It is found that albumin induces a time-dependent dissolution of Ti6Al4V in the presence of H2O2 in physiology saline. Potentiostatic polarisation measurements show that albumin supresses dissolution in the presence of H2O2 at short times (<24 h) but over longer time periods (120 h) it significantly accelerates corrosion, which is attributed to albumin-catalysed dissolution of the corrosion product layer resulting in formation of a thinner oxide film. Dissolution of Ti6Al4V in the presence of albumin and H2O2 in physiological saline is also found to be dependent on potential: the titanium ion release rate is found to be higher (0.57 µg/cm2) at a lower potential (90 mV), where the oxide capacitance and resistance inferred from Electrochemical Impedance Spectroscopy also suggests a less resistant oxide film. The study highlights the importance of using more realistic solutions, and considering behaviour over longer time periods when testing corrosion resistance of metallic biomaterials.There is increasing concern regarding the biological consequences of metal release from implants. However, the mechanisms underpinning implant surface degradation, especially in the absence of wear, are often poorly understood. Here the synergistic effect of albumin and H However, the peri-implant environment contains a variety of biological species which can act to modify corrosion behaviour, including proteins such as albumin and reactive oxygen species (ROS) such as hydrogen peroxide (H2O2)6. The effect of albumin alone, or H2O2 alone, on the corrosion of Ti alloys has been investigated16. However, the effect of combined exposure to both H2O2 and albumin on corrosion of Ti6Al4V has only recently been considered17. Yu et al. demonstrated that a combination of H2O2 and albumin can lead to significantly higher levels of corrosion of Ti6Al4V than either species alone17. In this paper, the mechanism proposed by Yu et al. to explain the synergistic action of albumin and H2O2 on Ti corrosion is explored, taking into account how the corrosion behaviour may change as a function of time.Implantable medical devices are commonly fabricated from titanium (Ti) alloys. Ti alloys have suitable mechanical properties for many biomedical applications and possess excellent corrosion resistance which is dependent on the presence of a stable passive oxide film on its surface2O219. Quantification of the levels of H2O2 in different environments in the body is complex and therefore there is a great variation in the way H2O2 exposures are simulated in in-vitro corrosion tests. ROS species have short half-lives and cellular production of ROS can take place over a wide range of time-scales, making quantification challenging20. However, it is accepted that peak concentrations of ROS can be reached when ROS release is confined in environmental niches, such as sub-cellular phagosomes or extracellularly between the cell and a material surface. H2O2 is known as a strong oxidant and can act to modify the corrosion resistance of Ti surfaces. Studies have been carried out to investigate the effect of H2O2 on corrosion of Ti alloys using concentrations ranging from 33 mM to 300 mM21. A number of studies have shown that H2O2 increases corrosion of Ti and its alloys by forming Ti(IV)-H2O2 complexes22, leading to formation of rougher, thicker and more porous surface oxide films.Inflammation is ubiquitous with healing following implant insertion, but can also be present chronically in the peri-implant tissues associated with failing devices. Inflammation is associated with the production (by immune cells that have infiltrated the peri-implant tissues) of ROS species such as superoxide anions and H4. It is generally agreed that albumin inhibits cathodic reactions on Ti by adsorbing onto the metal surface and covering the reaction sites and/or blocking mass transport of dissolved O217. However, it has also been claimed that albumin increases23, decreases23, or has no effect25 on anodic dissolution of Ti. Similar contradictory results have been found for other metallic biomaterials28. Metal-protein interactions are generally attributed to two effects: adsorption and chelation. In the case of adsorption, metal ion release is inhibited by a layer of adsorbed protein16. In the case of chelation, soluble metal-protein complexes are formed, enhancing the rate of dissolution30. Whilst albumin is the most prevalent protein in the tissue fluid, a wide range of proteins will interact with metal implant surfaces in-vivo. Different proteins can variably influence the corrosion susceptibility of the same metal surface31. For example, it has recently been shown that sequential exposure of albumin and fibrinogen to stainless steel modifies the corrosion rate32, and was attributed to displacement and exchange of the surface adsorbed albumin by the larger protein fibrinogen32.Proteins are known to interact with metal oxides and modify the corrosion resistance of implants. Albumin is the most abundant protein (4.2–5.3%) in blood plasma and extracellular tissue fluid2O2 has been shown to modify subsequent interactions with adsorbed proteins, including albumin. Nagassa et al. showed that exposing Ti surfaces to H2O2 enhanced surface roughness and resulted in greater serum albumin adsorption33. Sousa et al. demonstrated differential binding affinities of albumin to a sputtered TiO2 surface when compared with a TiO2 surface that had been modified by H2O2 immersion34. Yu et al. found a synergistic effect of albumin and H2O2 on Ti6Al4V corrosion by measuring the rate of metal ion release after a 2 week immersion in physiological saline containing both species17. It was shown that albumin accelerated dissolution of Ti6Al4V in presence of H2O2 during the long term exposure. However, short term electrochemical tests showed contradictory findings where albumin appeared to supress the anodic reaction of Ti6Al4V in presence of H2O2. To explain this contradictory behaviour, the author suggested a mixed potential theory where an “active” dissolution region was proposed at low potentials and adsorption of albumin would supress the corrosion potential by taking it into this more “active” region of Ti17. However, the hypothesis needs to be validated carefully and more direct measurements reflecting temporal effects on dissolution rates need to be carried out.Exposing Ti surfaces to H2O2 on corrosion of Ti6Al4V as a function of time. Despite the various simulated physiological media for corrosion testing, 0.9% NaCl was chosen as a basic testing electrolyte (directed by ASTM F1875–98 and F1801-97) to control surface interactions that may further impact on interpretation of the interaction between albumin, H2O2 and the Ti6Al4V surface35. Both short term (<24 h) and long term exposures (up to 120 h) are investigated by electrochemical techniques in order to reflect possible changes in degradation of the metal surface, which is often projected as a slow and continuous process following the implantation in the body. Additionally, to test the “active” dissolution theory proposed by Yu et al.17, ion release was measured as a function of potential and parallel EIS measurements were carried at the static potentials to characterise the metal/oxide/solution interface.The aim of this study is to understand the mechanism underpinning the synergistic effect of albumin and H2O2 in 0.9% NaCl compared with that in 0.9% NaCl alone; this is likely to be due to an increased cathodic current since H2O2 is a strong oxidant17. In a solution containing 4% albumin, in addition to 0.1% H2O2 in 0.9% NaCl, the OCP was consistently lower than that in the presence of NaCl and H2O2 alone, but higher than in NaCl alone. This is consistent with previous work by Yu et al.17, showing that the presence of albumin can decrease the rate of the cathodic reduction of H2O2, probably by adsorption on the surface.Figure 2O2 leading to a higher potential associated with a higher cathodic reaction rate (consistent with cathodic polarisation curves shown in reference17). Addition of H2O2 to NaCl also led to an increase in the anodic reaction rate above 400 mV (sufficiently above the OCP for the cathodic reaction to be negligible), with a small plateau from ~+400 to +600 mV followed by a steady increase. In the presence of NaCl, H2O2 and albumin, the anodic reaction above +400 mV was the same as that for NaCl and H2O2 alone, suggesting that albumin caused little or no change in the anodic reaction, but the cathodic reaction appears to be inhibited, explaining the relative values of the OCP shown in Fig. Potentiodynamic polarisation curves can be used to confirm the interpretation of the OCP behaviour. Figure 2O2 and combination of 0.1% H2O2 and 4% albumin. Nevertheless, after 120 h immersion, more pronounced two time constants were presented in all solutions, which could also be associated with a two layer structured passive film. It can also be found that the real part of the impedance , increased with immersion time which is consistent with their OCP behaviour as observed in Fig. 2O2 in 0.9% NaCl was lower than that the rest of the solutions at both measured time periods.Figure p)  and Rlf ; it should be noted that Rp is dominated by Rhf (oxide resistance). Figure p gradually increased with immersion time and subsequently approached steady state at around 70 h. It can be seen that the presence of H2O2 reduced Rp by more than an order of magnitude after the initial measurement. At 24 h, the value of Rp in the presence of both albumin and H2O2 was similar to that in the presence of H2O2. However, at longer times, Rp in the presence of albumin and H2O2 was significantly lower than that in the presence of H2O2 alone.The data shown in Fig. p) Fig.  is the s2O2 for 3 days at 37 °C. The mud-crack morphology of corrosion products suggests the formation of a thick corrosion product layer. Figure 2O2 without (c) or with (d) 4% albumin for 7 days at 37 °C. Figure Figure 2O2 or albumin. The potential was selected so that the effect on the anodic current density could be evaluated, but it should be noted that this value is a little higher than the interfacial potential under in vivo conditions. It can be seen that at an early stage , the current density increased after addition of solution to give a concentration of 0.1% H2O2 in 0.9% NaCl. For times up to 20 h, the solution with the additional 4% albumin had a lower current density compared with the control that contained only 0.1% H2O2 in 0.9% NaCl.Figure 2O2 alone, the initial increase in current density was found to reach a maximum value of >1 µA/cm2 at ~1.7 h followed by a steady decay of almost two orders of magnitude, reaching ~0.03 µA/cm2 at 70 h. This is likely to be a result of the accumulation of corrosion products similar to those shown in Fig. 2O2, the current density was found to be relatively constant after the initial increase without much of degradation. This is consistent with the observation of a much thinner corrosion product layer in Fig. 2O2-induced corrosion product, so the current reaches a steady state relatively quickly. A cross-over point in the current densities in these two solutions was observed at ~22 h and at 70 h, the current density in the presence of albumin and H2O2 was almost an order of magnitude greater than that in H2O2 alone.However, differences were revealed over longer exposure times. In the presence of Het al.17 proposed that the higher metal release in the presence of both H2O2 and albumin when compared with H2O2 alone, might be a consequence of albumin adsorption onto the metal surface, inhibiting the cathodic reaction and decreasing the potential to a proposed ‘active’ region for dissolution Ti6Al4V. To test this hypothesis, Ti6Al4V was exposed to 0.9% NaCl containing 0.1% H2O2 with and without 4% albumin for 24 h under potentiostatic control. Two potentials were chosen based on the OCP values observed in the two solutions after 24 h in Fig. 2O2 alone was close to 300 mV and in the presence of 0.1% H2O2 and 4% albumin a value closer to 90 mV was observed. Potential values of 90 mV and 200 mV were chosen so that they could be in the potential range of active/passive transition as proposed by Yu et al.17.Yu 2O2 and albumin in accelerating Ti ion release. However, in the presence of albumin and H2O2, Ti release was significantly higher at 90 mV than that at 200 mV (p < 0.001), supporting the hypothesis of Yu et al.17. In presence of H2O2 alone, slightly higher metal Ti ion release was observed at the lower potential, but the difference was much smaller (p < 0.01).Table 2 at 40 mV to ~930 kΩ-cm2 at 200 mV. Figure hf) and capacitance  Ti and Ti alloys23, and also protects it from dissolution induced by e.g. fluoride ions38. Adsorption of albumin films have been proposed to block dissolution. However, all of these experiments have taken place over short time periods, typically over no more than a few hours.At short time exposures (t < ~22 h), addition of 4% albumin suppressed the current density in the presence of H2O2 Fig. . Rapid a2O2 alone decreased significantly after the initial increase, most likely as a result of blocking of the surface by the peroxide corrosion product layer shown in Fig. 2O2 reduces corrosion resistance and thickens the outer porous layer21.After longer term exposures (up to 72 h), it can be seen from Fig. 2O2 and albumin resulted in a higher current density than that in the presence H2O2 alone, with a cross-over point at ~22 h. This is consistent with the observation of a much thinner corrosion product layer in Fig. In contrast, prolonged exposure in the presence of both Het al. via a kinetic perspective of albumin adsorption, adhesion and exchangeability (desorption)34. It was found that the more hydrophilic surface of TiO2 after H2O2 immersion adsorbed less albumin but with higher work of adhesion i.e. it was more strongly attached on the surface, compared with that of the more hydrophobic surface of sputtered TiO234. After the adsorption step, it was suggested that the exchangeability of albumin was changing with time: after 24 h the adsorbed albumin molecules on the H2O2-treated TiO2 surface seem to be less exchangeable than those adsorbed on the sputtered TiO2 surface; however after 72 h nearly all the adsorbed albumin molecules effectively exchanged with other albumin molecules, which suggested that longer time is needed for exchange of albumin molecules adsorbed on surface of H2O2-TiO2 complexes34. Therefore, the time dependent dissolution behaviour observed in the presence work is consistent with the desorption of albumin-metal complexes as it is a slow, rate determining process34, and can result in thinner oxides.The interaction between albumin and peroxide corrosion layer has been studied by Sousa p) of Ti6Al4V in in the presence of H2O2, Rp was significantly lower than that in NaCl alone after 24 h and both increased gradually with time, consistent with the formation of a thicker corrosion product layer. At 120 h, Rp in presence of H2O2 was over an order of magnitude lower than that in NaCl alone. Comparable values were measured for CP Ti in PBS where Rp decreased from 2 × 107 ohm-cm2 to 8 × 105 ohm-cm2 in the presence of 50 mM H2O2 (compared with 33 mM in the present work) after 125 h39. The decreased corrosion rate of Ti6Al4V in the presence of H2O2 over time has been attributed to growth of a corrosion product layer.The time-dependent data were confirmed by the EIS data shown in Fig. 2O2, Rp showed little change over time compared with the behaviour in H2O2 alone, and it can be seen from Fig. p in presence of H2O2 and albumin was significantly lower than that of H2O2 alone, consistent with the presence of a thinner corrosion product layer and increased dissolution. This observation correlates well with previous ICP-MS measurements carried out by Yu et al.17, where metal ion release was measured in identical solutions after 2-week incubation and the combination of H2O2 and albumin was found to give much higher Ti release than that of H2O2 alone. It suggests that the explanation for this observation is likely to be the increased dissolution of the peroxide corrosion product in the presence of albumin.In the presence of albumin and Het al., short term electrochemistry tests (t ~ 1 h) and long term (2 weeks) ion release measurements showed contradictory results regarding the effect of albumin on dissolution of Ti6Al4V in the presence of H2O2 in physiological saline. The investigation explained the observation using a proposed mixed potential theory, suggesting that albumin suppresses the cathodic reaction, decreasing the corrosion potential into a more “active” region for dissolution of Ti at lower potential17. In the present work, this hypothesis was tested by measuring metal ion release from Ti6Al4V at constant applied potentials. Table 2O2, the rate of Ti ion release was higher at lower potentials, consistent with the proposed “active dissolution” effect17. However, it is clear that the increased metal ion release at both potentials is substantially greater in the presence of albumin, indicating that the synergistic effect of albumin and peroxide observed at longer exposure times is dominated by the role of albumin in dissolving the peroxide corrosion product.In the work of Yu ests t ~  h and lo41. In the present work on Ti6Al4V, the major protein in the body, albumin, is shown to interact with the corrosion products of Ti in the presence of H2O2, which is produced in inflammatory processes. The level of H2O2 used in this work (0.1% = 33 mM) is at the lower end of those used in previous in vitro corrosion studies21. Concentrations of H2O2 measured in extracellular environments are frequently reported to be in the µM range43. However, both immune cells and microbial biofilms  create local micro-environments where H2O2 can be considerably concentrated reaching mM concentrations. For example, measurements of H2O2 concentration in surface biofilms of the oral microbe Streptococcus gordinni have been shown to increase with proximity to the biofilm with quasi-steady-state concentrations of 1.4 mM measured at 100 µm from the exposed microbial surface43. It was proposed that the concentration of H2O2 may reach much higher levels within the biofilm itself and at the biofilm substrate interface.Metal implants are exposed to an environment that is more complex than the physiological saline (0.9% NaCl) that is used in standard tests such as ASTM F1801-97 and ASTM F1875-98in vivo metal degradation45. It has been found that during inflammation, the cellular physiology produces a localized acidic45 and concentrated ROS environment42, which can lead to accelerated corrosion of implants and generation of metallic particulates and ions. These implant derived products have themselves been shown to elicit innate and adaptive cellular responses including release of pro-inflammatory cytokines46, leading in some cases to persistent peri-implant inflammation, bone resorption and aseptic loosening of the device48.More recently, the effect inflammatory cell induced corrosion has been recognised as an important mechanism for 49. It was found that albumin blocks dissolution in the first few hours of exposure but increased corrosion was observed over longer exposures49. As biomolecules such as albumin and H2O2 are present in inflamed peri-implant tissue, these observations raise the question of whether standard testing should be complemented by testing in more realistic environments, over more realistic time periods, and using enhanced corrosion characterisation methods rather than standard polarisation curves50.This work also highlights the need to examine the time-dependence of corrosion behaviour of metals used for biomedical implants, since in this case there is a clear switch in the observed behaviour at ~1 day. Similar time-dependent corrosion behaviour in the presence of albumin was also found in biodegradable Mg alloys in simulated body fluidTi6Al4V (ASTM Grade 5) was commercially sourced in disc shape specimens of 14 mm diameter and 1.2 mm thickness . The composition (max. wt%) was 0.05N, 0.08C, 0.015H, 0.3 Fe, 0.2O, 3.5–4.5V, 5.5–6.8 Al, with the balance Ti. Samples for electrochemical tests were cold-mounted in a non-conducting setting resin. To ensure a reproducible surface finish, all samples were wet polished from 800 to 4000 grit using SiC abrasive paper and finished with an OP-S colloidal silica suspension (0.04 μm) on MD-Chem cloth . Samples were cleaned thoroughly with deionised water , rinsed with 100% methanol and finally dried in cool air stream. To ensure consistency in the surface oxide layer between different samples, each sample was exposed in ambient lab air for a fixed time of 15 min following surface preparation prior to electrochemical experiments.2O2  was added to 0.9% NaCl to give a concentration of 0.1% (33 mM). Bovine serum albumin  was dissolved in 0.9% NaCl containing 0.1% H2O2 at concentration of 4%. Both reagent chemicals i.e. 30 wt% H2O2 and bovine serum albumin powder  solutions were prepared by dissolving NaCl  in deionised water. H2O2 with and without 4% albumin. Solutions were stirred during addition of species to ensure complete mixing. Solution of 0.9% NaCl containing H2O2 was added at ~0.6 h to give final concentration of 0.1% H2O2 and solution of 0.9% NaCl containing albumin was added at ~0.74 h to give final concentration of 4% albumin. The control solution without any addition of species was stirred at the same times.A Gill AC potentiostat (ACM instruments) was used for DC electrochemical tests. Samples were immersed in a test solution at the OCP for 1 h, followed by potentiodynamic polarisation from −50 mV (vs. OCP) at a scan rate of 1 mV/s. Each test was repeated three times to confirm consistency. In addition to potentiodynamic tests, potentiostatic polarisation at 400 mV was conducted after 1 h immersion at OCP in 0.9% NaCl, followed by sequential additions of 0.1% H−3 to 105 Hz. The acquisition rate was 10 points per decade. Measurements were taken after immersion in the solution for 24 h intervals up to 120 h. Additional samples were held at constant potentials for 20 h after which EIS was measured at the constant potential. A two time constant electrical circuit 52. Fitting of EIS data was performed by Gamry Echem Analyst software with simplex algorithm method and the quality of fitting was judged by chi squared value (χ2) < 1 × 10−3. Quantitative parameters were obtained. Polarisation resistance was calculated from the sum Rhf and Rlf, corresponding to resistances of inner compact and outer porous passive layer respectively. The impedance of a CPE is given by53:53.A Gamry PCI4 potentiostat (Gamry instruments) was used to measure EIS. EIS measurements were performed at open circuit potential perturbing with 10 mV RMC AC signal with a wide frequency range from 102O2 or combination of 0.1% H2O2 and 4% albumin. Solutions were then extracted and released ions of interests  were analysed by Butterworth Laboratories (UK) using inductive coupled plasma mass spectroscopy (ICP-MS) (Agilent 8800 ICPMS triple quad). The working quantitation limit for Ti is 10 µg/L, Al is 100 µg/L and V is 10 µg/L. 5 mL sample aliquots were measured and diluted up to 1000 mL with water and diluent containing 1.5 mL of Triton-x-100, 0.15 mL of 20% ammonia and 15 mL of 1.4 mM EDTA. The ion release results for each solution were compared across the potentials through one-way analysis of variance (ANOVA) at a significance level of α = 0.05. A total number of four repeats were performed for each condition.Samples were potentiostatically polarised at 90 or 200 mV for 24 h after 1 h immersion at OCP in 0.9% NaCl with presence of 0.1% H2O2 solution for 3 days in thermostatic chamber at 37 ± 1 °C. The H2O2 treated samples were then transferred to 0.1% H2O2 saline solutions with and without addition of 4% albumin for 7 days. Samples before and after incubation were examined by SEM JEOL 6060 at an acceleration voltage of 20 kV in both secondary electron (SE) and back scattered electron (BSE) mode.Ti6Al4V discs were mirror polished on both sides and ultrasonically cleaned with deionised water and methanol for 10 min respectively. Surface prepared discs were incubated in saline with 10% HThe datasets generated and analysed during the current study are available from the corresponding author on reasonable request.2O2 in 0.15 M NaCl.Albumin was found to have a time dependent effect on dissolution of Ti6Al4V in the presence of HAt short times (t < ~22 h), albumin supressed dissolution rates, but at longer times, the rate of dissolution increased to values greater than those found in absence of albumin.2O2 can be attributed to the enhanced dissolution of peroxide corrosion products in the presence of albumin, resulting in formation of a thinner oxide layer on the metal surface.The long term dissolution behaviour in the presence of albumin and H2O2 was found to depend on potential. At lower potential, a higher metal ion release rate was observed via ICP-MS measurements, and the oxide resistance and capacitance determined from EIS was consistent with a thinner oxide layer than that found at higher potentials.The rate of metal ion release in the presence of albumin and H2O2 in 0.15 M NaCl was observed to be significantly higher than that in 0.15 M NaCl alone, emphasising the need to test alloys for biomedical implants in more realistic physiological solutions. It is also essential to make measurements over longer time periods than those typically performed in standard tests involving conventional polarisation curves.The corrosion rate of Ti6Al4V in the presence of albumin and HSupplementary Information


The strength and specificity of protein complex formation is crucial for most life processes and is determined by interactions between residues in the binding partners. Double-mutant cycle analysis provides a strategy for studying the energetic coupling between amino acids at the interfaces of such complexes. Here we show that these pairwise interaction energies can be determined from a single high-resolution native mass spectrum by measuring the intensities of the complexes formed by the two wild-type proteins, the complex of each wild-type protein with a mutant protein, and the complex of the two mutant proteins. This native mass spectrometry approach, which obviates the need for error-prone measurements of binding constants, can provide information regarding multiple interactions in a single spectrum much like nuclear Overhauser effects (NOEs) in nuclear magnetic resonance. Importantly, our results show that specific inter-protein contacts in solution are maintained in the gas phase. Double mutant cycle (DMC) analyses can provide the interaction energies between amino acids at the interface of protein complexes. Here, the authors determine pairwise interaction energies using high-resolution native mass spectroscopy, offering a straightforward route for the DMC methodology. In this method, two residues of interest are mutated individually and in combination and the energetic effects of the mutations  are measured. If the effect of the double mutation differs from the sum of effects of the corresponding single mutations then an interaction, either direct or indirect, between the two residues is inferred. Under favorable circumstances, the deviation from additivity, termed the coupling energy, can be used to estimate the strength of intramolecular3 and intermolecular4 pairwise interactions in proteins or protein–ligand complexes. Coupling energies can also be used as constraints (much like nuclear Overhauser effects (NOEs) in nuclear magnetic resonance) to determine protein structures, e.g., when docking two proteins to each other5. Recent applications of the DMC method in molecular recognition studies include combining it with cryo-electron microscopy to identify the binding site of capsaicin in the TRPV1 ion channel6 and demonstrating an affinity-specificity tradeoff in PDZ–peptide interactions7. Here we describe a native mass spectrometry-based approach that simplifies the application and enhances the power of DMC analysis for studying protein interactions. Our results also show that specific inter-residue interactions are maintained in the gas phase.Methods such as X-ray crystallography, electron microscopy, and nuclear magnetic resonance can be used to determine the structures of protein complexes at high resolution. Nevertheless, complementary methods are needed for determining the energetics of binding reactions and for obtaining structural information in cases that are not amenable to analysis by these methods owing, e.g., to low solubility, large size, and/or high flexibility. One such complementary method that is widely used is double-mutant cycle (DMC) analysisi and Yj, and the two corresponding single mutants, Xm and Ym, . Likewise, the change in the binding free energy upon the mutation i→m, when residue j has already been mutated to m, may be expressed as ΔG(im→mm). The difference between ΔG(ij→mj) and ΔG(im→mm), which is equal to the difference between ΔG(ij→im) and ΔG(mj→mm), provides a measure of the coupling energy, ΔΔGint(ij), between residue i in protein X and residue j in protein Y. This coupling energy can be expressed in terms of binding constants as follows:Kij is the binding constant for the two wild-type proteins, Xi and Yj, Kmm is the binding constant for the two corresponding single mutants Xm and Ym and Kim and Kmj are the respective binding constants for Xi with Ym and Xm with Yj.The application of DMCs for detecting and quantifying inter-protein pairwise interactions between residue i in protein X and residue j in protein Y involves creating a cycle that comprises the four possible complexes that can be formed by the two wild-type proteins, XYm, Fig. . The chaGint(ij). The native mass spectrometry (MS) approach for DMC analysis that is described here circumvents the need for the determination of the individual binding constants and provides a way to measure ΔΔGint(ij) directly from a single spectrum. It is based on the realization that substitution of each of the binding constants in Eq. Kij = [XiYj]/[Xi][Yj]) leads to the following expression:Identification and quantification of an inter-molecular pairwise interaction using the DMC approach is labor intensive since it requires generating binding isotherms for the four complexes in each cycle using methods such as isothermal calorimetry or surface plasmon resonance. It is also error-prone because unavoidable errors in protein concentrations lead to errors in the estimates of the binding constants and, thus, to a cumulative error in the estimate of the value of the coupling energy, ΔΔ12. The high resolution afforded by the Orbitrap platform enabled us to determine the intensities of all four complexes simultaneously from a single mass spectrum. Hence, by mixing the two wild-type proteins, Xi and Yj, and the two corresponding single mutants Xm and Ym, and then measuring the intensities of all four possible co-existing complexes using native MS one can obtain a direct estimate of ΔΔGint(ij) that does not depend on knowing the concentrations of the free species .Native MS is a method based on the ability to transfer protein complexes to the gas phase without disrupting them13. Two DMCs were constructed: one for the interaction of Asn34 in Im2 with Ser84 in E9 and a second for the interaction of Asp33 in Im2 with Phe86 in E9. Phe86 is a main hot spot residue in the interface of E9 with Im913. In both cycles, all the mutations were to alanine as before13. The coupling energies of the two pairwise interactions were determined by the native MS-based approach for DMC analysis described here and, in comparison, by measuring all the required individual binding constants using isothermal titration calorimetry.The applicability of this native MS-based approach for measuring pairwise coupling energies was tested for the well-characterized interaction of colicin E9 endonuclease with the bacterial immunity protein Im2 with which it forms a 1:1 complex14. This was confirmed by showing that the affinity of the E9 Ser84→Ala mutant for Im2 is unchanged by the tail mutations . The positive coupling energy of more than 1 kcal mol−1 found for Asp33 in Im2 and Phe86 in E9 is consistent with a stabilizing and possibly cooperative anion–aromatic interaction between the O and OD2 atoms of Asp33 and the respective Cz and CE1 atoms of Phe86, which are separated in the complex structure by about 4.3 and 5.5 Å, respectively14. Cation-pi pairs in proteins have been documented widely but much less attention has been paid to anion–aromatic interactions between negatively charged side chains and the positive electrostatic potential associated with the ring edge of aromatic groups16. The negative coupling energy of about −1.7 kcal mol−1 reported for the interaction of Asp33 in Im2 and Phe86 in E9 in previous work13 suggests that the different buffer conditions employed in the two studies may have caused conformational switching between a favorable (negatively charged side chain with ring edge) and unfavorable (negatively charged side chain with ring face) anion–aromatic interaction.Given the relative concentrations of the four complexes that form each of the cycles, it was possible to calculate the coupling energies corresponding to them using Eq. 12. Here we have shown using DMC analysis that the interaction energies of specific residue pairs can also remain unchanged upon transfer to the gas phase. The native MS version of DMC analysis described here provides values of coupling energies without the need for multiple measurements of affinity constants and accurate determination of protein concentrations, thereby simplifying the measurements and reducing the experimental error. The method can be extended to work in a high-throughput fashion by mixing wild type and n different single mutants of one protein with wild type and m different single mutants of a second interacting protein. These n + 1 and m + 1 variants of the two proteins form m × n different mutant cycles that can yield m × n coupling energies that can serve as constraints for docking. Hence, it might be possible in the future to determine the structure of a complex from a single native mass spectrum if the structures of the free proteins are known.In summary, many previous studies have shown that protein–ligand and protein–protein complexes can be maintained in the gas phase17. The following mutagenic oligonucleotides were used together with the T7 universal primer as forward and backward primers, respectively.The plasmid pET21d containing the wild-type genes of E9 DNase and Im9 with a His tag or E2 DNase and Im2 with a His tag was used as a template for mutagenesis by restriction-free cloningPhe86→Ala in E9:5′-GTGTTTCAAAAGGTTATTCTCCGGCTACTCCAAAGAATCAACAGGTCG-3′;Asn34→Ala in Im2:5′-GGTGCTACTGAAGAGGATGACGCTAAATTAGTGAGAGAGTTTGAGCG-3′;Asp33→Ala in Im2:5′-GGTGCTACTGAAGAGGATGCCAATAAATTAGTGAGAGAGTTTGAGC-3′. The mutation Ser84→Ala in E9 was introduced together with two other mutations, His131→Ala and Arg132→Ala, designed to increase the difference between the mass of this mutant and those of the other variants without affecting its binding to Im2. This triple mutant was created using the following respective mutagenic oligonucleotides as forward and backward primers.Ser84→Ala:5′-GGTGCTACTGAAGAGGATGCCAATAAATTAGTGAGAGAGTTTGAGC-3′;His131→Ala and Arg132→Ala:5′-CCTAAGCGACATATCGATATTGCCGCAGGTAAGTAAAATGGAACTGAAGC-3′. The mutations were confirmed by DNA sequencing of the entire genes.E. coli BL21 (DE3) cells harboring the pET21d plasmid that contains the appropriate genes coding for E9 and Im9 with a His6 tag or E2 and Im2 with a His6 tag were grown in LB medium containing 100 µg ml−1 ampicillin at 37 °C until an O.D600 nm of 0.6 was reached and protein expression was then induced by adding 1 mM isopropylthio-β-galactoside. The cells were then grown for another 4 h at 37 °C and harvested. Cell pellets were stored at −80 °C until further use. Purification was carried out by resuspending the cells in 20 mM Tris buffer (pH 7.5) containing 100 mM NaCl, 5 mM imidazole, and 1 mM DTT (buffer A) to which was added the cOmplete, EDTA-free, protease inhibitor cocktail . The cells were then disrupted by sonication and the lysate was clarified by centrifugation. The supernatant was loaded on a 5 ml HisTrap HP column . The E9 DNase was dissociated from the column-bound Im9 and eluted by washing the column with buffer A containing 6 M guanidine hydrochloride. Im2 was eluted using a 10–200 mM imidazole gradient in buffer A containing 6 M guanidine hydrochloride. Fractions that contain the E9 and Im2 proteins were dialyzed overnight at 4 °C against water and lyophilized. The purified E9 was stored at −80 °C until further use. The Im2 protein was dissolved in 50 mM Tris buffer (pH 7.5) containing 50 mM KCl and 1 mM DTT (buffer B) and then further purified using a Superdex 75 column equilibrated with this buffer. The fractions containing Im2 were then loaded on a 5 ml HiTrap Q FF column . The Im2 protein was eluted from the Q FF column using a 0.05–0.5 M KCl gradient in buffer B. Fractions containing Im2 were combined, dialyzed overnight at 4 °C against water, and then lyophilized. The purified Im2 protein was stored at −80 °C until further use. The concentrations of E9 and Im2 proteins were determined using molar absorbance coefficients of 15,470 and 10,095 M−1 cm−1, respectively.Experiments were carried out using an ITC200 Microcal calorimeter. Lyophilized protein samples were dissolved in 250 mM ethylenediammonium diacetate (EDDA) buffer (pH 7.0) and then dialyzed overnight at 4 °C against this buffer. The protein samples were centrifuged and their concentrations determined before starting the ITC experiments. The titrations were carried out using a concentration of 0.04–1 mM E9 in the cell and a ten-fold higher concentration of Im2 in the syringe. Each ITC experiment comprised 26 injections with 3 min intervals of 1.5 µl Im2 at 25 °C. The heats of dilution were subtracted from the raw data and values of the binding constants were then determined by fitting the resulting binding isotherm to a 1:1 binding model using the ORIGIN program supplied by the manufacturer.18 and then sprayed into the instrument. Conditions within the mass spectrometer were adjusted to preserve non-covalent interactions, with the source operating in positive mode. The following experimental parameters were used: capillary voltage, 1.7 kV; HCD direct voltage, 30–70 eV; and inlet capillary temperature in the range of 160–245 °C. MS spectra were recorded at a resolution of 17,500 (at m/z 200).All the native MS experiments were performed using a Q Exactive Plus Orbitrap mass spectrometer  modified for optimal transmission and detection of high molecular weight ions. Before MS analysis, proteins were dialyzed against 0.25 M EDDA buffer (pH 7). Proteins forming the DMC of interest were then mixed at the desired ratios and incubated for 5 min at room temperature. Typically, an aliquot of 2 μl protein solution was loaded into a gold-coated nano-ESI capillary prepared in-house as previously described+ and (M+C2H10N2)+, were calculated using the PeakFit v4.12 software . In this program, peaks are fitted automatically to a series of Gaussians using a deconvolution approach. A numerical fitting procedure was repeated to minimize the deviation from experimental data as monitored by the coefficient of determination, R2. Typical R2 values were higher than 0.99. This analysis was repeated for the two detected charge states, i.e., 8+ and 9+. The reliability of the data deconvolution results was validated by comparison of the expected peak position calculated from the mass to charge ratio with the generated peakfit value. In addition, in cases of uncertainty due to the presence of bound ethylenediammonium adducts, the assignment was further confirmed by comparing the relative abundance of a specific peak in measurements performed at different ratios. For example, the peak at 2889 m/z can correspond to the complex of E9S84A with either wild-type Im2 or Im2N34A. This peak increases when the concentration of wild-type Im2 is doubled relative to the other three proteins Data supporting the findings of this study are available within the article and its  file andSupplementary InformationPeer Review File


Alternative polyadenylation is a rapidly emerging form of gene regulation, which in its simplest form, enables the generation of mRNA transcripts that code for the same protein but have 3′UTRs of different lengths and regulatory content. For oncogenes, shorter 3′UTRs would be preferred as a mechanism to evade miRNA regulation. The shortening of the 3′UTR of cyclin D1 in mantle cell lymphoma offers provocative insights into this process. Patient samples have revealed that 3′UTR shortening may occur due to mutations, or translocations that result in the generation of a chimeric 3′UTR. The truncated cyclin D1 3′UTRs resulting from alternative polyadenylation, use a premature canonical polyadenylation signal close to the stop codon that was generated either as a result of mutations or provided by another gene in the chimeric 3′UTR. The sequence of the polyadenylation signal in mantle cell lymphoma appears to be critical for 3′end formation of the cyclin D1 transcript. Shortening the 3′UTR allows cyclin D1 to potentially evade regulation by over 80 miRNAs that are predicted to bind to its full length 3′UTR. CCND1). Accidents that occur during recombination of the V(D)J segments of pre-B stage lymphocytes during differentiation give rise to the characteristic t translocation event that juxtaposes the CCND1 allele downstream of the IgH intronic regulatory enhancer (Eμ) element . Int. IntCCNDmer Fig. . Sanger mer Fig. . Howeverhe 3′UTR . We idennce Fig.  [5].Fig.CCND1 and a small segment of the adjacent 3′UTR was fused to a stretch of nucleotides derived from myotonic dystrophy kinase-related Cdc42-binding kinase (MRCK). This generates a chimeric 3′UTR, consisting of 361 nucleotides from both CCND1 and MRCK. The attached MRCK sequence contains a consensus PAS (AAUAAA) that is optimal for 3′end formation. Further studies showed that the sequence was the reverse complement of a portion of the MRCKs intron one region. Our results using a luciferase reporter containing this chimeric 3′UTR sequence showed that the CCND1/MRCK fusion gene is recalcitrant to regulation by three separate miRNAs [Using a combination of different techniques together with Sanger sequencing we found that in two out of three MCL cell lines as well as 8/13 MCL patient samples the full open reading frame of e miRNAs .CCND1 in MCL. In addition to abnormal transactivation of the promoter, truncations of the 3′UTR to generate transcripts that are recalcitrant to miRNA regulation result in higher cyclin D1 protein expression. We described a novel CCND1/MRCK fusion gene in MCL. In the CCND1/MRCK chimera, the truncated 3′UTR contains sequences from both genes, with the MRCK portion providing a canonical PAS to facilitate 3′end formation. This opens up a new potential avenue to generate therapeutics that will differentially target the MRCK sequences and are hence more specific for MCL without impacting normal CCND1 expression in other human tissues.There appears to be strong drive to express


The results reveal the rate determining steps in the corrosion mechanism during carburizing of steel. These steps are the diffusion of uncharged active gases in the stagnant–gas layer over the steel surface followed by the conversion of C into C4− and O into O2− at the gas–oxide interface simultaneously with the migration of Cr3+ from the metal-oxide interface to the gas-oxide interface. It is proposed that previous research on Al2O3 coatings may be the solution to producing effective coatings that overcome the corrosion challenges discussed in this work.Martensitic stainless steel parts used in carbonaceous atmosphere at high temperature are subject to corrosion which results in a large amount of lost energy and high repair and maintenance costs. This work therefore proposes a model for surface development and corrosion mechanism as a solution to reduce corrosion costs. The morphology, phase, and corrosion behavior of steel are investigated using GIXRD, XANES, and EIS. The results show formation of nanograin–boundary networks in the protective layer of martensitic stainless steel. This Cr In the energy conversion and production systems of the petrochemical industry, carburizing is a long–standing corrosion problem that has been known to produce approximately 137 quadrillion joules of lost energy8. For industries that use operating temperatures of about 600 °C or higher, stainless steels are widely used. The advantage is that a protective chromia scale can form on the surface of the steel when a selective oxidation of chromium occurs under low oxygen–partial pressure. At low oxygen–partial pressure, only very stable oxides such as chromia, alumina, silica, or some spinels can form. Chromia and alumina are well known for limiting or preventing carbon transport into the alloys12. For high temperature carburizing, the carbon transport through the oxide scale can involve a diffusion of carbon–bearing molecules such as CO and CO2 through pores or cracks, or a diffusion of carbon through preferential diffusion pathways such as grain boundaries or even clusters of metal particles assembled as nanonetworks formed in the oxide scale. The diffusion of carbon can lead to internal carburizing followed by internal precipitation of chromium carbide, chromium depletion, and chromia–scale failure caused by brittleness, metal dusting, and even pitting corrosion15. Although martensitic stainless steel is low cost and possesses good corrosion resistance in high CO2 atmosphere16, few works have reported on carburizing–induced corrosion of martensitic stainless steel11 and the corrosion mechanism in nanoscale is still unclear. An understanding of the nanoscale mechanism could connect the atomic and microscale mechanisms observed during carburizing to provide a complete model of the corrosion mechanism from the initial to final states. Our current study reports on this nanoscale mechanism and the complete corrosion mechanism corresponding to surface development of martensitic stainless steel in low oxygen–partial pressured carburizing. The 2 powerful superficial X–ray techniques used to examine the surface are grazing incidence X-ray diffraction (GIXRD) and X–ray absorption near–edge structure (XANES), and the electrochemical behavior is investigated by electrochemical impedance spectroscopy (EIS). Morphology, phase, phase portion and corrosion behavior of the steel surface can be investigated using these techniques to determine the surface development and complete corrosion mechanism  and can lead to a solution of the corrosion problem.Corrosion has had environmental and economic impacts for decades. The high cost of corrosion has been reported as approximately 3.5–4.5% of the United States’ GNP or more than 3% of the world’s GDP2O3–Cr7C3–nanograin film on top of the FeCr2O4 layer that has developed on the surface of the steel carburized at high temperature. The film possesses nanocrystallite–grain boundaries that join and form nanonetworks to transport ions involved in the corrosion reaction . Although the increasing Cr2O3 portion and grain growth can retard bulk ion transport, the steel still encounters corrosion due to ion transport through the nanograin–boundary networks. Adding EIS results to the analysis can allow the complete carburizing–induced–corrosion mechanism to be proposed. The rate determining steps involved in the corrosion mechanism are revealed. Once the corrosion mechanism is clarified, coatings that could be effective at preventing corrosion are considered based on past research.The GIXRD and XANES results show the generation of a mixed Cr17 (detailed in Methods and Supplementary Section 1). Carbon potential for carburizing is controlled by applying an electric current ranging between 100 to 300 W.Tests were performed to provide information about the initial stage of carburizing. In order to save time and money, these tests were performed in a well–controlled laboratory system rather than using real long–service parts in an industrial plant environment. The carburizing of a commercial martensitic stainless steel, Fe–13.60Cr–0.33C–0.37Mn–0.28Si–0.28Ni (wt%), was done in a low oxygen–partial pressured system using the current heating technique21. XANES is powerful element specific probe of local electronic and crystallographic environment due to the absorption by inherent electron excitation and transition processes. XANES probes bonding and the local structure in a short–range order around the absorbing atoms and is particularly sensitive to the geometric arrangement of the nearest and distant neighbors of the absorbing atoms25. EIS can be used to characterize materials and indicate their conductivity and corrosion behavior. EIS works in the frequency domain and determines the net impedance in an electrochemical process through the kinetics of charge transfer and electrochemical reaction29.The characterization of the steel surface was performed using GIXRD, XANES, and EIS. GIXRD is a nondestructive technique providing information about crystal structure, phase composition, grain or cluster size, crystallographic interplanar spacing, lattice parameters, and residual stress in the uppermost thin crystalline layer2O4. The peaks of rhombohedral Cr2O3 and orthorhombic Cr7C3 were observed in the spectra of steels carburized at 250 and 300 W. Peak broadening was not observed in the spectra. The average diameters or sizes of oxide and carbide grains along [hkl] are in the range of 9.1 to 27.5 nm (Supplementary Section 2).The high penetration depth of XRD caused high contribution of the substrate diffraction signal which diminished the signal from the surface compound therefore significant surface compound information was not obtained (Supplementary Section 2). For this reason, GIXRD was used at an average penetration depth of ~143 nm and the spectra are shown in Fig. 2O3; CrO2; CrO3; Cr7C3; and Cr23C6. Analysis determined Cr:Cr2O3 ratios of the best–fitted curves for uncarburized steel (96:4) and steels carburized at 100 W (96:4), 150 W (95:5), 200 W(95:5), and 250 W (65:35). The calculated curve with Cr2O3:Cr7C3:Cr ratio of 90:8:2 exhibited the best fit to the steel carburized at 300 W. The first and second peaks in the derivative spectra shown in the inset of Fig. 2O3 ratio of 65:35 and the Cr2O3:Cr7C3:Cr ratio of 90:8:2, respectively. The spectra results indicate that the steel carburized at 300 W possesses less absorption edge splitting and more white line shift which corresponds to the increased quantity of Cr2O3.In this study, Fe K–edge and Cr K–edge XANES were performed for the uncarburized and carburized steels. The Fe spectra of steels were almost identical and no additional distinguishing information was provided by the Fe K-edge results therefore only Cr K–edge spectra were reported. The experimental spectra and corresponding calculated curves are shown in Fig. An accelerated EIS test was performed in a highly conductive electrolyte (3.5 wt% NaCl). Potential, frequency, and current were controlled and recorded using an Autolab–PGSTAT302N potentiostat (Metrohm Autolab B.V.) incorporated with NOVA software.The electrochemical impedance of the steel was determined by fitting an equivalent circuit to the experimental data (Supplementary Section 4). Nyquist and Bode plots in Fig. QCPEcl) was observed in the high frequency region (1–10 kHz). The impedances of the steels carburized at 100, 150, 200, 250, and 300 W obtained from Nyquist and Bode plots were approximately 19, 3.8, 2.6, 3.1, and 5.5 kΩ cm2, respectively. Table The relaxation frequencies of the carburized steels, shown in the Bode plots, were in the approximate range of 10–1,000 Hz. The relaxation frequencies of the uncarburized steel and steels carburized at 100, 150, and 200 W were 6.87, 86.8, 28.1, and 49.4 Hz, while the time constants were 23.1, 1.8, 5.7, and 3.2 ms, respectively. The curves of steels carburized at 250 and 300 W exhibited two arcs located in the low and high frequency regions in the Nyquist plots. The small arc associated with low chemical–layer capacitance , it is proposed that the promotion of oxide and carbide formation in the results of this work, are due to increased diffusion of oxyen and carbon. The GIXRD spectra show an unclear amorphous characteristic of peak broadening30 indicating a polycrystallinity of the surface compounds. The average sizes of oxide and carbide grains means the compounds can be attributed to nanocrystallites. The sizes of the compounds tended to increase with increasing applied electric power which reflects grain growth promoted by increasing temperature.Cr, O, and C atoms were dissolved in BCC α–Fe at compositions lower than the solubility limits which led the steel to remain in a single phase solid solution. For the steel carburized at 200 W, the FeCr2O3 than steels at a lower carburizing power which corresponded to the detection of Cr2O3 by GIXRD. The spectra for uncarburized steel and steels carburized at 100, 150, and 200 W, correspond to the calculated curves with high Cr:Cr2O3 ratios and clearly exhibit Cr metal characteristic of absorption edge splitting. On the other hand, the steels carburized at 250 and 300 W exhibit minor edge splitting. The spectra and derivative spectra show positive shifts of the white line and absorption edge , respectively. It is evident that the shifts increased as the portion of Cr2O3 increased. The peak energy of the Cr white line, corresponding to a 1s → 4p transition31, can shift from Cr metal (∼6009 eV) towards Cr2O3 (∼6012 eV) when the Cr:Cr2O3 ratios decrease. The shift contributed by the increasing average valence of the transition metal33 (from 0 to 3 for Cr), was promoted by the increasing amount of Cr2O3 in the steel. The development of spectral features of steels carburized at 250 and 300 W became more closely associated to those of Cr2O3 as the amount of Cr2O3 in the steel increased with increasing carburizing power. Moreover, the increase in the amount of Cr2O3 could promote the oscillation of the spectrum located at energies of ∼6019 and ∼6024 eV. This means that low oscillation features of Cr metal can develop into high oscillation features of Cr2O3. The contribution of Cr7C3 to the spectrum of steel carburized at 300 W had a significant effect on the peak energy of the white line by decreasing the contribution of the Cr2O3 white line.The XANES results of phase analysis were slightly different from the GIXRD results which was due to a higher surface sensitivity of XANES. There was however some correspondence found between the results from the two techniques. The XANES calculated curves for steels carburized at 250 and 300 W indicated a higher proportion of Cr2O3 at ∼6012 eV. When features of the experimental spectra are softer or smoother than that of the calculated curves, it can reflect larger amounts of nano– or amorphous constituents in the material31. The nano– or amorphous constituents could be revealed by XANES but not by GIXRD since amorphous characteristic features such as peak broadening were not clearly observed in the GIXRD spectra shown in Fig. This decrease resulted in a peak energy for the white line of ∼6011 eV which is 1 eV lower than that of the reference Crχ2 ∼ 10−2–10−3, indicated that resistive and capacitive behaviors of the steels corresponded well to the Mansfeld equivalent circuit model (Supplementary Section 4). The calculated parameter of charge transfer resistance (Rct) was used to describe the corrosion resistance of the steel. Rct of the steels tended to decrease with increasing carburizing power up to 200 W and remained constant at approximately 3 kΩ cm2 for the steels carburized between 200–300 W. This constant Rct reflects a constant rate of redox reaction associated with the conversion process. The curves of uncarburized steel and steels carburized at 100, 150, and 200 W each exhibit an arc in the Nyquist plots. The curves reflect the occurrence of a single relaxation process in the steels. This work has shown that increasing carburizing power tended to decrease the magnitude of the arc diameter which indicates a decreasing capacitance and resistance of the steel. The relaxation frequencies of the steels suggest that they correspond to a rate determining step. It is proposed that during the EIS test, this rate determining step is that O2 diffusion  in a stagnant gas layer establishes an O2 concentration gradient over the steel surface34. The time constant of steels tended to decrease with increasing carburizing power. The results exhibit the same trend as our previous work for quenched–tempered martensitic stainless steel in which the steels carburized at 100, 150, and 200 W possessed impedances of 4.4, 2.8, and 3.7 kΩ cm2, respectively, and slightly lower relaxation frequencies (∼10 Hz) but in the same range 17. The decreasing impedance and arc diameter correspond to a decrease in O2–diffusion impedance. These decreases along with a decreasing time constant are the reasons for the increasing charge transfer35 and decreasing corrosion resistance of the steel.The data shown in Table 26. The Bode plot was used to distinguish the frequencies of the two relaxation processes and which were found to be in the ranges of 1–10 Hz and 1–10 kHz for conversion and migration processes, respectively.The curves of the steels carburized at 250 and 300 W each exhibit two arcs in the Nyquist plots which reflects two relaxation processes. It is proposed that these two relaxation processes were rate limiting steps in the overall processQCPEdl) was observed in the low frequency region (1–10 Hz) and was presumed to be associated with the accumulation of charges at the solution–oxide interface. This charge accumulation results in a decrease in the oxygen–reduction rate which corresponded to the trend noted in the gas conversion model. This model explains the generation of impedance for a redox reaction at a solution–electrode interface under the condition of uniform gas–partial pressured volume36. The conversion process was rate determining due to the accumulation of O2− which in turn causes the oxygen–reduction rate to decrease. The conversion process was the main contributer to the charge transfer associated with Rct of the steels. When the conversion process became the rate determining step at high carburizing power, Rct became the important parameter determining the corrosion resistance of the steel. Outward effective diffusion of Cr3+ (oxidized at the steel–oxide interface) in Cr2O3 is faster than inward effective diffusion of O2−. The Cr3+ migration process is therefore rate determining for the Cr2O3 formation reaction arising at the solution–oxide interface38. The small arc, which indicates the Cr3+ migration through the oxide layer, has a frequency of between 1–10 kHz which corresponds to the fact that migration is dependent on the structure of the material26. Therefore, the migration process depends on the quantity of the compounds generated at the steel surface during carburizing and their grain sizes. Accumulation and slow migration of ions in the chemical layer can be indicated by high values of both QCPEcl and chemical–layer resistance (Rcl). The chemical layer–relaxation frequencies (fcl) and calculated chemical layer–time constants (τcl) for the ion migration process of the steels carburized at 250 and 300 W were approximately the same at a value of 7.91 kHz and 0.02 ms, respectively. For the conversion process, the double layer–relaxation frequency (fdl) and calculated double layer–time constant (τdl) of the steels carburized at 250 and 300 W were approximately 2.95 and 1.68 Hz, and 54.0 and 94.7 ms, respectively. The results exhibit the same trend as our previous work for quenched–tempered martensitic stainless steel carburized at 250 W which possessed two relaxation processes with values for fcl and fdl of 4.5 kHz and 6.0 Hz, respectively17. The low fdl in the low frequency arc shown in the inset of Fig. 36. It is well known that time constants and resistances have a great influence on promotion of corrosion resistance of the steel. In this work, since the τcl of the steels were about the same, therefore we propose that the τdl contributed significantly to corrosion resistance. The high–frequency arc diameter of steel carburized at 300 W shown in the Nyquist plot was larger than the diameter of the steel carburized at 250 W. This indicates higher QCPEcl and Rcl due to a higher quantity of Cr2O3 and larger grain sizes as previously discussed in the XANES and GIXRD sections of this work. Higher quantities of Cr2O3 and larger grain sizes can retard the migration of ions because the steel now possesses a high chemical stability and less preferential diffusion pathways for grain–boundary diffusion.The arc associated with high double–layer capacitance  into the steel through two effects: concentration gradient and thermal activation. However, single bonds are unstable consequently C and O atoms would break their bonds with host Fe atoms and then re–bond with other Fe host atoms. This effect is called bond switching which occurs during inward diffusion39. The overall process can be described by the following reactions43:2O adsorption44, while the CO2 dissociation promotes oxygen uptake.By increasing the applied electric power for carburizing, the surface development of the steel can be compared to the stages in the carburizing process of actual “in service” parts. These parts are commonly serviced in low oxygen partial pressure in a carbonaceous atmosphere at a service temperature of approximately 600 °C or higher. The carburizing process can be divided into 4 sub–stages shown schematically in Fig. ing Fig. , C and O39. For the 2nd sub–stage of carburizing . The redox reaction between these ions creates stable bonds resulting in nucleation of FeCr2O4 at the steel surface. At this stage of oxide formation, the atomic vacancies can undergo self–assembly and generate nanovacancies at the oxide–steel interface45. The rate determining step in this reaction is suggested to be the diffusion of uncharged active gas  in a stagnant–gas layer  and the migration process  are the rate determining steps. Cr3+ generated at the oxide–steel interface migrate principally through the nanograin–boundary networks and react with O2− and C4− to form Cr2O3 and Cr7C3 in the region near the gas–oxide interface as presented in Fig. 2O3 and Cr7C3 are nucleated at the FeCr2O4 grain boundary by these reactions13:44. The mixed Cr2O3–Cr7C3 outer layer contains a higher proportion of Cr2O3 than that of Cr7C3  due to several reasons. A conversion of Cr7C3 to Cr2O3 occurs when the steel possesses high oxygen concentrations at near surface region13 and can be attributed to very low diffusion rates of carbon in Cr2O3 scale10, lower electronegativity of carbon than oxygen39, and less thermodynamic stability of Cr7C3. Due to the migration of Cr3+ ions, the steel can experience atomic vacancy injection at the oxide–steel interface during the growth of the mixed layer. The aggregation of injected vacancies in the form of nanocavities can lead to the formation of cavities at the oxide–steel interface promoting scale failure due to a weakening of the scale–steel adherence45. The migration process becomes one of the rate determining steps in the 3rd and 4th sub–stages  fed with argon (50 ml min−1 flow rate).The 1.7 × 1.0 × 0.2 cm specimens were prepared from commercial martensitic stainless steel: Fe–13.60Cr–0.33C–0.37Mn–0.28Si–0.28Ni (wt%). The material was received in an annealed state  with hardness of ∼220 HB. The microstructure of steel was constituted of randomly dispersed globular carbides in a ferrite matrix which corresponded to the steel treated in the annealed condition reported in literature51 and the calculated curves were determined using FEFF8.2 codes53. The FEFF8.2 codes utilize a full multiple scattering approach based on Abinitio overlapping muffin–tin potentials. The muffin–tin potentials used in the FEFF codes are self–consistent calculations with the Hedin–Lundqvist exchange–correlation function54. The XANES spectra of Cr were calculated in a spherical radius of 3.4 Å for Cr7C3 and 3.7 Å for Cr2O3 around the absorber Cr atom. The full multiple scattering calculations include all possible paths within a larger cluster radius of 5.0 Å for both Cr7C3 and Cr2O3. EIS measurement was performed using the Autolab–PGSTAT302N potentiostat (Metrohm Autolab B.V.) in 3.5% NaCl solution with a Ag/AgCl reference electrode and a platinum counter electrode incorporated with NOVA (1.11.0) software. The exposure surface area of the working electrodes was approximately 0.8–1.0 cm2. Before EIS measurements, the open circuit potentials (OCPs) of all working electrodes were monitored for 2,000 s until stable OCPs were achieved. The applied sinusoidal potential was held to 10 mV (r.m.s.) around the OCP and the frequency was controlled to between 100 kHz and 0.1 Hz. For each analysis condition, the EIS measurement was performed for at least three times by using a new sample in fresh solution. The closest single results to the averages of multiple data were chosen to be the representation of each analysis condition. The impedance and electrochemical parameters of the steel were determined by fitting the experimental data to the Mansfeld equivalent circuit model using NOVA (1.11.0) software.GIXRD was performed using D8 DISCOVER (Bruker Corporation) spectrometer with a Cu Kα X–ray source  at a 2° incident angle, 0° tilt angle, 0.02° step size, and 0.4 s step time. Cr K–edge XANES was carried out at the BL5.2: SUT–NANOTEC–SLRI XAS beam line of the Synchrotron Light Research Institute (Public Organization), Thailand. The total fluorescence yields were collected using a double crystal Ge (220) detector. The background correction and normalization of experimental spectra were done using the Athena program of IFEFFIT packageThe datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.Supplementary information


Cd59, with minimal change in Crry that controls the C3 convertase. Thus, innate immune complement activation in the circulation is not critical for development of hypertension and kidney damage due to increased sodium intake, and therapeutic manipulation of the complement system is not indicated in salt‐sensitive hypertension.Evidence indicates the immune system is important in development of hypertension and kidney disease. In the Dahl Salt‐Sensitive (SS) rat model, lymphocytes play a role in development of hypertension and kidney damage after increased sodium intake. Recent transcriptomic analyses demonstrate upregulation of the innate immune complement system in the kidney of Dahl SS rat fed a high‐salt diet, leading us to hypothesize that inhibition of complement activation would attenuate development of hypertension and kidney damage. Male Dahl SS rats on a low salt (0.4% NaCl) diet were instrumented with telemeters for continuous monitoring of arterial blood pressure. Animals received saline vehicle (Control) or sCR1, a soluble form of endogenous Complement Receptor 1  that inhibits complement activation. At Day 0, rats were switched to high salt (4.0% NaCl) diet and assigned to sCR1 (15 mg/kg per day) or Control groups with daily ip injections either days 1–7 or days 14–18. Urine was collected overnight for determination of albumin excretion. Treatment with sCR1, either immediately after high‐salt diet was initiated, or at days 14–18, did not alter development of hypertension or albuminuria. The sCR1 dose effectively inhibited total hemolytic complement activity as well as C3a generation. High salt caused an increase in message for complement regulator  Animal studies and human data indicate that the immune system is important in the development of hypertension and kidney damage  containing 0.4% NaCl from weaning. Experimental animals were fed 0.4% NaCl chow until 9 weeks of age and were then fed the same diet containing 4.0% NaCl for the remainder of the experimental protocol. The Institutional Animal Care and Use Committee of the Medical College of Wisconsin approved all studies.Experiments were performed on age‐matched, male Dahl SS/JrHsdMcwi (SS) rats obtained from inbred colonies at the Medical College of Wisconsin and provided food and water n = 6 animals (Control); (2) Dahl SS rats + ip sCR1 (15 mg/kg) from days 1–7 of high salt, n = 5 animals (sCR1); (3) Dahl SS rats + ip saline vehicle from days 14–18 of high salt, n = 7 animals (Control); (4) Dahl SS rats + ip sCR1 (15 mg/kg) from days 14–18 of high salt, n = 5 animals (sCR1). Based upon our previous observations indicating that immune mechanisms amplify salt‐sensitive hypertension in Dahl SS following an initial increase in blood pressure, the present experiments were designed to assess the importance of complement activation in the early  and the secondary  phase of hypertension.The influence of an elevated NaCl intake on the development of hypertension and renal damage in Dahl SS rats was assessed in rats randomly assigned to one of the following groups: (1) Dahl SS rats + ip saline vehicle from days 1–7 of high salt, Seven‐week‐old male rats, maintained on a low salt (0.4% NaCl) diet were instrumented with telemetry transmitters (DSI PA) for the continuous, direct measurement of arterial blood pressure as previously described  with daily ip injections on days 14–18 to test for the secondary/delayed effects of complement on disease progression. In this group of animals, serum was obtained at euthanasia from the abdominal aorta on day 22. Arterial pressure was also monitored daily during this experiment. Overnight urine collections were done on low‐salt diet as well as on Day 7, 14, and 21 of the high salt diet. Animals were euthanized with an overdose of sodium pentobarbital following 22 days on the high‐salt diet.sCR1  was provided at 5.4 mg/mL in PBS containing mannitol, and was dialyzed against endotoxin free saline (vehicle) prior to ip injection. sCR1 is a soluble form of the endogenous human complement regulator CR1 (CD35) with demonstrated ability to inhibit complement activation at the C3 and C5 convertases of the complement pathway, and demonstrated efficacy in numerous rat models of autoimmune and inflammatory diseases. The dose and frequency of sCR1 injection is based on the use of a similar treatment regimen with sCR1 in rats by Ramaglia et al.  and LillTotal hemolytic complement activity was measured in serum obtained from tail vein or abdominal aorta. Briefly, the assay involves serial dilution of serum and incubation with antibody‐coated sheep red blood cells for 1 h at 37°C. The antibody‐coated cells are lysed by complement, releasing hemoglobin. The inverse of the dilution of serum resulting in lysis of 50% of the cells is reported as the CH50. CH50 is a general measure of the overall function of the classical pathway of complement activation.μL based on signal intensity of 1 μL of standard pool of rat serum activated by yeast.The complement activation product C3a was measured by western blot as previously described  versus high salt (4.0%) diet was determined. Message for Crry and Cd59 obtained from high‐salt animals treated with saline vehicle or sCR1 were assessed by Student's t test. A probability value of P < 0.05 was considered significant.Data are expressed as the mean ± one standard error. Blood pressure and urine albumin excretion data were assessed for significance using a two‐way repeated measures analysis of variance (ANOVA) with a Holm‐Sidak post hoc test. Differences in CH50, C3a and fold changes in message for As depicted in Figure Serum was obtained from the tail vein of animals on day 8, one day after completion of the sCR1 treatment. As seen in Figure 3A, sCR1 treatment substantially and significantly decreased CH50 when measured from serum collected on day 8, indicating adequate inhibitory concentrations were achieved for 24 h following ip injection. CH50 in serum collected on day 21 from these animals had returned to normal values by time of euthanasia on day 21. When compared to control, sCR1 also significantly decreased serum C3a when measured in serum on day 8, 24 h after the last ip injection of sCR1 .A second group of animals were treated with sCR1 or saline vehicle on days 14–18 of the high‐salt diet as seen in Figure CH50 was measured 4 days after cessation of sCR1 treatment in this group of animals. As seen in Figure Crry and Cd59 was not affected by high‐salt diet. If message for the same regulators was assessed in aorta, high salt tended to decrease Crry in aorta (P = 0.06) and significantly increased Cd59 (P < 0.05). Thus, high salt caused minor decreases or no significant change in Crry that controls C3 convertase, with a large increase in Cd59 that controls formation of C5b‐9. Overall, the small decrease in Crry controlling the C3 convertase is not likely responsible for the ineffectiveness of sCR1 in altering hypertension or albuminuria with a high‐salt diet.Mammalian cells have numerous endogenous molecules that control complement activation to prevent deleterious effects of C3 deposition and formation of the membrane attack complex on our own cells. This includes the endogenous membrane proteins complement receptor 1‐related protein/gene y (CRRY) and CD59. CRRY is a rodent protein with activities of both human DAF (CD55) and MCP CD46). CRRY primarily controls activation at the C3/C5 convertase and C3 deposition by accelerating decay of the active enzyme as well as acting as a cofactor for inactivation of C3b and C4b, components of both the alternative and classical pathway convertases. CD59 prevents C9 from associating with C5b‐8, and thus operates to prevent formation of the terminal lytic pathway of complement activation, C5b‐9 (membrane attack complex). We hypothesized that high salt resulted in a reduction in complement regulators in the kidney cortex or vasculature resulting in increased C3 convertase activity and complement activation that overwhelmed the inhibitory capacity of the exogenous sCR1 administered. sCR1 also inhibits the C3/C5 convertase via decay acceleration of the enzyme as well as having cofactor activity for inactivation of C3b and C4b. As seen in kidney cortex (Fig. 6. CRRY pExcess complement system activation has been implicated in pathophysiology of numerous types of hypertension and kidney disease. Two recent transcriptomic studies had indicated that the complement pathway is upregulated in the kidney of the Dahl SS rat fed a high‐salt diet (Liu et al. As we have demonstrated, pharmacological or genetic blockade of the immune system does not prevent hypertension and related kidney damage in the Dahl SS rat, but attenuates the disease process (Mattson et al. sCR1 has been used in numerous animal models of autoimmune and inflammatory disease. It is a recombinant human molecule that has been demonstrated to be effective in inhibiting complement activation in the rat, mouse, and guinea pig (Regal et al. It is not known if high salt results in complement activation and C3 deposition in the kidney of the Dahl SS rat and this was not an endpoint measured in this study. A clear limitation of our study is the possibility that the important site of inhibition of complement activation may be locally in the tissues and the effectiveness of sCR1 at this tissue site is unknown. It is possible that stronger complement inhibitors targeted to the site of complement activation may be effective in salt‐sensitive hypertension. CR2‐CRRY or Ig‐CRRY are complement inhibitors that have clearly improved kidney function in a mouse model of lupus nephritis (Bao et al. Cd59 in the aorta was upregulated suggesting that activation of the terminal complement pathway was controlled. Our study is limited in that many other endogenous regulators of complement activation may influence the level of complement activation and were not assessed in the present study (Regal et al. Regulation of complement activation is essential to prevent excess complement activation from damaging our own cells. As with any enzymatic amplification system, the initiation and activation of the complement cascade is balanced by endogenous regulators that curb activation generally or locally. CRRY is one of the endogenous regulators that controls complement activation at C3 to prevent excessive cleavage of the C3 molecule into C3a and C3b. CD59 on the other hand prevents activation of the complement cascade at the C5b‐9 stage. If excessive complement activation occurred in response to high salt, and the endogenous regulators were simultaneously decreased, then addition of exogenous sCR1 may be insufficient to contain and control the complement system. However, minimal decreases in message for the complement regulators were evident at the C3 convertase level in the kidney, and Overall, this study indicates that complement activation is not critical for development of the salt‐sensitive phenotype, though changes in the complement system may be occurring. Thus, the immune cell infiltration that amplifies the disease phenotype and plays a critical role in the development of hypertension and kidney damage is not dependent on the innate immune complement system.None declared.


The LiO2 and LiO4 consist of a lithium layer sandwiched by an oxygen ring structure inherited from high pressure ε‐O8 phase, while Li2O3 inherits the local arrangements from ambient LiO2 and Li2O2 phases. These novel lithium oxides beyond the ambient Li2O, Li2O2, and LiO2 phases show great potential in improving battery design and performance in large battery applications under extreme conditions.The lithium–air battery has great potential of achieving specific energy density comparable to that of gasoline. Several lithium oxide phases involved in the charge–discharge process greatly affect the overall performance of lithium–air batteries. One of the key issues is linked to the environmental oxygen‐rich conditions during battery cycling. Here, the theoretical prediction and experimental confirmation of new stable oxygen‐rich lithium oxides under high pressure conditions are reported. Three new high pressure oxide phases that form at high temperature and pressure are identified: Li It is commonly known that the Li–O2 battery often forms insoluble discharge products, mainly oxides, which accelerate the degradation of the electrode and electrolyte, thus reducing the cycle stability and efficiency.2.Lithium‐ion batteries can store a large amount of energy in a relatively small volume and have been extensively used as rechargeable batteries for portable electronics. Over the last decade, lithium–air Li–O batterie2O2 and Li2O at the battery working temperature.3O4 nanoparticles as a discharge byproduct.There have been some studies on the lithium oxides at various conditions. Yao et al. investigated the thermal stabilities of Li3Cl and NaCl3 under pressure.2 and FeO2) formed from a high pressure and temperature redox reaction provide a new look at oxygen and hydrogen cycling in the deep earth interior.2O2 and mixture of Li2O2 with oxygen at high pressure, and found that the pure Li2O2 is robustly stable up to 57 GPa, while the mixture of Li2O2 with adequate oxygen environment produces a series of oxygen‐rich phases under high pressure and temperature. We also applied DFT calculations to search the possible thermodynamically stable phases, and found that the calculated results matched the experimental observations. This may inspire broader applications of these new forms of lithium oxides.Pressure as an efficient way to tune the lattice and electronic structure has been extensively used to explore novel material synthesis. Sodium chloride displays the polymorphisms Na22O) and lithium peroxide (Li2O2) are thermodynamically stable. During the charge–discharge cycling of Li–O2 batteries, Li2O2 plays the major role. It has been reported that a small amount of lithium superoxide (LiO2) nanoparticles can form on the surface of the Li2O2.2O2 (Sigma‐Aldrich) powder loaded in a diamond anvil cell without any pressure‐transmitting medium for a test at high pressure and room temperature. The Li2O2 powder was compressed to 57 GPa and subjected to synchrotron X‐ray diffraction (XRD) to follow the structural evolution as a function of pressure. Figure3/mmc symmetry to the highest pressure. As shown in Figure Bo = 94.77 GPa and derivative of Bo as 3.41.At ambient condition, both lithium oxide  was needed to heat the sample to the maximum temperature of 2200 K. A 2D raster scan was performed around the laser‐processed center of the sample, and the powder diffraction pattern was collected at each scanning location. We clearly saw totally different diffraction profiles in the laser‐processed center area, with the most diffraction peaks being spotty, indicating new structures had been synthesized in the form of many small single‐crystal grains. In the diffraction profile of the central 20 µm region where the laser heating was applied, one broad smooth powder ring appeared near 2θ = 7.8°, which was not evident outside this region. Upon opening the diamond anvil cell, we realized that the central area of the anvil had been damaged by the high‐power laser beams , and part of the carbon had been removed from the diamond culet surface.On the second run, Li2O2 powder was sandwiched between two thin layers of LiF pellets, and loaded with liquid oxygen in a diamond anvil cell. The sample was compressed to 50.0 GPa, followed by laser heating. The pressure inside the chamber dropped to 48.0 GPa after cooling to room temperature. With the thermal insulator LiF layers, only 20 W power was needed to heat the sample above 2000 K, and no damage was observed on the diamond surfaces. Synchrotron X‐ray diffraction was taken at the laser‐processed location. The diffraction profile changed dramatically from the previous two runs, indicating that new structures had been synthesized.On the third run, the Li2O2 powder and oxygen was compressed to 53 GPa without laser heating. The selected XRD patterns around 15 and 50 GPa are shown in Figure S2 (Supporting Information). All diffraction peaks can be characterized by the known ambient pressure Li2O2 phase (P63/mmc) and oxygen ε‐O8 phase  up to the highest pressure in this run.To clarify the high temperature requirement for synthesizing these new phases from previous three runs, we conducted one more room temperature compression experiment in a diamond anvil cell. Mixture of Li2O2 and O2, we predicted possible stoichiometry and crystal structure at 15 and 48 GPa, as shown in Figure2O3, LiO2, and LiO4 are stable phases, with LiO2 and LiO4 remaining stable even at 48 GPa. At 15 GPa, Li2O3 and LiO4 have Im‐3m and Ibam space groups, respectively. It is interesting to see that for LiO2, the transition pressure from the known ambient Pnnm phase to the new P4/mbm phase is at 12 GPa, close to our second run experimental condition around 15 GPa. The enthalpy plot of LiO2 is shown in Figure S3 (Supporting Information). The formation enthalpy of stable phases with respect to decomposition into Li2O2 and O2 is less than 0.1 eV per atom, and some phases possess total energy close to that of the stable phases. Thus, it is likely that the temperature effect could play a role in the thermodynamic stability.Crystal structure searching based on DFT was conducted to understand the energy landscape of the Li–O compounds in the experimental environments. With two end members, Li2 shows a tendency to become less stable at higher temperature.To understand the thermodynamic stability of each phase, we conducted phonon calculations. Clearly, these candidate phases show stable phonon dispersion relations . To compare the synthesizing condition with high temperature, we also tested the temperature effect up to 1000 K on the formation enthalpy at 48 GPa, using a quasiharmonic approximation. While other phases remain almost constant, LiO2 units. The effective charges on the Li/O2 unit in Li2O2 are +1.6 and −1.6, and the O—O bonding distance is 1.50 Å, compared to 1.21 Å in the solid oxygen. By taking more oxygen, the effective charge of the O2 unit is reduced to −1.07 (Li2O3), −0.82 (LiO2), and −0.41 (LiO4), forming superoxides. The corresponding bond length of O2 changes to 1.36, 1.31, and 1.25 Å, respectively, which are close to that of solid oxygen. The effective charge of Li remains almost constant and occupies similar volumes in all Li–O compounds. Therefore, we speculate that Li–O compounds under pressure take advantage of having smaller molecular volume of oxygen units by reducing electrons at antibonding states. In other words, Li–O compounds tend to form superoxides under pressure to lower the total energy by reducing their O2 volume. This trend can be also observed in the phonon dispersions curves . The calculated phonon dispersion of Li2O2 at 48 GPa was stable, a finding in good agreement with our pure Li2O2 high pressure experiments. Increasing oxygen concentrations in oxygen‐rich phases show higher vibrational modes of O2 units, and we can see that the O2 phonon band splits from the lower energy phonon bands.Bader charge analysis provided further insight into the bonding nature between Li and O4CO4 C1m1 phase predicted by Cancarevic et al.,2 (Pnnm) and LiO2 (P4/mbm) phases from our DFT calculations. Figure4CO4 (C1m1), LiO2 (Pnnm), and LiO2 (P4/mbm) phases. The distinguishing feature on the diffraction pattern is the very spotty diffraction rings, indicating micrometer‐sized grains with well‐annealed single crystals. The high temperature promotes the chemical reaction between Li2O2 and oxygen, and also allows the crystal growth of the new phases. The Bragg diffraction positions from these three phases match the d‐spacings from the powder diffraction. We conducted Reitveld refinement on the integrated 1D pattern with the above three phases. Although the intensity cannot be fitted very well due to the spotty patterns, the lattice parameters can be obtained with this refinement. Roughly, each phase occupies about 1/3 volumetrically. The fitting result and parameters of these three phases are displayed in Figure S5 and Table S1 (Supporting Information), respectively. With 14.4 GPa pressure and high temperature, the data indicate that the following redox reactions take place For interpreting the 14.4 GPa XRD results, we considered that the carbon that escaped from the diamond anvil surface may participate in the new compound formation. The powder diffraction profile fit well with the Li4CO4 and LiO2 are formed with further reduction and oxidation from pristine Li2O2 by carbon and oxygen, respectively. Both Pnnm and P4/mbm LiO2 phases are present at this pressure, which is close to the DFT calculation result on the Pnnm→P4/mbm transition pressure near 12 GPa. Cancarevic et al. proposed that the Li4CO4 (C1m1) phase could be synthesized in the pressure range 80–110 GPa.4CO4 from a mixture of Li2O and Li2CO3 but failed to obtain the predicted Li4CO4 phase up to 25 GPa and 1100 K.4CO4 from Li2O2 and vapor carbon at 14.4 GPa and 2200 K via a redox reaction, which provides a much lower pressure route to form lithium orthocarbonate. For comparison, we also took diffraction patterns at off‐center locations. In the bottom panel of Figure S5 (Supporting Information), the XRD profile taken at 30 µm away from the laser center is shown, where much lower heating temperature is expected. Very small amount of Li4CO4 was formed and some not well formed LiO2 P4/mbm and Pnnm phases (broad and low intensity peaks) were present, which indicates the temperature is an important parameter for the new LiO2 phase synthesis.Thus, the new compounds LiFigure2 (P4/mbm), LiO4 (Ibam), and Li2O3 (Im‐3m) along with high pressure ε‐O8 phase (C2/m)2O2 phase (P63/mmc). From the fitting volumetric percentage, the amount of LiO2 is quite small (3.27%), but the amount of Li2O2 has increased dramatically. The following chemical reactions can be considered Because at 48 GPa, the XRD Debye–Scherrer rings are smooth, we obtained good powder diffraction patterns for structural Reitveld refinement. As presented in 2 is mainly an intermediate product and largely converted to Li2O3 and LiO4 phases. At this high pressure, only the P4/mbm phase of LiO2 is observed, and the Li2O2 has the same structure as that at ambient pressure, consistent with the conclusion from run 1. Because of the thermal insulator layers of LiF, the diamond anvils survived, and no carbon was involved in the redox reaction at 48 GPa. In the above redox reactions, the LiO2 takes a new form as P4/mbm structure, whereas the LiO4 stays at Ibam phase. Additionally, the stable Li2O3 phase can be successfully detected from the powder diffraction. The strong peak at 2θ = 7° was attributed to the ε‐O8 phase.2 is a thermally stable phase under high temperature and pressure, it is largely combined with Li2O2 and O2 to form Li2O3 and LiO4, respectively, and LiO2 itself becomes less stable at high temperature and may decompose into Li2O2 and O2, or Li2O2 and LiO4. This decomposition might lead to only a small percentage of LiO2 at 48 GPa (only 3.27%).In the above redox reactions, the LiO2O3 phase in different pressures. Although the Li2O3 is predicted as a stable phase starting at 15 GPa by DFT calculation, we did not observe this structure with XRD; but at 48 GPa, more than 1/3 of the diffraction intensity was attributed to Li2O3. The DFT calculations did not indicate this phase at this pressure, as shown by the convex hulls in Figure 2O3 phase. At 14.4 GPa, the pressure and temperature are not enough to overcome the kinetic barrier to form Li2O3 phase, although thermodynamically it is stable; at 48 GPa, however, the pressure and temperature bring Li2O3 to a metastable phase, as we observed experimentally despite the DFT calculation not favoring formation of this phase at this pressure. Authors noticed a similar DFT calculation work was posted on arXiv, where some lithium‐rich lithium oxides are predicted as well.Comparing the DFT calculation and experimental results, we notice the discrepancy of Li3Figure2 (P4/mbm), Li2O3 (Im‐3m), and LiO4 (Ibam). Also, the Li2O2 phase (P63/mmc) is robustly stable up to 57 GPa at room temperature. The high pressure LiO2 and LiO4 phases can be considered as lithium atoms inserted between the layers of ε‐O8 phase, while the Li2O3 structure inherits the local atomic arrangements from the ambient Li2O2 and LiO2 (Pnnm) structures. At increasing temperature, the high‐pressure LiO2 (P4/mbm) phase losses its stability and converts to LiO4 and Li2O3 or decomposes to Li2O2 and O2. This variety of lithium oxides provides a rich field for discovering candidate electrode materials for lithium–oxygen batteries. For instance, the LiO4 phase could offer an opportunity of using the material as electrode for a close and novel lithium oxygen cell since LiO4 can provide the O2 needed in this case.In conclusion, under high pressure and temperature, several stable phases of oxygen‐rich lithium oxides can be synthesized (see 4High Pressure XRD Measurement: Synchrotron XRD measurements in the angle‐dispersive diffraction mode were conducted in a diamond anvil cell at beamlines 16BM‐D and 16ID‐B by the High Pressure Collaborative Access Team (HPCAT) at the Advanced Photon Source, Argonne National Laboratory. The monochromatic beam was focused to around 5–10 µm full width at half maximum, and the powder diffraction patterns were recorded by a Mar345 image plate at the 16BM‐D station and a Pilatus 1M detector at the 16ID‐B station. The 2D patterns were integrated to a 1D profile with Fit2d software, and the Rietveld structural refinements were conducted with the GSAS package.2O2 powder and liquid oxygen was compressed to 15 GPa, followed by high power laser heating to 2200 K. This heat treatment vaporized the carbon from the anvil surface, which did not have a thermal insulating layer, thus adding a small amount of carbon to the chemical reaction. The surface damage of the anvil after laser heating showed clear evidence of missing carbon from the anvil. The X‐ray wavelength used was 0.39 995 Å. On the third run, with the help of thermal insulator layers of LiF, no diamond anvil damage was observed after laser heating to above 2000 K. The X‐ray wavelength used was 0.40 663 Å. In both runs 2 and 3, no ruby was loaded in the diamond anvil cells to avoid the possible chemical reaction during laser heating. The pressures were calibrated with the diamond Raman signal. For the final comparison run on the mixture of Li2O2 and oxygen at room temperature up to 53 GPa, the X‐ray wavelength used was the same as third run. Since no heat treatment was involved, a small Ruby ball was loaded in the sample chamber as pressure calibrant.For the first run, the X‐ray wavelength was 0.4123 Å, and the pressure was calibrated by the ruby luminescence method. In the second run, a mixture of LiAb Initio Crystal Structure Searching: The first principle calculations were performed in the framework of density functional theory22s1) and 6 for O atoms (2s22p4). For crystal structure searching, we used USPEX with a plane‐wave basis set cutoff energy of 800 eV.Phonon Dispersion Curves from First Principles: Phonon calculations were conducted based on density functional perturbation theory2, Li2O3, and LiO4 at 14.4 and 48 GPa, as shown in Figure S4 (Supporting Information).SupplementaryClick here for additional data file.