Atmospheric chemistry considerations implicate an increase in CH4 sources, but process‐based estimates fail to reproduce the required amplitude. CH4 stable isotopes provide complementary information that can help constrain the underlying causes of the increase. We combine Earth System model simulations of the late preindustrial and LGM CH4 cycles, including process‐based estimates of the isotopic discrimination of vegetation, in a box model of atmospheric CH4 and its isotopes. Using a Bayesian approach, we show how model‐based constraints and ice core observations may be combined in a consistent probabilistic framework. The resultant posterior distributions point to a strong reduction in wetland and other biogenic CH4 emissions during the LGM, with a modest increase in the geological source, or potentially natural or anthropogenic fires, accounting for the observed enrichment of δ13CH4.The observed rise in atmospheric methane (CH 4 cycle as priors, with ice core observations in a Bayesian frameworkFor the first time we combine process‐based models of the CHFor the glacial we infer unchanged or increased geological sources, but a halved wetland flux, which is not captured by process modelingThis suggests missing processes in methane models, with potentially important consequences for confidence in future methane projections Its concentration has risen sharply over the past two centuries, reaching 1,799 ppbv by Common Era (CE) 2010  is among the largest such changes, with an increase from 375 ppbv to 680 ppbv  from −79 ± 4‰ to −98 ± 3‰  at the LGM and a reduction in wetland emissions (−41%) and CH4 lifetime (−32%). The latter is in conflict with most model‐based estimates.Fischer et al.  used a bδ13CH4 is positively correlated with the atmospheric concentration of CO2 and not CH4. This suggests that the observed variations of δ13CH4 likely arise through the effect of CO2 and climatic changes on the isotopic fractionation rates of CH4 sources or sinks , standard+LGM humans, which additionally incorporates an empirically based estimate of hunter‐gatherer fire activities during the LGM  values of 447 ppbv, −48.6‰, and −93.7‰, respectively, also shown in Figure 4 concentration and shift in isotopes. The low‐fire scenario in which all fire emissions are set to 10% of the late preindustrial results in a better prediction of the LGM CH4 change, but at the expense of the δ13CH4 and δD(CH4), which both shift to more isotopically depleted values. The standard‐fire+LGM humans has the opposite effect but is closer to the standard‐fire simulation. As a result, to simplify the presentation we only use the standard‐fire scenario  are potentially influenced by the isotopic signature of leaf carbon. For example, C3 and C4 plants exhibit very different leaf carbon isotope ratios. Termites are not observed to show any relationship between emitted δ13CH4 and the proportion of C3 versus C4 plants , CO2, climate, and dynamic vegetation separately cause changes of −0.29‰, −0.11‰, and 0.21‰, respectively . The CO2 forced change dominates in the tropics, whereas climate is more significant in midlatitudes. Both of these factors lead to negative excursions for the LGM relative to the preindustrial. The vegetation distribution change has a more widespread and mixed influence, with regions of negative shifts  in leaf δ13C in southern Africa, western north America, and Eurasia, and positive changes in South East Asia, South America, and Australia.To decompose the drivers of the leaf 4 emissions was calculated from the weighted sum of the simulated monthly leaf δ13C values, using either wetland CH4 emissions or monthly burned area (H17), see Figure δ13CH4 between tropical and extratropical wetlands and assumed no offset between δ13Cleaf and that of biomass burning  and similarly for biomass burning  were included along with the small change in fractionation factors due to the temperature dependence of methanogenesis and uptake in the box model. The zonally averaged values can be summed to give the global total when weighted by changing sources strengths in the three bands, given in Table δ13C on the atmospheric isotopic signature is relatively limited at 0.6‰. This does not capture a substantial fraction of the observed change and implies that changes to both the source mixture and the individual source signatures are required.The calculated changes in the isotopic signature of wetland 44 sources that are consistent with the ice core observations, we employ a Bayesian framework  values. Assigned observational uncertainties are 2 ppbv, 0.3‰, and 4.0‰ for CH4, δ13CH4, and δDCH4, respectively , (ii) both the concentration and δ13CH4 or (iii) δDCH4, and (iv) the concentration and both isotopes (blue). The top three panels show the fit to the LGM ice core observations in each case. In the lower panels, the distributions of the model parameters are shown . Prior distributions are shown by dashed lines.The MCMC algorithm was run for one million iterations, with the initial 250,000 iterations discarded  well. All four cases point to a reduced tropical wetland CH4 flux at the LGM. The posterior mean derived by considering all observations is 32.0 ± 5.0 TgCH4/year (mean ±1 standard deviation), and this is 59% of the prior mean (54.7 ± 15 TgCH4/year), or 44% of the preindustrial value. Northern wetlands are inferred to decrease to 22% of the preindustrial value when all observations are included, a reduction of 33% relative to the prior distribution. The prior and posterior are less divergent for termite, geological, and hydrate source strengths. The geological term is the only source with an increase at the LGM (posterior mean is 10.5 ± 2.5 TgCH4/yr), prior (10.0 ± 2.8 TgCH4/year). This emerges in both the all data and the concentration with δ13C cases. It is driven by the high δ13CH4 of geological sources (−33‰) and the model‐informed prior distribution (H17) on biomass burning (the other δ13CH4‐enriched source), which points to a reduction in biomass burning at the LGM.The results show that the MCMC algorithm reproduces the observed LGM methane concentration, 4 concentration alone or with one isotope type, are caused by the interplay between satisfying the prior information and the observations. For example, in the CH4‐only case, both the biomass burning and geological terms are reduced, because the isotopic constraint is absent and because this allows the wetland source to stay nearer to its prior distribution.In the remaining cases, the solutions that stem from only considering the CHδ13CH4 and δD(CH4) are not entirely consistent. Including the concentration with deuterium leads to an increased hydrate flux and a reduced biomass burning term, while δ13CH4 leads to the opposite behavior. Overall the reduced tropical wetlands are required to satisfy the concentration constraint, while relatively high biomass burning and geological sources are required by the δ13CH4. The main constraint from δD is more difficult to isolate. It acts in a compensatory manner between the biomass burning and hydrate terms.The inferences drawn separately from the 4 concentration and isotope observations replaced with the late preindustrial values . Though the results are likely model dependent , they suggest a limited role for changes in the isotopic discrimination of vegetation in the glacial‐interglacial changes in δ13CH4. This raises the question of why there is such a clear correlation between δ13CH4 and CO2 in the ice core record, which Möller et al.  also underestimates the changes in CH4 stable isotopes. Accounting for changes in isotopic discrimination of vegetation does not explain the observed LGM‐PI δ13CH4 shift.Current process‐based estimates suggest that a 50% reduction in sources is required to explain the CH4 emissions. Future work needs to better understand the climatic sensitivity of natural CH4 sources, including wetland and geological terms, and this study suggests that incorporation of CH4 stable isotopes into Earth System model simulations is one avenue for further progress.We applied a Bayesian framework to resolve the potentially conflicting information from models and observations. The results suggest that the concentration change was predominantly driven by wetlands, but the inferred emissions response is at the extreme end of model predictions. The isotopic change is either driven by a relatively limited reduction in biomass burning at the LGM, perhaps brought about by human activities (Kaplan et al., Supporting Information S1Click here for additional data file.


Pharmacological treatments can extend mouse lifespan, but lifespan effects often differ between sexes. 17‐α estradiol (17aE2), a less feminizing structural isomer of 17‐β estradiol, produces lifespan extension only in male mice, suggesting a sexually dimorphic mechanism of lifespan regulation. We tested whether these anti‐aging effects extend to anatomical and functional aging—important in late‐life health—and whether gonadally derived hormones control aging responses to 17aE2 in either sex. While 17aE2 started at 4 months of age diminishes body weight in both sexes during adulthood, in late‐life 17aE2‐treated mice better maintain body weight. In 17aE2‐treated male mice, the higher body weight is associated with heavier skeletal muscles and larger muscle fibers compared with untreated mice during aging, while treated females have heavier subcutaneous fat. Maintenance of skeletal muscle in male mice is associated with improved grip strength and rotarod capacity at 25 months, in addition to higher levels of most amino acids in quadriceps muscle. We further show that sex‐specific responses to 17aE2—metabolomic, structural, and functional—are regulated by gonadal hormones in male mice. Castrated males have heavier quadriceps than intact males at 25 months, but do not respond to 17aE2, suggesting 17aE2 promotes an anti‐aging skeletal muscle phenotype similar to castration. Finally, 17aE2 treatment benefits can be recapitulated in mice when treatment is started at 16 months, suggesting that 17aE2 may be able to improve aspects of late‐life function even when started after middle age. Demographic models predict that the number of people living to old ages in high‐income countries will increase  were either removed (gonadectomy) or exposed but remained in place (sham gonadectomy). These animals then began 17aE2 treatment at 4 months of age, or stayed on a control diet, and were euthanized at 25 months. Animals in cohort 2 did not undergo any surgery and were euthanized at 22 months. The main cohort of animals began 17aE2 treatment at 4 months, while a subset remained on the control diet until 16 months of age, but were then switched to 17aE2 treatment at 16 months of age. This allowed us to test whether a late onset treatment of 17aE2 can also produce functional benefits later in life, and to compare the anti‐aging effects of treatment onset at these two time points.22.1p = 0.001 for the interaction between 17aE2 treatment and time in a repeated measures ANOVA of monthly body weights), but were similar in intact (sham‐operated) animals of both sexes  were measured in gastrocnemius muscles collected from animals in cohort 1 and fixed in 10% buffered formalin immediately at dissection. Compared to samples taken from 6‐month‐old young controls, 25‐month‐old intact control mice showed a reduction in average muscle fiber CSA, an effect that was ameliorated in intact male mice treated with 17aE2 from 4 months Figure  .The gastrocnemius muscle is made up of a mix of muscle fiber types, although the large majority are type 2b fast‐twitch muscle fibers. Fast‐twitch muscle fibers typically show the greatest atrophy during aging, with fewer changes observed in slow‐twitch oxidative fibers , suggesting that variation in body weight between groups does not account for the improved grip strength in male mice treated with 17aE2. Rotarod performance was negatively related to body weight . When this negative relationship was accounted for by including weight as a covariate, mice treated with 17aE2 still showed improved rotarod performance relative to body weight . We note that the same relationship between body weight, rotarod performance, and 17aE2 treatment was observed in males in cohort 2 at 22 months. Combining both datasets to increase statistical power revealed a significant effect of 17aE2 treatment in male mice across both cohorts (p = 0.015), even when accounting for variation in weight by including body weight as a covariate.To test whether the effects of 17aE2 treatment on muscle aging are associated with improvements in late‐life physical function, we assessed forepaw grip strength and rotarod performance. Forepaw grip strength was assessed at 22 months in cohort 2 and was lower in these animals than in a comparable set of 6‐month‐old controls Figure a. We asst Figure c, althou2.4To test whether sex‐specific morphological responses to 17aE2 during aging were matched by sex‐specific biochemical changes in muscle, we conducted an untargeted analysis of primary metabolites in quadriceps muscle sampled at 25 months from Cohort 1. Using a 2‐way ANOVA to identify metabolites showing a sex‐specific response to 17aE2 in intact (sham‐operated) animals, we observed 8 metabolites that showed a significantly different response to 17aE2 treatment in each sex after correction for false discovery rate . This reflects an elevated abundance of amino acids in intact females on the control diet, but a switch under 17aE2 treatment, with intact males increasing amino acids and females showing a significant reduction  that explained 51% of the variance across the dataset and was significantly correlated with the abundance of all amino acids, although the relationship was strongest with serine and weakest with alanine Figure b, reflecn Figure b.2.5p = 0.003), further demonstrating that the male response to 17aE2 depends on the presence of male gonads. In females, ovariectomy prior to treatment also blocked the female‐specific declines in amino acids , indicating that the amino acid responses to 17aE2 in intact animals of both sexes were linked to the presence of male and female gonads.We used the principal component analysis to test whether the sex‐specific amino acid responses to 17aE2 were dependent on the production of gonadally derived hormones, by comparing metabolite responses to 17aE2 in mice that were gonadectomized at 3 months, prior to 17aE2 treatment, with responses observed in sham‐operated (intact) animals. While intact males show an elevation in amino acids with 17aE2 treatment, this effect is blocked in males that were castrated prior to drug treatment. This is reflected in the lack of response in PC1 to 17aE2 treatment in castrated males Figure b, and ths Figure b, and thp = 0.002). Specifically, males show an increase in relative LC3BII abundance after 17aE2 treatment , but not in the comparison with castrated to intact males (p = 0.18).Increased amino acids in muscle may represent a consequence of altered protein synthesis or breakdown, both of which can be regulated by the actions of gonadally derived hormones . The sex difference in 4EBP1 protein levels is also not observed in gonadectomized animals on the control diet .We also examined effects of 17aE2 on mTORC1 signaling, a key regulator of protein synthesis that has sexually dimorphic effects on physiology and aging in mice .We also assessed whether sex‐specific responses to 17aE2 in muscle structure and function were regulated by gonadal hormones, by comparing responses to 17aE2 in sham‐operated and castrated animals in cohort 1. Males castrated prior to 17aE2 treatment showed no increases in the weight of the quadricep muscle with 17aE2 treatment Figure , consist2.7In the second cohort of animals exposed to 17aE2 treatment, we evaluated a randomly selected subset of mice where treatment with 17aE2 began at 16 months of age. This allowed us to test whether the benefits of 17aE2 treatment for skeletal muscle aging and physical function could be recapitulated with a treatment beginning after middle age, an approach that may have advantages in some clinical settings if applied to humans. Male mice treated with 17aE2 from 16 months showed a larger gastrocnemius muscle weight at 22 months when compared to untreated animals, with this improvement being equivalent to that seen in individuals treated from 4 months  is not observed in quadriceps taken from animals with equivalent treatment sampled at 12 months of age . In cohort 2, a randomly selected subset of animals was maintained on the control diet until 16 months of age and then was switched to be treated with 17aE2 for the last 6 months of treatment.At 4 months of age, animals were randomly allocated to control or 17aE2 treatment. Animals in the control group remained on the 5LG6 diet, while animals allocated to 17aE2 had their diet switched to a food containing this drug at 14.4 ppm  and increased to 40 RPM gradually over a 5‐min period. The time at which the animal fell off the rotarod was used as a score, with each animal tested three times and the mean score used in analysis. The second cohort underwent the same testing protocol at 22 months of age. A subset of animals in cohort 2 were tested for grip strength using an EB1‐BIO‐GT3 grip strength meter with an EB1‐GRIP‐Mouse Grid. Subjects were removed from their cage by the base of the tail and suspended above the grid until their forepaws griped the grid. The tail was gently pulled in a horizontal direction away from the grid until the mouse released its grip. The maximal force was recorded. Each animal was tested six times with a 10 s rest between each. The mean of the six tests was used for analysis. All tests were conducted by an experimenter blind to treatment group and surgery status.4.3Animals were euthanized and tissues harvested during the morning after 18 hr of fasting. Tissues were weighed and then immediately frozen with liquid nitrogen and stored at −70°C unless otherwise stated. Deleted methodology for western blots, metabolomics, histology, and immunofluorescence is in the supplementary information.None declared. Click here for additional data file. Click here for additional data file.


This five-dimensional  approach enabled us to track the chemical evolution of many particles across the catalyst bed and relate these changes to the gas environment that the particles experience. Rietveld analysis of some 2 × 106 diffraction patterns allowed us to extract heterogeneities in the catalyst from the Å to the nm and to the μm scale  under different chemical environments. We are able to capture the evolution of the Ni-containing species and gain a more complete insight into the multiple roles of the CeO2-ZrO2 promoters and the reasons behind the partial deactivation of the catalyst during partial oxidation of methane.We report the results from the first 5D tomographic diffraction imaging experiment of a complex Ni–Pd/CeO 2 – ZrO2/ Al2O3 catalyst used for methane reforming.Multi-scale chemical imaging holds the potential to revolutionize our understanding of the relationships between structure and functionality in complex catalytic materials. Here the authors report the results from the first 5D tomographic diffraction imaging experiment of a complex Ni – Pd/ CeO Such materials are known to change with time under operating conditions, and in order to gain an insight into the structure–function relationships, it is highly desirable to study them in situ with spatially resolved techniques7. Non-destructive X-ray spectroscopic/scattering techniques are typically employed to study such materials, but it is the brilliant X-rays generated at synchrotrons coupled with state-of-the-art detectors and tomographic data collections that now allow the acquisition of spatially resolved signals from within the interiors of intact objects under operating conditions11. Operando chemical imaging in 5D by synchrotron X-ray Diffraction-Computed Tomography (XRD-CT) could emerge as a game-changing technique for the non-destructive investigation of functional materials in space and time under real process conditions14. This chemical tomographic technique, along with the complementary pair distribution function computed tomography technique, has been employed in several cases to study catalysts and more recently batteries, under in situ/operando conditions24. Such multi-scale chemical imaging tools hold the potential to revolutionise our understanding of the relationships between structure and functionality in complex, real world, catalytic materials ; information that can help us unravel the mechanisms underpinning catalytic reactions and catalyst deactivation and rationally design improved materials.Heterogeneous functional materials and devices, like catalytic solids, batteries and fuel cells tend to possess complex structures where the 3D spatial distribution of the various components is rarely uniform2) at gas-to-liquids (GTL) industrial plants (Fischer–Tropsch synthesis)27. The POX reaction is only mildly exothermic and it leads to a H2/CO molar ratio of 2, which is suitable for methanol and Fischer–Tropsch synthesis28. Given the promise of the POX reaction to decrease significantly the energy requirements of GTL plants, it is highly desirable to understand the spatio-temporal physico-chemical changes taking place in the real working catalyst. Such information is crucial in order to rationally design improved catalysts as the chemical  and temperature gradients in a reactor  can have a direct impact on the chemical state of the catalyst34.The catalytic partial oxidation of methane (POX) is considered a very promising alternative to the highly energy-demanding steam reforming of methane (highly endothermic reaction) to produce synthesis gas . However, Ni/Al2O3-based catalysts are usually prone to deactivation with time under POX reaction conditions for a variety of reasons; the most often reported in literature being carbon deposition on active Ni sites , sintering of Ni particles and solid-state reactions involving Ni 36. The CeO2–ZrO2 support promoters can offer improved oxygen storage capacity and redox properties, enhanced metal-support interaction (higher dispersion and stability of the Ni species) and increased catalytic performance at lower temperatures, while small amounts of noble metal  can enhance the reducibility of the Ni species through a hydrogen spillover mechanism41. In this work, we employed the XRD-CT technique to investigate the behaviour of a complex 10 wt.% Ni–0.2 wt.% Pd/10 wt.% CeO2–ZrO2/Al2O3 catalyst under different operating conditions.Ni/Al42. This new data collection strategy, coupled with the brilliant X-rays produced at the ESRF and the state-of-the-art Pilatus2M CdTe area detector allowed us to collect an XRD-CT dataset in <2 min ; the data collection rate is at least one order of magnitude faster than that previously reported  are allowed to move simultaneously min ie, 17 s; theq-space), and one dimension covering imposed chemical environment. Actually, one could consider this experiment covering more than five dimensions . The behaviour of a Ni–Pd/CeO2–ZrO2/Al2O3 catalyst was investigated during reduction and re-oxidation (redox experiment). In total, four 3D-XRD-CT datasets (each consisting of 30 XRD-CT datasets) were collected at the following operating conditions: ambient (1), 800 °C under He flow (2), 800 °C under 20% H2/He flow (3) and 800 °C under 20% O2/He flow (4). We present the results from the full Rietveld analysis of these datasets; the phase identification and Rietveld approach taken are described in the methods and Supplementary methods sections. The results represent the analysis of ~2 × 106 diffraction patterns, which is an order magnitude larger compared to that previously reported45.The in situ experiments were performed at beamlines ID31 and ID15A of the ESRF using the same catalyst , but its spatial distribution is not the same in all particles. We note, in the traditional 2D XRD-CT scan, it is not known whether the bottom, middle or top of each catalyst particle is probed, but as can be seen in Fig. 0 wt.%, b2 and ZrO2 phases are most interesting. As is clear in Fig. 2 map shows an egg-shell distribution . The same phenomenon is observed in the ZrO2 phase distribution volume shown in Fig. 2 phase (due to co-impregnation method used for the preparation of the CeO2–ZrO2/Al2O3 support), that is not the case. There is a strong signal generated by the CeO2 phase in the regions where ZrO2 is also present, but it does not diminish closer to the centre of the catalyst particles . As shown in Fig. 2/ZrO2 distributions are in full agreement with the rest of the XRD-CT datasets. The maps presented in the middle row of Fig. 2, ZrO2 and NiO phases, while the volume rendering of these data are presented in Fig. The CeO2 and ZrO2 phases. The range of the ZrO2 crystallite size is generally seen to be very narrow (ca. 8–12 nm), but in the regions where there are hotspots (areas of high concentration) of ZrO2 . For example, the catalyst particle rich in CeO2 shown in Fig. 2 phase distribution map), also corresponds to highly crystalline CeO2 . However, the range of CeO2 crystallite sizes is large in the other catalyst particles. In fact, the values of the CeO2 crystallite sizes are shown to follow an egg-yolk distribution. It can be seen in Fig. 2, the CeO2 crystallite size varies between 8 and 12 nm, while at/close to the catalyst surface, where there is ZrO2 present, the CeO2 crystallite size is less than half that value . This phenomenon is more apparent at the CeO2 crystallite size volumes presented in Fig. 2 crystallite size follows an egg-yolk distribution in all catalyst particles.Characteristic examples are the crystallite size maps and volumes of the CeOow ca. 8–2 nm, butow ca. 8–2 nm, butow ca. 8–2 nm, butow ca. 8–2 nm, but2 phase. Explicitly, the NiO concentration is relatively higher close to the surface of the catalyst particles. In these regions, the average NiO crystallite size is also typically higher compared to the inner core of the catalyst particles . This may imply that in this particle there is a different chemical interaction between the various components . It should be noted though, that no other crystalline phases were identified to be present in this particle.The NiO crystallite size distribution shows a similar behaviour to that observed for the ZrOles Fig. . Howevera of the CeO2 and ZrO2 unit cells. There are two distinct ranges regarding the values of the ZrO2 lattice parameter a. More specifically, as shown in Figs. 2 . In all other regions, where CeO2 is present, the ZrO2 lattice parameter a corresponds to higher values (>3.62 Å). This implies that there are actually two different ZrO2 phases present in the sample: (1) a high-purity ZrO2 present in areas of high concentration of ZrO2 and (2) a Zr-rich CexZryO2 phase (Ce incorporation in the ZrO2 unit cell leads to larger unit cell)49. Interestingly though, two distinct CeO2 crystalline species seem to be present in the catalyst particles too. As shown in Figs. 2 lattice parameter a near the centre of the catalyst particles is notably higher (5.38–5.40 Å) compared to closer to the surface of the catalyst (5.34–5.36 Å), where ZrO2 is present too. This implies that there is incorporation of a Zr4+ substituent onto a Ce4+ site in the CeO2 unit cell in the regions where both CeO2 and ZrO2 are present leading to Ce-rich CexZryO2 phase 40. Summarising the CeO2–ZrO2 results derived from the Rietveld analysis of the 3D-XRD-CT data of the fresh catalyst, it can be concluded that there are four distinct crystalline CeO2–ZrO2 species present in the catalyst  phase near the surface of the catalyst particles;Small crystallites of a Ce-rich Ce2 phase closer to the core of the catalyst particles;Larger crystallites of a higher purity CeOxZryO2 (x«y) phase near the catalyst surface;Small crystallites of a Zr-rich Ce2 phase where there is high concentration of ZrO2 .Larger crystallites of a higher purity ZrOThe maps shown in the bottom row of Fig.  sample:  a high-pare low <.6 Å. In −1). The catalyst remained at 800 °C under He flow (volumetric flow rate of 100 ml min−1) for 1 h while collecting a 3D-XRD-CT scan. The gas mixture was then switched to a 20% H2/He . Another 3D-XRD-CT measurement was then performed under these reducing conditions in an attempt to capture the state of the activated catalyst. Finally, the gas mixture was switched to 20% O2/He  and a last 3D-XRD-CT scan was performed .After the 3D-XRD-CT measurement was performed at ambient conditions, the temperature of the system was increased to 800 °C (temperature ramp rate of 20 °C per min) under the flow of He  is essential in order to avoid the NiO to NiAl2O4 transition and reduce the NiO to the desired, active metallic Ni phase, as the formation and growth of the NiAl2O4 phase is a temperature-driven phenomenon taking place even under inert chemical environment (He flow). However, the phase distribution maps of NiO, NiAl2O4 and CexZryO2 revealed that NiO is mainly present near the surface of the catalyst particles (where the most of the CexZryO2 phases are present) while NiAl2O4 is closer to their core. This provides direct evidence that one of the roles of the CexZryO2 phases is to stabilise the NiO phase and suppress the formation of the undesired NiAl2O4 phase ). The crystalline metallic Ni phase is seen to form quickly and is present even in the first XRD-CT data collected under H2 flow (bottom of the sample volume probed—XRD-CT scan 1). However, the continuous growth of the Ni diffraction signal and decrease of the NiO and NiAl2O4 peaks  imply that NiO and NiAl2O4 were still present during the acquisition of this 3D-XRD-CT measurement . In order to decouple these phenomena, we performed another diffraction experiment where we show that the NiO/NiAl2O4/Ni concentration gradients shown in Fig. 23.The results from the Rietveld analysis of the 3D-XRD-CT data collected during reduction  are presented in Fig. 2O4 phases along the catalyst bed can also be readily observed. A closer inspection of the results also reveals that the NiAl2O4 phase, which is known to be more difficult to reduce compared to the NiO phase, remains present at positions of the catalyst bed where the NiO diffraction signal has already diminished35. Of course, as also shown in the 30 XRD-CT-summed diffraction patterns presented in Fig. The phase distribution volumes shown in Fig. xZryO2 mixed oxides seem to be the predominant phases compared to the respective higher purity CeO2 and ZrO2 phases. This is implied from the CeO2 phase distribution volume shown in Fig. 2 is seen to be mainly present near the surface of the catalyst particles, similar to the ZrO2  phase distribution volume (egg-shell distribution). It should be emphasised that the most important result obtained from this 3D-XRD-CT dataset is related to the correlation between the Ni and the CexZryO2 phase distribution maps. Specifically, as shown on the right side of Fig. xZryO2 phases are present. This result provides direct evidence that the most important role of the CexZryO2 promoters is to enhance the reducibility of the NiO and the formation of the active catalyst component, the metallic Ni phase.The CeQ values than expected (ca. Q = 2.85 Å−1) corresponding to a smaller unit cell than a pure Pd one  and that Pd exists as a NixPdy phase  rather than a high-purity Pd phase; the formation of NixPdy alloys has been previously reported in other catalytic systems55.Finally, it should be mentioned that metallic Pd is observed at the 3D-XRD-CT data collected under reducing conditions. The phase distribution volume of Pd shown in Fig. 2/He. The re-oxidation process of metallic Ni to NiO is really fast as the metallic Ni peak is absent in all 30 XRD-CT datasets and only the NiO and NiAl2O4 phases can be observed . As discussed previously, NiO is the only observed crystalline phase in the fresh catalyst, while NiAl2O4 is the predominant Ni-containing phase in the 3D-XRD-CT dataset collected at 800 °C under He flow. Under these conditions, the NiO follows mainly an egg-shell distribution, similar to the CexZryO2 phase (where y > x), while the NiAl2O4 phase is mainly present closer to the core of the catalyst particles. This information provides direct evidence that the CexZryO2 suppresses/delays the formation of the undesired NiAl2O4 phase.The evolution of the Ni-containing crystalline phases during this 5D tomographic diffraction imaging redox experiment is summarised in Fig. 2O4). This is also illustrated in panel b where it is shown that first the NiO is reduced and then the NiAl2O4 phase (probing from bottom to top of the bed). A similar time effect is shown during the re-oxidation experiment as the top of the bed (last scan) contains less NiO compared to the bottom of the bed (first scan) indicating that the NiO to NiAl2O4 transition can also be captured.As discussed previously, under reducing conditions, a Ni chemical gradient is observed along the length of the bed, which is a time effect  to form the metallic Ni phase. The catalyst bed was then exposed to a POX reaction mixture , which was kept constant for the duration of the experiment and seven XRD-CT scans were collected in total.The last experiment was an operando XRD-CT experiment of the same catalyst during the partial oxidation of methane. The same protocol was used for the pre-treatment of the catalyst as in the redox experiment. Specifically, the temperature of the system was increased under the flow of He up to 800 °C (temperature ramp rate of 20 °C per min) and then the catalyst was activated under the flow of 20% H2O4 remain only in traces for the duration of the experiment, while Al2O3 is not changing; for these reasons, these maps are not shown here and emphasis is given on the active catalyst components. More importantly, the crystalline Ni phase is seen to be very stable for the duration of the POX experiment (ca. 3 h). We also note there is no evidence for formation of crystalline Ni carbide phases 56.In Fig. 4/O2 molar ratio is 2:1 (stoichiometric ratio for the POX reaction)59. Here, a higher CH4/O2 molar ratio of 4:1 was used in an attempt to force the catalyst to deactivate faster and capture the corresponding solid-state changes. As it is shown in Fig. 2O4 mentioned previously. However, as is shown in the bottom part of Fig. The typical reaction mixture used to test the performance of POX catalysts is a gas mixture where the CHxZryO2 phases is seen to decrease with time under reaction conditions although one may argue that the decrease is not very significant (ca. 10 % decrease after 3 h). The Pd phase  is also seen to decrease as a function of time under the POX reaction conditions. It should be noted though that no other crystalline Pd-containing species were observed in the XRD-CT data collected during the POX experiment. The mass spectrometry data acquired during the POX experiment are presented in Supplementary Figure 55. From the phase distribution maps presented in Fig. xZrx1−O2 are less prone to graphite formation; in these particles, the CexZr1−xO2 forms a protective layer near the surface of the catalyst particles. As mentioned previously, the POX reaction experiment lasted for ~3 h and it can be seen that the diffraction signal from this phase doubled during this time period , leading to 84% CH4 conversion and almost 100% H2 yield. However, exposing the catalyst to the CH4/O2 molar ratio equal to 4:1 for 3 h, partially deactivated the catalyst as it did not regain its initial performance when the reaction mixture was switched back to 2:1. As shown by the in situ XRD-CT data, this is highly likely to be due to the graphite formation  by X-ray chemical imaging studies and these have been shown to yield far greater insight into the structure of single particles or else a handful of particles in a cross-section21. Despite these important advantages, there will always be doubts as to whether a single particle/group of particles are truly representative of their ilk in terms of composition and/or behaviour . The biggest challenge in this regard is to be able to follow changes in the catalyst particle structure as a function of their location in a reactor. It is well known that catalyst particles experience variation in reactant/product composition both radially and longitudinally, thereby rendering attempts to correlate structure–function relationships in a reactor difficult and in some cases impossible. The 3D chemical imaging results presented herein obtained in real time and under operando conditions therefore represent significant step in attempting to extract more meaningful correlations between structure and function in catalytic and other functional materials.It is well known that time-resolved in situ or operando X-ray spectroscopic or scattering studies provide information on evolving structure activity relationships in functional materials with researchers in the field of heterogeneous catalytic materials shown historically to be keen to exploit their potentialxZryO2 crystalline species present in the sample. This information could only be obtained by collecting the 3D-XRD-CT dataset and performing the Rietveld analysis. It should be emphasised that the analysis of the reconstructed data (>2 × 106 diffraction patterns) although very challenging, is now possible on reasonable timescales so as to obtain unprecedented physico-chemical information regarding the state of the catalyst under different operating conditions. We were able to create, for the first time, 3D maps corresponding to phase distributions, crystallite sizes and unit cell lattice parameters, demonstrating the multi-length scale information that can be obtained from performing such an experiment and the corresponding data analysis. As such, we were able to effectively track the evolution of the crystalline Ni-containing species under different chemical environments . NiO was the only crystalline Ni-containing phase present in the fresh catalyst and it was shown that a reducing environment is necessary in order to avoid the formation of the undesired NiAl2O4 phase at high temperatures. It was shown that the role of the CexZryO2 promoters is to suppress this conversion and enhance the reduction of the Ni oxides to the catalytically active metallic Ni. It should be emphasised that this chemical information could have not been obtained with any conventional technique but only from the spatially resolved diffraction signals present in an XRD-CT dataset.Specific to this study, we have shown how the Rietveld analysis of the reconstructed XRD-CT data allowed us to differentiate between four different Ce2O4 concentration gradients were observed along the catalyst bed during reduction but not during re-oxidation. This phenomenon was attributed to the water formation during the reduction of the NiO/NiAl2O4 species to metallic Ni. It was also shown that the re-oxidation of the catalyst at 800 °C under 20% O2/He flow can have a strong impact on the NiO crystallite size. Metallic Ni was seen to be the main crystalline Ni-containing phase of the catalyst under POX reaction conditions, but sintering of the Ni crystallites took place as a function of time under reaction conditions. More importantly though, crystalline graphite was seen to form and continuously grow under POX reaction conditions which, as the laboratory catalytic activity data collected under the same conditions indicate, caused the partial deactivation of the catalyst.The reduction process was seen to be significantly slower compared to the re-oxidation. Specifically, significant Ni/NiO/NiAlWe have shown that it is possible to track the chemical evolution of many particles across a catalyst bed and relate these changes and variations to the non-uniform gas composition the catalyst particles experience. The insights and understanding gained from this in situ study can be used to rationally design improved methane-reforming catalysts. With the advancements in synchrotron brightness, detector performance, sample environment (new reactor cells) and data analysis , multi-dimensional chemical imaging techniques are bound to become increasingly easier to perform and one can readily foresee that they will replace conventional in situ XRD and X-ray imaging as the preferred method for characterising functional materials and devices .2–ZrO2/Al2O3 catalyst was prepared by sequential impregnation method. The CeO2–ZrO2/Al2O3 support was prepared by the co-impregnation method62. The microspherical (γ + δ)-Al2O3 with granules size of ~500 μm was used. Approximately 500 µm of (γ + δ)-Al2O3 was impregnated by aqueous solution of salts (cerium nitrate Ce(NO)3 · 6H2O and oxychloride of zirconium ZrOCl · 8H2O) at the required ratio. The CeO2–ZrO2/Al2O3 was dried at 120 °C for 6 h and calcined under air at 850 °C for 6 h with a heating rate of 2 °C per min. The 10 wt.% CeO2–ZrO2/Al2O3 support was impregnated by aqueous solution of nickel nitrate salt Ni(NO3)2 · 6H2O of the appropriate concentration. Then, Ni/CeO2–ZrO2/Al2O3 was dried at 120 °C for 6 h and calcined in air at 500 °C for 4 h with a heating rate of 2 °C per min. The Ni/CeO2–ZrO2/Al2O3 was impregnated by aqueous solution of palladium nitrate Pd(NO3)2 salt of the appropriate concentration. The catalyst were then dried at 120 °C for 6 h and calcined in air at 500 °C for 4 h with a heating rate of 2 °C per min. The catalyst samples used in this study were kindly provided by the Boreskov Institute of Catalysis (BIC).The 10 wt.% Ni–0.2 wt.% Pd/10 wt.% CeO2–ZrO2/Al2O3 catalysts ; the catalyst loading was 35 mg for the operando experiment . In each case, the reactor was mounted into a gas delivery stub, itself mounted to a standard goniometer . The goniometer was fixed to a rotation stage set upon a translation stage to facilitate the movements required for the CT measurement. At ID31, heating was achieved by virtue of two Cyberstar hot air blowers heating each side of the catalytic reactor while at ID15A a furnace designed for CT experiments was used. Temperature calibration was performed before all experiments using a thermocouple by measuring the temperature at the catalyst bed. During the operando XRD-CT measurements, the outflow gasses were monitored by mass spectrometry using an Ecosys portable mass spectrometer. The mass spec line was inserted inside the capillary from the top. The mass spec capillary is dragging with a constant flow rate of 20 ml min−1.The three catalytic reactors investigated in this study consisted of 10 wt.%Ni–0.2 wt. Pd/10 wt.% CeO2–ZrO2/Al2O3 catalyst were acquired at different operating conditions: (1) at room temperature, (2) at 800 °C under He flow, (3) at 800 °C under 20% H2/He flow (reduction step) and (4) at 800 °C under 20% O2/He flow (re-oxidation step). Each 3D-XRD-CT scan composed of 30 XRD-CT scans, each one collected at a different vertical position . Each XRD-CT scan lasted ~2 min. The tomographic measurements were made with 100 translation steps (translation step size of 25 μm) covering 0–180° angular range, in steps of 2.5° . The detector calibration was performed using a CeO2 NIST standard. Every 2D diffraction image was converted to a 1D powder diffraction pattern after applying an appropriate filter  to remove outliers using in-house developed MATLAB scripts63. The final XRD-CT images  were reconstructed using the filtered back projection algorithm.XRD-CT measurements were made at beamline station ID31 of the ESRF using a 70 keV monochromatic X-ray beam focused to have a spot size of 25 × 25 μm. 2D powder diffraction patterns were collected also using the state-of-the-art Pilatus3 X CdTe 2 M hybrid photon counting area detector. The total acquisition time per point was 15 ms (exposure time of 11 ms and readout time of 4 ms). Four 3D-XRD-CT scans of the Ni–Pd/CeO−1) up to 800 °C with a ramp rate of 20 °C per min. An XRD-CT scan was collected and then the inlet gas mixture was switched to 20% H2/He (50 ml min−1). Eight successive XRD maps were collected covering the whole bed . Each XRD map lasted ~5 min. Finally, a 3D-XRD-CT scan was collected after the XRD maps. The 3D-XRD-CT scan composed of 10 XRD-CT scans, each one collected at a different vertical position .XRD mapping and XRD-CT measurements were made at beamline station ID15A of the ESRF using a 91 keV monochromatic X-ray beam focused to have a spot size of 40 × 20 μm . 2D powder diffraction patterns were also collected also using the state-of-the-art Pilatus3 X CdTe 2 M hybrid photon counting area detector. The total acquisition time per point was 10 ms. Initially, an XRD-CT scan was performed at the middle of the catalyst bed at ambient conditions. The XRD-CT scan was made with 151 translation steps (translation step size of 40 μm) covering 0–180° angular range, in steps of 1.44° . Calibration and processing (5% trimmed mean filter) was carried as described in the previous section  with X-ray detection again using the Pilatus3 X detector. The temperature of the system was increased under the flow of pure He (30 ml min−1). The inlet gas was then switched to a 20% H2/He gas mixture . Finally, the catalyst bed was exposed to a POX reaction mixture , which was kept constant for the duration of the experiment.XRD-CT measurements were made at beamline station ID31 of the ESRF using a 70 keV monochromatic X-ray beam focused to have a spot size of 20 × 20 μm. Here, the total acquisition time per point was 20 ms. Tomographic measurements were made with 225 translation steps (translation step size of 20 μm) covering 0–180° angular range, in steps of 1.125° . Calibration and processing was carried as described in the section above  with X-ray detection again using the Pilatus3 X detector. The temperature of the reactor was then increased to 800 °C with a ramp rate of 20 °C per min under the flow of He . Unless stated otherwise, the Rietveld analysis of the XRD-CT data presented in this work was based on the intensity of the scale factors and should be treated as a semi-quantitative analysis. Rietveld analysis was performed using the summed diffraction pattern of each XRD-CT dataset prior to the Rietveld analysis of the XRD-CT data in order to have a good starting model before performing the batch Rietveld analysis.Quantitative Rietveld refinement was performed using the reconstructed diffraction patterns using the TOPAS software, on a voxel by voxel basisDescription of Additional Supplementary FilesPeer Review FileSupplementary InformationSupplementary Movie 1Supplementary Movie 2Supplementary Movie 3Supplementary Movie 4


This self‐floating property is simply realized by a carbonization process other than the complicated surface modification process. The simple synthesis procedure helps to increase not only the solar absorption but also the retention of W18O49 in the porous net structure. This composite absorbs almost the whole solar spectrum and generates localized heat at the surface, which is beneficial for water evaporation. Its water evaporation rate is 6.6 times higher than that of pure water. It has a stable cyclic performance over ten cycles under the illumination of simulated sunlight (500 W Xe lamp). Its flexibility makes it easy to reuse and transfer, which is evaluated by the bending deformation test. The W18O49/carbon foam composite is a prospective material in solar energy conversion field, and the preparation procedure is feasible to scale‐up.A flexible, self‐floating W Seawater desalination is a very promising means to gain fresh water.2O3),2−xS),3−x)To enable efficient solar desalination, the broadband and efficient light absorption of this self‐floating composite is the primary condition.18O49 as efficient light absorbing material. And the porosity enables the melamine foam a high loading capability of W18O49. The final seawater desalination composite was fabricated by calcining the melamine foam loaded with W18O49 in N2 at 500 °C. The composite is beneficial for solar steam generation due to its capability of self‐floating at the water surface and good flexibility.Melamine foam is an optimal substrate due to its high porosity, low density, and hydrophilicity. Whereas, melamine foam will sink after absorbing water and this could be tuned by carbonization process . Thus, we choose the porous melamine foam as the substrate with W2The X‐ray diffraction (XRD) patterns were recorded by PANalytical B.V. Empyrean X‐ray Powder Diffraction with Cu Kα radiation over a range of 10–70° (2θ) with 0.02° per step. Scanning electron microscope (SEM) images were performed with JSM‐6700F electron microscope. X‐ray photoemission spectroscopy  was performed using monochromatic Al Kα radiation (1486.6 eV). Solid UV–vis absorption was performed by using Lambda 950 spectroscope. The thermal image was taken by Fluke thermal imaging camera.3Figure18O49/carbon foam composite. These two XRD patterns almost have the same diffraction peaks except from the additional peaks at ≈23° and 48° which could be clearly observed in Figure 18O49 ,18O49 is successfully loaded into the carbon foam. Furthermore, monoclinic W18O49 crystal could withstand lots of oxygen vacancies due to its unique crystal structure. And the oxygen vacancies can be tuned through the environment of redox reaction.2 atmosphere does not change the original crystal phase.18O49 is exactly caged into the carbon foam (W18O49 was dyed in red and the carbon foam was dyed in green). As a matter of fact, the most common morphology of W18O49 is nanowire due to its preferential growth direction along the 〈010〉 direction.18O49 nanowires aggregate and interweave with the porous net of the foam during the annealing process, which exactly insures the retention of W18O49 in the porous foam during its practical application. This is important and essential for its practical and long‐term application.The top and side view SEM images of the composite intuitively revealed that WFigure18O49/carbon foam exhibits the highest absorbance which could be attributed to two reasons. The first one is the porous net structure which increases the light scattering and the other one is the embedded strong solar absorption material. We have reported a photothermal conversion material due to its reduced tungsten (W5+ or W4+), which directly affect its photothermal conversion efficiency at a large extent.5+ or W4+) is being annealed in inert or reductive atmosphere.18O49 in N2 not only helps the aggregation of W18O49 but also increases its light absorption. As shown in Figure2 atmosphere, the total amount of reduced tungsten (W5+ or W4+) increase from 16.7% to 45% , which could dramatically increase its solar absorption. And this is consistent with the UV–vis–NIR absorption result.The melamine foam changes from white to black in color after the carbonization process. As a result, compared to the melamine foam, the absorbance of the carbon foam during the whole UV–vis–NIR spectrum increases dramatically as shown in 5+ or W4+), the W18O49/carbon foam composite should have an excellent photothermal conversion performance. Figure18O49/carbon foam after being irradiated by a 808 nm laser. The top view thermal image (irradiated from top) illustrates that the heat is evenly distributed throughout the horizontal plane and the side view thermal image (irradiated from side) reveals that the heat is concentrated at the top of vertical plane. That is to say, the bottom carbon layer could reduce the heat loss by thermal diffusion which is unnecessary for evaporation. The side view thermal image also reveals a better photothermal conversion performance of W18O49 than that of the carbon foam because the up temperature is higher than the down temperature when they are irradiated at the same condition. Moreover, it also reveals that the W18O49 is just deposited on the surface of the carbon foam which is consistent with the side view SEM image . And its good cyclic stability and flexibility makes it a prospective material in solar energy conversion field.In summary, we have successfully fabricated a self‐floating W518O49Synthesis of W: W18O49 nanomaterials were prepared by using a solvothermal method.6 (100 mg) was dispersed in 1‐butanol (60 mL) with sonication for 3 min. The obtained blue solution was transferred into a 100 mL Teflon‐lined autoclave and kept at 200 °C for 24 h. The product was washed with water and ethanol four times by centrifugation and then dried in the vacuum overnight.18O49/Carbon FoamSynthesis of W: 50 mg as‐prepared W18O49 nanomaterial was dispersed in 10 mL water by ultrasonic treatment to form a homogenous dispersion. The W18O49 dispersion was quickly loaded into the melamine foam by dipping into the dispersion. The composite of W18O49 and melamine foam was annealed in N2 to form the self‐floating W18O49/carbon foam composite. And the mass ratio of W18O49 to the carbon foam was about 1.25 (50 mg W18O49 to a 0.005 g cm−3 density of carbon foam with 2 × 2 × 2 cm3). The W18O49 was just loaded at the surface of the carbon foam and its loading thickness was about 0.5 cm.Solar Steam Generation: The self‐floating composite was evenly floated at the surface of 100 mL water in the beaker which was coated with thermal insulation material. The beaker was steadily placed in an electronic balance which records the mass change of the whole system during the simulated solar light illumination. The solar irradiation was simulated by a solar simulator xenon lamp at a power of 500 W.The authors declare no conflict of interest.


Radiation‐induced lung injury (RILI) is the major complication of thoracic radiation therapy, and no effective treatment is available. This study explored the role of high‐mobility group box 1 (HMGB1) in acute RILI and the therapeutic effect of glycyrrhizin, an inhibitor of HMGB1, on RILI. C57BL/6 mice received a 20 Gy dose of X‐ray radiation to the whole thorax with or without administration of glycyrrhizin. Severe lung inflammation was present 12 weeks after irradiation, although only a mild change was noted at 2 weeks and could be alleviated by administration of glycyrrhizin. Glycyrrhizin decreased the plasma concentrations of HMGB1 and sRAGE as well as TNF‐α, IL‐1β and IL‐6 levels in the bronchoalveolar lavage fluid (BALF). The expression of RAGE was decreased while that of TLR4 was significantly increased at 12 weeks, but not 2 weeks, after irradiation in mouse lung tissue. In vitro, the expression of TLR4 increased in RAW 264.7 cells after conditioning with the supernatant from the irradiated MLE‐12 cells containing HMGB1 but showed no change when conditioned medium without HMGB1 was used. However, conditioned culture had no effect on RAGE expression in RAW 264.7 cells. Glycyrrhizin also inhibited the related downstream transcription factors of HMGB/TLR4, such as NF‐κB, JNK and ERK1/2, in lung tissue and RAW 264.7 cells when TLR4 was activated. In conclusion, the HMGB1/TLR4 pathway mediates RILI and can be mitigated by glycyrrhizin. GL has been shown to be a natural inhibitor of HMGB1 by binding to the HMGB1 protein directly and inhibiting its biological activity.To explore the role of HMGB1 in the development of RILI and the radioprotective mechanism of GL, we performed studies and tried to find whether GL can mitigate RILI by inhibiting the HMGB1/TLR4 signalling pathway.22.1Adult female C57BL/6 mice  were obtained from the Experimental Animal Center of Rui Jin Hospital in Shanghai, China. All animal experimental procedures were approved by the Animal Ethics Committee of Rui Jin Hospital and were performed in accordance with the Guide for The Care and Use of Laboratory Animals published by the National Institutes of Health. The mice were maintained under a 12‐h light/dark cycle with standard food pellets and water available ad libitum. Before irradiation, the mice were randomly divided into four groups: normal control (C group); GL without irradiation ; irradiation only (R group); and irradiation plus GL .i.p.) 1 hour before irradiation and every day until week 4 and then three times per week until week 12.After anaesthetization by intraperitoneal administration of 50 mg/kg 1% sodium pentobarbital, each mouse was fixed on an organic glass pedestal with the body fully stretched. The whole thorax received a single 20 Gy photon dose . The irradiation field was 40 × 2 cm, according to the breadth of the pedestal and the lung size of 8‐week‐old mice. Before irradiation, the irradiated area of each mouse was verified under X‐ray simulator to ensure that the head and abdomen were shielded. GL  was administered intraperitoneally  were stained with haematoxylin and eosin (H&E) and evaluated by two experienced pathologists who were blinded to the experimental treatment groups. The pneumonia scores were assessed by evaluating the thickness of the alveolar septa and infiltrated inflammatory cells in the alveolar and interstitial space, as previously reported.2.3g for 15 minutes, and the supernatant was frozen at −80°C for ELISA. After removing the supernatant, the pellet was resuspended in 0.5 mL of PBS for cell counting and then stained with the Diff‐Quick method with cytospins for differential cell counts for statistical analysis.The mice were killed, the trachea was fully exposed, and a small plastic catheter was inserted into the trachea. After irrigation with 0.5‐1 mL of PBS, bilateral BALF was collected, and the process was repeated 2‐3 times until the total BALF volume reached approximately 1 mL. The collected BALF was centrifuged at 15002.42.The mouse alveolar epithelial cell line (MLE‐12) and mouse monocyte cell line (RAW 264.7) were purchased from ATCC (Manassas). MLE‐12 cells were cultured with F12/DMEM supplemented with 10% foetal bovine serum  in a 6‐cm culture dish; the medium was replaced with serum‐free medium 2 hours before irradiation. Cell irradiation was also performed under Linac with an 8 Gy dosage. RAW 264.7 cells were cultured with DMEM with 10% FBS. All cells were cultured at 37°C in a humidified atmosphere of 95% and 5% CO2.55 RAW 264.7 cells were seeded in the upper chamber of 8‐µm porous transwell inserts without Matrigel in serum‐free DMEM with or without GL. After co‐culturing for 24 and 48 hours, the upper chambers were removed and fixed with 4% paraformaldehyde for 20 minutes at room temperature. Then, cells were stained with crystal violet, and after washing with PBS, non‐migrated cells were removed from the transwell chambers with a cotton swab. We counted the number of migrated cells from five different microscope fields for statistical analysis.Cell migration assays were performed using a transwell migration chamber (Corning). MLE‐12 cells were seeded in a 24‐well cell culture plate with 10% F12/DMEM with or without HMGB1 peptide‐free lipopolysaccharide immediately after irradiation; transwell inserts were suspended in the individual wells. A total of 1 × 102.6To mimic the process of RILI in vitro, we applied the supernatants from irradiated MLE‐12 cells as conditioned medium and observed the effect on RAW 264.7 cells, which represent infiltrated immunocytes in vivo. HMGB1 expression was detected in the irradiated supernatants by ELISA 24 and 48 hours after replacing the original medium of RAW 264.7 cells when the confluence was approximately 70% and the cells were maintained in culture for an additional 24 hours.2.7Tissue samples and cells were homogenized with RIPA lysis buffer . Protein concentrations were determined by using a BCA protein assay kit. The proteins were separated on a 12% SDS‐polyacrylamide gel and transferred to a PVDF membrane. After blocking with 5% bovine serum albumin (BSA) in TBS, the membrane was incubated with primary antibody at 4°C overnight. The secondary antibody was conjugated to horseradish peroxidase and incubated at room temperature for 1 hour. Immunoreactivity was detected with an enhanced chemiluminescence kit (Beyotime Biotech) by a ChemiDoc™ MP System (Bio‐Rad). The following antibodies were used: HMGB1 , RAGE , RAGE , TLR4 , TLR4  and β‐actin ; pNF‐κB/NF‐κB, pJNK/JNK and pERK/ERK were all purchased from Cell Signaling Technology and incubated at a dilution of 1:1000.2.8Total RNA was extracted from frozen lung tissue using TRIzol Reagent (Invitrogen) according to the manufacturer's instructions. Then, the total RNA was reverse‐transcribed into CDNA with a PrimeScript™ RT reagent kit with gDNA Eraser . Real‐time PCR was performed by monitoring the intensity of fluorescence in real time using SYBR Green dye  with a StepOne™ Real‐time PCR machine (Applied Biosystems). The PCR primer pairs used were as follows: β‐actin, forward, 5′‐ATGACAACTTTGGCATTGTG‐3′ and reverse, 5′‐CATACTTGGCAGGTTTCTCC‐ 3′; HMGB1, forward, 5′‐TTGTGCGAAAAGAAGTGC AG‐3′ and reverse, 5′‐TACAAACACAGCCTCCCACA‐ 3′; RAGE, forward, 5′‐TGGCTCGAATCCTCCCCAAT‐3′ and reverse, 5′‐CCTCCTTCCCTCGCCTGTTA‐3′; and TLR4, forward, 5′‐GAGCCGGAAGGTTATTGTGGTAGTG‐3′ and reverse, 5′‐TCAAGGACAATGAAGATGATGCCAGAG‐3′.2.9The BALF concentrations of TNF‐α, IL‐1β and IL‐6 were measured using ELISA kits (Beyotime Biotech), and the plasma levels of HMGB1 and sRAGE were detected by an IBL ELISA kit (Japan) and R&D ELISA kit (American), respectively, according to the manufacturer's directions.2.10t tests. Statistical significance was defined as P < .05.Data are represented as the mean ± SEM from at least three independent experiments. All variables were analysed by one‐way ANOVA or Student's 33.1Radiation caused obvious pulmonary inflammation at 12 weeks, indicated by macrophage, lymphocyte and neutrophil infiltration in the alveoli and interstitium via histopathology. For mice treated with GL, infiltrated immunocytes in lung tissue were decreased significantly at 12 weeks after irradiation, as shown in Figure 3.2Although lung inflammation at 2 weeks was mild, infiltrated cells increased noticeably in the BALF. GL decreased the total number of infiltrating immunocytes in the BALF and changed the distribution of inflammatory cell types. Figure 3.3At 2 and 12 weeks after irradiation, TNF‐α, IL‐1β and IL‐6 in the BALF increased significantly compared to those without radiation exposure, and GL inhibited the release of these pro‐inflammatory cytokines after irradiation Figure .3.4At the latent and inflammation stages of RILI, both mRNA in lung tissue and plasma levels of HMGB1 increased significantly, while the change in HMGB1 protein in the lung tissue was inconsistent, with a mild change at 2 weeks after irradiation but a significant decrease at 12 weeks. Glycyrrhizin can inhibit HMGB1 synthesis and release both in the latent and inflammation stages of RILI Figure .3.5At the latent stage of RILI, both RAGE and TLR4 expression in the lung tissue changed slightly, but during the inflammation stage, RAGE levels decreased while those of TLR4 increased significantly. Radiation caused increased TLR4 mRNA expression at the inflammation stage but had little effect on RAGE mRNA at both the latent and inflammation stages of RILI. In contrast to the change in RAGE in the lung tissue, radiation caused the soluble RAGE in plasma to increase persistently along with the damage to alveolar epithelial cells Figure .3.6To mimic the chemotaxis of irradiated lung tissue to peripheral blood mononuclear cells, we used MLE‐12 and RAW 264.7 cells as alveolar epithelial cells and peripheral monocytes, respectively, in vitro. MLE‐12 cells were irradiated with 8 Gy dosage, and HMGB1 was detected 48 hours after irradiation but not 24 hours. Transwell assays showed that migrating RAW 264.7 cells increased significantly at 48 hours when HMGB1 was released, but not at 24 hours when HMGB1 was still located intracellularly. We also found that the addition of HMGB1 peptide promotes the migration of RAW 264.7 cell in control and radiation groups as well as glycyrrhizin inhibits the migration stimulated by HMGB1 (Figure 3.7To exclude the interference of different cell types between the inflammation stage and latent stage in vivo, we examined the receptors of HMGB1 in irradiated MLE‐12 cells and RAW 264.7 cells after conditioned culture. Radiation had no direct effect on the expression of RAGE and TLR4 in MLE‐12 cells. The supernatant of the irradiated MLE‐12 cells had a moderate effect on the expression of RAGE in RAW 264.7 cells. However, the expression of TLR4 increased significantly after culture with supernatants of irradiated MLE‐12 cells containing the released HMGB1 at 48 hours post‐irradiation. Glycyrrhizin inhibited the expression of TLR4 in RAW 264.7 cells, which was activated by supernatants from irradiated MLE‐12 cells at 48 hours post‐irradiation (Figure 3.8Many transcription factors are involved in the HMGB1/TLR4 signalling pathway; we examined the expression of NF‐κB, JNK and ERK1/2 in vivo and in vitro when TLR4 was activated. We found that pNF‐κB, pJNK and pERK1/2 increased significantly in lung tissues during the inflammation stage of RILI and in RAW 264.7 cells when cultured with conditioned medium containing HMGB1. Interestingly, glycyrrhizin can block the activation of these downstream transcription factors in vivo and in vitro Figure .4The incidence of clinical RILI is observed in up to 30% of lung cancer patients receiving thoracic irradiation and approximately 10%‐15% in other thoracic malignancy patients.Radiation‐induced lung injury has a relatively long latent stage that exhibits no obvious pathological changes, but many cytokines have already been investigated in vivo and throughout the whole process of RILI.HMGB1 is one of the most important DAMP molecules. Some reports have shown that radiation causes HMGB1 release and serves as an extracellular immunoregulator. Some researchers have observed the release of HMGB1 after irradiation and that blocking its release can alleviate RILI,Glycyrrhizin, a natural inhibitor of HMGB1, blocked the release of HMGB1, TNF‐α, IL‐1β and IL‐6 after whole lung irradiation. It also decreased the infiltrating immunocytes in the BALF and lung tissue, which indicated that HMGB1 was an important mediator and a potential therapeutic target of RILI.RAGE and TLR4 are the receptors to which HMGB1 binds directly. RAGE is moderately expressed in many cell types but highly expressed in alveolar epithelial cells, while TLR4 is expressed mainly in immunocytes.To exclude the effect of cell numbers at different stages in vivo, we mimicked the process of RILI in vitro. As our research showed, macrophages and lymphocytes are the main infiltrating cells in the inflammation stage. We used RAW 264.7 cells as the infiltrating cells in the alveolar interstitium and MLE‐12 cells as the alveolar epithelial cells. To further illuminate the effect of HMGB1, we examined the supernatants of irradiated MLE‐12 cells and found that the level of HMGB1 was increased at 48 hours but not at 24 hours post‐irradiation. The transwell results showed that migration increased significantly after co‐culture for 48 hours only when HMGB1 was released. To confirm that this chemotaxis is mediated by HMGB1, we added HMGB1 to the lower chamber of the transwell system and found that migrated cells were more obvious than the control cells. This migration can also be inhibited by GL, and HMGB1 can rescue the migration inhibited by GL. Interestingly, radiation did not alter the expression of RAGE and TLR4 on MLE‐12 cells directly. However, replacement of the medium of RAW 264.7 cells with the supernatants of irradiated MLE‐12 after 48 hours, which contained HMGB1, significantly increased the expression of TLR4 in RAW 264.7 cells. Glycyrrhizin can block the migration and inhibit the expression of TLR4 in immunocytes in vivo and in vitro. These results demonstrated that HMGB1 recruits immunocytes to the damaged sites in the lung tissue and mediates inflammation via TLR4 in immunocytes during the progression of RILI.In contrast to the decrease in RAGE in the lung tissue after irradiation, the level of sRAGE in plasma remained high because of the continuous damage to alveolar epithelial cells in RILI. Soluble RAGE is the C‐terminal splice variant of RAGE that is shed from the plasma membrane via proteolytic cleavage by matrix metalloproteinases. It can be secreted into plasma when damage occurs, so it reflects the severity of cell injury to some extent. As RAGE is highly expressed in alveolar epithelial cells, the plasma level of sRAGE is considered a biomarker for ARDS, ventilation and ventilator‐associated pneumonia and COPD.Intracellular transcription factors downstream of HMGB1/TLR4 that mediate sterile inflammation include JNK, ERK1/2 and NF‐κB, etc.Glycyrrhizin, an inhibitor of HMGB1, can bind HMGB1 directly without interfering with other chemokines.In conclusion, our research indicated that HMGB1 is a vital mediator of acute RILI and that its inhibitor GL can mitigate RILI via the HMGB1/TLR4 signalling pathway. The radioprotective effect of GL deserves further investigation and has potential clinical translational value.The authors declare that they have no competing interests.Jia‐Yi Chen and Lei Zheng conceived and designed the research. Lei Zheng conducted most of the experiments, analysed the data and wrote the main part of the paper. All authors participated in parts of the research.


The Hedgehog (Hh) signaling pathway is crucial for vertebrate embryonic development, tissue homeostasis and regeneration. Hh signaling is upregulated in basal cell carcinoma and medulloblastoma and Hh pathway inhibitors targeting the Smoothened (SMO) protein are in clinical use. However, the signaling cascade is incompletely understood and novel druggable proteins in the pathway are in high demand. We describe the discovery of the Hh‐pathway modulator Pipinib by means of cell‐based screening. Target identification and validation revealed that Pipinib selectively inhibits phosphatidylinositol 4‐kinase IIIβ (PI4KB) and suppresses GLI‐mediated transcription and Hh target gene expression by impairing SMO translocation to the cilium. Therefore, inhibition of PI4KB and, consequently, reduction in phosphatidyl‐4‐phosphate levels may be considered an alternative approach to inhibit SMO function and thus, Hedgehog signaling. Cell‐based screening identifies a thienopyrimidine derivative as an inhibitor of Hedgehog signaling by selective inhibition of phosphatidylinositol 4‐kinase IIIβ (PI4KB), reduction of PI4P levels and prevention of Hedgehog‐mediated ciliary localization of Smoothened. Thus, PI4KB may be a relevant target protein for inhibition of Hedgehog signaling. Gli1, Ptch1 and Ptch2.The Hedgehog (Hh) signaling pathway is essential during vertebrate embryonic development as well as for tissue homeostasis and regeneration in adults. Crucial events during pathway activation include binding of the Hh ligand to the Patched (PTC) receptor, transduction of the signal to the seven‐pass transmembrane protein Smoothened (SMO) and subsequent relocalization of SMO from intracellular vesicles to the cilium.Hh signaling is upregulated in cancers of the skin We monitored Hh‐induced osteogenesis as a cell‐based screening for inhibitors of the Hh pathway. Subsequent target deconvolution and validation led to the discovery of the Hh pathway inhibitor Pipinib, which selectively targets PI4KB over the other three PI4‐kinases. Inhibition of PI4KB by Pipinib reduces intracellular PI4P levels, in particular at the Golgi apparatus, and impairs translocation of SMO to the cilium as well as subsequent pathway activation. Our results indicate that PI4KB may be a relevant target protein for pharmacological inhibition of Hh signaling.1  or cyclic substituents  and introduction of heteroatoms  or of tertiary amines (Table 9–11) leads to reduction of bioactivity. In two cases, moderate cytotoxicity was observed (Table 5 and 11). Replacement of the methyl ester in R2 by a nitrile group (Table 12–14) and wider variation of the substitution pattern of the aromatic ring (Table S1) also yielded compounds with lower activity. Introduction of a substituent into the thiophene ring was not beneficial either (Table S1).In order to identify novel inhibitors of Hh signaling, 336,639 compounds were subjected to an osteoblast differentiation assay that monitors differentiation of multipotent C3H10T1/2 mesenchymal progenitor cells to osteoblasts upon activation of the Hh pathway by treatment with the SMO agonist Purmorphamine.1  to which the full‐length protein is proteolytically converted upon pathway inactivation  revealed eight potential targets (Table S3), of which phosphatidylinositol 4‐kinase IIIβ (PI4KB) showed the highest inhibition (76±2 %). Pipinib did not inhibit the enzymatic activity of Syk and PRKD1 and PRKD2 (Table S2). Of the potential targets, GRK7 was excluded because mouse cells were used in the osteoblast differentiation assay and mouse orthologs of GRK7 do not exist  but not the isoenzymes PI4KA (phosphatidylinositol 4‐kinase IIIα), PI4K2A (phosphatidylinositol 4‐kinase IIα) or PI4K2B (phosphatidylinositol 4‐kinase IIβ) up to a concentration of 50 μm . Kinetic measurements and analysis demonstrated that Pipinib is an ATP‐competitive inhibitor of PI4KB activity  identified PI4KB as the only target protein of Pipinib  and GT‐CFP led to reduction of the Golgi intensity of YFP‐PH(FAPP1) within 10 min . Using the CRISPR‐Cas9 system, we attempted to disrupt the Pi4kb gene. Unfortunately, Pi4kb knockout cells could not be generated, most likely because PI4KB is an essential gene.Ptch1 and Gli1 after pathway stimulation with Purmorphamine  at the same concentration reduced gene expression only by 44 and 17 %  was proposed as small‐molecule mediator of signal propagation from PTC to SMO. We demonstrate that phosphatidylinositol 4‐kinase IIIβ (PI4KB) generates PI4P that is relevant for Hh signaling. The mechanism by which the Hh signal is transduced from PTC to SMO and by which PTC controls SMO in mammalian cells has been described as “the major mystery of the Hh field”.Drosophila, loss‐of‐function mutations have shown that the lipid kinase SST4, the ortholog of mammalian PI4KA, is necessary for SMO translocation to the membrane upon Hh pathway activation.In Our results demonstrate the relevance of PI4KB for Hh signaling as previously indicated by genetic evidence.The authors declare no conflict of interest.As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.SupplementaryClick here for additional data file.SupplementaryClick here for additional data file.SupplementaryClick here for additional data file.SupplementaryClick here for additional data file.


Chalcogenide glasses are well known for their high ionic conductivity nature and wide compositional flexibility. As the GeSbSe material has high glass forming ability, it is expected that the materials can be doped with a high amount of foreign element (in the present case Ag). For all of these reasons, the GeSbSe materials can be expected as a potential candidate for solid state electrolyte for ionic batteries. The ionic conductivity behavior of Agx(Ge16Sb12Se72)100-x chalcogenide glass system. From the impedance data, real and imaginary parts of conductivities were extracted and plotted as a function of applied frequency. The interpretation of the current article data were given in “Percolation behavior of Ag in Ge16Sb12Se72 glassy matrix and its impact on corresponding ionic conductivity” [1].In the present article, you will find the impedance data of Ag Specifications tableValue of the data•The current impedance data is very useful for comparing similar ion conducting disordered materials.•The random-walk model data fitting to experimental values shows an importance of random-walk model.•The data shows influence of Ag doping concentrations on impedance behavior.•It also shows the behavior of conductivity with respect to temperature.•The current data shows the importance of impedance spectroscopy and random-walk model to get more depth information about ionic conduction in disordered materials.1σ1) and imaginary (σ2) parts of conductivities extracted using random-walk model from impedance data of Ag15 as a function of temperature. 2x(Ge16Sb12Se72)100-x with 0 ≤ x ≤ 25, were prepared by the melt quenching method. The exact composition and corresponding label is given in All of the samples, having nominal composition Ag-3 Pa). The sealed ampule was loaded into a rocking tube furnace and heated with a rate 2 °C/min up to 950 °C before dwelling at 950 °C for 12 h in the rocking furnace. After 12 h, the temperature was slowly cooled down to 730 °C and at 730 °C ampules were quench into water, and later annealed at a temperature close to Tg for 4 h, followed by slow cooling down to room temperature. The obtained glass rods after annealing were shown in 2O3 powder with varying sizes such as 6 µm, 3 µm, and 1 µm. The obtained disks after polishing were also shown in For impedance measurements, the polished disks were sputtered with gold on both sides for a good electrical contact with the electrodes as well as to form a blocking electrode.All of the elements  used for the synthesis were of high purity 5 N. A batch of 10 g, with an appropriate amount of each element was mixed together to get a more homogeneous mixture and sealed in a quartz ampule (with the inner diameter of 10 mm) under high vacuum  with an accuracy of ± 1 °C. The input capacitance and impedance of the instrument were lower than 8 pF and higher than 100 GΩ, respectively. The obtained impedance data were first converted into its corresponding real (ρ1) and imaginary (ρ2) resistivity data to take account of sample dimensions using following relations,Impedance spectroscopy (IS) measurements were carried out on a PGSTAT 302 N with the FRA32 and NOVA 10 software. The cell used stainless disk-like electrodes sputtered with gold to block the AgThe obtained values were plotted in the form of nyquist plot shown in σ1) and imaginary (σ2) parts of conductivities using following relations.To observe the temperature influence as well as to see the conductivity behavior, Ag15 were selected as a case example and impedance data were collected in the temperature range of 295 K to 368 K. The obtained impedance data were first converted to its corresponding resistivity using above relation. The obtained resistivity values were further converted into corresponding real (The obtained data were plotted in