About 80% of the committed oxygen loss occurs below 2000 m depth, where a more sluggish overturning circulation will increase water residence times and accumulation of respiratory oxygen demand. According to the model results, the deep ocean will thereby lose more than 10% of its pre-industrial oxygen content even if CO2 emissions and thus global warming were stopped today. In the surface layer, however, the ongoing deoxygenation will largely stop once CO2 emissions are stopped. Accounting for the joint effects of committed oxygen loss and ocean warming, metabolic viability representative for marine animals declines by up to 25% over large regions of the deep ocean, posing an unavoidable escalation of anthropogenic pressure on deep-ocean ecosystems.Less than a quarter of ocean deoxygenation that will ultimately be caused by historical CO 2 emissions would halt upper ocean oxygen loss, it would continue in the deep ocean for 100 s of years.Ocean warming and changing circulation as a result of climate change are driving down oxygen levels and threatening ecosystems. Here the author shows that though immediate cessation of anthropogenic CO Theory and models nevertheless predict that global-mean surface temperatures would essentially stop rising further and remain relatively stable for many decades to centuries once CO2 emissions are stopped5. This is also implicit in the concept of transient climate response to cumulative CO2 emissions (TCRE7), which provides the scientific rationale for relating temperature targets to remaining carbon budgets. Radiative forcing from non-CO2 greenhouse gases and aerosols can lead to some variations in the relation between cumulative CO2 emissions and warming9; however, given their shorter atmospheric lifetimes, temperature stabilization appears possible even for non-zero emissions of these substances8. As the explicit call for balancing sources and sinks of greenhouse gases in the Paris Agreement in 201510, the goal of achieving net zero CO2 emissions to stabilize global-mean surface temperatures has gained substantial traction in climate politics and scenario development. Accomplishing this goal must, however, not be regarded as automatically stopping the increase in climate damages.The Earth system is not in equilibrium with current levels of atmospheric CO2 emissions are stopped, many components of the Earth system will continue to respond to the anthropogenic perturbation with their inherent response timescales and inertia16, producing committed impacts long after emissions are stopped. The uptake of CO2 by the ocean essentially gives rise to a slow multi-centennial decline of atmospheric CO2 and the associated radiative forcing. According to current models, the remaining radiative forcing during this phase of ocean adjustment to the anthropogenic perturbation is closely balanced by oceanic heat uptake4. The relative stability of global-mean surface temperatures within a few tens of degrees upon ending CO2 emissions5 thus comes at the expense of increasing acidification and ocean warming11. Ocean warming and associated thermal expansion of seawater adds to committed sea-level rise13, which primarily results from committed melting of inland ice15. Committed changes in terrestrial ecosystems have also been reported16. This study investigates committed changes in marine oxygen levels that are already declining at a previously unexpected pace in response to anthropogenic CO2 emissions18, and that act, together with warming and acidification, as key stressors on marine ecosystems19. Such committed changes will also have to be accounted for when assessing ecological and socio-economic impacts caused by anthropogenic CO2 emissions20, even if they materialize well after emissions are stopped and global-mean surface air temperatures have stabilized.Even though global-mean surface temperatures are expected to remain stable when CO21, which is calibrated to simulate observed climate properties and oxygen distributions23, is employed to examine what would happen if CO2 emissions were stopped by the end of year 2020. Forced with historical CO2 emissions until 2010 and emissions corresponding to the Reference Concentration Pathway (RCP) 8.5 high-emission scenario24 until year 2020 and closely agreeing with the actual emission data available until now1, the model simulates atmospheric CO2 levels of 411 μatm and global annual-mean surface air temperatures 1.03 °C above pre-industrial in year 2020, in agreement with observations. The simulated ocean heat uptake is consistent with the recent observational estimates of 1.29 ± 0.79 × 1022 J yr−1 for the entire water column over the period 1991–201625, for which the model yields 1.15 × 1022 J yr−1 and of 33.5 ± 7.9 × 1022 J for the upper 2000 m over the period 1960–201526, for which the model yields 31.6 × 1022 J. The simulated oceanic carbon uptake of 29.7 Pg C for the period 1994–2007 also agrees well with the recent observational estimate of 29 ± 5 Pg C27. After emissions are stopped by the end of 2020, simulated surface air temperatures increase by another 0.04 °C within 7 years before slowly leveling off at about 0.01 °C above the year 2020 temperatures towards the end of the twenty-first century and staying within ±0.02 °C at this level until year 2650  is larger than all the CO2 the ocean has taken up until 2020 (634 Gt CO2), whereas the committed future ocean heat uptake until year 2650 (2.7 × 1024 J) is even three times as large as the heat taken up until year 2020 (0.9 × 1024), leading to another 16 cm of unavoidable thermosteric global sea-level rise. The simulated oceanic oxygen inventory declines by 5.0 Pmol O2 (1.8%) from pre-industrial year 1770 to 2020. This is consistent with the decline found in other Earth system models32, but somewhat slower than inferred from observations that suggest a 4.8 ± 2.1 Pmol decline between 1960 and 2010 alone17. The simulated oxygen decline continues for several hundred years after stopping emissions in 2020 and asymptotically reaches a total loss of 20.3 Pmol O2 until year 2650 , whereas low-oxygen waters, sometimes called hypoxic (O2 < 70 mmol m−3) or suboxic (O2 < 5 mmol m−3), are generally found beneath the surface mixed layer but within the upper few hundred meters. Between years 2020 and 2650, the volume of hypoxic and suboxic waters shows only a relatively small and steady committed increases by 8% and 15%, respectively  finds that the oceanic oxygen loss at the end of the increase in atmospheric CO2 (3.6 Pmol) increases almost fivefold to 17.5 Pmol after another 650 years of constant atmospheric CO2 levels29. Interestingly, that model also simulates a deep Southern Ocean convection event and subsequent onset of ventilation of the deep ocean from the south, providing some confidence in the robustness of the results reported here.Results of the Earth system model of intermediate complexity used in the current study indicate that ocean deoxygenation will continue for several centuries, and that, in fact, the committed loss of marine oxygen is more than three times larger than the oxygen loss realized until now. A simulation with a full Earth system model with prescribed doubling of atmospheric CO−3, which lose more than half of their volume between years 2020 and 2650, on the expense of volume gains at lower oxygen classes between 200 and 230 mmol m−3, and between 130 and 180 mmol m−3  than average (30%) to the total oxygen loss in deep waters  and indicator of metabolically viable marine environments, declines by 10% to 25% over much of the deep ocean below 2000 m between year 2020 and 2650  as long as sufficient dissolved oxygen is available. In regions where oxygen concentrations fall below a threshold of 5 mmol O2 m−3, nitrate is used as an electron acceptor . No other electron acceptors are simulated and remineralization stops whenever nitrate runs out, which does not happen in the model runs used here.The University of Victoria Earth System Climate Model31).The ocean component is coupled to a single-level energy-moisture balance model of the atmosphere, a dynamic–thermodynamic sea ice component, and a terrestrial vegetation and carbon-cycle component. Continental ice sheets are prescribed and kept fixed in the present configuration. All model components use a common horizontal resolution of 1.8° latitude times 3.6° longitude. The current model version does not consider any fluxes across the water-sediment interface and also does not account for fluxes related to weathering on land. Oceanic phosphorus is thus strictly conserved. As the atmosphere contains about a hundred times as much oxygen as the ocean, any feedback of marine oxygen changes on atmospheric oxygen is neglected as in earlier studies  as a result of stabilizing feedbacks in the nitrogen cycle47.In the configuration employed here, eutrophication via runoff or atmospheric deposition of nutrients is not considered and ocean deoxygenation is thus entirely caused by effects related to anthropogenic CO38 is defined as the ratio of O2 supply to the temperature-dependent resting O2 demand of an organism and it combines temperature and pO2 as indicators of metabolically viable marine environments. Here, not Φ itself but only changes of Φ are considered48, employing the scripts provided by ref. 49:E0 describes the effect of temperature on the critical pO2, i.e., the threshold oxygen partial pressure required for maintaining the resting metabolic rate (hypoxia vulnerability in ref. 40) to temperature, which is different for different species. Parameter kB is the Boltzmann constant and pO2 is the partial pressure of oxygen.The metabolic index ΦE0, the species average of ref. 40 is taken (E0 = 0.4 eV). E0 varies considerably among species40 and may be different for deep-sea species, for which such information is not yet available. As an extreme example, a small negative value of E0 = −0.2 eV, indicating an increase in the critical pO2 with decreasing temperature, was found for a species living at the lower oxycline of the oxygen minimum zone in the eastern tropical Pacific50 (where pO2 tends to increase downwards with decreasing temperature). As changes in Φ are dominated by changes in pO2 rather than temperature, the results are relatively similar even for a very low value of E0 = −0.2 eV (Supplementary Fig. For Supplementary InformationPeer Review File


Physics of Weyl electrons has been attracting considerable interests and further accelerated by recent discoveries of giant anomalous Hall effect (AHE) and topological Hall effect (THE) in several magnetic systems including non-coplanar magnets with spin chirality or small-size skyrmions. These AHEs/THEs are often attributed to the intense Berry curvature generated around the Weyl nodes accompanied by band anti-crossings, yet the direct experimental evidence still remains elusive. Here, we demonstrate an essential role of the band anti-crossing for the giant AHE and THE in MnGe thin film by using the terahertz magneto-optical spectroscopy. The low-energy resonance structures around ~ 1.2 meV in the optical Hall conductivity show the enhanced AHE and THE, indicating the emergence of at least two distinct anti-crossings near the Fermi level. The theoretical analysis demonstrates that the competition of these resonances with opposite signs is a cause of the strong temperature and magnetic-field dependences of observed DC Hall conductivity. These results lead to the comprehensive understanding of the interplay among the transport phenomena, optical responses and electronic/spin structures. Previous work has proposed that the anomalous and topological Hall effects, associated with Weyl nodes, should have a signature in optical conductivity. Here, using THz optical spectroscopy, the authors assign these two effects to optical conductivity resonances, arising near band anti-crossings, in thin films of MnGe. Several mechanisms classified into either intrinsic or extrinsic ones have been proposed so far. The intrinsic mechanism based on the band structure affected by relativistic spin-orbit coupling (SOC) was first proposed by Karplus and Luttinger, and subsequently formulated in terms of the Berry phase arising from the electronic band topology5. The enhanced Berry curvature owing to the crossing point, i.e., Weyl node7, is considered to cause the large AHE; this character is contrasted to the extrinsic origin such as skew scattering and side-jump mechanisms, which are irrelevant to the electronic band structure10. In addition to the topologically protected Weyl point, the accidental crossing points of electronic band structure also generate the Berry curvature where the SOC opens a finite gap (band anti-crossing). The subtle balance of the competition among these crossing points near the Fermi level can produce intriguing behaviors such as non-monotonic temperature dependence and giant magnitude of intrinsic AHE17. On the other hand, when the magnetic structure, to which conduction electron is coupled, is composed of noncoplanar spins with scalar spin chirality (SSC) or finite skyrmion number, the so-called topological Hall effect (THE) emerges even without SOC26. The THE is often argued in terms of emergent magnetic field in analogy to ordinary Hall effect, when the spin-chirality period is large enough compared with the lattice spacing like the conventional skyrmion-lattice phase22. However, the electron mean-free path exceeds the size of spin modulation period, e.g., skyrmion-lattice spacing, the THE should be considered in the momentum space in terms of Weyl electrons as in the case of AHE, rather than the real-space picture of emergent magnetic field26. Thus, the direct experimental evidence for the essential role of the Weyl nodes for such nontrivial behaviors of both AHE and THE remains to be experimentally elucidated.Understanding of unconventional electromagnetic phenomena stemming from the quantum topological natures is a central issue in contemporary condensed matter physics. The anomalous Hall effect (AHE) caused by spontaneous breaking of time reversal symmetry invokes a long debate regarding the microscopic origin in the past decadesσxy(ω)32. The interband optical transitions across each band-crossing point lead to the resonance structures in σxy(ω), whose DC limit approaches the Hall (AH and/or TH) conductivity observed as the transport phenomena. Accordingly, the σxy(ω) spectra quantitatively clarify the contribution from each band crossing point distinctly to AHE and THE. Since the enhanced Berry curvature of the crossing points near the Fermi level causes the large AHE and THE, the low-energy optical transition should play the essential role.The spectroscopic study in terms of the optical Hall conductivity will be a powerful approach to this issue. Similarly to the ordinary Hall effect arising from the cyclotron resonance, the AHE and perhaps the THE as well should exhibit the characteristic resonance structures in the optical Hall conductivity spectrum 36, as shown in Fig. 35. On the other hand, the inherently large SOC in this compound also produces the conventional AHE approximately in proportion to the field-induced magnetization. Therefore, MnGe provides a good arena to spectroscopically identify and distinguish the Berry curvatures generated around the Weyl nodes by the SOC as well as by the SSC (skyrmion number), which respectively show up as AHE and THE. We demonstrate that the terahertz resonances of the band anti-crossing points (Weyl nodes) close to the Fermi level are assigned distinctly to the origins of AHE and THE and that the competition of these resonances causes the strong temperature and magnetic-field dependence of DC Hall conductivity as observed.In this work, we investigate the optical transitions associated with the giant AHE and THE in MnGe thin film in terms of the terahertz optical Hall conductivity. MnGe is a member of the B20-type alloys with the chiral crystal structure Fig.  and beloσxy of the MnGe thin film used in this study when magnetic field H is applied normal to the film plane, i.e., H||[111]. At relatively high temperatures, e.g., above 30 K, σxy increases with H, followed by the saturation, which well corresponds to the magnetization curve as a hallmark of AHE  in addition to the longitudinal optical conductivity σxx(ω) (see Methods). The real part of σxx(ω) shows a steep increase towards zero frequency at low temperatures  spectra represent the conduction electron dynamics as expressed by the Drude model ε0, 1/τ and ωp are the dielectric constant in vacuum, scattering rate and plasma frequency, respectively. The scattering rate 1/τ, i.e., the width of the zero-frequency peak in Re σxx(ω), decreases monotonically with decreasing temperature and reaches ~ 1 meV at 4.5 K, indicating good metallic conduction and high quality of the sample  thin film with the thickness of 85 nm fabricated on the insulating Si(111) substrate . We obtained the complex spectra of the magneto-optical Faraday rotation in the magnetic field applied along the MnGe [111] axis, i.e., normal to the film plane Fig. . We noteres Fig. . In the σxx(ω) spectra, the Faraday rotation and Hall conductivity spectra show prominent features at low temperatures and at moderate magnetic fields. At 40 K and 7 T, the Faraday rotation spectrum θF(ω) is almost constant, and the magnitude is consistent with the DC Hall angle  and Im σxy(ω), are deduced from σxx(ω), θF(ω) and ellipticity ηF(ω) (see Methods). The dramatic spectral changes in the Hall conductivity spectra σxy(ω) are observed with the changes of magnetic field   shows a shallow dip at ~2.3 meV and the steep rise towards lower frequency, which are enhanced at higher magnetic fields  should converge to zero at ω = 0 due to the causality constraint  Fig. . On the lds Fig. . Since tω = 0 plane in Fig. −1cm−1 at 10 K from 380 Ω−1cm−1 at 40 K with decreasing temperature, which is followed by the decrease towards the lowest temperature (4.5 K). This low-temperature anomaly in DC Hall conductivity coincides with the appearance of the resonance structure in σxy(ω)  and Im σxy(ω) are flat and consistent with the DC Hall conductivity . With decreasing temperature, the resonance structures rapidly develop while keeping σxy(ω) above 4 meV almost unchanged. It is thus concluded that the dramatic change of transport Hall conductivity at low temperatures and moderate magnetic fields (at least up to 7 T) stems from the resonance structures in terahertz region (< 4 meV). These low-energy resonances in σxy(ω) indicate the interband optical transitions across the band (anti-)crossing points or Weyl nodes, which suggests the dominant role of the Berry-curvature mechanism for the both AHE and THE in MnGe. We note that the ordinary Hall effect hardly contributes to the σxy(ω) in this energy region  Fig. . At 40 KH(k) is expressed as follows30;σ0 and σi  are the identity and Pauli matrices, respectively, and μ is the chemical potential measured from the band crossing point. Accordingly, we assume that the band dispersion is quasi two-dimensional and  = , so that it has the level splitting of 2|m| at the crossing point  with use of the general expression given by the Kubo formula;Jx(y) is the current operator given by f(εn) is the Fermi distribution function, n〉 are the energy and the Bloch wave function of the nth band, respectively, and δ is the phenomenological damping constant. c(k) (k)) is the annihilation (creation) operator. The energy-dependent Hall conductivity σxy(ω) is thus given by28;e, h and a are the elementary electric charge, Planck constant and lattice constant, respectively. Re σxy(ω) and Im σxy(ω) in Eq.  and Im σxy(ω)  and σxyβ(ω), respectively. The constant term σxyconst. represents the low-energy tail of higher-energy resonances above the present energy window. We note that in the chiral metal MnGe the band crossing with intense Berry curvature is the  Weyl point, which is robust against some perturbation7; the minimal Hamiltonian is given by, k vector interconnecting two Weyl points  (green), σxyβ(ω) (orange) and σxyconst. (light blue), quantitatively reproduces the experimental spectral features; the dispersive shape with the sign change in Re σxy(ω) (red) and the large positive peak (~1 meV) in Im σxy(ω) (blue). The similar results were obtained for the spectra at other magnetic fields and temperatures. The fitting parameters at 4.5 K are summarized in Fig. fα,β and σxyconst. monotonically increase by applying the magnetic field, while the resonance energies of α-band (2μα ~ 0.8 meV) and β-band (2μβ ~ 1.4 meV) show little magnetic-field dependence. These small energy scales of resonance energies (1 meV ~ 11.6 K) are consistent with the strong temperature dependence of the Hall responses below 30 K, because the low-energy structures should be smeared out by thermal agitations at high temperatures.To understand the spectral feature of the terahertz resonances, we introduce the two-band model describing the intrinsic AHE and THE with minimal assumptions Fig. : The modint Fig. , inset. entclass1pt{minimaity Fig. . Accordints Fig. . Here, wσxyα(ω) and σxyconst., and negative one, σxyβ(ω). Therefore, the negative resonance, σxyβ(ω), can be ascribed to the THE, while the positive resonance σxyα(ω) to the positive AHE. The positive σxyconst. may possibly have both contributions from the AHE and THE whose resonances locate at higher energies, but appears to be mostly dominated by the AHE process in the present case because of its nearly M(H)-linear behavior +σxyconst.} and Re{σxyβ(ω = 0)} as indicated by filled and open circles at 4.5 K  + σxyconst. (filled circles) quantitatively reproduces the monotonic increase of the AHE estimated from the transport data  (open circles). In addition, the rapid diminishment of the THE at higher temperatures  is consistent with the σxyβ(ω = 0) obtained from the terahertz measurement. These observations confirm the whole consistency of our theoretical analysis and assignment, thus demonstrating that the emergence and competition of these two anti-crossing points are a major origin of the strong magnetic-field dependence and temperature dependence of DC Hall conductivity.The signs of the DC AH and TH conductivities are observed to be positive and negative, respectively Fig. . The ter5 K Fig.  and 20 K5 K Fig. , and plo26. In such the short-period spin order with SSC, the THE is ascribed to the Berry curvature arising from the band crossings similarly to AHE, while the AHE and THE can be associated with different band crossings as suggested by the recent study26; the Berry curvature for the AHE arises from the band crossings composed of the equal- and opposite-spin band pairs impartially while that for the THE only of the opposite-spin band pairs. This momentum-space picture of the THE is not applicable to the archetypal skyrmion-hosting material MnSi with much longer magnetic period (~18 nm). In MnSi, the magnitude of the THE, that is much smaller than the present case, is well explained in terms of the real-space picture that the conduction electrons are deflected by the emergent magnetic field carried by skyrmions23; namely, there is no necessity to invoke the momentum-space Weyl nodes.It should be emphasized that the observation of the resonance of the THE indicates that the formation of the topological spin texture modifies the electronic band structure. For MnGe thin film, the magnetic period (~3 nm) is shorter than the mean-free path (~3.6 nm at 2 K) estimated from the simple free-electron model  substrates with use of the molecular beam epitaxy technique. The details of the growth procedure and characterization are described in Supplementary Note The magneto-resistivity and Hall resistivity were measured by using Physical Property Measurement System (Quantum Design). Concerning the analysis for the decomposition of the AHE and THE, see Supplementary Note σxx(ω) = Re σxx(ω) + i Im σxx(ω) of the thin film;t (ω), d, Z0 and ns are the complex transmittance, the thickness of the film, the impedance of free space (377 Ω), and the refractive index of the Si substrate, respectively.In the THz-TDS, laser pulses with the duration of 100 fs from a mode-locked Ti: sapphire laser were split into two paths to generate and detect THz pulses with using the photoconductive antenna. Transmittance spectra of the thin film were obtained by measuring the transmission of both the sample and bare substrate. We used the following standard formula to obtain the complex conductivity Ey(t), which is polarized perpendicular to the incident light Ex(t), was measured in the crossed-Nicols configuration by using wire-grid polarizers. To eliminate the background signal, we anti-symmetrized Ey(t) with respect to positive and negative magnetic fields. The Fourier transformation of the THz pulses Ex(t) and Ey(t) gives the Faraday rotation θF(ω) and ellipticity ηF(ω); Ey(ω)/Ex(ω) ≃ θF(ω) + iηF(ω) for the small rotation angles.The rotatory component of the transmitted THz pulses σxx(ω) and Faraday rotation and ellipticity spectra θF(ω) + iηF(ω), the Hall conductivity spectra were calculated from the following formula; 28. To estimate the DC-limit rotation angle θF(ω = 0) in Fig. σxx(0) and σxy(0) obtained from the transport measurements.By using the longitudinal conductivity spectra Supplementary Information


The development of chemoresistance reduces the efficacy of anti‐cancer drugs. Cervical cancer is still one of the most common cancer types in developing countries. The oncogenic protein high mobility group AT‐hook 2 (HMGA2) is involved in the development and progression of tumors, although its role in chemoresistance of cervical cancer remains unclear. Here, we report that HMGA2 is highly expressed in cervical cancer and negatively correlated with cisplatin‐induced cell death. We performed liquid chromatography‐tandem mass spectrometry to demonstrate that HMGA2 has high potential to interact with casein kinase II A1 (CSNK2A1). Moreover, we observed that HMGA2 co‐localizes with CSNK2A1 in the nucleus by immunofluorescence. Binding of HMGA2‐CSNK2A1 was detected by immunoprecipitation assays. In addition, we identified that cisplatin induces an interaction between CSNK2A1 and HMGA2, thereby promoting the phosphorylation of HMGA2. CX‐4945, a CSNK2A1 inhibitor, could inhibit the phosphorylation of HMGA2 and sensitize tumor cells to cisplatin. Our results reveal that CSNK2A1‐dependent HMGA2 phosphorylation may partially underlie cisplatin‐resistance in cervical cancer, suggesting that HMGA2 phosphorylation may have potential as a predicative biomarker and therapeutic target to improve chemotherapeutic efficacy. The oncogenic protein high mobility group AT‐hook 2 (HMGA2) participates in the development and progression of tumors. Cisplatin may enhance the interaction between HMGA2 and serine‐threonine kinase casein kinase II A1, thereby causing enhancement of HMGA2 phosphorylation in a time‐ and dose‐dependent manner, which in turn affects the bcl‐2/bax pathway and the resistance of cervical cancer cells to cisplatin. CCK‐8cell counting kit‐8CSNK2A1casein kinase II A1DAPI4′,6‐diamidino‐2‐phenylindoleDDPdiamminedichloroplatinumHMGA2high mobility group AT‐hook 2qRT‐PCRquantitative real‐time polymerase chain reactionCervical cancer is the fourth most common cancer in women around the world, with an estimated half a million women diagnosed annually, and more than 300 000 women having died from this disease . AlthougCisplatin, an extensively employed chemotherapy agent in clinical practice , exerts As an architectural transcriptional factor, the oncogenic protein high mobility group AT‐hook 2 (HMGA2) is highly expressed in the early embryo and malignant tumours, such as prostate cancer , as wellCSNK2A1 and CSNK2A2 genes, respectively) and two regulatory  subunits [Casein kinase II (CK2) is a highly conserved serine‐threonine kinase, it phosphorylates hundreds of cellular proteins and participates in the regulation of immunity, lipid metabolism and transcriptional elongation , 15, 16.subunits . Of thessubunits , 19. CSNTo address this gap, we investigated the expression of HMGA2 in cervical cancer tissues and cell lines, and explored the role and potential mechanisms of HMGA2 in cisplatin resistance. Here, we report that the phosphorylation level of HMGA2 was associated with the chemoresistance in cervical cancer cells. Cisplatin could increase the phosphorylation of HMGA2 by enhancing the interaction between CSNK2A1 and HMGA2. Our findings support the potential role of HMGA2 as a novel target for cisplatin resistance and suggest the feasibility of combining cisplatin and HMGA2 inhibition for the improved chemotherapy of cervical cancer.2 at 37 °C. The anti‐Flag M2 agarose and monoclonal mouse antibody against Flag were obtained from Sigma‐Aldrich . The polyclonal rabbit antibody against HMGA2 was purchased from the Abcam . The polyclonal rabbit antibodies against GFP, CSNK2A1, Bcl‐2, Bax and β‐actin were purchased from the Proteintech . Phospho‐Ser/Thr antibody was acquired from Cell Signaling Technology . Cisplatin was obtained from MedChemExpress . The Escherichia coli strain BL21 (DE3) was purchased from TransGen . Glutathione Sepharose 4B was purchased from GE Healthcare . CX‐4945  was purchased from Selleckchem .HEK‐293T (293T) cells, human normal cervical epithelial cell line (H8), human cervical cancer cell lines  and cisplatin‐resistant cell line HeLa/diamminedichloroplatinum (DDP) were purchased from the Chinese Academy of Science . All cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% (v/v) fetal bovine serum in a humidified atmosphere of 5% COThe plasmid containing HMGA2 was obtained by cloning the full coding sequences for the wild‐type into the vector of pcDNA3.0 vector or pLVX‐IRES, then confirmed by sequencing. The pLVX‐IRES‐HMGA2 was co‐transfected with the virus packing particles (PMD2.G and psPAX2) into the 293T cells for 3 days, then collected and the supernatants containing lentivirus were concentrated. Next, the HeLa cells were infected with the supernatants. After 48 h, we added the hygromycin to the infected cells, which were subsequently cultured for another 2 weeks. Next, the overexpression of HMGA2 was validated in the survived cells by a western blot assay. The HMGA2 knockdown stable cell lines were obtained as described previously . The seqWe first extracted the total RNAs with the TRIzol reagent , then synthesized the complementary DNA using a cDNA Synthesis Kit  in accordance with the manufacturer’s instructions. The gene for HMGA2 was amplified using the primers 5′‐CAGGAAGCAGCAGCAAGAAC‐3′ (forward) and 5′‐GCCTCTTGGCCGTTTTTCTC‐3′ (reverse), the endogenous gene for GAPDH was amplified using the primers 5′‐GAAGGTCGGAG‐ TCAACGGATT‐3′ (forward) and 5′‐GAAGGGGTCATTGATGGCAAC‐3′ (reverse). The operation process, reaction conditions and the interpretation of the results matched those described previously .At different time points, cells were harvested and treated with the lysis buffer containing the protease inhibitor and then centrifuged. The concentration was detected in accordance with the manufacturer’s instructions . Then, we used SDS/PAGE to separate the proteins and transferred them to the poly(vinylidene difluoride) membranes. The target proteins were detected and visualized using specific primary antibodies and appropriate secondary antibodies.The ethics committee of Shanghai General Hospital approved our study. Written informed consent was obtained from each of the patients who provided cervical cancer tissues. The study methodologies conformed to the guidelines set by the Declaration of Helsinki. The expression of HMGA2 in peri‐tumor and tumor tissues was measured by an immunohistochemistry assay as described previously . Human tCell proliferation was examined using a cell counting kit‐8 assay (CCK‐8) . After treatment with cisplatin, cells were seeded into the 96‐well plates and then incubated for 24, 48 and 72 h, respectively. At different time points, the culture medium was removed and changed to Dulbecmodified Eagle’s medium with CCK‐8 solutions, followed by culturing for 1 h. The optical density in each well was measured at 450 nm via a spectrometer.The stable cell lines of HMGA2‐overexpression or HMGA2‐knockdown and the control cells were treated with cisplatin, and then the cell death rates were detected using an annexin V‐fluorescein isothiocyanate/propidium iodide apoptosis detection kit  as described previously .After being transfected with the plasmids and treated with cisplatin for different time points, cells were harvested, lysed and centrifuged, then part of the supernatant was selected as the lysis part, whereas others were immunoprecipitated with anti‐Flag M2‐agarose overnight at 4 °C, then centrifuged, washed and blended with the loading buffer. TRhe sample was used for IP part. Protein samples were then analyzed by a western blot assay.in vitro phosphorylation assay was conducted as described previously [E. coli BL21 (DE3) and purified using Glutathione‐Sepharose 4B. Flag‐CSNK2A1 and GST‐HMGA2 complex were incubated at 30 °C in kinase buffer for half an hour. The phosphorylation of HMGA2 was analyzed by a western blot assay using anti‐p‐Ser/Thr antibody.An eviously . BrieflyCells were seeded on the coverslips in 24‐well plates and transfected with plasmids if needed. After pretreatment as required, cells were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X‐100, blocked with 2% bovine serum albumin, incubated with the primary antibodies, then stained with Alexa Fluor 488‐ or 568‐conjugated secondary antibodies. 4′,6‐diamidino‐2‐phenylindole (DAPI) was used for nuclear staining. Immunofluorescence was observed via fluorescence microscopy.spss, version 20.0 . Statistical significance was performed using Student's t‐test. P < 0.05 was considered statistically significant.All experiments were repeated at least three times. Data are shown as the mean ± SD, and processed with The function of HMGA2 in cervical cancer is unclear. Based on our previous study of prostate cancer, we detected the expression of HMGA2 in different cervical cancer cell lines and the normal cell line. As shown in Fig. To evaluate whether HMGA2 has an impact on the chemoresistance of cervical cancer, we established stable HMGA2 knockdown cell lines with the specific plasmids and evaluated the HMGA2 expression by a western blot assay Fig. . Then, wTo better understand the potential mechanisms of HMGA2 involved in cisplatin resistance, we transfected Flag‐HMGA2 plasmids into 293T cells, then treated cells with cisplatin. The phosphorylation status of HMGA2 was determined by immunoprecipitation and western blot assays with the specific antibody. As shown in Fig. in vitro phosphorylation assay. The results demonstrated that CSNK2A1 phosphorylated HMGA2 in vitro  with that in HeLa cells. The HMGA2 phosphorylation level was increased in HeLa/DDP cells Fig. . Next, wThe incidence of cervical cancer has decreased markedly in developed countries over recent decades, although it remains high in developing countries as a result of unhealthy lifestyles . Drug reet al. [As a high mobility group protein, HMGA2 has diverse functions in biological processes , 29. In et al.  found thet al. , which set al. , 33. In Interestingly, as shown in Fig. The novelty of the present study is that we first demonstrated the role of the interaction of HMGA2 and CSNK2A1 in cisplatin resistance for cervical cancer. However, our study is limited by a lack of information regarding potential phosphorylation sites, for which additional investigations will be carried out in the near future.In the present study, we first reported the phosphorylation of HMGA2 played an important role in cisplatin resistance for cervical cancer, which prompts a new direction for investigating the effects of HMGA2 in the future. Furthermore, we found that the kinase CSNK2A1 interacted and regulated the function of HMGA2 through its activated phosphorylation. In sum, we have demonstrated that the CANK2A1/HMGA2/Bcl‐2/Bax axis modulates the sensitivity of cervical cancer cells to cisplatin, and potentially provides new therapeutic targets for overcoming chemoresistance in cervical cancer.The authors declare that they have no conflicts of interest.SZ performed most of the experiments, and also drafted and revised both the figures and the manuscript. WD performed the cell culture. XH performed the cell proliferation detection. YJ re‐edited the manuscript and provided suggestions on the revision. SXQ supervised the project and modified the manuscript.Fig. S1. Effects of cisplatin on the expression of HMGA2.  HeLa cells were treated with or without cisplatin for 24 h, then a western blot assay was conducted to evaluate the expression of HMGA2.  HMGA2 expression were tested in the cisplatin resistant cells (HeLa/DDP). Data were analysed using Student's t‐test and are presented as the mean ± SD of three independent experiments. Ns, not significant.Click here for additional data file.


The challenge in the construction of such SMSI remains to be the inertness of SiO2. Herein, a simple and scalable method is developed to prepare 2D silica (2DSiO2) supported Cu catalysts with SMSI by carefully manipulating the topological exfoliation of CaSi2 with CuCl2 and thereafter calcination. The prepared Cu‐2DSiO2 catalysts with the unique encapsulated Cu nanoparticles exhibit excellent activity and long‐term stability in high‐temperature CO2 hydrogenation reactions. This feasible and low‐cost solution for stabilizing Cu catalysts might shed light on their realistic applications.Cu‐based catalysts exhibit excellent performance in hydrogenation reactions. However, the poor stability of Cu catalysts under high temperatures has restricted their practical applications. The preparation of stable Cu catalysts supported by SiO 2 catalysts at high temperatures has been a long‐sought‐after goal for many catalytic reactions that demand low cost and high throughput. Herein, a cost‐effective and feasible route is developed to prepare a new Cu/2DSiO2 catalyst with strong metal–support interaction that exhibits ultrahigh stability at high temperatures, which is not enjoyed by the traditional impregnation method.Achieving long‐term stability of Cu/SiO When it comes to the hydrogenation of carbon–oxygen species, Cu+ species could also act as effective promoters by polarizing the carbon–oxygen bonds to activate the reactants. However, deactivation commonly occurs for conventional Cu‐based catalysts via sintering of metal particles under high temperature and redox reactions of copper species of mutable valences (Cu0 and Cu+) in the strongly reducing or oxidizing atmospheres. This has restricted the industrial implementations of these Cu‐based catalysts.Cu‐based catalysts have been tremendously studied in hydrogenation reactions as Cu exhibits prominent activity in the dissociation of Heactants.12 Whe2 has aroused tremendous interest due to the high abundance, low toxicity, feasible processability, and low cost of SiO2. However, reports regarding the construction of SMSI, a widely practiced and effective means to stabilize metals, are rare for Cu/SiO2 catalysts due to the relative inertness of SiO2 compared with reducible oxides like CeOx, TiOx, etc. Classical SMSI refers to the physical coverage/encapsulation of metal nanoparticles with oxide overlayers to prevent the migration. To aid the encapsulation by SiO2, previous arts regarding the synthesis of stable Cu/SiO2 catalysts with SMSI required evaporation of the corrosive ammonia, careful regulation of pH, and surfactants. Moreover, most of these Cu catalysts were tested at mild temperatures below 350 ℃, so their stability for those reactions performed at high temperature and high conversion is still a mystery. Therefore, it still remains a challenge to design feasible and low‐cost processes to prepare Cu/SiO2 catalysts with SMSI and thereby stable catalytic activity, especially under high temperature.The exploration of stable Cu catalysts supported by SiO of SiO2.35 How2 supported Cu catalysts (Cu‐2DSiO2) with high stability. This Cu‐2DSiO2 catalyst was obtained by tuning the topological exfoliation chemistry of CaSi2 with CuCl2 which directly dispersed Cu nanoparticles between 2D silicon nanosheets (2DSi), followed by calcination to transform the composites into active Cu‐2DSiO2 catalysts. The endothermic CO2 hydrogenation to CO was utilized as a model reaction to determine the catalytic performance of the prepared Cu catalyst at high working temperatures. The Cu‐2DSiO2 catalysts exhibited superior activity and stability during a prolonged test in comparison to control catalysts prepared through the traditional impregnation method. We demonstrate that the unique Cu–O–Si encapsulation generated from SMSI are responsible for the superiority of these Cu‐2DSiO2 based samples. This new Cu on SiO2 catalyst that can be prepared in large quantity and with low cost sheds light on its practical applications.Herein, we proposed a new strategy to prepare 2DSiO22.12 has been reported to exfoliate CaSi2 into Si nanosheets, which demonstrated decent performances as anodes for Li‐ion batteries. The excess amount of CuCl2 was used as the sacrificial reagent, and discarded after isolation of 2DSi. In contrast, as demonstrated in Figure2 with the designed amount of CuCl2. We discovered that the category of the Cu species obtained  depended on the concentration of the CuCl2 precursor (C0) when the fixed amounts of CaSi2 and solvent were used. First, when C0 equals 0.2 m, Cu nanoparticles with relatively large size (60 nm) supported by 2DSi could be obtained through the reaction of CaSi2 + CuCl2 → Cu + 2Si + CaCl2  are added to the reactants, the as‐formed copper nanoparticles are etched through a comproportionation reaction (Cu + CuCl2 → 2CuCl). This etching strategy was used as an effective method to reduce the size of Cu by carefully controlling the amount of CuCl2 precursor. Partially etched Cu nanoparticles supported by 2DSi could be obtained when appropriate amounts of CuCl2 were used as the precursor, shown in Figure C0 grows, suggesting the reduced proportion of Cu (0) species  transformed into CuCl. Therefore, 0.2085 m was selected as the appropriate C0 for the preparation of small‐sized copper nanoparticles encapsulated by 2DSi. The remnant by‐product CuCl was removed by NH3·H2O. The solid products (Cu‐2DSi) were collected and then calcined at 400 ℃ under air (denoted as Cu‐2DSiO2‐400). The Cu‐2DSiO2‐400 precursor exhibited a layered structure  is much smaller than that of the unetched Cu nanoparticles obtained using 0.2 m CuCl2 (60 nm), shown by Figure 2‐400 precursor was further reduced at 500 ℃ under H2 to be transformed into an active Cu catalyst (the corresponding sample is denoted as Cu‐2DSiO2‐400r). XRD results suggest that the CuO species has transformed into metallic copper 2·3H2O on 2DSi and a commercial SiO2 support, respectively. These two samples were annealed at 850 ℃ in air, followed by reduction at 500 ℃ in H2. The corresponding samples were denoted as Cuim/2DSiO2r and Cuim/SiO2r, respectively. The copper nanoparticles were in the metallic states in both samples, confirmed by the XRD results  reaction which favors high temperature and thereby requires high thermal stability of the catalyst. As shown in Figure2‐400r exhibits a much higher CO2 conversion (19.1%) than Cuim/2DSiO2r (12.9%) and Cuim/SiO2r (10.1%) with the same loading amount of Cu. This should be attributed to the much smaller size of the Cu nanoparticles in Cu‐2DSiO2‐400r in comparison to Cuim/2DSiO2r and Cuim/SiO2r. Furthermore, the superiority of Cu‐2DSiO2 based catalysts could be further improved to as high as 22.7% (35.9 mmol g–1 h–1), by enhancing the calcination temperature under air from 400 ℃ to 850 ℃ (the corresponding sample was denoted as Cu‐2DSiO2‐850). After the same reduction treatment at 500 ℃ under H2 (denoted as Cu‐2DSiO2‐850r), the size of the Cu nanoparticles . Therefore, the higher CO2 conversion of Cu‐2DSiO2‐850r (22.8%) than Cu‐2DSiO2‐400r (19.1%) cannot be attributed to the size difference. According to the Arrhenius plots, the Cu‐2DSiO2‐400r and Cu‐2DSiO2‐850r catalysts showed similar apparent activation energy for CO production, suggesting the same reaction mechanism occurred on these two samples  or CO rate of 4442 mmol gCu–1 h–1  was obtained, which is comparable to the recently reported high‐performance Cu catalysts for RWGS reaction.As shown above, the Cu‐2DSiOs Figure . Therefo2 conversion of all the Cu catalysts were below the equilibrium value under this testing condition, which guaranteed the stability property of these samples was valid  which might be ascribed to the special metal–support interaction (MSI) between Cu and 2DSiO2. Particularly, Cu‐2DSiO2‐400r exhibited a small drop in the conversion during the first 2 h, and then an everlasting rising stage, whereas Cu‐2DSiO2‐850r exhibited almost no drop in conversion at any stage: a rising stage from the beginning, and then stabilized CO2 conversion after 33 h. TEM results reveal that the Cu nanoparticles of these two samples kept similar size distribution after the 54 h test  experiments were performed to study the surface chemical states. Cu 2p X‐ray photoelectron spectra (XPS) spectra show that two peaks of Cu 2p1/2 (953.6 eV) and Cu 2p3/2 (933.6 eV) with a satellite peak centered at around 943.2 eV exist for Cu‐2DSiO2‐850, indicative of the surface Cu2+ species of Cu‐2DSiO2‐850  and Cu 2p3/2 (932.4 eV) with the disappearance of the satellite peak. These results indicate the Cu species in these two samples, which correspond to the actual active sites during the reaction, are Cu+ or Cu0. This was further verified by the X‐ray induced Auger electron spectra (XAES) of the Cu LMM region  for the Cu‐2DSiO2‐850 series is higher than that for the Cu‐2DSiO2‐400 series, suggesting the higher electronegativity of Cu species for the Cu‐2DSiO2‐850 sample. This should be ascribed to the stronger metal–support interaction for Cu‐2DSiO2‐850 through the formation of Cu–O–Si species. The unique Cu–O–Si species with SMSI were also verified by the Si 2p and O 1s XPS spectra  at 400 ℃ and 850 ℃, denoted as Cu(removed)‐2DSiO2‐400 and Cu(removed)‐2DSiO2‐850, respectively. The shifts of Si 2p and O 1s peaks to higher binding energies were also observed for Cu(removed)‐2DSiO2‐850 in comparison to Cu(removed)‐2DSiO2‐400, suggesting that the higher oxidation state of Si at the higher calcining temperature is the major contributor to such shift, as the influence of Cu–O–Si was excluded in these samples by removal of Cu. However, the binding energies of Si and O for Cu(removed)‐2DSiO2‐n were still higher than Cu‐2DSiO2‐n (n = 400 or 850). This should originate from the contribution of Cu–O–Si species with SMSI, which keeps the 2DSi from transforming entirely to bulk SiO2. Nevertheless, the atomic ratios of Si to O determined by the XPS spectra approaches 1:2 for Cu‐2DSi‐850r, suggesting the major component of the support in Cu‐2DSi‐850r is 2D SiO2. The chemical structures of the surface Cu species were further studied using Raman microscopy  show characteristic curves of metallic Cu  spectra and the fitting results of them show that the Cu–O for Cu‐2DSiO2‐400 locates at ≈1.97 Å, longer than that of Cu‐2DSiO2‐850 (≈1.91 Å). This indicates that the interaction between Cu and O for Cu‐2DSiO2‐850 is stronger than that for Cu‐2DSiO2‐400, which should account for the unique and stable Cu–O–Si species for Cu‐2DSiO2‐850. The lower coordination number (CN) of metallic Cu in Cu‐2DSiO2‐400r (6.5) than that of Cu‐2DSiO2‐850r (8) is in accordance with the smaller size of Cu nanoparticles of Cu‐2DSiO2‐400r in comparison to Cu‐2DSiO2‐850r. The values of CN and distance of Cu‐O for Cu‐2DSiO2‐400r and Cu‐2DSiO2‐850r kept similar after long‐term high‐temperature reaction, suggesting the high stability of these two samples which should be ascribed from SMSI.Since the XPS and Raman (microscope) experiments mainly characterize the surface of the catalysts, the X‐ray absorption spectroscopy (XAS) studies were further performed to study the chemical states of the integrated Cu components of the samples. The Cu K‐edge X‐ray absorption near edge structure (XANES) spectra show that the near‐edge absorption energy of Cu‐2DSiOu Figure . Therefo2‐400r which was fitted to two distinct peaks located at 2113 and 2125 cm–1, corresponding to Cu0–CO and Cu+–CO, respectively. In contrast, the asymmetry of the CO adsorption curve for Cu‐2DSiO2‐850r is much more apparent with the fitted Cu0–CO and Cu+–CO peaks located at 2124 and 2136 cm–1, respectively. The blue shift of the adsorption peaks for Cu‐2DSiO2‐850r should be ascribed to its unique Cu–O–Si species with SMSI. The CO adsorption results indicate that Cu+ exists in Cu‐2DSiO2‐400r and Cu‐2DSiO2‐850r which could not be observed from the XRD patterns as Cu2O crystallites  experiments were performed to determine the reducibility of these Cu catalysts  in comparison to the other Cu catalysts. Different from Cu‐2DSiO2‐400, asymmetric curves that can be fitted to two peaks were found for the other Cu catalysts in the TPR profiles. The split peaks should be ascribed to the reduction of smaller and larger Cu nanoparticles, respectively, in which the peak at the higher temperature range corresponds to the larger particles. The hard reducibility of Cu‐2DSiO2‐850 is revealed by its highest reduction temperature among the as‐prepared Cu catalysts. This indicates that the interaction between Cu and the support for Cu‐2DSiO2‐850 is the strongest. Therefore, the resulting unique Cu–O–Si species has led to its excellent catalytic performance.Hs Figure . The H2‐atalysts.51 DifFigure2 based catalysts possessed an encapsulation and confined structure mainly composed of Si0 and Cu0 that can only be obtained by the exfoliation method. The encapsulation of Cu by 2DSiO2 could improve the stability of the Cu‐2DSiO2 based samples by restricting the sintering of Cu nanoparticles during high‐temperature calcination and tests. The Si0 and Cu0 species in close contact at the Cu–Si interface can easily form unique Cu–O–Si species during oxidation, generating SMSI for the Cu‐2DSiO2 based catalysts, in accordance with the ultrahigh stability during the prolonged test. The gradually enhanced production in the preliminary stage could also be attributed to the increased porosity of the support resulting from the corrosion of silicon oxides by the high‐temperature steam in the product gases  level activity for RWGS reaction. The strategy to construct Cu‐2DSiO2 catalysts could provide useful guidance for the design of other‐metal‐based M‐2DSiO2  catalysts with ultrahigh activity and stability.This work introduced a new and highly stable Cu catalyst supported by 2DSiO42), ammonium hydroxide solution , copper nitrate trihydrate (99.99%), and the silicon oxide support of high surface area (0.125 in pellets) were purchased from Sigma‐Aldrich, Aladdin, Macklin, and Alfa Aesar, respectively. Anhydrous copper chloride , ethanol , and hydrochloric acid  were purchased from Sinopharm Chemical Reagent Co., Ltd. Milli‐Q water  was used in all experiments.All the chemicals were used as received without further purification. Calcium silicide  m. 80 mL of the CuCl2 ethanol solution were added into a round‐bottom flask. The solution was stirred at 600 rpm and heated to 60 ℃, followed by the addition of 0.8 g of CaSi2. The mixture was kept stirring at 60 ℃ for 3 h. After cooling to room temperature, the solid in the mixture was collected and washed with ethanol twice under centrifugation. The supernatant was removed and the precipitate was dried under vacuum.CuCl2 was dissolved in ethanol to form a solution of 0.2085 m. 80 mL of the CuCl2 ethanol solution was added into a round‐bottom flask. The solution was stirred at 600 rpm and heated to 60 ℃, followed by the addition of 0.8 g of CaSi2. The mixture was stirred at 60 ℃ for 3 h. After cooling to room temperature, the solid in the mixture was collected and washed with ethanol twice under centrifugation. The supernatant was removed and the precipitate was dried under vacuum. 300 mg of the dried powder  was mixed with 30 mL of NH3•H2O solution. The suspension was transferred to a shaking bed  for stirring for 30 min. The mixture was then centrifuged followed by the removal of the supernatant. The precipitate (Cu‐2DSi) was washed with ethanol twice under centrifugation and then dried under vacuum. The Cu‐2DSi was then calcined at n ℃ (denoted as Cu‐2DSiO2‐n) under air, followed by reduction at 500 ℃ for 2 h under H2 atmosphere (denoted as Cu‐2DSiO2‐nr). The Cu loading of Cu‐2DSiO2‐nr was 15% based on the inductively coupled plasma optical emission spectrometer (ICP‐OES) results.CuCl2 powder was dispersed in 200 mL of hydrochloric acid precooled to ‐25 ℃ under N2 atmosphere. The mixture was then kept at ‐20 ℃ under stirring in an inert atmosphere for 7 d. The suspension was filtered under vacuum. The filter liquor was removed and the solid product was washed with acetone by filtration under vacuum. The feed ratio of the Cu precursor and the 2DSi support was calculated based on the loading amount of Cu for Cu‐2DSiO2‐nr. To be specific, 0.1673 g of Cu(NO3)2•3H2O was dissolved in 20 mL of ethanol under stirring. 0.25 g of the 2DSi powder was added to the solution and kept under stirring at 80 ℃ until the disappearance of liquid. The powders were dried under vacuum and then calcined at 850 ℃ for 2 h in air, and the corresponding sample was denoted as Cuim/2DSiO2. The Cuim/2DSiO2 sample was then reduced at 500 ℃ for 2 h under H2 atmosphere, and the obtained catalyst was denoted as Cuim‐2DSiO2r.2D silicon (2DSi) was prepared through a reported recipe. 4 g of CaSi2 support was calculated based on the loading amount of Cu for Cu‐2DSiO2‐nr. To be specific, 0.3377 g of Cu(NO3)2•3H2O was dissolved in 20 mL of ethanol under stirring. 0.5044 g of the silicon oxide support (grinded to penetrate through an 80‐mesh sieve) was added to the solution and kept under stirring at 80 ℃ until the disappearance of liquid. The powders were dried under vacuum and then calcined at 850 ℃ for 2 h in air, and the corresponding sample was denoted as Cuim/SiO2. The Cuim/SiO2 sample was then reduced at 500 ℃ for 2 h under H2 atmosphere, and the obtained catalyst was denoted as Cuim/SiO2r.The feed ratio of the Cu precursor and the SiO2 hydrogenation under atmospheric pressure were carried out in a quartz tube reactor  was reduced in the flow reactor in situ at 500 ℃ under H2 for 2 h. The temperature was kept at 500 ℃ and the flowing gas in the reactor changed from H2 to the reaction atmosphere (CO2: H2: N2 = 5: 5: 5 mL min‐1) after the reduction process. The testing longed for 54 h for all the Cu catalysts. For Cu‐2DSiO2‐400 and Cu‐2DSiO2‐850, the CO production rates at various temperatures were also collected by reducing the reaction temperature after the long‐time testing to acquire the Arrhenius plot (Table 2 (XCO2) was defined as:F is the flow rate of reactants or products (mol min‐1), C is the concentration of reactants or products (%).Flow reactor studies for CO‐1 under Ar (20 mL min‐1). The catalyst was kept at 400 ℃ for 30 min under Ar and then cooled to room temperature. The flowing gas changed from Ar to 10%H2/Ar. After that, the reactor was heated to 800 ℃ at a heating rate of 10 ℃ min‐1. The signal of the gas product was recorded online with a TCD (Agilent).20 mg of the Cu catalyst was put inside a flow reactor and heated to 400 ℃ at a heating rate of 10 ℃ min2 for 0.5 h. The flowing gas was changed from H2 to Ar and the reactor was cooled to room temperature. The inlet flow was switched to CO of 20 mL min‐1. The adsorption species on the surface of catalysts were detected online.In situ diffuse reflection infrared Fourier transform spectroscopy (DRIFTS) experiments of CO adsorption were performed with an in situ DRIFTS reaction cell  and an FTIR spectrometer (Thermo Nicolet iS50). Approximately 20 mg of the as‐reduced catalysts (grinded to penetrate through an 80‐mesh sieve) were packed into the in situ cell. The catalyst was further reduced in situ in the cell at 400 ℃ under Hα X‐ray source (hν = 1486.6 eV) and binding energies referenced to C1s (284.8 eV). The metal content of different samples was measured by an Inductively coupled plasma optical emission spectrometer (ICP‐OES) . X‐ray diffraction (XRD) was measured on an XRD‐700 X‐ray diffractometer from Shimadzu. Raman spectra were obtained with a Raman spectrometer (Horiba LabRAM HR Evolution) equipped with a 532‐nm‐laser excitation source.TEM images, STEM images, and EDX mappings were obtained with an FEI Talos F200X (200 kV) transmission electron microscope. X‐ray absorption spectroscopy experiments were performed at the 4B9A beamline of the Beijing Synchrotron Radiation Facility (BSRF). X‐ray photoelectron spectroscopy (XPS) and X‐ray induced Auger electron spectroscopy (XAES) were obtained using a Thermo Scientific K‐Alpha Scanning ESCA Microprobe instrument  equipped with an Al K2 hydrogenation and the CO rate under each condition is the average value of the corresponding points.The particle size of Cu for different Cu samples was measured by nanomeasurement: at least 100 particles were analyzed for each sample. Two to four data points were recorded for each testing condition in thermocatlytic COThe authors declare no conflict of interest.2 TPR, Raman experiments and analyzed the XAS results. D.Z. carried out the TEM and CO adsorption experiments. M.X. and C.Z. carried out the TEM characterization. K.F., B.Y., D.Y., and L.H. provided suggestions for the manuscript. W.S., D.Y., B.Y., L.H. provided the financial or equipment support for this work. S.W., W.S., and G.A.O. wrote the paper. All authors commented on the final manuscript.S.W. and K.F. contributed equally to this work. W.S., S.W., K.F., B.Y., and G.A.O conceived and designed the experiments. S.W. carried out and analyzed the synthesis, catalysis, XRD, XPS, and ICP experiments. K.F. carried out the XAS, HSupporting InformationClick here for additional data file.


Mycoplasma hyopneumoniae was evaluated on three pig farms.The field efficacy of a bivalent vaccine containing porcine circovirus type 2b (PCV2b) and M. hyopneumoniae infections between 84 and 126 days of age while concurrent porcine circovirus‐associated disease and clinical M. hyopneumoniae infection between 70 and 105 days of age. Each farm vaccinated pigs with a single dose of a bivalent vaccine at 10 days of age while unvaccinated pigs were administered a single dose of phosphate buffered‐saline at the same age.Three pig farms were used, two of which had a history of subclinical PCV2 and clinical p < .05) when compared with unvaccinated animals. The amount of PCV2d loads in blood and M. hyopneumoniae loads in nasal swabs of vaccinated animals were also significantly lower (p < .05) when compared with unvaccinated animals. Immunologically, vaccinated groups elicited a significantly higher (p < .05) level of protective immunity against PCV2d such as neutralizing antibodies and interferon‐γ secreting cells (IFN‐γ‐SC), as well as protective immunity against M. hyopneumoniae such as IFN‐γ‐SC when compared with unvaccinated animals. Pathologically, vaccination significantly lowered (p < .05) the scores of M. hyopneumoniae‐induced pneumonia and PCV2‐associated lymphoid lesions when compared with unvaccinated animals.Vaccination improved growth performance and reduced clinical scores significantly (M. hyopneumoniae infection under field conditions.The evaluated bivalent vaccine provided good protection against PCV2d and  M. hyopneumoniae infection under field conditions.Bivalent vaccine was effective in improving growth performance and protecting pigs against PCV2d and  PCV2 is a very small DNA virus which can be divided into at least eight different genotypes (‘a’ to ‘h’) based on its sequence identity in open reading frame 2 (ORF2)  and Mycoplasma hyopneumoniae are the smallest self‐replicating bacteria and are distinguished by the lack of a cell wall that is present in most bacteria  was first introduced into the Asian market. In particular, this bivalent vaccine is clinically interesting because it contains the PCV2b genotype. Genetically, PCV2b and PCV2d  are more closely related than PCV2a and PCV2d .Coinfection with PCV2 and 22.1M. hyopneumoniae, while all piglets from this production system were immunized against both pathogens. Farms A and C were selected based on history of concurrent subclinical PCV2 infection and enzootic pneumonia. Herds approximately 12–18 weeks of age displayed subclinical PCV2 infection and enzootic pneumonia characterized by respiratory signs and growth retardation which were mainly associated with a marked increase in the mortality rate  from 21 to 140 days of age. Lungs were examined at the slaughterhouse where it was confirmed that 12 of 20 Farm A pigs and 15 of 20 Farm C pigs had mycoplasmal pneumonia lesions based on scoring from a previous method  at approximately 13–18 weeks of age. A lung examination was performed at the slaughterhouse, which confirmed that 8/10 pigs had mycoplasmal pneumonia lesions. Submitted cases from Farm B met the definition of PCVAD  between May and October of 2019. Farms A, B and C were each a 380‐sow, 160‐sow and 430‐sow (respectively), farrow‐to‐finish swine farm with an all‐in‐all‐out production system. All three farms are located in Chungcheongnam‐do. Sows from these selected farms had not received vaccines against PCV2 or AD Chae,  includin2.2M. hyopneumoniae  at 10 days of age. An equal volume of phosphate‐buffered saline  was injected in the same anatomical location in pigs of the unvaccinated  groups. Both vaccinated and unvaccinated pigs were comingled before they were randomly distributed into four different pens kept within one room. Each pen contained 10 pigs with a similar proportion of each treatment per pen. Pens were identical in design and equipment which included free access to a feed and water trough. Standard farm procedures were followed regarding the feeding and watering of study animals. Blood and nasal swabs were collected at 0 (10 days of age), 18 (28 days of age), 39 (49 days of age), 81 (91 days of age) and 102 (112 days of age) days post‐vaccination (dpv). All methods used in this study were approved by the Seoul National University Institutional Animal Care and Use Committee.The experimental design for the field study strictly adhered to the guidelines set by QIA. QIA guidelines required that 20 piglets  were selected and assigned to each group of vaccinated and unvaccinated animals. In an effort to minimize sow variation, four to six 7‐day‐old piglets were randomly selected from each sow and assigned evenly to either the vaccinated or unvaccinated group using the random number generation function in Excel . The pigs in the vaccinated  groups were injected intramuscularly in the right side of the neck with 1.0 ml of the bivalent vaccine containing PCV2b and 2.3Pigs were monitored and scored weekly for clinical signs as previously described , 70 (60 dpv), 112 (102 dpv) and 175 (165 dpv) days of age. The average daily weight gain  was analysed over three time periods: (1) between 10 and 70 days of age, (2) between 70 and 112 days of age and (3) between 112 and 175 days of age. ADWG during the different production stages was calculated as the difference between the starting and final weight divided by the duration of the stage. Data for dead or removed pigs were included in the calculation.2.5DNA was extracted from serum samples by use of a commercial kit . Real‐time PCR was used to quantify the number of genomic DNA copies for PCV2d . Real‐time PCR was used to quantify the number of genomic DNA copies for 2.7M. hyopneumoniae antibodies with commercial ELISA kits . Serum samples were considered as positive for anti‐PCV2 antibodies if the reciprocal ELISA titre was > 350 and as positive for M. hyopneumoniae antibody if the sample‐to‐positive (S/P) ratio was ≥ 0.4 in accordance with the manufacturer's instructions for each kit. Serum samples were also tested for neutralizing antibodies (NA) against PCV2d  was used to test for both PCV2 and 2.8M. hyopneumoniae and PCV2d‐specific interferon‐γ secreting cells (IFN‐γ‐SC). Peripheral blood mononuclear cells (PBMC) were stimulated using the aforementioned challenge M. hyopneumoniae and PCV2d strains  at the Seoul National University  to estimate the percentage of the lung affected by pneumonia. Scoring was performed out of 100 total possible points over the entire lung as follows: 10 points each to the right cranial lobe, right middle lobe, left cranial lobe and left middle lobe; 27.5 points each to the right caudal lobe and left caudal lobe and 5 points to the accessory lobe . The severity of peribronchiolar and perivascular lymphoid tissue hyperplasia was assessed by scoring mycoplasmal pneumonia lesions (0 to 6)  to obtain the quantitative data. For the analysis of PCV2, 10 fields were randomly selected, and the number of positive cells per unit area (0.95 mm2) was determined as previously described  clinical scores between 25 and 74 dpv when compared with the unvaccinated group. Clinical scores were significantly lower (p < .05) in the Farm B vaccinated group between 32 and 74 dpv, and at 95 and 102 dpv when compared with the unvaccinated group. Farm C clinical scores were significantly lower (p < .05) between 32 and 95 dpv in the vaccinated group when compared with the unvaccinated group  in the ADWG was observed on Farm A between vaccinated and unvaccinated group between 10 and 70, between 70 and 112 and between 112 and 175 days of age. The ADWG in the Farm B vaccinated group during the 10–70 and 112–175 days period was significantly higher (p < .05) than that of the unvaccinated group. The overall growth performance of all three farms (10 to 175 days of age) of vaccinated groups was significantly higher (p < .05) than that of unvaccinated group  number of genomic copies of PCV2d in their blood at 39 dpv compared with that of unvaccinated animals. Vaccinated pigs from all three farms had a significantly lower (p < .05) number of genomic copies of PCV2d in their blood between 81 and 102 dpv compared with that of unvaccinated animals  at all three farms. Vaccinated pigs from Farms A and C had a significantly lower  number of M. hyopneumoniae genomic copies in their nasal swabs when compared with unvaccinated animals at 39 and 81 dpv (Farm A), and 81 dpv (Farms B and C)  Figure .3.6p < .05) in the vaccinated group when compared with the unvaccinated group at 18–102 dpv in three farms. The mean frequencies of PCV2‐specific IFN‐γ‐SC remained at basal levels (< 20 cells/106 PBMC) in both groups until 0 dpv. Thereafter, the mean number of PCV2‐specific IFN‐γ‐SC was significantly higher (p < .05) in the vaccinated group when compared with the unvaccinated group from 18 to 81 dpv (Farms A and C) and from 18 to 102 dpv at Farm B , significant differences between vaccinates and non‐vaccinates were not detected at any of the three farms in regard to anti‐PCV2 antibodies. The PCV2 ELISA titres Figure  and PCV2B Figure .3.7M. hyopneumoniae antibodies were not detected in any of the groups or farms. Vaccinated animals from Farms A and C had significantly higher (p < .05) M. hyopneumoniae ELISA S/P ratios at 39 and 81 dpv (respectively) when compared with unvaccinated animals. Farm B vaccinated animals had a significantly higher (p < .05) M. hyopneumoniae ELISA S/P ratios at 18 and 39 dpv when compared with unvaccinated animals. The mean number of M. hyopneumoniae‐specific IFN‐γ‐SC remained at basal levels (< 20 cells/106 PBMC) in both groups until 18 dpv  in the vaccinated groups when compared with the unvaccinated groups from 18 to 81 dpv (Farm A) and from 39 to 81 dpv (Farms B and C) , anti‐v Figure . The mea) Figure .3.8p < .05) of M. hyopneumoniae‐induced pneumonia and PCV2‐associated lymphoid lesions when compared with unvaccinated pigs. Vaccinated pigs on three farms also had significantly lower numbers of PCV2 antigen‐positive cells within lymphoid lesions when compared with unvaccinated pigs.The results of lung and lymphoid lesion score are summarized in Table 4M. hyopneumoniae resulted in a statistically significant improvement in growth performance when compared with unvaccinated pigs in all three farms, regardless of whether the farm was experiencing a subclinical PCV2 and clinical M. hyopneumoniae concurrent infection (Farms A and C), or concurrent PCVAD and clinical M. hyopneumoniae infection (Farm B). Although Farm B received a PCV2a vaccine, PCVAD was not eradicated. Commercial PCV2a‐based vaccines have proven to provide cross‐protection against PCV2d  could interfere with piglets’ active seroconversion after vaccination  or NA (> 7 log2) titres seemed to show interference with the development of the humoral immune response after vaccination. Most of the piglets (> 80% in three Farms) had lower ELISA (< 9 log2) or NA (< 7 log2) titres at the time of vaccination (data not shown). The data presented in this study support that the bivalent vaccine can elicit PCV2‐specific NA and IFN‐γ‐SC even in the presence of MDA. It is therefore concluded that a negative MDA effect on active immunization after vaccination could not have impacted the efficacy of the bivalent vaccine.The most common age of piglet PCV2 vaccination occurs either at weaning or immediately after weaning (around 3–4 weeks of age). In contrast, the evaluated bivalent vaccine containing PCV2b and M. hyopneumoniae also continues to be debated because of the interference of MDA. M. hyopneumoniae may infect pigs within the first 3 weeks of life . Therefore, an earlier vaccination against M. hyopneumoniae at less than 21 days old may be necessary in order to vaccinate piglets to prevent the onset of a natural infection. A number of studies have been conducted to evaluate early piglet vaccination. One experimental challenge study proved that vaccination of pigs at 7 days old with M. hyopneumoniae was effective in reducing lung lesions even in the presence of MDA at a titre considerably higher than what is typically seen in the field  of the Research Institute for Veterinary Science (RIVS) from the College of Veterinary Medicine and by the BK 21 FOUR Future Veterinary Medicine Leading Education and Research Center.All of the methods were previously approved by the Seoul National University Institutional Animal Care and Use, and Ethics Committee. Sample collection was carried out according to the animal welfare code of Korea.https://publons.com/publon/10.1002/vms3.420.The peer review history for this article is available at


C6H3(CF3)2‐3,5, Arf). These receptor systems are capable of capturing gaseous CO, and in the case of the ‐BArf2 system this can be shown to occur in reversible fashion at/above room temperature. For both systems, the binding event is accompanied by migration of one of the aryl substituents to the electrophilic carbon of the CO guest. Experiments utilizing an additional equivalent of PtBu3 allow the initially formed (non‐migrated) CO adduct to be identified and trapped (via demethylation), while also establishing the reversibility of the B‐to‐C migration process. When partnered with the slightly less Lewis acidic ‐BArf2 substituent, this reversibility allows for release of the captured carbon monoxide in the temperature range 40–70 °C, and the possibility for CO sensing, making use of the associated colourless to orange/red colour change.We report two BNB‐type frustrated Lewis pairs which feature an acceptor‐donor‐acceptor functionalized cavity, and which differ in the nature of the B‐bound fluoroaryl group (C Acridan‐derived receptors featuring an acceptor‐donor‐acceptor functionalized cavity offer a metal‐free approach for binding carbon monoxide. Reversibility can be tuned through borane Lewis acidity and the binding event signalled colorimetrically. Recently, a number of low‐valent main group compounds have been developed with a suitable orbital manifold to allow for “transition‐metal like” capabilities in the coordination of CO, including isolable carbenes and phosphinidenes, and transient species such as borylenes and silylenes.1 unit. Acc1 via lithiation followed by quenching with the respective chloroborane, ClBArf2 or ClB(C6F5)2, yielding strongly coloured 2 (orange) or 3 (red) in 86–90 % yield  and a 19F NMR spectrum consistent with only one type of CF3 or C6F5 group. In the case of 2, the spectroscopic data could be corroborated by X‐ray crystallography  adduct of 3 with tBu3PNNO (see Supporting Information).Frustrated Lewis pairs featuring a BNB arrangement of the acid/base functions can readily be synthesized from acridan dibromide 4 and 5; Scheme 2, the orange colour of the toluene solution disappears on shaking under a CO atmosphere, and the colourless crystalline product 4 precipitates from the reaction mixture after a few minutes in 70–80 % isolated yield. 5 can be synthesized in a similar way , in this case using Me4Si to recrystallize the product. The 1H NMR spectra of the adducts (4 and 5) are very similar, both showing two singlets for the C(CH3)2 unit and two different aromatic CH3 resonances, together with a (shifted) lower‐field signal associated with the NMe function. The 19F NMR spectra are consistent with the presence of four distinct CF3 (for 4) or C6F5 environments (for 5), implying a significant reduction in symmetry accompanying the binding of CO (see Supporting Information). In the case of 4, the nature of the product could be confirmed unambiguously by X‐ray crystallography  Å, cf. 1.128 Å for CO and ca. 1.23 Å for the C=O bonds in organic carbonyl compounds).When treated with CO (at room temperature and ca. 1 atm pressure), both BNB systems give similar products  to generate zwitterion 6  are consistent with C6F5 units bound to three‐ and four‐coordinate boron centres, respectively. The structure of 6 was ultimately determined by X‐ray crystallography  Å) and planar arrangement of atoms around carbon (sum of angles=360.0°) are consistent with the formation of a C=N double bond, and consequent formulation of 6 as an iminium borate species.By contrast, heating 6 Scheme . The 1H y Figure , and con4 and 5 at elevated temperatures reflects differences in the Lewis acidities of boranes bearing C6F5 and Arf functions. Aryl migration is shown to be reversible in both cases (see below), and the more modest Lewis acidity associated with the Arf substituent ultimately translates into weaker binding of the CO molecule, and the ability of 2 to reversibly capture carbon monoxide. 5, by contrast, is prone to rearrangement before a comparable CO release can occur.We postulate that the differing behaviour of 2 or 3 with carbon monoxide, we hypothesize that the first‐formed species in the uptake of CO is a simple BNB⋅CO adduct of the form postulated in Figure f systems indicate that the free energy of 4 is only marginally lower than that of this intermediate, Int (ca. 2 kJ mol−1 at the PBE1PBE/TZVP level). With this in mind, we attempted to trap Int via reactions with Lewis bases. While the addition of CO to a solution containing 2 and DMAP  simply leads to the isolation of the corresponding B‐bound adduct of 4  and PtBu3.Although not directly observable in the reaction of either 8 and 9 are characterized by resonances corresponding to the [MePtBu3]+ cation (e.g. δP ca. 49 ppm), and each features two signals in the 11B NMR spectrum at δB<0, indicative of O‐ and C‐ligated four‐coordinate boron centres (e.g. δB −1.1 and −14.9 ppm for 9). The structures of both salts could also be confirmed crystallographically  by the addition of PtBu3. This observation is consistent with the idea that the B‐to‐C aryl migration process is chemically reversible in each case—a factor that, in the case of the Arf system, ultimately underpins the reversibility of CO uptake.From a mechanistic perspective it is important to note that phosphonium salts f2 system, CO uptake can be shown by VT‐NMR studies to occur reversibly. For both systems, the binding event is accompanied by migration of one of the aryl substituents to the electrophilic carbon of the CO guest. Further experiments employing a bulky tertiary phosphine allow the initially formed (non‐migrated) CO adducts to be identified and trapped (via demethylation), while also establishing the reversibility of the B‐to‐C migration process. In conjunction with the slightly less Lewis acidic ‐BArf2 substituent, this reversibility allows for release of the captured carbon monoxide in the temperature range 40–70 °C, with an associated colourless to orange/red colour change.In summary, we report on two BNB‐type frustrated Lewis pairs featuring an acceptor‐donor‐acceptor functionalized cavity. These receptor systems are capable of capturing gaseous carbon monoxide; in the case of the ‐BArDeposition Numbers 2082376, 2082377, 2082378, 2082379, 2082380, 2082381, and 2082382 contain the supplementary crystallographic data for this paper. These data are provided free of charge by the joint Cambridge Crystallographic Data Centre and Fachinformationszentrum Karlsruhe Access Structures service www.ccdc.cam.ac.uk/structures.The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.SupplementaryClick here for additional data file.