the cholesterol content of the rat erythrocyte membrane has not been reported, and also, the CO2 permeabilities seen here for rat red cells are lower than those for human red cells.This would be in agreement with the present observation of a lack of an effect of pCMBS on PCO2 of rat red cells might – at least partially – be caused by the reduced role of CO2 channels in rats compared to human erythrocytes.Instead, the lower POverall we draw the following conclusions:−3 cm/s at 37 °C−3 cm/s at the same temperature. This may be surprising in the view of the PCO2 that is lower for rat red cells than it is for human red cells (see above). It may be considered that in the case of HCO3− uptake or release, the membrane constitutes the major diffusion resistance since PHCO3− is almost 50 times lower than PCO2, and since the intracellular diffusion of HCO3− in comparison is much faster . If then the product of P x cellular surface area is decisive for the kinetics of HCO3−, we would, with the numbers given by−3 cm/s × 146 µm2 for humans and of 2.1 × 10−3 cm/s × 100 µm2 for rats, respectively. In other words, these products agree within about 15%, and, as in the case of CO2 uptake kinetics, we thus expect similar kinetics of HCO3− exchange in both rat and human red cells. The inhibitory profile of PHCO3− seen in Applying the present mass spectrometric technique, we have previously reported the bicarbonate permeability of normal human red blood cells to be 1.2–1.3 × 102, which can effectively be inhibited by DIDS and by phloretin. In studies on the role of gas channels for red cell CO2 exchange, the difficulty may arise that both these inhibitors inhibit CO2 permeation as well as HCO3− permeation. Both these processes contribute to overall CO2 exchange, although with quite different kinetics. This problem can, at least in vitro, be solved by complementing studies with DIDS or phloretin with experiments using Dibac, which very efficiently inhibits PHCO3− without affecting PCO2. This should allow one to dissect the effects from inhibition of PCO2 from those resulting from inhibition of PHCO3−.We present clear evidence that the red cell membrane of the rat possesses protein channels for CO