The proposed structure has a monolayer of Cu2O - Au core-shell particles on a Au substrate with 1 nm thick graphene spacer layer. Mie simulations are used to optimize the aspect ratios of core-shell particles to shift their plasmon resonances to NIR region using MieLab software. Further, Finite Difference Time Domain (FDTD) simulations using Lumerical software are used for the design of the multiparticle layered SERS substrate as MieLab software works only for single particle systems. Designed structure is shown to provide high field enhancement factor of the order of 108 at an excitation of 1064 nm thus ensuring the possibility of using the proposed structure as efficient NIR SERS substrate which could probably be used for various NIR sensing applications.Near Infra-Red Surface Enhanced Raman Spectroscopy (NIR SERS) has gained huge attention in recent years as the conventional visible SERS suffers from overwhelming fluorescence background from the fluorophore resulting in the masking of Raman signals. In this paper, we propose a novel multi-layered SERS substrate- (Cu Out of these, SERS was used in the trace analysis of various chemicals, finding potential applications in fields such as biochemistry, medical diagonostics, food contamination detection, water safety, trace level explosive detection and forensics6. Raman signals of the analyte molecules get enhanced in SERS by the amplification of light due to the generation of localized surface plasmons in the gaps and sharp edges of metallic nanoparticles, micro or nanopatterned structures or on the roughened metallic surfaces10. Large enhancement in Raman signals is achieved when the plasmon resonance wavelength almost matches with the excitation wavelength which generally falls within the visible to NIR region (400 to 850 nm)11. Visible light excitation in SERS may result in photobleaching, plasmonic heating and also in an overwhelming fluorescence background as most of the fluorophore analytes have strong emission within the visible region resulting in the masking of Raman signals12. These problems can be overcome by using larger wavelength excitations (NIR lasers)13. Tunability in plasmon resonances to match with the exciation wavelength can be achieved by the modification of metallic or metal hybrid nanoparticle morphologies or using core-shell particles14. Different metal-dielectric, dielectric-metal, metal-metal and multilayer metal-dielectric structures are shown to have large tunability of plasmon resonances from visible to NIR region16. Various imaging techniques can be used for nanoscale level imaging of such structures18. Plasmonic resonances of core-shell particles are highly sensitive to core radius, shell thickess and refractive indices of core and shell19. High refractive index dielectric core enables efficient coupling between spherical and cavity plasmons leading to large tunability of symmetric modes (longer wavelength modes)20. Cu2O is a high refractive index semiconductor (~2.6 in NIR) which along with metallic shell provides larger visible to NIR tunability in plasmonic resonances compared to other semiconductors21. Current interest in the design of SERS substrates is in metal- dielectric-metal multilayer structures with a thin graphene spacer layer which enables efficient coupling of plasmons with the metallic substrates22.Rapid growth in biosensing and bioimaging research fields in the recent past involved potential development of novel nanostructured materials and various imaging techniques that involved optical probes, specialty fiber optics, Surface Enhanced Raman Spectroscopy (SERS)24. Longer excitation wavelength of 1064 nm is hence chosen to avoid the overwhelming fluorescence background from fluorophores. SERS enhancement in graphene monolayer and NIR SERS by hollow Au nanotags are proposed earlier26. Plasmon resonances below 800 nm are made use of in the above works which is far below the excitation wavelength 1064 nm. Here we propose a layered NIR SERS substrate with monolayer of Cu2O - Au core-shell particles on Au substrate separated by 1 nm thick graphene layer. Layered structure is designed in such a way that the plasmon resonance exactly matches with 1064 nm which can provide enhanced NIR SERS properties. Mie computations and FDTD simulations are used to explore the potential of the proposed structure in NIR SERS applications. Mie calculations using MieLab software is used for the plasmon resonance study of single core-shell particle systems. MieLab software can only be used for the study of single particle systems. Due to this limitation, further modelling of multi core-shell particle embedded SERS substrate is carried out using Lumerical FDTD simulations. Mie theory used for the study of absorption efficiencies of core-shell particles is discussed in the next section.The aim of our present work is to model a novel and efficient NIR SERS substrate with large SERS field enhancement factor values at NIR excitation wavelength of 1064 nm. In general, fluorophores used in biological applications - Thioflavin, Cyanine dyes etc. have strong emission within 400 nm to 750 nm wavelength range2O – Au core-shell particles in the near infra-red region27. According to Mie theory,Qsca, Qabs and Qext are the scattering, absorption and extinction efficiencies of particles, z is the size parameter =, R is the radius of the particle, λ is the wavelength of light, ns is the refractive index of the spherical particle and nm is the refractive index of the medium and al and bl are the Mie coefficients.Mie theory in the modified form for core-shell particles is used for the calculation of the absorption efficiency of Cua and outer radius b) with core refractive index m1 and shell refractive index m2, Mie coefficients al and bl are calculated using the modified relations21,For core-shell particles  is found to be comparable with the value obtained using MieLab simulation (0.15). Figure Q2O are obtained from Johnson and Christy data29 and Querry et al.30 respectively. We have performed Mie calculations on various aspect ratios of Cu2O-Au core-shell particles. Table 2O-Au core-shell particles with core radius 40 nm, 50 nm and 60 nm and with Au shell thickness in each case varying from 2 nm to 30 nm in steps of 4 nm. Plasmon resonance tunability with increase in Au shell thickness for Cu2O (40 nm)-Au, Cu2O (50 nm)-Au and Cu2O (60 nm) –Au is observed to be from 1325 nm to 505 nm, 1500 nm to 675 nm and 1630 nm to 712 nm, respectively.For the ease of simulation we have used MieLab for single particle studies. It is not possible to use MieLab for multiparticle systems. Hence we have used FDTD for further modelling of layered substrates and visualization of electric field. Refractive index data required for simulation for Au and Cu2O - Au core-shell particle with core radius 50 nm and Au shell thickness (2 nm to 30 nm) are found to have tunable plasmonic resonance within the desired biological window of 650 nm to 1350 nm  - Au core-shell particles, plasmon resonances are found to be tunable from 675 nm to 1500 nm which falls within the desired NIR biological window. Absorption efficiency increases with increase in shell thickness till 14 nm beyond which the Qabs reduces . From Fig. 2O (50 nm) - Au core-shell particle with shell thickness 6 nm, plasmon resonance is at 1040 nm which is very close to the desired excitation of 1064 nm.Among the different aspect ratios, Cu nm Fig.  and the ces Fig.  since th2O (50 nm) - Au core-shell particle at 1064 nm  is designed on Au substrate of thickness 50 nm.We have tried further to improve the NIR absorption efficiency by distributing Cu2O (50 nm) - Au (6 nm) core-shell particles is studied in each case using two field monitors along XZ and YZ directions. Field intensity between the particles is observed to be the highest with three layers of graphene spacer which corresponds to an approximate thickness of 1 nm  – Au core-shell particles are selected for designing the NIR absorber due to their large Qabs values in the NIR region. Optimized Au shell is 6 nm for maximum absorption at 1064 nm for single Cu2O (50 nm) - Au core-shell particle. But the absorption scenario becomes entirely different when Cu2O - Au core-shell particles are distributed over the Au substrate. Plasmonic coupling between Au substrate and core-shell particles via graphene spacer layer significantly modifies the effective absorption by the designed structure. Hence the NIR absorption properties are further studied for different Au shell thickness values for layered structure. Single layer of Cu2O (50 nm) - Au particles are uniformly distributed over the graphene layer and the structure is then irradiated with NIR source (power = 1 mW). Two different power monitors are used to collect the transmitted and reflected signals from the structure when irradiated with plane wave source (λ = 0.7 µm to 2 µm). Perfectly matched layer (PML) boundary condition is employed in all simulations. The mesh size is taken as 0.2 nm which is well below the thickness of the spacer layer. The incident polarization is parallel to the substrate.Figure 2O (50 nm) particles without Au substrate and Cu2O (50 nm) particles on Au substrate. Figure 2O (50 nm) in the presence and absence of Au substrate. Plasmonic field due to Au substrate is found to have a huge influence on the NIR absorption of Cu2O and is reflected as a clear enhancement in the NIR absorption over the entire NIR region. To prove the enhanced plasmonic coupling of core-shell particles with the substrate in the presence of spacer graphene layer, we have modelled two systems in which two Cu2O (50 nm) - Au (6 nm) particles are distributed on Au substrate with and without 1 nm thick spacer graphene layer. The excitation wavelength is chosen as 1064 nm in FDTD simulations. Field distribution in the XY and YZ directions without and with the spacer layer are shown in Fig. In order to confirm the influence of Au substrate on NIR absorption, FDTD simulations are performed for two different configurations – Cu2O (50 nm) - Au core-shell particles with different Au shell thicknesses. Huge enhancement in absorbance is observed in the NIR region due to the plasmonic coupling between Cu2O - Au with Au substrate through graphene spacer layer. We could observe a gradual enhancement in NIR absorption over the entire range 0.7 µm to 1.6 µm when the Au shell is added which is the highest at 30 nm beyond which it is a constant. NIR absorption in the window 0.9 µm to 1.1 µm for (Cu2O (50 nm) - Au) – Graphene - Au layered structure increases by 18% compared to (Cu2O (50 nm)) – Graphene - Au layered structures. Increase in absorption in the specified window is attributed to the effective plasmonic coupling between Au substrate and core – shell particles. Local field around the core – shell particle has contribution from both the localized plasmonic field and the incident electric field. Enhancement in NIR absorption due to Au shell results in the formation of hot spots with stronger local electric field confinement between the core-shell particles. It is inferred from simulations that the hotspots provide significant enhancement factor values which are proportional to the fourth power of locally generated electric field. Generation of such strong hotspots enable SERS detection of biomolecules even with very low Raman cross sections. Our specific interest is in the region 0.9 µm to 1.1 µm where we could observe a new plasmonic peak originating at higher Au shell thicknesses due to the effective plasmonic coupling between the particles and the substrate. At lower Au shell thickness values, the dipolar and quadrapolar resonances of core-shell particles are far apart where dipolar resonances fall in the IR and quadrapolar resonances fall in the UV region. As the thickness of Au shell increases, quadrapolar resonances get red shifted towards visible region while blue shift occurs to dipolar resonances to visible region making both the resonances much closer. This results in the coupling between these modes causing enhanced effective plasmonic absorption over a wide range and formation of new plasmonic peaks. Such a strong plasmonic absorption within the NIR biological window is highly useful for sensing applications particularly sensing based on NIR surface enhanced Raman spectroscopy (SERS). The possibility of using (Cu2O - Au) – graphene - Au layered structures as NIR SERS substrate using FDTD simulations is studied further.Further, FDTD simulations are performed on the layered structure shown in Fig. 2O (50 nm) – Au particles when they are kept on Au substrate with graphene spacer layer. Scattering enhancement factor here is defined as (E/E0)4 where E is the maximum local electric field and E0 is the incident electric field. Gap between Cu2O (50 nm) - Au particles and also that between Au substrate and Cu2O (50 nm) - Au particles are maintained as 1 nm in all simulations. This enabled the plasmons from the particles as well as the substrate interfere constructively to produce highly enhanced local electric field regions within the gaps called hotspots. The excitation source of wavelength 1064 nm to study the NIR SERS property of the layered structure is Total - field scattered - field. It helps to separate the scattered field from the incident field and helps to avoid the reflections from the simulation boundaries.FDTD simulations are further used to study the scattering enhancement in Cu0)4 where E is the local maximum electric field and E0 is the amplitude of input source electric field. With the addition of Au shell, EF is found to increase from 1.24 × 103 to 1.86 × 108 at larger shell thicknesses and the increase is observed to be very fast till the thickness value reaches 22 nm  which is a measure of SERS efficiency of substrate is defined as  - Au (22 nm)) is found to show large electric field enhancement factor of the order of 108 at 1064 nm excitation. Graphene spacer layer enables efficient plasmonic coupling between the upper layers of core-shell particles with Au substrate. Field enhancement factor values which are better than the reported NIR SERS substrates suggest the possibility of using the structure as efficient NIR SERS substrate for various applications. General drawbacks of visible SERS such as fluorescence background, photo bleaching and plasmonic heating which adversely affect the intensity of SERS signals can be overcome by the proposed structure as it is designed for a longer wavelength of 1064 nm.We have proposed a novel metal - dielectric – metal layered NIR SERS substrate which can find immense potential in NIR based bio sensing applications. NIR SERS substrate with Cu2O – Au core – shell particles. MieLab is a computational package which simulates the scattering of electromagnetic field of multilayered spheres based on Yang’s algorithm27. Absorption efficiency (Qabs) values are calculated for various aspect ratios of Cu2O – Au core – shell particles towards their optimization for maximum absorption in the near Infra-red (NIR) region. Simulations are performed for three different Cu2O core radii −40 nm, 50 nm and 60 nm, respectively. Au shell thickness is varied from 2 nm to 30 nm in steps of 4 nm for each core radius value. Refractive index values of Cu2O and Au are provided as input parameter for simulations.MieLab software is used for the study of plasmon resonance tunability in Cu2O – Au core – shell particles. FDTD method is based on the classical solution of Maxwell’s curl equations. This method solves Maxwell’s equations on a discrete spatial and temporal grid (Yee cell)32. Perfectly matched layer (PML) boundary condition is used throughout the simulations. Plane wave source (λ = 0.7 to 2 µm) is used as the irradiation source. Simulations are performed at a mesh size of 0.2 nm. (Cu2O – Au)- Graphene – Au layered structure with optimized dimensional parameters are modelled as surface enhanced Raman scattering (SERS) substrate using lumerical FDTD module. Two frequency domain field monitors are used for collecting the transmitted and reflected signals when irradiated with the plane wave source. SERS enhancement factor (EF) is simulated for the optimized geometry of the substrate at an excitation wavelength of 1064 nm using FDTD simulations.Finite difference time domain (FDTD) simulation using lumerical FDTD software is used for visualizing the electric field enhancement at the vicinity of Cu


Rangifer tarandus) bones from the sites of Kastelhöhle-Nord and Y-Höhle indicates human occupation of Switzerland was most likely restricted to between 23,400 and 22,800 cal. BP. This timeframe corresponds to Greenland Interstadial 2, a brief warming phase, supporting the hypothesis that human presence was facilitated by favourable climatic episodes. Carbon, nitrogen and sulphur stable isotope analysis of the fauna provides palaeoenvironmental information for this time period. These findings contribute to our understanding of human activity in ice-marginal environments and have implications for understanding cultural connections across central Europe during the LGM.Central Europe during the Last Glacial Maximum (LGM) was dominated by polar desert and steppe-tundra biomes. Despite this, a human presence during this time period is evident at several locations across the region, including in Switzerland, less than 50 km from the Alpine ice sheet margin. It has been hypothesised that such human activity may have been restricted to brief periods of climatic warming within the LGM, but chronological information from many of these sites are currently too poorly resolved to corroborate this. Here we present a revised chronology of LGM human occupation in Switzerland. AMS radiocarbon dating of cut-marked reindeer ( Despite this, human occupation during this period is evidenced from two locations in Switzerland; Kastelhöhle-Nord and Y-Höhle5 , which may have facilitated human occupation in Switzerland6. If this hypothesis is to be tested, more precise chronological and environmental information regarding the human activity at Kastelhöhle-Nord and Y-Höhle is required. This information is also crucial to situate the Swiss LGM archaeological record within the wider debate surrounding the extent of human occupation in central Europe during the LGM, the environmental conditions under which it occurred, and the extent to which different cultural groups may have interacted6. Here we present a revised chronology of human occupation in Switzerland during the LGM based on new radiocarbon dates, and environmental interpretations based on stable isotope analysis  of reindeer bone collagen.Switzerland during the Last Glacial Maximum  are recorded in marine or ice core records11. Associated time intervals range from c. 25,000 to 18,000 BP, but broadly centre upon 21,000 BP11. Within Switzerland, the last glacial advanced occurred sometime after 30,000 BP1. Local geomorphological evidence and speleothem δ18O minima suggest maximum ice extent may have been reached between 26,500 and 22,000 BP, while the onset of rapid ice decay started by c. 19,000 BP13. Climate models and proxy data indicate a cold dry climate existed north of the Alps during this time period, while the formation of Alpine speleothems suggests warm air masses from the south delivered precipitation to the northern Alpine slopes8. North of the mountains, continuous periglacial permafrost conditions existed, with tundra and steppe landscapes dominated by graminoid and forb vegetation15. Nonetheless, the Alpine LGM was punctuated by sub-millennial climate fluctuations related to changing temperature and precipitation regimes, which influenced local environmental conditions and ice extent18. Within Switzerland a positive excursion in speleothem δ18O occurred between 23,230 ± 37 BP and 22,759 ± 47 BP2, linked to changing precipitation patterns, and this appears to have coincided with a phase of recession in the Alpine ice sheet18. The timings of these local environmental changes matches with the Greenland warm period of GI-2 , within dating uncertainties. This suggests that the warming in Switzerland may have been associated with continental-scale processes. The Greenland record of GI-2 is interrupted by a brief climatic cooling lasting around 200 years  and centring on approximately 23,100 BP9. The expression of this event in the Swiss record is less clear but may occur between 22,971 ± 46 BP and 22,888 ± 50 BP2.The LGM is a time-transgressive period within Marine Isotope Stage 2 (MIS2). The nomenclature is variously used to refer to the episode during which maximum global ice volume was reached, when minimum global sea level occurred, or when the lowest oxygen isotope values  bones from Kastelhöhle-Nord (n = 3) and Y-Höhle (n = 1) represent the only directly dateable archaeological evidence from this time period in Switzerland6. Both cave sites are located within 10 km of one another, on the north-eastern edge of the Jura Mountains, less than 50 km from the maximum ice sheet extent. Lithics found in association with the reindeer bones at Kastelhöhle-Nord have been attributed to the Badegoulian/Early Magdalenian6, a Late Upper Palaeolithic culture found in western-central Europe. In contrast, the cut-marked reindeer bone from Y-Höhle was found in a secondary context with no association to other archaeological remains5. Lithics of Badegoulian character are also found in Germany 19, while broadly contemporaneous evidence of human activity is also present in southern Germany (Mittlere Klause), and further east in Austria (Kammern-Grubgraben and Langmannersdorf), the Czech Republic  and possibly Poland 23. The latter are usually attributed to Epigravettian cultures of eastern central Europe21. Hence, a great amount of uncertainty surrounds the cultural and chronological relationship between these sites, as well as the environmental conditions under which they were occupied, but it has been postulated that brief climatic warming events may have facilitated the periodic expansions of human populations into northern central Europe, and cultural contact across it6.The link between GI-2 and human activity in Switzerland has previously been postulated25. Ultrafilters remove the low molecular weight fraction from the sample, thus more thoroughly removing modern organic contaminants24. However, not all studies conclude that ultrafiltration is a necessary part of the radiocarbon bone sample preparation procedure27, and without stringent cleaning protocols their use may risk introducing additional contaminants into the sample29. Certainly, it is necessary to employ rigorous quality checks to the radiocarbon dating process, regardless of the method of sample preparation used31. Following such quality controls, the application of the ultrafiltration method has been shown to have particularly significant implications for the dating of bone collagen from Palaeolithic contexts, where the contribution of even the smallest amount of contamination can have significant consequence for the obtained date34. Considering these developments, it is timely to re-evaluate the radiocarbon chronology of human occupation in Switzerland during the LGM. As such, the four previously dated cut-marked reindeer bones from Kastelhöhle-Nord intermediate horizon and Y-Höhle were targeted for radiocarbon dating using the current ultrafiltration sample preparation methodology and quality control criteria used by the Oxford Radiocarbon Accelerator Unit (ORAU).One of the most significant developments in the radiocarbon dating of bone collagen in the last 20 years has been the routine inclusion of an ultrafiltration step in the sample pre-treatment procedure at some laboratories41. In this study we use carbon (δ13C), nitrogen (δ15N) and sulphur (δ34S) isotope ratios in reindeer bone collagen to examine paleoenvironmental conditions during the period of human activity at Kastelhöhle-Nord and Y-Höhle. Carbon isotope ratios are largely determined by dietary behaviour, and reindeer δ13C values are known to be systematically enriched in comparison to other herbivore species due the consumption of lichen43. Dietary δ13C values are also influenced by atmospheric CO2 δ13C value and concentration, and by environmental parameters such as temperature, moisture availability and density of vegetation cover46. Bone collagen δ15N values are linked to both dietary specialisation48 and to environmental conditions32. Soil and plant δ15N values are influenced by climatic variables such as temperature and precipitation, mediated through soil processes50. In particular, soil maturity, nutrient availability and microbial activity have been cited as having a strong control on herbivore bone collagen δ15N values, with permafrost and proximity to ice sheets likely playing a significant role in the generally low δ15N values observed in Late Pleistocene Europe39. Bone collagen δ34S values largely reflect the varying forms of sulphur present in soil, which is related to mineral input from the underlying geology, deposition of sulphates from groundwater and the atmosphere, and microbially-mediated fractionation processes51. As such, δ34S values are spatially variable and often considered a tool for exploring mobility and landscape utilisation53. However, bone collagen δ34S values may also hold significant promise as a palaeoenvironmental proxy representing changing hydrological and microbial processes55. In this study, bone collagen stable isotope analyses are performed on the 4 dated specimens, plus a further 7 reindeer bones from Kastelhöhle-Nord intermediate horizon.Bone collagen can also be analysed for its stable isotope compositions, which are powerful tools for investigating past ecology and terrestrial environments, and have been employed widely in Late Pleistocene archaeological research56. All samples had C:N atomic ratios between 3.2 and 3.4, and carbon and nitrogen content between 41–45% and 14–16%, respectively, comparable to in vivo collagen58. Sulphur content ranged between 0.14–0.19%, while C:S and N:S atomic ratios were between 625–785 and 190–245, indicating good sample integrity59.Collagen preservation at both sites was excellent; all samples produced collagen yields between 2.9% and 14.3%, which is comparable to the amount of collagen produced from modern samples prepared using the same methodology14C BP shall be used for this sample in the subsequent discussion of the results. The overall range represented by the new radiocarbon dates is 19,300 ± 90 14C BP to 19,121 ± 60 14C BP , and with a period of change to Alpine precipitation patterns . This phase of Alpine environmental change likely corresponds to Greenland Interstadial 2 , although the dating uncertainties do no permit correlation with absolute certainty. Nonetheless, the age range of the faunal radiocarbon dates does appear to correlate well with the local, short-lived episode of climatic amelioration and glacial recession , indicates that human activity during this time period was likely not restricted to a single occupation event. Indeed, while the Y-Höhle sample was found in a secondary context with no association to other archaeological remains5, the Kastelhöhle-Nord samples were found in association with a lithic assemblage of 228 artefact5, which could represent multiple periods activity at the site. The impact of the cooler phase that occurred within GI-2  cannot be assessed given the short duration of the event relative to the dating uncertainties.The slightly older date from Y-Höhle compared to Kastelhöhle-Nord  and lower Austria (Kammern-Grubgraben) have been interpreted as indicating long-distance contact across western and eastern central Europe during this time, and this is supported by the overlapping radiocarbon chronologies of the sites 19. More broadly, radiocarbon dating of archaeological material from Mittlere Klause in southern Germany, Langmannersdorf in Austria and Stranska Skala IV in the Czech Republic also span this time interval22.Although sparse, the archaeological record of central Europe after c. 25,000 BP does attest to human activity persisting during the LGM part of MIS 2, at least sporadically14C BP to 19,070 ± 60 14C BP , significantly refining the likely duration of human activity from a previous estimate of 19,380 ± 90 14C BP to 17,350 ± 190 14C BP23. This revised chronology shows striking overlap with the Swiss dates, and with GI-2. By comparison, Wiesbaden-Igstadt, Mittlere Klause, Langmannersdorf and Stranska Skala IV all have radiocarbon chronologies that span this time interval, but with age ranges too broad to evaluate whether a more certain relationship with GI-2 can be established. Indeed, the only ultra-filtered date from Mittlere Klause (OxA-9856 18590 ± 260 14C BP4) was later withdrawn due to concerns over potential contamination60. It would certainly be of interest to re-date this sample and revisit the chronology of other sites to further evaluate the timing of human activity in central Europe during the LGM.Recent chronological investigations at Kammern-Grubgraben (Layers 2–4) have produced a series of 6 ultrafiltered AMS dates ranging from 19,330 ± 70 13C values obtained from the Swiss reindeer samples are characteristic of a diet incorporating a significant proportion of lichen38. Lichens are typically enriched in 13C by c. 2–4‰, relative to C3 vascular plants growing within the same environment61, and this enrichment is reflected in reindeer skeletal δ13C, relative to other herbivores with a diet based solely on C3 vascular plants43. For many modern reindeer herds lichen is an important food source, particularly in the winter months when reindeer forage for the resource under snow cover62. It is likely that the ability to utilise lichen as a significant dietary component facilitated the survival of reindeer close to the ice sheet margins. Their use of this habitat could also explain the reason for human presence in the region during the LGM. However, it is not possible to evaluate whether reindeer, or indeed people, remained year-round in the vicinity of Kastelhöhle-Nord and Y-Höhle. Both sedentary and long-distance seasonal migratory behaviours are observed in modern reindeer herds65 and a similar diversity of behaviours has been argued for European Pleistocene populations69. It is therefore possible that human presence in northern Switzerland represents only short-term, perhaps seasonally-restricted, exploitation of local reindeer herds during periods of favourable environmental conditions.The δ15N and δ34S values provide information on the environment in which they lived. In the context of the Jura Mountains during the Late Pleistocene, soil maturity has been suggested as the controlling factor on reindeer δ15N isotope ratios, with low values (≤2‰) indicating recently deglaciated, nutrient limited landscapes and higher values (≥3.5‰) indicating reindeer were utilising refugial areas, where soils were more developed38. The majority of the LGM Kastelhöhle-Nord and Y-Höhle reindeer display intermediary δ15N values (+2.5‰ to +3.9‰). We suggest that the observed δ15N values could be produced in an environment where climatic/permafrost conditions limited soil nutrient cycling, which would have produced low environmental δ15N values, but where landscape stability facilitated the development/survival of soils and hardy vegetation enough to sustain reindeer populations. There is a smaller amount of variation in the δ34S values for the Swiss reindeer than in other LGM populations70, suggesting the Swiss animals had a more homogenous geographical range. However, it is interesting to note that of the four directly dated samples, the oldest (from Y-Höhle) has the lowest δ34S value (–12.6‰), while the three slightly younger Kastelhöhle-Nord samples have an average δ34S value of –8.2 ± 0.4‰, potentially indicating a temporal change in local environmental conditions or migratory behaviour.Regardless of whether the reindeer represent a sedentary or migratory population, their δ13C and δ15N values between locations is observed, suggesting reindeer ecology and behaviour plays a significant role in the derived isotopic signatures  and highest (≥5‰) δ15N values that are observed in the Middle Rhine region of Germany and southwest France respectively, are absent from the Swiss and French Jura and the Massif Central samples. While the lack of the highest δ15N values suggests an environment in which soil nutrient cycling was limited, probably due to overall low environmental temperatures, the lack of the lowest δ15N values also suggests periglacial processes likely did not dominate the environmental δ15N signature38. Significant location-based differences are apparent in the δ34S values . Reindeer δ34S values reflect the soil δ34S values upon which they fed. Soil δ34S values are related to underlying lithology and can be altered through changing rates of mineral weather and soil-bedrock interactions, controlled by hydrological conditions and bacterially-mediated reduction and oxidation processes55. The surface lithology of the regions compared  are underlain by similar late Mesozoic and Cenozoic limestone and sandstone deposits72, which are unlikely to vary in δ34S values to the extent observed in the reindeer data. This suggests environmental parameters are influential in the observed signal. Low environmental δ34S values have been related to low oxygen/water-logged soil conditions, though the processes that govern these fractionations are complex and not yet fully understood75. It is therefore possible that the lower δ34S values observed in the Swiss samples, compared to the French samples, could indicate an environment that had experienced wetter/more oxygen limited soil conditions. Such conditions could have been brought about by changing Alpine precipitation regimes, as identified in local speleothem records2, or by localised permafrost thaw related to the  increased temperatures of GI-2.When results are compared to published reindeer collagen stable isotope data from other regions in Europe during the LGM, considerable overlap in δres Fig. . The higues Fig. , althoug6. Stable isotope analysis of reindeer bone collagen, directly associated with human activity, further supports this interpretation. While the data indicates the landscapes in which humans hunted reindeer remained comparatively cold, it also suggests relatively stable environmental conditions. Compared to other European reindeer data, the higher carbon isotope values suggest a greater reliance on lichen by the Swiss reindeer, indicating that other vegetation types may have been more limited. The low sulphur and intermediary nitrogen isotope values could represent the influence of reduced/changing soil bacterial activity related to a brief respite in the most severe climatic conditions associated with the global LGM and local glacial advance. Combined, this data supports the hypothesis that human activity in Switzerland was made possible by a phase of climatic warming that occurred during the part of MIS2 that is more broadly characterised by global glacial advance.The refined chronological model presented here, based on current available archaeological evidence, indicates that the LGM human occupation of Switzerland was likely restricted to a brief period of local climatic warming, most probably associated with Greenland Interstadial 2. This supports the hypothesis put forward by Terberger and Street23, offer a tantalising insight into what could be a relatively brief episode of LGM human activity across central Europe, facilitated by the warmer environmental conditions that GI-2 provided. Whether the other known LGM archaeological sites in central Europe also correspond to this warmer period, or whether they represent different episodes of human activity during the LGM, can only be further examined through better chronological characterisation of these sites. Additionally, we stress the importance of also obtaining location-specific palaeoenvironmental records when making inferences about the types of landscapes in which human occupation took place.While the chronologies of many of the central European LGM archaeological sites are currently too imprecise to permit robust comparison, the striking similarities between the revised chronology for Switzerland and that recently published for Kammern-Grubgraben in Austria31, which is based on a modified version of the Longin75 method. All samples were treated with 0.5 M hydrochloric acid (HCl) at 4 °C until fully demineralised (24hrs – 2 weeks), then thoroughly rinsed with ultrapure water. Demineralised samples were then gelatinised in pH3 HCl solution at 75 °C for 48hrs and filtered using a pre-cleaned Ezee-filter. The filtrate was passed through a pre-cleaned 15–30 kD ultrafilter, with the >30 kD fraction collected and lyophilised.0.2 to 1.3 g of bone sample was collected from each specimen using a dental drill. Collagen extraction was performed at University College London (UCL) using a modified version of the Oxford Radiocarbon Accelerator Unit (ORAU) collagen extraction procedure (AF)13CVPDB = –26.4‰, δ15NAIR = –4.5‰), USGS41 , USGS43 , IAEA-S-2 , and IAEA-S-3  were analysed76. Results are reported as per mil (‰) relative to the internationally accepted standards VPDB, AIR and VCDT. Measurement uncertainty was determined to be ±0.1‰ for δ13C, ±0.2‰ for δ15N, and ±0.3‰ for δ34S on the basis of repeated measurements of an in-house bone collagen standard and a certified fish gelatin standard .For stable isotope analysis, 1.2–1.5 mg aliquots of purified collagen were weighed into tin capsules and analysed using a Delta V Advantage continuous-flow isotope ratio mass spectrometer coupled via a ConfloIV to an EA IsoLink elemental analyser  at the Scottish Universities Environmental Research Centre (SUERC). For every ten unknown samples, three in-house standards that are calibrated to the International Atomic Energy Agency (IAEA) reference materials USGS40 (L-glutamic acid, δet al.31. Approximately 5 mg of purified collagen was weighed into a tin capsule that had been baked at 500 °C for 12 hours. Samples were combusted using an elemental analyser coupled to an isotope ratio mass spectrometer, employing a splitter to allow for collection of the CO2 77. Samples were graphitised by reduction of collected CO2 over an iron catalyst in an excess H2 atmosphere at 560 °C79. 14C dates were measured on the Oxford AMS system using a caesium ion source for ionisation of the solid graphite sample24. To denote the bone pre-treatment at UCL rather than at ORAU, all measured dates were given “OxA-V-wwww-pp” numbers, where “wwww” indicates the wheel number, and “pp” is the position of the sample on the wheel24. As bone pre-treatment was performed in the laboratory at UCL dates were corrected for potential background contamination within this laboratory using two known-age reference samples80. Corrected dates are denoted by adding a “C” to the end of the date code assigned by ORAU. Uncorrected measured date values as well as further details of the correction calculations are provided in Supplemental Information 3. Details of the pre-treatment procedure used in the previous dating of these samples is given in Supplementary Information Radiocarbon dating was performed at ORAU using their standard procedures, as described by Brock Supplementary information.


Zea mays L.) production in Northeast China is vulnerable to climate change. Thus, exploring future adaptation measures for maize is crucial to developing sustainable agriculture to ensure food security. The current study was undertaken to evaluate the impacts of climate change on maize yield and partial factor productivity of nitrogen (PFPN) and explore potential adaptation strategies in Northeast China. The Decision Support System for Agrotechnology Transfer (DSSAT) model was calibrated and validated using the measurements from nine maize experiments. DSSAT performed well in simulating maize yield, biomass and N uptake for both calibration and validation periods  < 10%, −5% < normalized average relative error (nARE) < 5% and index of agreement (d) > 0.8). Compared to the baseline (1980–2010), the average maize yields and PFPN would decrease by 7.6–32.1% and 3.6–14.0 kg N kg−1 respectively under future climate scenarios (2041–2070 and 2071–2100) without adaptation. Optimizing N application rate and timing, establishing rotation system with legumes, adjusting planting dates and breeding long-season cultivars could be effective adaptation strategies to climate change. This study demonstrated that optimizing agronomic crop management practices would assist to make policy development on mitigating the negative impacts of future climate change on maize production.Maize ( Zea mays L.) is one of the most important cereal crops to support food security. However, the global average maize yields have declined by approximately 3.8% over the last decades due to climate change1. Northeast China is the dominant maize production area in China and plays a unique role in food security. Maize planting area and production in this region account for 31.5% and 32.8% of the total for China, and 6.8% and 7.4% of the global total in 2018, respectively3. However, Northeast China is one of the most vulnerable regions to climate change, as annual mean temperature has been significantly increasing by 0.38 °C per decade, and precipitation has decreased slightly with more frequent droughts and floods during the last 50 years5. Previous studies have showed that the negative impacts of climate change on maize yield were mainly associated with the warming and increased drought frequency during the growth periods in Northeast China8. Maize potential and attainable yields were estimated for a reduction of 2.1% and 8.0%, respectively in Northeast China from 1961 to 2009 due to climate change using the Agricultural Production Systems Simulator (APSIM) model7. Maize yield gaps are mainly affected by management practices  over the period 1961–20109. Thus, adjustment of management practices  played an important role to close yield gap, improve nutrient use efficiency (NUE) and minimize environmental risks under climate change. Lv et al.7 demonstrated that the improved cultivars and agricultural practices could mitigate the negative impacts of climate change from 1961 to 2009 relative to the yields of the cultivar planted in the 1960s. Thus, exploring effective adaptation measures in Northeast China is crucial to improving maize production and maintaining environmental health under future climate change.Maize 15. Fertilizer application rate and timing should be adjusted to meet the nutrient demands of crop growth and avoid nutrient loss when crop biomass decreases due to water and temperature stress over time under climate change15. For example, He et al.14 explored the response of maize yields to fertilizer application rate under future climate scenarios, which suggested that the nitrogen (N) rate of 150 kg N ha−1 would be suitable for high maize yields in Canada based on the Decision Support System for Agrotechnology Transfer (DSSAT) model. Crop rotation diversity is beneficial to improving soil physical quality, nutrient availability and soil microbial diversity, which contributes to high crop yield and low environmental risk18. Ma et al.19 predicted that including legumes in rotation would be advocated for mitigation under a changing climate, which would increase crop yields by about 5% for rainfed agricultural systems in Australia. Therefore, exploring adaptation strategies based on comprehensive agronomic management practices are essential to promoting sustainable maize production in Northeast China under future climate change.Many potential adaptation strategies have been explored to mitigate the negative impacts of maize yield under climate change conditions. Several studies indicated that adjustment of crop phenology could be beneficial for mitigating yield loss under future climate scenarios 21. The DSSAT model is a widely used tool for testing cropping technologies, assessing management practices, and exploring climate change mitigation stategies24. The DSSAT model has been successfully used to optimize field management practices to achieve high crop yield, improve understandings of crop physiology, soil management and weather effects on crop growth and environmental quality26, and explore the responses of crop production to climate change and develop effective adaptation strategies27. Although the DSSAT model has been used to simulate the impacts of climate change on maize yield, no detailed adaptation strategies were assessed in simulations by considering comprehensive management practices for improving maize production and N use efficiency in Northeast China. Therefore, the objectives of this study were (1) to calibrate and evaluate the DSSAT model using the measured maize yield, biomass and N uptake from nine field experiments in Northeast China; (2) to simulate the climate change impacts on maize yield and partial factor productivity of N (PFPN) during two future periods of 2041–2070 and 2071–2100, relative to the baseline (1981–2010) under Representative Concentration Pathways (RCP) 4.5 and 8.5 scenarios; and (3) to explore potential adaptation strategies to reduce the negative impacts of future climate change on maize yield and N use efficiency.Process-based models are valuable tools in evaluating management practices and climate change impacts on crop production, soil water balance and carbon (C) & N dynamics in diverse agroecosystemsFor calibration, the simulated maize yield, biomass and plant N uptake matched well with the measured data under the optimum nutrient application (OPT) treatment at all sites. The average statistical values of nRMSE ≤ 6.0%, −4.8% ≤ nARE ≤ 0.4% and d ≥ 0.79 indicated “good” to “excellent” agreements between the simulated and measured maize yield, biomass and plant N uptake Table , 2. For −1, 44.6 to 33.7 kg N kg−1 and 44.2 to 36.2 kg N kg−1 at LN, JL and HLJ respectively under future climate scenarios  and two future periods [2050s (2041–2070) and 2080s (2071–2100)] based on two climate change scenarios (RCP4.5 and RCP8.5)  rotation under both baseline and future climate scenarios  mineralization and crop needs15. Crop rotation diversity is an important management practice to increase crop yield and reduce environment pollution by modifying the soil environment across a wide range of soil types and climatic conditions18. Smith et al.15 predicted that yields increased for rotational maize under climate change due to higher SOC and reduced crop water stress in Canada, meanwhile, the lower simulated N runoff loss was found in rotational maize and higher nitrous oxide (N2O) emissions, but the lower annual N2O emissions were observed during the entire rotation. Our simulation showed that the negative impacts of future climate change on maize yields could be mitigated under the maize-soybean rotation system mainly due to the improved soil fertility, soil water utilization and nutrient use efficiency41. Previous study indicated that maize yield had stronger responses with legume rotation than other cereal crops 39.It has been reported that the negative impacts of climate change on crop productivity could be mitigated by effective agronomic adaptation techniques and seed genetic improvementcy Table  led to r10, in Burkina Faso for maize11, in the west and northwest Iran for wheat12 and in Canada for spring wheat and maize14. These adverse impacts of climate change could be partially offset by optimal planting date, mainly due to the ability to match crop growth with changed temperature and rainfall distribution. Our results indicated that delaying planting date would be beneficial for maize yield under future climate change across all sites  and thermal time from silking to physiological maturity (P5) are the most sensitive periods for maize yields21. In the CSM-CERES-Maize model, the cumulative thermal time from emergence to tassel initiation and daily development rate of maize from silking to maturity could be calculated based on P1 and P5 parameters45. Reasonable increase of the values of the parameters P1 and P5 would mitigate the negative impacts of future climate change on maize yields based on the DSSAT simulation, implying that longer growing season cultivars should be developed in order to benefit the maize crop from longer growing seasons under warmer future climates and to cope with heat stress. Our study was in agreement with He et al.14 who indicated that breeding longer growing season cultivars with high thermal degree days could result in higher maize yield under future climate scenarios based on modelling using the DSSAT model.Changing planting date is considered an effective adaptive strategy to mitigate the negative impacts of climate change on crop yields on a global scale, such as in Northeast China for maizetes Fig. . This is47. In this study, the dynamic measurements of soil inorganic N (NO3−–N and NH4+–N) were not available to calibrate and evaluate soil N simulations during the field experimental periods, which may cause uncertainty in long-term C & N feedback under climate change scenarios. In the model simulation, soil parameters were assumed to remain constant in the future, but certain soil physical properties  may change under different management practices  which could further affect maize yield and soil process25. Basso et al.48 and Smith et al.15 indicated that soil C & N dynamics and water status could be affected if soil physical properties were re-initialized in a long-term simulation. In addition, most crop models including the DSSAT model do not incorporate the explicit simulation of the heat impacts on male and female flowering, fertilization of female flowers, and kernel abortion which may lead to more uncertainties in predicting crop production under future climate scenarios49. Furthermore, although the DSSAT model performed well in simulating crop yield and nutrient cycling under various soil and crop management practices, the model does not simulate the direct impacts of pests and/or diseases, extreme weather  and complex nutrient transfer processes50, which could lead to the uncertainties in the simulation.Overall, changes in simulated maize yield under future scenarios could be explained based on crop-climate-soil interactions. However, the uncertainties in projections of climate change impacts on crop growth in agro-ecosystems are large and unavoidable as they inherited from uncertainties in climate and crop models, accuracy in model calibration and evaluation, and change in socio-economic emission scenarios−1 with two time-splitting at LN and JL and at 150–180 kg ha−1 at HLJ would result in high maize yields and NUE under future climate scenarios. Maize-soybean rotation with lower fertilizer application rates could be beneficial to obtaining higher maize yields compared to maize monoculture for both baseline and future climate scenarios. Late planting could mitigate the negative impacts of climate change on maize partially due to the increased precipitation in July and August in the future. Developing longer growing-season cultivars should be recommended to obtain stable and high maize yield under future climate change conditions. Optimized agronomic crop management practices could be considered as effective adaptation strategies to climate change for maize production in Northeast China.The well calibrated DSSAT model was proven a capable tool for assessing climate change impacts on maize yield and NUE and exploring adaptation strategies under RCP 4.5 and RCP 8.5 scenarios in Northeast China. Our study indicated that maize yields and NUE would significantly decrease under future climate scenarios at LN, JL and HLJ provinces compared to the baseline scenario if no adaptation measures were taken in the simulations. Optimized fertilization rate at 180–210 kg ha53. The typical maize cultivars at these sites were selected to represent regional differences of the climatic conditions. These experimental sites were initially established to optimize fertilization management with high quality datasets including maize yield, biomass, nutrient uptake and soil properties which provided detailed inputs to run the DSSAT model. Thus, using modelling approach in major maize planting areas with typical cultivars is feasible to explore potential adaptation management practices under future climate change in Northeast China. Detailed field experimental practices for each site are shown in Table Field experiments were conducted from 2011 to 2016 in Northeast China, where is the main maize production region comprising of Liaoning (LN), Jilin (JL) and Heilongjiang (HLJ) provinces. There were nine experimental sites including Chaoyang (LNCY) and Changtu (LNCT) in LN; Liufangzi (JLLFZ), Taojia (JLTJ) and Chaoyang (JLCY) in JL; and Qiangan (HLJQA), Shuangcheng (HLJSC), Binxian (HLJBX) and Harbin (HLJHRB) in HLJ Table . In this54. For the OPT treatment, the N fertilizer (urea) application rate ranged from 150 to 211 kg N ha-1 at the experimental sites. 1/3 and 2/3 of the total rate was applied as basal fertilizer and side-dressing respectively at the jointing stage at LN and JL sites, whereas 40% and 60% of the total fertilizer was applied as basal and side-dressing respectively at HLJ site. Additionally, the ratio was changed to 1/4, 2/4 and 1/4 at the sowing, jointing and tasselling stages from 2015 to 2016 at JL sites. For the FP treatment, N fertilizer application rate varied from 153 to 280 kg N ha-1 at the experimental sites which was 100% applied as basal fertilizer. More detailed management practices for each experiment site are shown in Table −1) was calculated based on grain yield/fertilizer N rate. Detailed measurement information can be referred to Xu et al.54.Two treatments were set for each experiment consisting of optimum nutrient application (OPT) from Nutrient Expert system and farmers’ practice (FP)http://dssat.net/) is a mechanistic dynamic model combined with the Crop System Model (CSM), two soil C & N models (the CERES-based and the CENTURY-based soil models) and a soil water balance model (Ritchie method), which simulate crop growth, soil water balance and soil C & N cycling with daily time-step under different cropping systems, management practices and climate conditions45. The CENTURY-based module was employed to simulate soil C and N processes because it was more suitable for long-term sequence simulations55. The CSM-CERES-Maize module was used to simulate maize growth in the experiment years and predict the impacts of climate change scenarios on maize growth. Additionally, the CSM-CROPGRO-Soybean module was used to simulate soybean growth (maize-soybean rotation) under climate change scenarios for management adjustments23.The Decision Support System for Agrotechnology Transfer  the local daily climate data ; (b) initial soil condition ; (c) field management practices ; (d) crop cultivars. The weather data was obtained from the local weather station at the each experimental site. The annual and seasonal mean climate variables from 2011 to 2016 are shown in Supplementary Table 21, which mainly include parameters for determine critical phenology stages  is delayed for each hour where the photoperiod is greater than 12.5 h and P5, thermal time from silking to physiological maturity), grain filling , and phylochron interval between successive leaf tip appearances (PHINT). The calibrated cultivar coefficient is listed in Supplementary Table 3−–N and NH4+–N) are only required to be setup before the first year simulation, then soil water, C and N flows could be continuously transferred from the beginning to the end of the simulation automatically56. In this study, the calibrated DSSAT model was then employed to predict the impacts of climate change on maize yields and N use efficiency. The cultivar parameters for soybean were used in the maize-soybean cropping system under future climate change based on the calibration from Liu et al.23 In the simulation of maize-soybean rotation, the parameterizing information for rotation maize  was consistent with the monoculture maize simulation. The Sequence Analysis mode was used to simulate crop rotations, which could carry-over the soil water and nitrogen processes from one crop to another57. Two sequences were created (maize-soybean and soybean-maize rotations) with the same weather conditions to ensure that each phase of the rotation presents in each year. Based on the previous study23, on average, soybean was planted on May 1 and harvested on September 30 for each year at all sites, and N fertilizer (urea) was applied at 20 kg N ha−1 as basal application.In this study, the DSSAT model parameters were calibrated using the measurements from OPT treatments for all years at each experimental site Table . Cultiva60.i is the simulated value, Mi is the measured value, i = 1, …, n is the number of measured values, and The model performance statistics can be used to evaluate the difference between simulated and measured data, including root mean square error (RMSE), normalized root mean square error (nRMSE), normalized average relative error (nARE), and index of agreement (d) value see Eqs. –4 below559. The model performance was satisfactory for yield and biomass if nARE < ± 15%60. The value of nARE (%) value indicates underestimation when the nARE < 0 or overestimation when the nARE > 0 compared to the measured values. For d value, d ≥ 0.9, 0.8 ≤ d < 0.9, 0.7 ≤ d < 0.8 and d < 0.7 illustrates “excellent”, “good”, “fair” and “poor” match, respectively23.For nRMSE, an “excellent”, “good”, “fair” and “poor” model performance is claimed when the nRMSE ≤ 10%, 10 < nRMSE ≤ 20%, 20% < nRMSE ≤ 30% and nRMSE > 30%, respectively61. This method relies on empirical relationships between observed climate data and data from global climate models (GCMs). The simulated monthly data by the GCMs were statistically downscaled to the specific sites using the inverse distance-weighted (IDW) interpolation method. A bias correction procedure was applied during this step to correct biases in the site-based monthly GCMs values. And then daily climate variables  were temporally scaled for each site from the spatially downscaled monthly data through the WGEN stochastic weather generator. Unlike other statistical downscaling methods that require complicated data  and high computational cost , statistical downscaling method is reliable and rapid mainly due to the use of historical observation data to modify the monthly GCMs data and low computational cost. These climate data obtained from this method could match with crop models in order to explore the future climate impacts on agricultural systems. Due to the complexity of the climate system, the future climate data was produced by Climate System Models from Beijing Climate Centre BCC-CSM1.1 (BC1) and BCC-CSM1.1 (m) (BC2) which were obtained from the Coupled Model Inter-comparison Project phase 5 (CMIP5) to drive the DSSAT model in this study. The BC1 model is a fully coupled global climate-carbon model with an interactive vegetation and global carbon cycle, which includes the atmospheric component BCC Atmospheric General Model version 2.1 (BCC_AGCM2.1) with a horizontal resolution of T42 (approximately 2.8125° × 2.8125° transformed grid), 26 levels in a hybrid sigma/pressure vertical coordinate system with the top level at 2.914 hPa, ocean component Modular Ocean Model version 4 (MOM4)-L40, land component BCC Atmosphere and Vegetation Interaction Model version 1.0 (BCC_AVIM1.0), and sea ice component [sea ice simulator (SIS)]62. The BC2 model is established to increase the horizontal resolution of the atmospheric component and land component to T106 (approximately 1.125° × 1.125° transformed grid), forming BCC_AGCM2.2 and BCC_AVIM1.1, respectively based on the BC1 version63. Compared to the BC1 model, the BC2 model with finer horizontal resolution performed better for some climate characteristics. For example, the spatial variability of the simulated climatological monthly precipitation is closer to the observations and tropical sea surface temperature annual cycles in the tropical ocean are generally more reasonable63. In this study, both climate models (BC1 and BC2) were used to explore climate change impacts on maize production in Northeast China.In this study, climate change datasets were generated based on the statistical downscaling methodhttp://data.cma.cn/en). Two Representative Concentration Pathway scenarios  based upon radiative forcing levels of 4.5 and 8.5 w/m2 at the end of the 21st century, respectively were selected for two future periods from the 2050s (2041–2070) and the 2080s (2071–2100). Historical meteorological data from one representative experiment in each province is selected to be used for downscaling and bias-correction of the future climate data from the GCMs. The calibrated parameters for maize cultivars of FY9, NH101 and GF1 at LN, JL and HLJ sites respectively were used in the climate scenario simulations  and sidedressing 54; (b) Maize-soybean rotation was considered compared to the maize monoculture23; (c) Planting dates were shifted early/late with a 10 day interval based on the default 14; (d) Maize cultivar with longer growing seasons were developed based on two thermal time parameters of P1 [thermal time from seedling emergence to the end of the juvenile phase (degree days > 8 °C)] ranging from 160 to 425 °C.d. with a 50, 30 and 25 °C.d. interval and P5  ranging from 656 to 980 °C.d. with a 50 °C.d. interval at LN, JL and HLJ, respectively14. For default parameters, planting date  and fertilizer application rate  as basal at LN, JL and HLJ sites will be used in the future climate scenarios, respectively.Four adaptation strategies were explored to mitigate the negative impacts of climate change on maize production based on sensitivity analysis under baseline and future climate scenarios in Northeast China. The management practice scenarios include the following: (a) fertilizer N application rates were simulated from 0 to 300 kg N haSupplementary Information.


These heat treatments resulted in the formation of tetragonal σ precipitates that formed heterogeneously at different elements of the microstructure. The raw data of the present article include backscattered electron (BSE) micrographs where σ precipitates can be observed within grains, at grain boundaries, and triple points of the FCC matrix. From these images, the dimensions of the five largest precipitates observed within grains and those of the five largest allotriomorphs are provided for different times and temperatures in tables. As the σ precipitates are more enriched in Cr and depleted in Ni than the surrounding matrix, Cr-depleted (Ni-enriched) zones form in the FCC matrix next to the precipitates and widen at rates determined by diffusion. To document the evolution of the corresponding concentration profiles with time, electron dispersive X-ray spectroscopy (EDX) was used and the data are reported in Excel files. From these concentration profiles, the widths of the diffusion affected zones for Ni and Cr were systematically determined at different temperatures and times, apparent diffusion coefficients were deduced and all these data are provided in tables. The research data reported here have a fundamental value and document the growth kinetics of σ precipitates within grains and at grain boundaries. These data may help to establish a model able to predict how the precipitation kinetics of σ particles in FCC HEAs is affected by the alloy grain size and how the microstructure  evolves with time and temperature. This approach may also be extended to austenitic steels and superalloys.A data compilation related to the growth kinetics of a topologically closed-packed (TCP) phase is reported. A high-entropy alloy (HEA) with a composition of Cr Specifications Table•26Mn20Fe20Co20Ni14 (in at.%) high-entropy alloy (HEA). These data may be useful for other researchers in the community of HEAs and compositionally complex alloys (CCAs). Moreover, they constitute benchmark data that could be used to refine existing models dealing with the precipitation kinetics of TCP phases in polycrystalline FCC alloys.The fundamental research datasets reported here are related to the growth kinetics of σ precipitates in an initially single-phase FCC Cr•The microstructural data compilation reported here  may be used to improve existing algorithms allowing the automated analysis of microstructures.•The temporal evolution of concentration profiles at interfaces (FCC/σ and FCC grain boundaries) may serve as a benchmark dataset for phase-field modelling.•i.e., they could help to understand how to tune their chemistry to impede their formation thermodynamically and/or kinetically.Our data may be used to improve the understanding of the precipitation kinetics of TCP phases in complex engineering alloys such as superalloys and austenitic steels, •The apparent diffusion coefficients reported here could be useful for other researchers working in the field of diffusion and are interested in how these coefficients are affected by chemistry and dislocation density.1e.g., how can one explain solid solution strengthening in these alloys Recently, high-entropy alloys (HEAs) allowed to study several fundamental questions, P42/mnm) in an initially single-phase Cr26Mn20Fe20Co20Ni14 HEA (composition in at.%) with an average grain size of 50 µm. Anneals between 600 °C and 1000 °C for times from 0.05 h to 1000 h followed by air cooling were performed to study the growth kinetics of σ precipitates that formed within grains and at grain boundaries.The data compilation of the present article includes microstructural and compositional data related to the precipitation of σ precipitates  in an alloy annealed at 700 °C for 1000 h. This Excel file has five sheets labelled from “GB1” to “GB5” representing individual datasets recorded at different locations. In each Excel sheet, the distances (in µm) between the first point of the EDX line scan and the subsequent points are listed in column A. The corresponding local concentrations in Cr, Mn, Fe, Co, and Ni are provided in at.% in columns B to F, respectively. As another example, the Excel file “1000C_00.1h_FCC-sigma” comprises five EDX line scans  that were recorded at five distinct FCC/σ interfaces in an alloy annealed at 1000 °C for 0.1 h (6 min). One end of each EDX line scan was always located far away from σ particles and FCC grain boundaries. At this extremity, the measured FCC composition should reflect the instantaneous composition of the FCC matrix and the values obtained at different times and temperatures are given in The concentration profiles, forming at interfaces and resulting from the precipitation of the σ phase, were recorded by EDX perpendicularly to interfaces (FCC/σ interfaces and FCC grain boundaries) after different anneals. The From the concentration profiles, the characteristic widths of diffusion affected zones that form at σ/FCC interfaces and FCC grain boundaries were determined using the methodology illustrated in Figs. 3a,b of Ref. WDAZ ≈ 2 (D t)1/2 where WDAZ is the width of the diffusion affected zone after annealing for a time t and D is the apparent diffusion coefficient. The values obtained for Cr and Ni are reported in Apparent diffusion coefficients between 600 °C and 1000 °C were deduced from the widening kinetics of the diffusion affected zones provided in Tables 8–11 using 226Mn20Fe20Co20Ni14 (in at.%) HEA was solution annealed at 1080 °C for 15 min followed by air cooling resulting in a single-phase FCC alloy with a mean grain size of ∼50 µm. Small pieces (∼8 × 8 × 2 mm3) were subsequently annealed in evacuated quartz tubes at temperatures between 600 °C and 1000 °C for times up to 1000 h followed by air cooling. These pieces were then prepared metallographically and electropolished following the procedure reported in ZIP file attached to this paper). This allowed to follow the growth kinetics of σ precipitates observed within grains of the FCC matrix and those located at the grain boundaries. For each BSE micrograph, the radii of the five largest precipitates observed within grains were measured resulting in a total of 20 measurements (four BSE micrographs × five precipitates). Despite the fact that some of the σ precipitates observed within grains showed some facets, they were treated as spherical and their mean radii are provided in lGB) and average half-thickness (thGB) are reported in The CrZIP file attached to this paper. The EDX experiments were performed such that at least one extremity of each concentration profile was located in the FCC phase as far as possible from σ particles and grain boundaries. Thus, the FCC composition reflecting the instantaneous solute concentration, Concentration profiles resulting from σ-phase precipitation in an FCC matrix were recorded across interfaces (σ/FCC interfaces and FCC grain boundaries) after different heat treatments by energy-dispersive X-ray (EDX) spectroscopy using a detector of type Oxford X-Max 80 SDD in a JEOL JSM-7200F SEM at an acceleration voltage of 15 kV, a probe current of 14 nA, and a working distance of 10 mm. EDX point analyses were carried out at discrete points (5 s each) along test lines made at right angle to grain boundaries and σ/FCC interfaces using a step size in the range [0.04 - 0.2 µm] depending on the width of the diffusion affected zones in the vicinity of these boundaries. For each heat treatment, at least five EDX concentration profiles were recorded at FCC/σ interfaces as well as FCC grain boundaries and the corresponding data can be found in the WDAZ) given in Tables 8–11 for different temperatures, apparent diffusion coefficients (D) for Cr and Ni were deduced using the following equation of Ref. WDAZ ≈ 2 (D t)1/2 where t is the annealing time. These apparent diffusion coefficients for Cr and Ni are provided in Using the temporal evolution of the width of the diffusion affected zones (The author declares that he has no known competing financial interests or personal relationships, which have, or could be perceived to have, influenced the work reported in this article.


New clinical trials for diabetic macular oedema (DMO) are being designed to prove superiority over aflibercept when this agent is already very effective in improving visual acuity (VA) and DMO. The aim of this study was to determine the optimal inclusion–exclusion criteria for trials to aim for superiority in visual outcomes with newer agents. As Phase 1 studies are short duration, we aimed to evaluate the early response of aflibercept in a real-world cohort initiated on monthly aflibercept for 3 consecutive injections and observed the effects at 4 months. The sub-optimal responders were pre-defined based on different cut-offs for VA and central sub-field thickness (CST). 200 patients with treatment naïve DMO treated with 3 loading doses of aflibercept were included in the study. We found that those presenting with baseline VA of 35–54 ETDRS letters (n = 43) had higher proportion of sub-optimal responders compared to other categories (p < 0.001). Patients with baseline CST of less than 400 µm (n = 96) responded less well functionally and anatomically to loading dose than eyes with baseline CST of 400 µm or more , indicating that eyes with CST ≥ 400 µm is another inclusion criteria. There was minimal correlation between change in CST and change in VA at 4 months (r = − 0.27), suggesting that both these inclusion criteria are non-exclusive. However, for maximal efficacy, patients that meet both these inclusion criteria are more likely to show benefit from an alternative intervention. New trials should aim to include patients with treatment naïve DMO with VA between 35–54 letters and CST of 400 µm or more when aflibercept is used as the comparator. The standard treatment of eyes with visual impairment due to DMO is repeated intravitreal anti-vascular endothelial growth factor (anti-VEGF) injections until disease stability is achieved8. In clinical trials, treatment with any of the three currently available antiVEGF agents, bevacizumab, ranibizumab or aflibercept in eyes with visual impairment due to DMO resulted in approximately 50% of eyes gaining more than 2 lines of vision over 2 years and 30% gaining 3 or more lines of vision9. Protocol T showed that aflibercept is more efficacious when considering the change in visual acuity (VA) over time especially in the loading phase and it is also more effective in causing resolution of the macular fluid4. Therefore, in clinical trials on new agents, aflibercept should ideally be used as the comparator.Centre-involving diabetic macular oedema (DMO) is a sight threatening complication of diabetes characterised by macula fluid that can be qualitatively and quantitatively assessed using optical coherence tomography (OCT)10.Currently, several novel agents are being investigated for their efficacy in DMO. These drugs should ideally show superior response and/or durability whilst maintaining easy delivery and similar or lower costs compared to aflibercept to be more advantageous. A key challenge is the design of clinical trials for these agents. As proof-of-concept studies are short , novel agents should ideally show an early superior VA outcome within 4–6 months for the drug manufacturers to plan a future superiority trial. Early VA responses at 12 weeks have been explored to assess long-term outcomes with conflicting results in trials of new agents aiming for superior visual outcomes10. In Protocol U, when treated with ranibizumab loading dose before randomisation approximately 45% of the patients defined as having persistent oedema improved, suggesting that most eyes assumed to have persistent oedema respond to anti-VEGF agent when optimally treated.It is also clear from the trial design of Protocol U that there is always room for improvement in eyes with DMO when sub-optimally treated with anti-VEGF agents11. Moreover, the DRCR protocol W showed that the majority of eyes with good VA defined as Snellen 20/25 did not show a deterioration of VA over 2 years, with only about 17% needing initiation of aflibercept injections during this period12.There are other challenges that also need to be considered. For example, the recent analysis by Bressler et al., showed that there is no correlation between VA outcomes and changes in central subfield thickness (CST) at 12, 52, and 104 weeks after antiVEGF agents for DMOTherefore, choosing the ideal cohort to evaluate novel agents is clearly a necessity to be certain that the effects of these agents are superior to current therapies. Sub-optimal response to aflibercept in the loading phase in real-life is not well studied. Although post-hoc analysis of Phase 3 RCTs are available, these do not represent all patients with DMO especially those with good presenting VA.The aim of this study was to determine the early response to aflibercept loading phase of 3 injections in treatment naïve DMO in real-life to extrapolate the inclusion criteria for investigating novel agents. Real-life studies are better for this study because we require an unselected DMO cohort to define the inclusion criteria and where suboptimal responses may be identified better than in the best care scenario seen in the strict protocols of a randomised controlled trial.What is the ideal visual acuity inclusion criterion for clinical trials on new investigational products for DMO when the comparator is aflibercept?What is the central macular thickness that will potentially respond better to another treatment?Are there any anatomical features that need to be excluded to ensure optimal response?The research questions included:This was a retrospective, single-centre study. The study was approved by the Clinical Effectiveness Department, Moorfields Eye Hospital, London (Study Number 577) and informed consent from study participants was waived. The study adhered to the Declaration of Helsinki.We conducted a search of the Moorfields Eye Hospital electronic patient records (Open Eyes) for patients diagnosed with new onset DMO commenced on aflibercept therapy between December 2016 and September 2019.Centre-involving DMO was diagnosed clinically, confirmed by fundus fluorescein angiography (FFA) and spectral domain optical coherence tomography (SD-OCT). To be included in this study, the eyes had to complete monthly intravitreal aflibercept therapy for 3 months and follow-up visit at 4 months. The presence of DMO was defined as central subfield thickness (CST) ≥ 300 µm on SD-OCT  due to intraretinal oedema and/ or subretinal fluid due to DMO. Vitrectomised eyes and eyes previously treated with panretinal photocoagulation (PRP) were included. Although macular laser was not an exclusion criterion, there were no patients with previous macular laser.Exclusion criteria for the study eye included the following: (1) any intravitreal injections of anti-VEGF agents or corticosteroids prior to initiation of aflibercept; (2) presence of other ocular disease that may confound the results ; and (3) patients who had inadequate retinal imaging at baseline and 4 months.For eligible patients, the following baseline data were collected from their medical records: age, gender, ethnicity, type of diabetes, VA measured in Early Treatment Diabetic Retinopathy (ETDRS) letter score in clinics, SD-OCT central subfield thickness and total macular volume and severity grade of diabetic retinopathy. The VA measurements were also collected 1 month after 3 loading injections. All SD-OCT scans at baseline and after 3 loading injections were double graded for the following morphological parameters: predominant type of DMO ; cyst location ; cyst size ; disorganisation of retinal inner layers within the central 1 mm, state of subfoveal external limiting membrane (ELM) defined as absent, disrupted or intact in central 500 μm, state of subfoveal ellipsoid zone (EZ) defined as absent, disrupted or intact in central 500 μm, presence/absence of posterior vitreous detachment, presence/absence of vitreomacular abnormalities, presence/absence of epiretinal membrane (ERM), and presence of hyperreflective foci .Limited early response was defined utilizing both functional and anatomical parameters. Limited functional response was defined as a gain of less than 5 letters and anatomical limited response was defined as < 10% decrease or increase in central subfield thickness after 3 loading injections.Descriptive data were presented as means and standard deviation (SD). Wilcoxon Rank test was used to compare outcome variables between baseline and follow-up visits. Baseline VA was stratified into four subgroups: 70 ETDRS letters or better; 55–69 letters, 35–54 letters and < 35 letters, and baseline and 1-month post loading VA within each subgroup were compared using Wilcoxon Rank test. All parameters were compared between baseline and one-month post loading phase. The morphological parameters were compared between those eyes that responded versus limited responders using Pearson chi-squared test or Fisher’s exact test when the expected value in any cell is 5 or less. Statistical analysis was performed using STATA software .Two hundred eyes that received intravitreal aflibercept loading doses for new onset DMO between December 2016 and September 2019 and had complete dataset were included in the analyses. At baseline, the mean age of the study cohort was 62.8 (SD 12.2) years, 64% were males and of multiethnic descent  and 91% of the cohort had type 2 diabetes.The mean baseline VA (SD) was 62.3 SD 15.2) ETDRS letters. The mean change in VA for the whole cohort was + 4.8 (SD 10.2) ETDRS letters after the 3 loading injections. 107 (53.5%) did not gain 5 or more letters after the loading dose. Table 5.2 ETDRSThe majority of the patients who presented with VA categories 55–69 letters (Snellen 20/80 to < 20/40) and 35–54 letters (Snellen 20/200 to < 20/80) showed significant improvement in VA. Higher standard deviation was observed in the lower VA categories. Although, the maximum VA gain was observed in the category of < 35 letters, the numbers of patients who presented in this category is low.Table Baseline CST categorised into < 400 μm and ≥ 400 μm is shown in Fig. When considering the whole cohort, 107 (53.5%) did not gain 5 or more letters after the loading dose. These patients had mean baseline VA of 66 letters (SD = 15.3), approximately 5 letters higher than the mean of the whole cohort and 76.3% of the patients presented with 70 letters or better. On excluding the patients with 70 or more letters at baseline, 46 patients gained less than 5 letters. In this group, 19/46 (41%) were in the 35–54 (41%) and 55–69 letter (50%) groups at baseline. However, although the 55–69 letter group had considerable numbers of non-responders based on this definition, 57.4% of patients in this group improved to 70 letters and the standard deviation was lesser than the 35–54 letter category. So, the chance of superiority in this group is lesser than the 35–54 letter group.The mean baseline CST of the limited responders (decrease of less than 10% of baseline CST) was 382 µm (SD = 108) and not dissimilar to the mean baseline CST of 420.9 µm (SD = 115.3) for the whole group. The mean CST after 3 injections was 314.7 µm (SD = 100.7) for the whole cohort (p < 0.001), with a mean reduction of CST by 106.2 µm (SD = 112.2). In contrast, the mean CST after 3 injections in the anatomical limited responder was 391 µm (SD = 122) (p = 0.123) and the mean change of CST was only 9 µm (SD = 54.3).13. The correlation in the limited responders with baseline < 70 letters (n = 46) was − 0.02  Fig. 13. The cWe also compared the disease characteristics of the responders and limited non-responders defined as gaining less than 5 letters in both the whole cohort and in the 35–54 and 55–69 letters category  were less likely to have CST reduction of 10% or more. Although these anatomical changes can be used as imaging biomarkers to predict response, EZ layer is often masked by shadowing of intraretinal fluid at initiation of treatment and may re-constitute or re-appear post macular fluid resolution. Loss of integrity of EZ layer is a poor prognostic marker of VA but it is difficult to ascertain the integrity of EZ layer in the presence of cystoid macular oedema adjacent to the outer retina and is therefore best to avoid this point within the inclusion–exclusion criteria.When we consider the diabetic retinopathy status, a higher proportion of patients with co-existing PDR did not achieve 5 letter gain after 3 aflibercept loading doses but this group did not show any difference in response in reducing CST by more than 10% suggesting that this difference is not related to presence of DMO. This effect may be due to co-existent macular ischaemia or abnormalities at vitreo-retinal interfaces such as epiretinal membrane, which is more prevalent in eyes with PDR than NPDR. Therefore, in future trials, a sub-analysis of outcomes based on DR status is recommended.The strength of this study is that we have assessed the response of 3 loading injections of aflibercept in patients in clinical practice and therefore our findings are more generalisable compared to clinical trial cohorts. Our findings on CST and VA correlation resemble that of previous similar analysis done in clinical trials further justifying that the observations noted in this study may be used to plan future studies.14.This study is limited in that it did not assess potential associations of DMO with other factors such as the presence of macular ischemia and level of glycaemic control, which have both been shown to be directly associated with poor VA outcomesIn conclusion, we have characterised the baseline features of limited early responders to aflibercept loading dose to allow newer drugs to be evaluated on a more appropriate patient cohort. We found that those with baseline VA between 35–54 ETDRS letters and CST >  = 400 μm are the ideal cohort that will most likely benefit from trials with other agents.


Above ΔΨm values of 140 mV, the production of ROS in mitochondria increases exponentially. Stress signals like hypoxia, stress hormones, and high glutamate or glucose in neurons increase the cytosolic Ca2+ concentration which activates a mitochondrial phosphatase that dephosphorylates COX. This dephosphorylated COX exhibits no allosteric ATP inhibition; consequently, an increase of ΔΨm and ROS formation takes place. The excess production of mitochondrial ROS causes apoptosis or multiple diseases.Psychosocial stress is known to cause an increased incidence of coronary heart disease. In addition, multiple other diseases like cancer and diabetes mellitus have been related to stress and are mainly based on excessive formation of reactive oxygen species (ROS) in mitochondria. The molecular interactions between stress and ROS, however, are still unknown. Here we describe the missing molecular link between stress and an increased cellular ROS, based on the regulation of cytochrome c oxidase (COX). In normal healthy cells, the “allosteric ATP inhibition of COX” decreases the oxygen uptake of mitochondria at high ATP/ADP ratios and keeps the mitochondrial membrane potential (ΔΨ In cells, the mitochondrially produced •O2− is rapidly converted into H2O2 by the action of superoxide dismutases present in the matrix and the intermembrane space i spikes that are amplitude and frequency encoded [Calcium represents a universally important messenger in all multicellular life . The cyt encoded –75. In a encoded .2+ concentration of about 0.1 μM in normal relaxed cells is generally correlated with a low ΔΨm of 100–120 mV ([references in [41]). Multiple stress signals such as hypoxia, stress hormones, and various chemicals have been shown to increase ΔΨm to high values via increased cytosolic Ca2+ concentrations. This increase of ΔΨm, which occurs often transient, is named “hyperpolarization” of ΔΨm. Numerous studies have demonstrated the hyperpolarization of ΔΨm by various compounds [m was measured, followed by an increase of ROS, and finally neuronal death. Mitochondrial hyperpolarization represents an early and reversible step in T cell activation and apoptosis [m values have also been described by synthetic cannabinoids in human proximal tubule cells [m in HepG2 and Huh7 human hepatocarcinoma cells and HCC4006 human lung adenocarcinoma cells [m and ROS formation, and at high doses apoptotic cell death [m was furthermore shown with protamine sulfate [The low cytosolic Caompounds . The neuompounds  in humanpoptosis . Transiele cells , by statma cells , and by ll death . Hyperpo sulfate  and grap sulfate .2+ concentration, followed by mitochondrial ROS formation and eventually followed by apoptosis and cell death. But also psychosocial stress results in the increase of cytosolic Ca2+ concentration, as shown in isolated cardiomyocytes [d-aspartat-receptor was related to an increase of cytosolic Ca2+ and the formation of ROS [All these compounds increase primarily the cytosolic Camyocytes , in platmyocytes , hippocamyocytes , urothelmyocytes , and carmyocytes . The neun of ROS , 91.2+ and hyperpolarization of ΔΨm was so far unknown in the current literature [m at low values (below 130 mV). The increase of cytosolic Ca2+ concentration by various stress signals from 0.1 to 1–10 μM [m and increased production of ROS.In the sequence of reactions between stress and multiple diseases, shown in Fig. terature , 6, 92.  1–10 μM  activate 1–10 μM , 69, folm via the “allosteric ATP inhibition of COX,” which prevents the formation of high and deleterious amounts of ROS. All types of stress signals which increase the cytosolic Ca2+ concentration, dephosphorylate COX, and switch off the allosteric ATP inhibition, leading to an increased ROS production via increasing the ΔΨm, thus finally resulting in apoptosis, or in the long run to multiple diseases.From these results, we conclude that the health in all higher organisms is based on the maintenance of a low mitochondrial membrane potential ΔΨm), attenuated mitochondrial superoxide production (ROS), and in turn neuronal death of cultured HT22 cells following glutamate exposure and oxygen-glucose deprivation [The physiological significance of the “allosteric ATP inhibition of COX” was recently verified by experiments of Hüttemann and coworkers, who discovered inhibition of COX activity by near-infrared light of 750- and 950-nm wavelength. The near-infrared light reduced the mitochondrial membrane potential (ΔΨrivation . In livirivation .m to values resulting in deleterious mitochondrial ROS production. Another way to decrease ΔΨm and ROS generation in mitochondria is by using uncoupler of oxidative phosphorylation, which induces a backflow of translocated protons at the inner mitochondrial membrane [These results with living rats coincide with the conclusion derived from results on the allosteric ATP inhibition of COX that attenuation of COX activity prevents the increase of ΔΨmembrane .m increases. The allosteric ATP inhibition of COX represents an essential mechanism of higher aerobic organisms to avoid the increase of ΔΨm and thus the formation of deleterious ROS during rest. Without this mechanism, long-living organisms would suffer from various diseases and would die early due to accelerated aging.The “allosteric ATP inhibition of COX” is based on the feedback inhibition of COX by ATP, which binds to the matrix side of the “supernumerary” subunit IV. This subunit is lacking in bacteria , but it


Our computational study identifies a crystalline phase of CaO3 by reaction of CaO and O2 at high pressure and high temperature conditions; ensuing experiments synthesize this rare compound under compression in a diamond anvil cell with laser heating. High-pressure x-ray diffraction data show that CaO3 crystal forms at 35 GPa and persists down to 20 GPa on decompression. Analysis of charge states reveals a formal oxidation state of −2 for ozone anions in CaO3. These findings unravel the ozonide chemistry at high pressure and offer insights for elucidating prominent seismic anomalies and oxygen cycles in Earth’s interior. We further predict multiple reactions producing CaO3 by geologically abundant mineral precursors at various depths in Earth’s mantle.High pressure can drastically alter chemical bonding and produce exotic compounds that defy conventional wisdom. Especially significant are compounds pertaining to oxygen cycles inside Earth, which hold key to understanding major geological events that impact the environment essential to life on Earth. Here we report the discovery of pressure-stabilized divalent ozonide CaO 3) is stabilized at the high pressures and temperatures characteristic of the lower mantle, with implications for the deep Earth’s chemistry.Calcium and oxygen are abundant elements in the Earth’s mantle, largely present as calcium oxide. Here the authors show, by experiments and computations, that calcium ozonide (CaO Also notable are recent experimental and theoretical studies that have led to the discovery of unconventional iron oxides with unusual oxidation states7 in FeO2 (ref. 8), Fe2O3 (ref. 9), and Fe5O6 (ref. 10), opening avenues for making and exploring iron oxides with peculiar properties like unusual chemical valence and bonding interactions. Such results also restore the redox equilibria inside Earth and place oxygen reservoirs at greater depths than previously thought. These findings offer insights for elucidating large-scale geological activities that may have influenced events related to the origin of life on Earth.Pressure and temperature are key thermodynamic variables that prominently influence material structure and properties. Diverse high-pressure and high-temperature (HPHT) conditions simulated in computation and generated in laboratory-based experimental devices offer exciting opportunities for new material discovery and exploration of otherwise inaccessible deep-Earth environments. Recent years have seen the advent and rapid advance of computational structure search and characterization of pressure-stabilized compounds with unusual stoichiometries, such as Na-Cl11. In accordance with their respective valence electron counts, calcium and oxygen are expected to preferably form CaO12, which is present throughout the mantle. The oxidation state of O in CaO is O2− at ambient conditions. Meanwhile, calcium peroxide14 also can stabilize at ambient or high pressures15. In general, oxygen species with oxidation states higher than −2 can be synthesized in superoxide (O2−), peroxide (O22−), and ozonide (O3−) compounds and play prominent roles in oxidation chemistry16. Among these compounds, ionic ozonides are regarded as a species with unusual reaction processes and properties, and are scarce due to their high reactivity, thermodynamic instability, and extreme sensitivity to moisture in ambient environments17.Calcium and oxygen are two of the most abundant elements widely distributed in Earth’s mantle3 containing unusual divalent ozone anions that shed light on ozonide chemistry at extreme conditions, and the results offer insights for understanding deep-Earth chemical reactions that are relevant to oxygen cycles inside our planet.In this work, we report on the discovery from a combined theoretical and experimental study a high-pressure phase of CaO19, which has been successful in resolving crystal structures of a large number and variety of materials at high pressure20. Here, we explore calcium oxides in the oxygen-rich regime, seeking compounds that do not exist under ambient conditions. Studies of mantle rocks have shown that oxygen fugacity of the upper mantle is relatively high21, thus connecting the present work to prominent geological topics concerning oxidation states of minerals and oxygen storage and cycles inside Earth. We have performed structure searches on CamOn  with maximum simulation cells up to four formula units (f.u.) at each composition, and this procedure identifies two stable Ca–O compounds, a CaO2 phase at 30 GPa and an unusual stoichiometric CaO3 phase at 50 GPa. This result distinguishes CaO2 and CaO3 as two viable oxygen-rich calcium oxides. Furthermore, the CaO4-containing superoxide group (O2−) is found to stay above but close to the convex hull, making it energetically more favorable with respect to the dissociation route into CaO + O2 above 34 GPa and thus may be experimentally synthesized at high temperature −1 anion in KO3. It is also seen that the two terminal O atoms carry more negative partial charges (0.65e per O) than the bridge O atom (0.21e) within each O3 anion in CaO3, because the central O interacts much less with Ca cations than with the terminal O anions. To the best of our knowledge, the presently identified [O3]−2 ionic charge state has never been seen in other ozonides17.To decipher the nature of bonding and charge states in CaO30 to elucidate electronic configurations in O3− and O3−2. Results  and smaller volume shrinkage of −3.76% render MgO3 unstable against decomposition into MgO and solid O2 in C2/m symmetry, in agreement with previous reports31.The reactivity of CaO and Od in Eq.  at 30 GP3 involving several minerals abundant in Earth’s mantle as reactants:21, thus conducive to these reactions in forming CaO3.The newly discovered calcium ozonide is expected to have major implications for geoscience. In this context, we have examined additional viable routes producing CaOmentclass2pt{minimmentclass2pt{minim3 at ~20 GPa, which corresponds to pressures at the boundary of Earth’s upper and lower mantle. Previous studies revealed that several minerals such as CaCO3 (ref. 32), MgCO3 (ref. 33), and CO2 (ref. 34) can dissociate and produce oxygen in this pressure range, offering an abundant source of O2 for these proposed reactions inside Earth’s mantle. The resulting compound CaO3, which was not previously considered, provides an alternative mechanism to explain seismic anomalies near 660 km depth in Earth’s mantle where pressure is ~20 GPa36.Our calculations show that the reactions described in Eqs.  and 2)  2) produ3 and FeOOH by FeO2 and CaO under H2O-saturated conditions and the equilibrium pressure of this reaction is about 90 GPa  and temperature (1800 K) was proposed8 to exist in Earth’s lower mantle below 1800 km. Our calculations show that once CaO and H2O are thrusted to deeper than 1800 km, they can react with FeO2 and produce CaO3 + FeOOH. Due to the higher density of FeOOH compared to that of the mantle37  was piped into the container. Liquefied O2 infused into the sample chamber as the pressure medium and precursor. The samples were pressurized to 35–40 GPa and heated up to ~3100 K by an offline double-sided laser-heating (wavelength 1064 nm) system at HPSTAR and HPSynC of the Advanced Photon Source (APS), Argonne National Laboratory. Temperature was obtained from fitting the thermal radiation spectra to the Planck radiation function right after the reported chemical reaction has occurred in the DAC sample chamber. Laser spots at HPSTAR and HPSynC are approximately 20 μm in diameter. Pressure was calibrated by the fluorescence of ruby balls placed inside the sample chamber41. Optical absorption was monitored during and after the compression process. Synchrotron XRD data were also collected at 35–40 GPa and during the ensuing decompression process at the 13-BMC (λ = 0.4337 Å), GeoSoilEnviroCARS, Argonne National Laboratory and BL15U1 at Shanghai Synchrotron Radiation Facility (λ = 0.6199 Å). The X-ray probing beam size was about 15 µm at the bending beamlines, and 5 µm at the undulator beamlines.High-purity CaO  powder or Ca piece  were compressed into thin plates of 50 μm × 50 μm × 15 μm dimensions and loaded in a DAC with a culet of 300 μm. The sample chamber has a 100 μm diameter hole drilled in a pre-indented rhenium or steel gasket (38 μm thickness). The DAC was placed in a sealed container immersed in liquid nitrogen. O42 as implemented in its same-name CALYPSO code19 , which is based on a global minimization of free energy surfaces in conjunction with ab initio total-energy calculations. Structural optimization, electronic structure, and phonon calculations were performed in the framework of density functional theory within the generalized gradient approximation43 as implemented in the VASP code44. The electron–ion interaction was described by the projector augmented-wave potentials45, with 3s23p64s2 and 2s22p4 configurations treated as the valence electrons of Ca and O, respectively. The dynamic stability of the predicted new phases was verified by phonon calculations using the direct supercell method as implemented in the PHONOPY code46. Crystal structures were visualized with VESTA47.Our structure prediction is performed using CALYPSO  methodologySupplementary InformationPeer Review File