The doped In2O3 could effectively improve the gas-sensing properties of the prepared materials to nitrogen oxides (NOx) at room temperature. The 1% In2O3 doped CuO/ZnO sample (1 wt% In2O3–CuO/ZnO) showed the best gas-sensing properties whose response to 100 ppm NOx reached 82%, and the detectable minimum concentration reached 1 ppm at room temperature. The prepared materials had a good selectivity, better response, very low detection limit, and high sensitivity to NOx gas at room temperature, which would have a great development space in the gas sensor field and a great research value.For the first time, ordered mesoporous ZnO nanoparticles have been synthesized by a template method. The electroplating after chemical plating method was creatively used to form copper film on the surface of the prepared ZnO, and then a CuO film-decorated ordered porous ZnO composite (CuO/ZnO) was obtained by a high-temperature oxidation method. In Gas sensors can detect various inflammable, explosive, toxic, and harmful gases accurately and effectively. The development of efficient, low cost, highly sensitive, highly selective and convenient chemical sensors can precisely recognize and measure the presence of toxic gases and human exposure levels, which have become a significant endeavour for the well-being and welfare of people, safety and process control in industrial applications . As gas-2O3 [Among various kinds of developed oxide gas-sensing materials, ZnO has attracted great attention. ZnO is a functional n-type semiconductor, which has been considered as an essential semiconductor gas-sensing material because of its high chemical stability, suitability to doping, harmlessness and economic nature ,9. Howev2O3  and so o2S using a range of p–n composite nanostructures [et al. [CuO is a p-type semiconductor with a low band gap of 1.2–2.0 eV, and its p-type characteristic provides a route to form p–n junctions with n-type metal oxides . CuO hasructures –20. Compructures ,21. Rai  [et al.  reported [et al. .et al. [2 sheets gas sensor [2-based sensor [2-based gas sensor [2 nanosheets sensor [et al. [et al. [et al. [et al. [The gas-sensing properties of materials are closely related with the density and species of surface adsorptive gases. Therefore, for high performances of gas-sensitive materials they need to have a larger Brunauer--Emmett--Teller (BET) surface area. Late et al.  presentes sensor , 2D WS2-d sensor ,27, sings sensor , SnSe2 ns sensor  and 2D bs sensor ,31. Poros sensor . Liu et  [et al.  reported [et al.  reported [et al.  reported [et al.  reported [et al. .2O3 was loaded in the prepared CuO film–ZnO by ultrasonic-assisted method to sensitize the room temperature gas-sensing performance of the prepared materials. The fluffy porous structure was helpful for gas diffusion and surface reaction, which led to the sensor exhibiting faster response. The copper film was more uniform when the electroplating after chemical plating method was used to form copper film, and the n–p heterojunction between CuO film and ZnO matrix played an important role in the enhancement of the sensor response and selectively. The In2O3 played a sensitizer role to sensitize the surface reaction. In2O3 was a catalyst, it reduced the activation energy of the oxidation–reduction reaction on the surface of composites, efficiently separated the oxygen molecules, improved the activity of gas-sensitive materials, increased the free electron amount and expedited the reaction rate. As such, the gas sensitivity of the composites was enhanced. The prepared material greatly reduced the working temperature of the sensor, which would have a great development prospect and great research value in the sensor field.In this study, ordered mesoporous ZnO nanoparticles have been synthesized by a template method. The electroplating after chemical plating method was creatively used to form copper film on the porous ZnO surface, and then a CuO film-decorated ordered porous ZnO composite (CuO film–ZnO) was obtained by a high-temperature oxidation method. In2.2.1.2·2H2O was purchased from Tianjin Guangfu Fine Chemical Research Institute in China. Sodium hydroxide  was purchased from Beijing Chemical Works Research Institute in China. Potassium sodium tartrate tetrahydrate  was purchased from Tianjin Guangfu Technology Development Co. Ltd in China. Formaldehyde solution  was purchased from Tianjin Fuchen Chemical Reagents Factory in China. Ethylene diamine tetraacetic acid disodium  was purchased from Shenyang Reagent Factory in China. Copper(II) sulfate pentahydrate  was purchased from Sinopharm Chemical Reagent Co. Ltd in China. Ethanol and deionized water were used for all processes of washing and dissolution.All chemicals used in our work were of analytical grade, and used without any further purification. Cetyltrimethylammonium bromide  was purchased from Changzhou Xinhua Reagent Research Institute in China. Zn(Ac)2.2.2.2.1.2· 2H2O was dissolved into the solution with stirring. The pH of the solution was adjusted with LiOH to alkaline; the reaction then proceeded at 80°C for 2 h. The product was filtered, washed with deionized water and dried at room temperature to get the powder sample. The original sample was calcined at 500°C for 4 h, and the ordered mesoporous ZnO material was finally obtained.One gram of CTMAB was added into 480 ml of deionized water, and the mixture was vigorously stirred until the solution was homogeneous at 80°C. About 4.92 g of Zn(Ac)2.2.2.The electroless plating solution was prepared according to The prepared powder was put into semipermeable membrane, and the negative pole was inserted into the powder. The powder was immersed in the plating solution . The she2.2.3.2O3 decoration amounts on the gas-sensing performance, a series of In2O3 decorated CuO film–ZnO composites were synthesized. In a typical procedure, 0.1 g prepared CuO film–ZnO product was dispersed in 20 ml deionized water with the aid of ultrasonication. A certain amount of In(NO3)3 (In(NO3)3 concentration: 10 wt%, 15 wt%, 20 wt% and 25 wt%) was added in the above solution. The reaction system was dispersed in ultrasonic for 10 min, and the suspension liquid was aged for 2 h at room temperature. The suspension liquid was filtered, washed with deionized water and dried for 10 h at 60°C. The prepared samples were heated for 4 h at 500°C with a warming rate of 2°C min−1 to afford the products. The In-loaded contents were obtained with an inductively coupled plasma–optical emission spectrometer (ICAP 6000 Series), and the corresponding doping contents of In2O3 were calculated which were 0.5, 1, 3 and 6 wt%, respectively.In order to study the effect of different In2.3.α radiation (λ = 1.5418 Å and operating at 30 kV and 20 mA). Transmission electron microscopy (TEM) images were taken with a JEOL 2010 TEM instrument. The scanning election microscopy (SEM) images were taken with a JEOL JSM-5600 L. Physical adsorption of nitrogen was performed with a Micromeritics ASAP2010M volumetric adsorption analyser at 77 K. A sample was degassed in vacuum at 573 K for 12 h before measurement. Surface area was calculated based on the BET model, while pore size was computed using the Barrett–Joyner–Halenda method.Powder X-ray diffraction (XRD) patterns were collected with a Siemens D5005 diffractometer using Cu–K2.4.In this study, an interdigitated Au electrode was selected for the gas-sensing detection. The sample was disposed as follows: about 3 mg of the prepared sample was dispersed in ethanol, which was dispersed ultrasonically for about 20 min. The sample material was spin-coated on the interdigitated electrode surface to form a thin film, which was dried to obtain the gas sensor. The parameters of interdigitated gold electrode and schematic diagram of the prepared gas sensor are shown in Ra and Rg were the resistances of the sensors in the air and target gas, respectively. The response and recovery time were defined as the time taken by the sensors to achieve 85% of the total resistance change in the case of adsorption and desorption, respectively [The sensor was welded onto a socket, and the electrical properties of the sensor were measured by a JF02E intelligent gas-sensing analysis system. The sensor response was defined asectively –40.3.3.1.2O3–CuO/ZnO sample. The sharp diffraction peaks indicated the highly crystalline characteristic of 1 wt% In2O3–CuO/ZnO samples. All of the diffraction peaks were consistent with the standard card of CuO (JCPDS No. 44-0706), which showed that the CuO has been deposited on the surface of the ZnO. The diffraction peaks of wurtzite ZnO (JCPDS No. 36-1451) could be seen in the prepared material. In the XRD pattern, the characteristic peaks of the In2O3 could also be seen. It showed that the In2O3 has been doped into the prepared CuO film–ZnO matrix material.2O3–CuO/ZnO samples. From figure 3a, the peaks of C, Zn, O, Si and Cu could be seen in CuO/ZnO sample. The sample was first placed on a silicon wafer when the sample was tested, which led to the highest Si content peak. The C and O were mainly from the conducting resin. In figure 3b, the indium (In) content peaks could be seen in 1 wt% In2O3–CuO/ZnO sample, which could prove the existence of the In2O3 in 1 wt% In2O3–CuO/ZnO sample, and the In2O3 has been successfully assembled into the CuO film–ZnO matrix. The Pt content peak was attributed to the Pt conductive agent sprayed on the surface of the sample. There were no peaks of other elements in samples, which indicated that the prepared samples were pure.2O3–CuO/ZnO samples. From figure 4a, the CuO/ZnO sample showed a whole flower-like appearance, whose surface was smooth. From figure 4b, the 1 wt% In2O3–CuO/ZnO sample showed an analogous flower-like structure, which obtained from the petals of flower-like CuO/ZnO sample fallen off, the analogous flower-like structures were heaped with irregular flaky grain.a shows the TEM image of the 1 wt% In2O3–CuO/ZnO sample, which was taken parallel to the direction of the channels. From figure 5a, it could be seen that there were a lot of ordered pores in the sample. Figure 5b shows the high-resolution TEM (HRTEM) image of the 1% In2O3–CuO/ZnO sample. From figure 5b, it could be seen that the lattice fringes of the sample were very clear, which were the lattice fringes of CuO. In the HRTEM images, the ordered pores could be very clearly seen under the CuO lattice fringes, which indicated that the CuO thin films were formed on the surface of the prepared sample.Figure 53.2.a shows the gas response bar charts of different In2O3-doped concentration samples to different concentrations of NOx. The study found that the sensitivity of each sample decreased when NOx concentration decreased from 100 to 5 ppm. The results showed that In2O3 could significantly enhance the response of the materials to NOx, and the 1 wt% In2O3–CuO/ZnO sample had the best response to NOx among five prepared  In2O3–CuO/ZnO sensor samples in this study. At room temperature, the response and response time of the sensors with different In2O3 contents to 100 ppm NOx gas are shown in the electronic supplementary material, table S1.Figure 62O3–CuO/ZnO sensor are shown in figure 6b. The resistance sharply decreased when the NOx gas was injected, and the resistance recovered to its initial value when the NOx gas was discharged. When the prepared sensor was in air, oxygen molecules were adsorbed on the surface of the sensor; the adsorbed oxygen captured electrons from the conductance band to produce negatively charged oxygen species. These chemisorbed oxygen species would act as surface acceptors, trapping electrons and increasing surface resistance of the sensor. When the NOx gas was injected, the NOx would react with the formed oxygen ions, which released the trapped electrons back to the sensor surfaces, and the resistance of the sensor was sharply decreased. It indicated that the prepared sensing material was a p-type semiconductor material.Under the current operating conditions, the response–recovery curves of 1 wt% Inc shows the response and response time curve of the 1 wt% In2O3–CuO/ZnO sample to 100–1 ppm NOx at room temperature. When the NOx gas was injected into the sensing chamber, the resistance of the 1 wt% In2O3–CuO/ZnO sensor rapidly decreased, and reached the minimum resistance value in a short time. The 1 wt% In2O3–CuO/ZnO sensor exhibited a fast and reversible response to NOx, the response time being only 7 s to 100 ppm NOx which was much shorter than that reported in a lot of literature [x concentration from 100 to 1 ppm, and the corresponding response time increased. It was related to the gas concentration, gas diffusion and adsorption on the surface of the nanomaterials. With decreasing of NOx concentration, the NOx gas partial pressure declined in the reaction chamber which led to gas phase reaction velocity being slower, and the response time increased. It is worth noting that the minimum detection limit of 1 wt% In2O3–CuO/ZnO gas sensor was only 1 ppm at room temperature, the corresponding response still reached 17% and the corresponding response time was 136 s.Figure 6terature ,39,41–432O3–CuO/ZnO gas sensor, the responses of 1 wt% In2O3–CuO/ZnO sensor to different gases  were measured at room temperature. The corresponding test results are shown in figure 6d. The 1 wt% In2O3–CuO/ZnO gas sensor had a relatively high response to NH3. When the NH3 concentration was 100 ppm, the response reached 48%. However, the response to NH3 of the sensor was also significantly lower than the response to the same concentration of NOx. The response of other gases  was closer to 0%. The experimental results showed that the 1 wt% In2O3–CuO/ZnO gas sensor had good selectivity.In order to study the selectivity of 1 wt% Inx gas response in this study. The result is shown in The humidity is one of the significant factors in the gas-sensing process, and the degree of humidity influences the sensing performance of oxide semiconductors. However, the humidity has little effect on NO2O3–CuO/ZnO sample. The response of the prepared sample remained stable after six months. It indicated that the prepared 1 wt% In2O3–CuO/ZnO sample had good stability.3.3.−, O2− and O2−) and the adsorbed target gas molecules. These reactions lead to the resistance variation of the gas sensor. Generally, the n-type oxide semiconductor materials (such as ZnO) response mechanism involves the interaction between the detected gases and chemisorbed oxygen ions on the surface of the materials, leading to the resistance change of the sensor [2−, O− and O2−) at grain boundaries, and a thick space charge layer will be formed. The negative surface charges will generate a higher surface potential barrier, which leads to a high electrical resistance [x gas, the NOx gases attract the electrons from sensor materials, which can be attributed to their high electron affinity. The process leads to electrons of the sensor materials transfering to the surface NOx, and forming NO2− (from NO2) and NO− (from NO). The process traps electrons from the conduction band or donor level of materials, which leads to electron density reduction and the hole carrier density increase in the sensor. The hole carriers result in a decrease in resistance of the semiconductor layer. It will lead to a thinner space-charge layer, and reduce the potential barrier at the grain boundaries and cause a decrease in the sensor resistance [The gas-sensing behaviour of ZnO results from the surface chemical reactions. The reactants are the chemisorbed oxygen species  and NO2−, and release the trapped electrons back into the conduction band. The process can be expressed as follows [Eg) of ZnO is 3.4 eV, and its electron affinity (c) is 4.35 eV. The band gap of CuO is 1.35 eV, and its electron affinity is 4.07 eV. From a thermodynamic perspective, the electrons will transfer from the conduction band of CuO to ZnO, while the holes will oppositely migrate from the valence band of ZnO to CuO [Also, the target gases NO follows :3.3NO2+O to CuO . The proO to CuO .The higher sensitivity of CuO/ZnO composite can be attributed to the electronic sensitization induced by CuO ,51. Therx easier to react with adsorbed oxygen ions on the surface, which increases gas sensitivity performance.In addition, high specific surface area of sensing material usually has positive effects on the sensing response . The por2O3. Generally, the n–n (or p–n) type heterojunction-based interfacial barriers between In2O3 and ZnO (or CuO) nanocrystals are another mechanism accounting for the enhanced sensing performance [2O3 side along with a notch at the host side. The discontinuity band sets barriers for electrons in both conduction band and valence band. The heterojunction interfacial barriers can strongly modify the charge transport behaviours of carriers because the carrier concentration varies exponentially with the barrier height. Further studies are required to determine the mechanism through which the In2O3-doped semiconductor interfacial barriers promote the sensing performance towards gases [2O3 should not be overlooked. The catalytic activity of In2O3 accelerates the dissociation of oxygen molecules, and causes a spillover of the adsorbed oxygen ions on the surface of 1% In2O3–CuO/ZnO composite. More adsorbed oxygen ions provide more sensing activity sites and shows high response [2O3 is very little, the p–n heterojunction or n–n/p–p homo-type heterojunction cannot be formed, and the In2O3 only plays a role of sensitizer to sensitize the surface reaction. With the In2O3 content increasing, the sensitization is enhanced. But, the sensitized mechanism is changed when the doped In2O3 quantity exceeded the limit, which can be explained with heterojunction mechanism, and the response of the sensor will be the lower when the In2O3 content is not enough to form the continuous heterojunction.On the surface of the prepared CuO/ZnO composite, the heterogeneous structure can be formed between CuO and ZnO by ultrasonic method, and the gas-sensitive properties can be enhanced by Informance ,65–67. Ids gases . Also, tresponse ,69–71. I2O3–CuO/ZnO sensor may be for the following reasons. First, the fluffy porous structure is helpful for gas diffusion and surface reaction, which lead to the sensor exhibiting faster response. Second, n–p heterojunction between CuO and ZnO plays an important role in the enhancement of the sensor response and selectively. The CuO has a lower Fermi level than ZnO, and ZnO would receive electrons from CuO, leading to the formation of an accumulation layer at the CuO/ZnO interface. The increase in electrons on the surface of ZnO is able to facilitate adsorption of oxygen molecules and a decrease in resistance. Third, the doping of In2O3 is also helpful for remarkable gas-sensing performance of 1 wt% In2O3–CuO/ZnO composite. The reasons include electron interactions between In2O3 and ZnO, the catalytic activity of In2O3 accelerates the dissociation of oxygen molecules, and causes a spillover of the adsorbed oxygen ions on the surface of 1 wt% In2O3–CuO/ZnO composite. More adsorbed oxygen ions provide more sensing sites, and show high response. The mechanism is intuitively explained in In this study, the remarkable gas-sensing performance of the prepared 1 wt% In4.2O3 is loaded into the prepared CuO/ZnO by an ultrasonic-assisted method, affording an In2O3–CuO/ZnO composite. The prepared 1 wt% In2O3–CuO/ZnO composite shows the best gas-sensing performance, in which the In2O3 doping content is only 1 wt%. For 1 wt% In2O3–CuO/ZnO sample, its response time is only 7 s to 100 ppm NOx, and the corresponding response reaches 82%. It is worth noting that the minimum detection limit of 1 wt% In2O3–CuO/ZnO gas sensor was only 1 ppm at room temperature, the corresponding response still reached 17% and the corresponding response time was 136 s. The response of the 1 wt% In2O3–CuO/ZnO gas sensor is much higher, the minimum detection limit is much lower, the selectivity and stability are much better and the corresponding response time is much shorter at room temperature than that reported in a lot of literature.An ordered mesoporous ZnO nanomaterial has been synthesized by a template method. The prepared porous ZnO nanomaterial is used as matrix to prepare a porous Cu film–ZnO composite by the electroplating after chemical plating method, and finally a CuO film-decorated ordered porous ZnO composite is obtained by a high-temperature oxidation method. Then, In


In the case of Zn2+ species, the availability of imidazole nitrogen donors enhances metal complex stability. Interestingly, an opposite tendency is observed for Cu2+ complexes at above physiological pH, in which amide nitrogens participate in binding.Consecutive histidine repeats are chosen both by nature and by molecular biologists due to their high affinity towards metal ions. Screening of the human genome showed that transcription factors are extremely rich in His tracts. In this work, we examine two of such His-rich regions from forkhead box and MAFA proteins—MB3 (contains 18 His) and MB6 (with 21 His residues), focusing on the affinity and binding modes of CuThe online version of this article (10.1007/s00775-017-1512-x) contains supplementary material, which is available to authorized users. Histidine-rich (His-rich) motifs are present in many peptide domains and consist of multiple His residues, which can efficiently bind metal ions –4. They The widespread occurrence of histidine repeats in nature is very exciting from the evolutional, biological, chemical and medical point of view. Understanding their interactions with metal ions is not only chemically intriguing, but is also the fundamental step towards using them in potential medicinal applications –29. Sing2+ and Zn2+ tot = 2 × 10−4 M] were prepared in a 1:1 MeOH/H2O mixture at pH 6 (by adding an appropriate amount of NaOH). The samples were infused at a flow rate of 3 μL/min. Before each experiment, the instrument was calibrated externally with the Tunemix mixture. Data were processed by application of the Bruker Compass DataAnalysis 4.0. program.High-resolution mass spectra were obtained on Bruker MicrOTOF-Q spectrometers  equipped with an Apollo II electrospray ionization source with an ion funnel. Spectrometer was used for measurements on Cu2+ and Zn2+ complexes were calculated basing on two titration curves carried out over the pH range 2–11 at 298 K in a total volume of 3 cm3. The potentiometric titrations were performed using a Dosimat 665 Metrohm titrator connected to a Metrohm 691 pH-meter and a Metrohm LL Unitrode glass electrode. The glass cell was equipped with a magnetic stirring system, a microburet delivery tube and an inlet–outlet tube for argon. The pH-metric titrations were performed in 30% DMSO solution of HClO4 at 0.1 M NaClO4 ionic strength (both ligands are insoluble in pure water solution). Solutions were titrated with 0.1 M carbonate-free NaOH. Electrodes were calibrated daily for hydrogen ion concentration by titrating HClO4 with KOH in the same experimental conditions as above. Purities and the exact concentrations of ligand solutions were determined by the Gran method 4+, [LH5]5+, [LH6]6+ ligand species, respectively .Electrospray ionization mass spectrometry (ESI–MS) confirmed the purity of the studied MB3 ligand , the most accurate fit of titration curves for the Cu0, the mo15L]15+, with a maximum concentration at pH 4.2 . The shift of the d–d band from 620 to 568 nm in pH range 4–6 suggests the coordination of a fourth nitrogen atom resulting in the {3Nim, 1N−} binding mode for the [CuH13L]13+, [CuH10L]10+ and [CuH7L]7+ species. The coordination of an amide nitrogen is provided by the appearance of intense d–d bands in CD spectra at 530–650 nm range . The coordination of amide nitrogen to Cu2+ is also supported by the increase intensity of the characteristic band at 313 nm [−2L]2− is {2Nim, 2N−} supported by the shift of the d–d band from 570 to 551 nm  and appearance of intense d–d band at around 500 and 640 nm on CD spectra . Comparison of the experimental EPR spectra for Cu2+–MB3 systems at pH 6 and 9–10 show the differences in superhyperfine splitting patterns and especially the changes in the values of 2+ ion, respectively 4+, [LH6]6+, [LH7]7+ and [LH8]8+ ligand species, respectively .Electrospray ionization mass spectrometry confirmed the purity of the studied MB6 ligand , CD  and EPR . The first Cu2+ complex detected with the MB6 ligand is [CuH19L]19+, with maximum concentration already at pH 3.8. In this complex, two imidazole nitrogens are coordinated to Cu2+, which is supported by the d–d band at 687 nm for pH 4 . Next species, [CuH18L]18+ result from the deprotonation and copper(II) ion binding to the third histidine imidazole—the d–d band shifts from 687 to 618 nm in the {3Nim} complex. Moreover, EPR parameters are in good agreement with the 3N binding mode . At pH around 5, a clear shift of the maximum absorption in the direction of shorter wavelengths is observed in the UV–Vis spectra , indicating a {2Nim, 1N−} binding mode (one of the imidazoles is substituted by an amide). For [CuH14L]14+, [CuH12L]12+, [CuH10L]10+, [CuH7L]7+ and [CuH6L]6+ species, present at pH range 5.2–6.5, no significant changes are observed in the UV–Vis and CD spectra, suggesting that the {2Nim, 1N−} donor set does not change and the deprotonations correspond to the proton loss of histidines which are not involved in Cu2+ binding. Similar to the case of the Cu2+–MB3 system, due to the precipitation observed at pH around 6.5–9.3, we were not able to record spectroscopic spectra in this pH range also for Cu2+–MB6 complexes. However, because of the presence of ethylene glycol in EPR measurements, which enhanced the complex solubility, we were able to obtain EPR parameters that also confirmed the {2Nim, 1N−} copper binding mode at physiological pH , but the answer is not as trivial as it may seem. To discuss complex stability, we compared so-called competition plots—they are based on the calculated formation constants and describe a hypothetical situation, in which equimolar amounts of the metal ion and two multi-histidine ligands are present in solution at different pH values  and a snake venom peptide fragment with nine consecutive histidines (Ac-EDDHHHHHHHHH-NH2) [2+ with higher affinity than the His6-tag and the 9-His fragment .What is the impact of the presence of the extraordinarily high number of histidines on the stability of the complexes they form with Cues Figs. , 6. We cHHH-NH2) , 42. In 6-tag, below pH 6, the outcome is easy to interpret—MB3 and MB6 form more stable complexes with Cu2+ . This is in good agreement with what we already show—less histidines there are in the sequence, the sooner the amides start to participate in the binding and the more stable the complex is. Why do amide nitrogens start bind to Cu2+ at lower pH in the case of shorter fragments, with respect to the multihistidine MB3 and MB6 ligands? We can hypothesize that this is due to (1) the presence of so-called polymorphic binding states, where the metal “moves back and forth” along such regions [The comparison becomes far less trivial in the case of copper(II), which is able to deprotonate and bind to amide nitrogens. For the His2+ Figs. c, 6c. Fo2+ Figs. a. The re regions .Supplementary material 1 (PDF 1227 kb)Below is the link to the electronic supplementary material.


OH)2D3 and vitamin D receptor (VDR) on peripheral CD4+/CD8+ double‐positive (DP) T lymphocytes in systemic lupus erythematosus (SLE). MRL‐LPr/LPr mice with SLE (n = 20) and normal MRL mice (n = 20) were assigned into the control group 2D3), the 1,25(OH)2D3 group 2D3), the VDR‐knock‐in + 1,25(OH)2D3 group 2D3) and the VDR‐knockout group 2D3) (n = 10 per group). Levels of T lymphocytes were measured by flow cytometry. The mRNA and proteins expressions of inflammatory factors were measured by qRT‐PCR and ELISA. Extracellular signal‐regulated kinase‐1/2 (ERK1/2) expression was measured by Western blotting. Compared with normal mice, SLE mice showed reduced levels of CD4+, CD4+/CD8+ ratio, and DP lymphocytes. The levels of SLE‐related indicators all increased significantly, followed with severe skin ulcers and urinary system infection. With the increase in time, skin ulcers and urinary system infection were significantly improved, levels of CD4+, CD4+/CD8+ ratio, and DP lymphocytes increased, and levels of SLE‐related indicators all decreased in the 1,25(OH)2D3 group. There were no significant changes in bioindicators in the control and the VDR‐knock‐in + 1,25(OH)2D3 groups. The symptoms of SLE gradually occurred in the VDR‐knockout group. This study demonstrates that VDR and 1,25(OH)2D3 could elevate CD4+/CD8+ DP T lymphocytes and reduce expressions of inflammatory factors, thus inhibiting the development and progression of SLE.This study aims to explore effects of 1,25( Systemic lupus erythematosus (SLE) is an autoimmune disorder of connective tissues affecting multiple systems and is associated with a variety of clinical manifestations 2D3, a steroid hormone whose synthesis involves the catalytic 1α‐hydroxylase that transforms 25(OH)D3 to 1,25 (OH)2D3, a key factor in maintaining autoimmune tolerance, and participates in a variety of cell functions including differentiation, proliferation and apoptosis 2D3 inhibited the maturation of dendritic cells (DCs) and the activation of Th1 cells in SLE patients 2D3 and mediating its biological functions VDR gene polymorphism is associated with clinical manifestations of SLE 2D3 and VDR on the peripheral CD4+CD8+ DP T lymphocytes in a mouse model of SLE, in order to determine whether 1,25(OH)2D3 and VDR are involved in the pathogenesis of SLE.The most active vitamin D metabolite is 1,25(OH)Animal experiments were designed with the consent of the animal ethics committee, and all the studies were conducted strictly in accordance with international standards 2D3 and the VDR‐knock‐in + 1,25(OH)2D3 groups were fed with 5 μg/kg of 1,25(OH)2D3  daily by gastric tubes. The mice in the control and the VDR‐knockout groups were fed normally. All mice were fed for 8, 16 and 24 consecutive weeks.Six‐ to eight‐week‐old MRL‐LPr/LPr mice with SLE and normal MRL mice  were selected. There were 20 mice for each type, with half females and half males, weighing 20–24 g. All mice were housed in clean animal rooms at 22–25°C, with normal circadian rhythm. And all mice were given free access to food and drinking water. SLE VDR‐knock‐in mice and normal VDR‐knockout mice models were established and identified by Seajet Scientific Inc. . The grouping of mice is shown in Table After fed for 8, 16 and 24 weeks, the body weights of all mice were measured. Then, the mice were anaesthetized with intraperitoneal injection of 10% chloral hydrate in saline . The blood samples were drawn from retinal venous plexus. Heparin was added into the blood samples to prevent clotting. The samples were divided into two parts, one was used for the determination of T lymphocytes by flow cytometry, and the other was used to detect the expressions of inflammatory cytokines and biochemical indicators. Both kidneys in the mice were collected. Part of the kidney samples was used to detect the expressions of inflammatory cytokines by quantitative real‐time polymerase chain reaction (qRT‐PCR) and Western blotting. The other part of kidney samples was fixed in 10% neutral formalin, washed with PBS, dehydrated with gradient alcohol, processed with xylene and embedded in paraffin . The embedded samples were cut into 5‐μm‐thick slices and were used for the observation of tissue morphology.Tissue diluent  was mixed with blood samples with a 1:1 ratio. Equal volume of lymphocyte separation medium (Invitrogen Inc.) was added carefully. The mixture was centrifuged at 626 g for 30 min. at 20°C in a Thermo centrifuge. After the centrifugation, there were four layers of visible cells in the centrifuge tube. The second layer that contained milky lymphocytes was collected and washed by adding 5 ml cell washing buffer (Invitrogen Inc.). The samples were then fully mixed and centrifuged for 10 min. at 2000 r.p.m. The supernatant was discarded, and the remaining precipitation was mononuclear cells. The cells were resuspended in 80 μl of immunomagnetic buffer solution and 20 μl of magnetic beads  were added into the tube. The suspension was maintained at 4°C in the dark for 15 min. and centrifuged at 1000 r.p.m for 10 min. The supernatant was discarded, and 500 μl of buffer solution were added to resuspend the cells. The cells were then transferred into a magnetic separation column (Miltenyi biotec) which was washed twice with the buffer solution. At the end, the magnetic field was removed and the T lymphocytes were collected with 1 ml of RMPI‐1640 medium .6/ml. A volume of 100 μl cell suspension was collected in the Eppendorf tube for the measurement. Fluorescein isothiocyanate (FITC)‐labelled anti‐IgG1 antibody  and phycoerythrin (PE)‐labelled anti‐IgG antibody  of 10 μl each were added into the negative control. In the testing samples, 10 μl of monoclonal antibody for CD4  or CD8  was added and incubated in the dark for 20 min. A volume of 100 μl haemolysin  was subsequently added and incubated in the dark for 15 min. The mixture was centrifuged again, and the supernatant was discarded. The samples were then measured using flow cytometry . The cells were sorted with a flow rate of 4000 cells per second. The number of analysed cells should not be less than 2000. The results were analysed with the CellQuest software. The changes in CD4+, CD4+/CD8+, DP, CD8+ and double‐negative (DN) cells were analysed with the CS2000 software.The collected T lymphocytes were diluted to 1 × 10Blood urea nitrogen (BUN) was detected by continuously monitoring of the urease level according to the manufacturer's instruction of a reagent kit . In brief, 10 μl of samples was added into a test tube, and 10 μl of calibration solution was added into a control tube. The colorimetric values of the two tubes were immediately measured after mixing using a Thermo spectrophotometer. The absorbance of the mixture was measured at 30 sec. (A1) and 90 sec. (A2), and the serum level of BUN (mmol/l) was calculated as (A2 of sample − A1 of sample)/ × concentration of the calibration solution.The serum creatinine (Cr) was detected by a picric acid method, according to the manual of the reagent kit (Rongsheng biotech). In brief, 100 μl of sample solution was added into a test tube, and 100 μl of calibration solution was added into a control tube. The tubes were incubated in a 37°C water bath for 30 sec., and the initial absorbance (A1) and the absorbance at 90 sec. (A2) were measured at the wavelength of 505 nm. The level of Cr (μmol/l) was calculated as (A2 of the sample − A1 of the sample)/ × concentration of the calibration solution.The ELISA measurements were conducted in strict accordance with the manual of the experimental kit . In brief, the ELISA kit was equilibrated at room temperature for 20 min. before using it to prepare the experimental solutions. After the standard was dissolved, 100 μl of standard solution was added into the reaction plate to produce the standard curve. A volume of 100 μl sample solution was added into each well, and the plate was incubated at 37°C for 90 min. After washing the plate, 100 μl of promptly made working solution containing biotinylated antibodies was added into the wells and incubated at 37°C for 60 min. After the second washing, 100 μl of promptly made solution containing enzyme‐bound reactants were added and incubated in the dark at 37°C for 30 min. The plate was then washed consecutively for three times, and 100 μl of substrate solution were added and incubated in the dark at 37°C for 15 min. Finally, stop solution was quickly added into the plate to terminate the reactions, and within 3 min., the optical density (OD) values were measured at a wavelength of 450 nm using a multifunction plate reader . The standard curve was drawn based on the measured OD values, and the plasma levels of interleukin‐4 (IL‐4), IL‐10, IL‐17 and interferon‐γ (INF‐γ) were calculated from the standard curve.Ct) values were obtained to calculate the relative gene expressing using 2−∆∆Ct, where ∆Ct = Ct of target gene − Ct of internal control, and ∆∆Ct = ∆Ct (sample group) − ∆Ct (control group) TRIzol (Invitrogen Inc.) was used to extract mice renal RNA. The RNA concentration and purity were measured with NanoDrop2000 (Thermo Fisher Scientific Inc.). According to the gene sequences published in the Genbank database, PCR primers were designed using the Primer5.0 software and were synthesized . The primer sequences are shown in Table Mouse renal proteins were extracted and the protein concentrations were measured by a BCA Kit (Boster). The extracted proteins were denatured by adding a buffer solution and boiled for 10 min. at 95°C. The proteins were then separated using 10% polyacrylamide gel electrophoresis (PAGE) (Boster). During PAGE, each well was added with 40 μg of samples, and the electrophoresis voltages were 80 V in concentrated gels and then 120 V in separation gels. The transferring membrane was polyvinylidene fluoride (PVDF). Wet transfer was used with a transferring voltage of 100 mV. The operation time of transfer was 60 min. and sealed at room temperature and in 5% bovine serum albumin (BSA) for 1 hr. The primary antibodies were all used at a 1:1000 dilution, including phosphorylated extracellular signal‐regulated kinase‐1/2 , total ERK1/2  and β‐actin . During primary antibody staining, the samples were incubated overnight at 4°C. After the staining, the samples were rinsed three times by a Tris‐buffered saline and Tween‐20 (TBST) solutions, with each rinse taking 5 min. Secondary antibodies were then added and incubated at room temperature for 1 hr. The samples were rinsed three times again by TBST, with each rinse taking 5 min. The samples were then developed using a chemical luminescence reagent, and the grey values of the target bands were analysed by the ImageJ software using β‐actin as the internal control.The pathological morphology of sample tissues was observed by HE staining, which was beneficial to increase the colour difference among different parts of the tissue and cell structures. Paraffin sections of renal tissues were dewaxed using xylene (Boster) and hydrated using gradient alcohol (Boster). After staining with haematoxylin (Boster) for 10 min., the slides were washed with water. The slides were subsequently differentiated with a hydrogen chloride and ethanol solution (Boster) for 30 sec., and stained in blue using ammonia (Boster) for 30 sec. Finally, the slides were washed twice with water, counterstained by eosin for 3 min., dehydrated with alcohol and made transparent using xylene (Boster). The slides were mounted in neutral resin and placed under a microscope  to observe the pathology of renal tissues.Immunoglobulins IgG, IgA, IgM, C3 and C1q were labelled with FITC. Renal biopsy slides were dewaxed and processed in 3% catalase for 10 min. After subsequent treatment by pepsin for 10 min., the slides were incubated in Gibco calf serum at 37°C for 1 hr. The slides then underwent immunofluorescence staining by adding corresponding fluorescent antibodies including IgG (1:100 dilution), IgA (1:100 dilution), IgM (1:80 dilution), C3 (1:50 dilution) and C1q (1:50 dilution). All antibodies and reagents were purchased from DAKO Company . During antibody staining, the slides were placed in a wet box and incubated at 37°C for 45 min. After the staining, the slides were washed with TBST and then dried at room temperature. The slides were then mounted by adding a glycerol buffer DAKO Company. In order to detect the intensity of immune complex precipitation, the slides were imaged under a fluorescent microscope.anova), and the differences between two groups were compared using LSD t‐test. P < 0.05 indicated the difference was statistically significant.SPSS18.0 statistical software was used for statistical analyses. Measurement data were presented as mean ± standard deviations. Differences of measurement data of the normal distributions among multiple groups were analysed using one‐way analysis of variance 2D3 group, the weight of mice was also raised with the increase in time, but the difference was not significantly different. In addition, the severity of loosening hair, skin ulcers and urinary system infection was not significantly improved. In the control group, the weight of mice was increased along with the time, and there was no skin ulcer or urinary system infection. In the VDR‐knockout group, the weight of mice was decreased with the increase in time, and the fur became loose. In addition, skin ulcers and urinary tract infections occurred +CD8− single‐positive (CD4+SP), CD4+/CD8+ and CD4+CD8+ DP T lymphocytes were decreased in SLE mice, while the levels of CD8+CD4− single‐positive (CD8+SP) and CD4−CD8− DN T lymphocytes were increased in SLE mice . In the VDR‐knock‐in + 1,25(OH)2D3 group, the levels of CD4+SP, CD4+/CD8+ and DP T lymphocytes were elevated, while the levels of CD8+SP and DN T lymphocytes were reduced along with the time . At 24 weeks after the treatment, there were no significant differences between the 1,25(OH)2D3 and the control groups. In the VDR‐knock‐in + 1,25(OH)2D3 and the control groups, the T lymphocyte parameters had no significant changes along with the time . However, in the VDR‐knockout group, the levels of CD4+SP, CD4+/CD8+ and DP T lymphocytes were decreased, while the levels of CD8+SP and DN T lymphocytes were increased along with time  . The levels of BUN and Cr were significantly reduced along with the time in the 1,25(OH)2D3 group. At 24 weeks after the treatment, there were no significant changes in levels of BUN and Cr between the 1,25(OH)2D3 and the control groups. In the VDR‐knock‐in + 1,25(OH)2D3 and the control groups, the levels of BUN and Cr had no significant changes along with the time . However, in the VDR‐knockout group, the levels of BUN and Cr were increased along with time  . In the VDR‐knock‐in + 1,25(OH)2D3 group, the expressions of IL‐4, IL‐10, IL‐17 and INF‐γ were significantly reduced along with the time . At 24 weeks after the treatment, there were no significant changes in expressions of IL‐4, IL‐10, IL‐17 and INF‐γ between the 1,25(OH)2D3 and the control groups. In the VDR‐knock‐in + 1,25(OH)2D3 and the control groups, the expressions of IL‐4, IL‐10, IL‐17 and INF‐γ had no significant changes along with the time . However, in the VDR‐knockout group, the expressions of IL‐4, IL‐10, IL‐17 and INF‐γ were increased along with time  . In the 1,25(OH)2D3 group, the expressions of IL‐4, I L‐10, IL‐17 and INF‐γ mRNA were decreased in SLE mice along with the time . At 24 weeks after the treatment, there were no significant changes in expressions of IL‐4, IL‐10, IL‐17 and INF‐γ mRNA between the normal and SLE mice. In the VDR‐knock‐in + 1,25(OH)2D3 and the control groups, the expressions of IL‐4, IL‐10, IL‐17 and INF‐γ mRNA had no significant changes along with the time . However, in the VDR‐knockout group, the expressions of IL‐4, IL‐10, IL‐17 and INF‐γ mRNA were gradually increased along with the time  . In the 1,25(OH)2D3 group, the p‐ERK1/2 expression was reduced, the t‐ERK1/2 expression was elevated, and the ratio of p/t‐ERK1/2 was decreased along with the time . At 24 weeks after the treatment, there were no significant changes in p‐ERK1/2 expression between the 1,25(OH)2D3 and the control groups. In the VDR‐knock‐in + 1,25(OH)2D3 and the control groups, the expressions of p‐ERK1/2 and t‐ERK1/2 as well as the ratio of p/t‐ERK1/2 had no significant changes along with the time . However, in the VDR‐knockout group, the p‐ERK1/2 expression was gradually increased, the t‐ERK1/2 expression was gradually decreased, and the ratio of p/t‐ERK1/2 was elevated along with the time  . At 24 weeks after the treatment, renal injuries of SLE mice were improved, and there were no significant changes in inflammatory cell infiltration between the normal and SLE mice. In the VDR‐knock‐in + 1,25(OH)2D3 and the control groups, renal injuries and inflammatory cell infiltration had no significant changes along with the time . However, in the VDR‐knockout group, the degree of renal injuries and inflammatory cell infiltration were increased along with the time, and SLE symptoms gradually occurred in the mice of VDR‐knockout group  3 and C1q at the same time, which is a characteristic immune pathological change for lupus nephritis. In addition, microscopically visible immune complex deposition in a variety of different sizes was seen in the glomerular endothelium, epithelium, mesangial and tubular basement membrane. In the 1,25(OH)2D3 group, the levels of immunoglobulin and complement deposition were decreased gradually along with the time . At 24 weeks after the treatment, there were no significant changes in the levels of immunoglobulin and complement deposition between the normal and SLE mice. In the VDR‐knock‐in + 1,25(OH)2D3 and the control groups, the level of complement deposition had no significant changes along with the time . However, in the VDR‐knockout group, the level of complement deposition was gradually increased along with the time, and SLE symptoms gradually occurred in the mice of VDR‐knockout group   Figure .2D3 and VDR on peripheral blood T lymphocytes in SLE. The results showed that 1,25(OH)2D3 and VDR were associated with the occurrence of SLE, suggesting that they may become new targets for the treatment of SLE.The purpose of this study is to elucidate the effects of 1,25(OH)2D3 negatively related to the prognosis of SLE SLE is a systemic autoimmune disease with clinical heterogeneity, and some specific genes can increase the susceptibility to SLE 2D3 significantly improved the health of SLE mice, while the health conditions of normal mice which are insufficient in VDR deteriorate gradually along with the time. 1,25(OH)2D3, the active form of vitamin D, mainly functions on calcium homeostasis and bone metabolism. It is located in 12q of the chromosome and includes more than 470 sites of single nucleotide polymorphism (SNP), with some SNPs upregulating the level of 1,25(OH)2D3VDR is expressed in all types of cells of the immune system 2D3, which are mainly activated by 1,25(OH)2D3 inducing the phosphorylation of some signal transduction molecules, such as protein kinase C (PKC) and ERK1/2; 1,25(OH)2D3 can modulate the PKC activity, thus activating the Raf in the upstream of the ERK1/2 pathway, which lead to the phosphorylation of ERK1/2 et al. 2D3 can rapidly induce the phosphorylation of PKC and ERK1/2 in growth zone cells, and the induction of 1,25(OH)2D3 on ERK1/2 phosphorylation is mediated by PKC. According to Deeb et al., 2D3 involve 1,25(OH)2D3 binding with the cytosolic and membrane VDR to activate PKC through phosphorylation, thus activating ERK pathway through Ras‐Raf‐MEK‐ERK cascade. A previous study demonstrates that 1,25(OH)2D3 interferes the ERK signalling pathway to inhibit tumour cell growth 2D3 dosage inhibited the activity of SLE VDR polymorphisms and SLE severity VDR deficiency showed deteriorating health conditions, indicating that the VDR deficiency may increase the risk of SLE.In this study, feeding with 1,25(OH)4+SP, CD4+/CD8+ and DP in SLE mice were decreased, while the number of CD8+SP and DN cells was increased, indicating certain immune abnormalities. The ability of 1,25(OH)2D3 to suppress inflammation has been linked to its capacity to regulate DC and T‐cell functions + and CD8+ T cells, and these changes can further induce SLE; in SLE patients, the number of CD4+ T cells was decreased, the number of CD8+ T cells was increased, and the ratio of CD4+/CD8+ T cells was continued to decline In this study, it was found that as compared to normal mice, the levels of CD+ T cells can be divided into two distinct subgroups (Th1 and Th2) based on different expressions of cell factors In this study, it was also shown that as compared with normal mice, the protein and mRNA expressions of IL‐4, IL‐10, IL‐17 and INF‐γ in SLE mice were significantly increased. Mature CD42D3 and VDR participated in the occurrence of SLE by regulating cytokine secretion in different subsets of T cells, and they may become new drug targets during the treatment of SLE. However, there are still limitations in this research, and the exact mechanisms of 1,25(OH)2D3 and VDR remain elusive, which require further studies.In summary, this study found that 1,25(OH)None.


However, these variables display a puzzling asynchrony. Well-dated records of Southern Ocean temperature will provide crucial information because the Southern Ocean is likely key in regulating CO2 variations. Here, we perform multiple isotopic analyses on an Antarctic ice core and estimate temperature variations at this site and in the oceanic moisture source over the past 720,000 years, which extend the longest records by 300,000 years. Antarctic temperature is affected by large variations in local insolation that are induced by obliquity. At the obliquity periodicity, the Antarctic and ocean temperatures lag annual mean insolation. Further, the magnitude of the phase lag is minimal during low eccentricity periods, suggesting that secular changes in the global carbon cycle and the ocean circulation modulate the phase relationship among temperatures, CO2 and insolation in the obliquity frequency band.The δD temperature proxy in Antarctic ice cores varies in parallel with CO 2 through glacial cycles. Here, the authors show that a 720,000-year Antarctic temperature record is affected by variations in obliquity-induced local insolation that are associated with phase modulation of eccentricity cycle.The Antarctic temperature record displays a puzzling asynchrony with changes in CO The values of a temperature proxy, the hydrogen isotopic composition (δD), in the Antarctic EDC ice core2 have varied in parallel with CO2 concentrations over the past 800 thousand years 3. However, δD apparently leads CO2 variations. For example, during the last termination (TI), the start of Antarctic warming has been estimated to be synchronous with CO2 increase4 or to lead CO2 increases by 800 ± 600 years5 on the East Antarctic Plateau. The lead is ca. 2000 years at a West Antarctic site6. Over the past 420 kyr, the Vostok ice core shows that the Antarctic δD temperatures lead the CO2 variations by 1.3 ± 1.0 kyr7. During the lukewarm interglacials (430–650 kyr BP), Antarctic δD leads CO2 by 1900 years, and the correlation between CO2 and δD is weaker (r2 = 0.57), as determined from the EDC core8.Precise knowledge of the relationship between changes in temperature, atmospheric CO2 variations10. A box model, for example, estimated a ca. 60% increase in CO2 during TI that is attributable to direct and indirect temperature effects, such as changes in sea ice cover and vertical mixing in the Southern Ocean9. On millennial time scales, a multi-proxy study suggests that an antiphased hemispheric temperature response to ocean circulation changes resulted in Antarctic temperatures leading global temperatures and CO2 during TI11. On orbital time scales, the latitudinal temperature differences between Antarctica and the surrounding ocean may arise from annual mean insolation (AMI) variations because the amplitude of AMI variations is largest in the polar regions and smallest in the mid-latitude regions16. The AMI is paced only by the 41-kyr periodicity of the Earth’s obliquity17. Indeed, a strong 41-kyr cycle that is likely related to the local AMI variations was recognized in the Antarctic temperature record obtained from the Vostok ice core in the 1980s15. However, uncertainties in the age models of the ice cores and ocean sediments limit our understanding of the potentially different time lags between Antarctic and Southern Ocean temperatures relative to AMI changes. Therefore, well-dated surface temperature records with high temporal resolution from the Southern Ocean provide crucial information.Although the mechanisms underlying the coupling and the phase lags remain unclear, the Southern Ocean region, rather than Antarctica, is thought to play the central role in regulating COTsite), it is also affected by the extent of fractionation-associated rainout from the water vapour as it is transported from its moisture source in the ocean to the site where the precipitation occurs. After correcting the δD variations for this moisture source effect, the ΔTsite record shows a stronger correlation with CO2 over the past 350 kyr18, suggesting a potential bias in the use of δD as a temperature proxy. However, the effects of moisture source corrections are unknown prior to 420 kyr BP19. The lack of long records prevents elucidation of the potential influence of the 400-kyr eccentricity cycle on the relationship among AMI, CO2 and Antarctic and ocean temperatures.In addition to these climatic mechanisms, the validity of δD as a temperature proxy should be considered. While this proxy depends on Antarctic site temperatures  using new δD and deuterium excess records from the Antarctic Dome Fuji (DF) ice core, which spans the past 720 kyr. The new ΔTsite and ΔTsource records enable us to compare the pattern and timing of temperature variations between the Antarctic Plateau and its oceanic moisture source using the same age scale from that of the DF ice core. Our data extend the longest (420-kyr) record of ΔTsite19 by 300 kyr, and they reveal the impact of moisture source corrections during the lukewarm interglacials. The correlation between ΔTsource and CO2 is stronger than that between δD and CO2. Over the entire periods covered by the extended records, changes in local solar insolation, paced by obliquity, strongly affects ΔTsite. At the obliquity periodicity, the changes in ΔTsite and ΔTsource are delayed with respect to AMI. Further, the magnitude of the phase lag is minimal during the period of low orbital eccentricity, suggesting that the phase modulation is related to secular changes in the global carbon cycle and ocean circulations.To investigate the relationship between temperatures and CO20 to obtain a 720-kyr deuterium excess (d = δD–8δ18O) record. The δD and δ18O values of the ice between the depths of 2399.5 and 3035.1 m (297–715 kyr BP) were measured at 10-cm intervals. The new DF2 data were combined with earlier data from the first Dome Fuji core (DF122) and were plotted on the AICC2012 time scale23. The relationships among δD, d, ΔTsite and ΔTsource were quantified using a mixed cloud isotopic model (Methods). In this study, temperatures are expressed as deviations, Δ, from the modern value (the average of the past 2 kyr). ΔTsource is a proxy for temperature in moisture source regions22, and the major moisture source for modern precipitation at DF is estimated to lie in the South Atlantic and Indian Oceans between 38 and 65˚ S, based on backward-trajectory calculations26 2  over the past 720 kyr. With their higher sampling resolution (10 cm vs. 55 cm), the DF data depict the paleoclimate signals in greater detail than the EDC core. However, the deepest part appears to have been smoothed by ice diffusion, a process also observed in the 11-cm samples from the EDC core that correspond to Marine Isotope Stage (MIS) 11  to 4.0 °C (MIS 9.3) when 2-kyr mean values are used , as suggested by the EDC δD variations2.The DF Δord Fig. . Millenn °C MIS 1.2 to 4.0d record is shown in terms of the logarithmic definition of d . The use of these values produces glacial–interglacial variations in ΔTsource that are larger than those presented by previous studies29, resulting in good agreement between the DF ΔTsource record and a stacked SST record30 over the past 150 kyr22. Here, to evaluate the reliability of our ΔTsource estimates over multiple glacial cycles, we constructed a stacked sea surface temperature (SST) record based on 17 individual deep-sea sediment cores obtained in the source region , and these correlations are significantly stronger than that between δD and CO2 . The only exception for the strong coupling between ΔTsource and CO2 is observed during MIS 12.2, during which the ΔTsource shows exceptional cooling  is stronger than that between δD and CO2 . Rather, the correlation between CO2 and ΔTsource suggests a close link between surface temperature changes in the Southern Ocean and the global carbon cycle.The similarities and differences between temperatures and CO δD Fig. . Indeed,CO2 Fig. . Within  δD Fig. . These rTsource and CO2, ΔTsource lags CO2 slightly in most frequency bands   and the moisture source region (38–66° S). The ΔTgrad record is negatively correlated with the obliquity  Fig. . A decreity Fig. . This reTsite and ΔTsource was evaluated by filtering the records in the obliquity band . Thus, the difference is significantly reduced, and roughly consistent with the ΔTsite/ΔTsource ratio. Remaining difference would be balanced by other compensation mechanisms, such as the transport of heat from the low to the high latitudes36, and latitudinal contrasts in the cloud feedback, which is negative near 60° S and positive at low latitudes37.The impact of the differences in AMI on the amplitudes of Δand Fig. . The ran °C Fig. , which inge Fig. . Althougion Fig. . AccountTsite is delayed with respect to AMI (or obliquity) by 4.1 ± 2.7 kyr at the obliquity period  over the past 100–720 kyr23. Large uncertainties were expected during MIS 11-12 because of the lack of age markers23. However, an abrupt decrease in δ18O values seen in Chinese stalagmites dated independently using the U/Th technique38 coincides with an abrupt increase in EDC CH439 at Termination V, which supports the accuracy of the AICC2012 age scale.As seen in the Δcle Fig. . On aver18O record also shows a similar varying phase with respect to obliquity40 11. Thus, the timing at which CO2 begins to rise during a termination would be determined by when the Northern Hemisphere ice sheet begins to melt. When eccentricity is small, the summer insolation maxima are small. Thus, if obliquity rises beyond the threshold of melting, a moderate climate forcing could cause warming enough that the southern margin of the North American ice sheet begin to retreat41. Therefore, from 350 to 450 kyr BP, the obliquity component of CO2 began to rise rapidly without a phase difference relative to obliquity.The phase modulation of COity Fig. , which r2 to increase, occurs at the time of low eccentricity45 , which is a proxy for deep-water ventilation in the South Atlantic Ocean, shows weaker deep-water ventilation during glacial times with a clear 100-kyr glacial cycle;49 this gradient also displays a secular 400–500-kyr-long trend in the glacial minima from MIS 6 to MIS 14  during the next 10 kyr would cause temperatures on the East Antarctic Plateau to decrease by 1–2 °C in the obliquity frequency band. The obliquity-paced solar radiation is also important as a driver of changes in glacier mass balance in Antarctica52. Thus, it is crucial to understand the climate mechanisms that underlie the apparent 400-kyr phase modulation of AMI relative to temperatures, CO2 and ice volume.Our temperature data reveal that the asynchrony between Antarctic temperature and CO53 at the DF station in East Antarctica. The drilling site is located at a summit of the East Antarctic ice sheet and has a current annual mean air temperature of −55 °C. Extensive analyses of the deepest part of the ice (3000.0–3035.0 m) indicate that the bottom ice originated from meteoric water with altered chemical compositions54.The DF2 ice core was drilled to near bedrock at a depth of 3035.2 m18O values were performed for 6349 samples, which were obtained at depths ranging from 2399.5 to 3035.0 m. Each sample was 10 cm in length, which corresponds to a mean temporal resolution of ~70 yr. To remove surface contamination, 13 wt% of the surface was removed from each 24-g ice sample with a ceramic knife. The ice samples were melted at room temperature in sealed polyethylene bags. The water was then transferred to glass bottles and preserved in a frozen state. Both δD and δ18O values were measured using an automated equilibration method55 at the National Institute of Polar Research, Japan. The δ18O values of the ice samples (δ18Oice) were used in this study, whereas the δ18O values of ‘sawdust’ samples (δ18Osaw) were used in the previous DF article20. The sawdust was collected during ice cutting at the DF site. The average difference (=δ18Osaw–δ18Oice) and its variations (±1σ) were 0.31 ± 0.18‰ (n = 15), respectively. The small enrichment in δ18Osaw was caused by sublimation during sampling and/or transport of the samples from Antarctica to Japan. In addition, the resolution of this study is five times higher than that of the 50-cm sampling used in the previous DF2 δ18Osaw data20.Measurements of δD and δδ notation: δ = Rsample/RVSMOW−1, where Rsample and RVSMOW are the isotopic ratios (D/H and 18O/16O) of the sample and Vienna Standard Mean Ocean Water (VSMOW), respectively. To improve the precision of the measurements, 45% of the samples were measured two to four times. The analytical precisions (1σ), which are based on the differences between the duplicate analyses of individual samples, were 0.44‰ (δD), 0.05‰ (δ18O) and 0.52‰ (d).The isotope data are given using conventional 58, the resolution was ca. 400 years, and some parts were discontinuous. The overlapping portion (2400–2500 m) of the DF1 and DF2 records exhibited remarkable similarity59 because the horizontal distance between the two sites was only 43 m. The combined record was obtained by replacing the DF1 data58 with the DF2 data within the depth interval where the two cores overlap22 and adding the DF2 record. In this study, the DF records were plotted on the AICC2012 age scale60. To transfer the DF data to the AICC2012 age scale23, volcanic tie points between DF and EDC were used for 0–216 kyr BP61. For 216–720 kyr BP, the isotopic profiles were synchronized using the Match software package62. The glacial inceptions were excluded from the predetermined tie points20 because of potential differences in the patterns of the two isotopic profiles63. The largest mismatch between DF2 and EDC was found during 600–610 kyr BP. Isotope matching was impossible for this part of the record because of substantial differences in the observed trends.To obtain a complete isotopic record over 720 kyr, we used published data sets from the first Dome Fuji (DF1) core for shallow depths ranging from 0.0 to 2399.5 m. For the portion of the record derived from the DF1 core64. However, the δD and δ18O variations in Antarctic ice cores2 depend on the extent of fractionation-associated rainout from atmospheric water vapour along the pathway from the oceanic moisture source to the precipitation site, as well as the site temperature (ΔTsite). To estimate the site air temperature (ΔTsite), including a correction for the temperature of the moisture source regions (ΔTsource), a linear inversion method was used. This method has previously been applied to the Vostok core29, the EDC core65 and the DF core22. The sensitivities of the δD and d values to DF site air temperatures (ΔTsite) and the temperatures within the moisture source region (ΔTsource) were estimated using a mixed cloud isotopic model66. The multiple linear regression is expressed using the following equations:Traditionally, δD is used as a proxy for surface air temperatures, based on the spatial linear correlation between δD measurements and Antarctic temperatures18Osw)67 using the Rayleigh equation68. For the temperature sensitivity parameters, γsite, γsource, βsite and βsource, we used the values given by Uemura et al.22. Based on Monte Carlo simulations performed using the Vostok core, the uncertainties in the glacial–interglacial magnitudes of ΔTsite and ΔTsource are 1.1 °C and 0.8 °C, respectively29. The uncertainties for the DF site should be similar to those at Vostok because both are located in the inland portion of East Antarctica.where Δ represents the deviation from the modern , and the subscript ‘corr’ indicates the ice core record corrected for the isotopic composition of ocean water  by the t-value (97.5% confidence level). The stacked SST profile and uncertainty intervals are shown only for the period during which multiple data records (4 or more) were available . The albedos of the DF region and the moisture source region are 0.78 and 0.46, respectively.The albedo climatology was derived from data collected by the TIROS-N satellite. This data set was obtained from the climate data archive of the Joint Institute for the Study of the Atmosphere and Ocean  of CO2 with δD, ΔTsite and ΔTsource are 0.68–0.73, 0.76–0.80 and 0.75–0.79, respectively. For the lukewarm period, the ranges of the correaltion of CO2 with δD, ΔTsite and ΔTsource are 0.60–0.70, 0.72–0.79 and 0.77–0.83, respectively. Thus, the correlation between ΔTsite (and ΔTsource) and CO2 is significantly stronger than that between δD and CO2. The correlation between ΔTsource and CO2 is slightly higher than that between ΔTsite and CO2, but it is statistically insignificant.The uncertainty of the linear correlation coefficient was estimated using the Fisher 87  with a bandwidth of 0.00488).Spectral analyses were conducted with the Blackman–Tukey method 30% lag) using the Analyseries software package0% lag us87 Fig. . The ampσphs). The relative lags between the temperature proxies were as reliable as possible because they were estimated from the same ice sample. The phase analysis between ΔTsite and ΔTsource . Determining the exact phase lag between CO2 concentrations and temperatures was complex because the air was younger than the surrounding ice. A phase analysis between the temperature proxies and CO2  Ice-age versus orbital-age phase analysis error (σorb). To compare with the variation in obliquity, the absolute age uncertainty of AICC2012 for ice must be considered. The uncertainty of AICC2012 is ±2.6 kyr on average during the past 100–720 kyr23. The phase lag and confidence intervals (90%) of ΔTsite, ΔTsource and ΔTgrad with respect to obliquity were 4.1 ± 0.7, 7.5 ± 1.3 and 1.9 ± 0.3 kyr, respectively. For example, a combined error estimate for ΔTsite that is based on the root mean squared error is ±2.7 kyr. Therefore, the 4.1-kyr lag of ΔTsite against obliquity was significant during most of the studied period. The error in the gas-age versus orbital-age phase analysis is similar to σorb because the uncertainty of the AICC2012 age scale for gas is ±2.6 kyr, on average, during the past 100–720 kyr23.The uncertainties associated with the phase analysis were of three types: (i) ice-age versus ice-age phase analysis error . The data are also available from the authors.The raw data used in this study are available at the NOAA’s National Centers for Environmental Information (NCEI), which is formerly the National Climate Data Center (NCDC) data archive (Supplementary Information


Speciation of Hg2+ and MeHg+ has been achieved by in vitro approaches with fluorogenic probes supported in cultured cells. 2+ and MeHg+ have been prepared and used for the chemical speciation of both cations in aqueous solution as well as in HEK293 cells. The best selective speciation of Hg2+ and MeHg+ has been achieved by in vitro approaches based on fluorogenic probes supported in cultured cells, due to the particular sensitivity of the HEK293 cells to permeation by Hg2+, MeHg+ and the fluorogenic probes. In particular, MeHg+ was selectively detected in cell nuclei by probe JG45.Selected new fluorogenic probes that interact in different ways with Hg MeHg+ has a high mobility in the body due to formation of a complex with the amino acid cysteine. Since this complex of MeHg+ and cysteine resembles the structure of the large and neutral amino acid methionine, it can enter the cell and exit as a complex with reduced glutathione, thus forming water-soluble complexes in tissues.ii) species have to be continuously monitored. There are many chemical probes that are useful for the fast detection of Hg2+ contamination. Usually, they work by complexation of Hg2+ by colorimetric or fluorogenic reagents.2+, which subsequently undergo a colour or fluorescence change.+,2+ and MeHg+,+-induced neurotoxicity promotesii) species,+ by mimicking its behaviour in cells, opening the way to biomimetic selective molecular probes for Hg(ii) species. Following this idea we decided to design and test some new sulfur-containing fluorogenic probes for their ability to selectively interact with Hg(ii) species. We have previously prepared Hg2+/MeHg+ colorimetric dosimetersii) cations. Following this idea we now report the use of new fluorogenic probes for the selective detection and speciation of Hg2+ and MeHg+ in aqueous–organic mixtures and in HEK293 cells.Environmental contamination by mercury is a serious issue because of the large amounts of mercury released into the environment by human activities such as coal-fired power stations, primary metals production and the chlor-alkali industry. Another major concern is the persistence of mercury in the environment, not only as the volatile mercury metal but also as the water-soluble mercury(ii) Hg+ and met2+/MeHg+. The probes were prepared in fair yields by reaction of previously synthesized compounds 1a–d and dimethylphosphinothioic chloride 2a or O,O-diethyl phosphorochloridothioate 2b in the presence of Hünig's base  and water  by adding one to four equivalents of 5 × 10–3 M solutions of selected cations, as perchlorate or triflate salts, or anions, as tetrabutylammonium salts, in water and recorded all changes that the fluorogenic probes underwent with each analyte under a common TLC-UV light, λ = 366 nm, by qualitative measurements (photographs). As examples, photographs of BD116 and BD119 are shown in 2+ in mixtures of methanol–water 20 : 80 v/v or acetonitrile–water 20 : 80 v/v with comparable efficiency. In contrast, the remainder of the probes were less efficient fluorogenic probes in those mixtures of solvents or showed partial interaction with competing thiophilic cations such as Ag+ and Au3+, when used in mixtures of solvents with decreasing proportion of water. None of the probes was sensitive to the presence of common anions neither in organic solvents nor in their mixtures with water.The ketone derivatives BD116, BD118 and JG7 are creamy solids giving colourless solutions whereas the dicyanomethylene derivatives BD119, BD117 and JG30 are yellow solids giving pale yellow solvatochromic solutions in the most common organic solvents. The solvent effects were experimentally studied in terms of the Lippert–Mataga equations in selected examples, showing a strong influence of general solvent effects and the absence of hydrogen-bonding interactions contributing to the Stokes shifts  and a 2-fold increase of the quantum yield. Fitting of the emission intensity titration curves to a 1 : 1 complex equation gave a stability constant K = (1.9 ± 0.1) × 104 M–1 for the reversible complexation between BD116 and Hg+. Replication of the titration five times afforded a detection limit of (5.0 ± 1.0) × 10–6 M for the determination of Hg2+. Titrations of BD116 with MeHg+ in the same conditions showed a 6-fold increase of emission band centred at 480 nm. Fitting of the emission intensity titration curves to a 1 : 1 complex equation gave a stability constant of K = (5.7 ± 0.4) × 104 M–1, higher than the value obtained in the previous case, and replicates of the titration gave a detection limit of (9.9 ± 1.1) × 10–6 M for the determination of MeHg+, lower than the previous case. 2+ or MeHg+. In turn, titrations of BD119 with Hg2+ in the same conditions showed a 6-fold increase of the emission band . Fitting of the emission intensity titration curves to a 1 : 1 complex equation gave a stability constant of K = (5.5 ± 0.5) × 103 M–1, much lower than the value obtained for the complex BD116/Hg2+ . In all cases the curves reached a maximum for X2+Hg = X+MeHg = 0.5, confirming the formation of 1 : 1 complexes.For quantitative experiments we selected a concentration value of 2.5 × 102+ and for BD116/MeHg+ were monitored by means of the ultra-fast T-jump technique in conducting solutions having a constant ionic strength (I) of 0.1 M NaClO4, a concentration of the probe 2.5 × 10–5 M in MeOH–H2O 20 : 80 v/v and under metal cation excess conditions ranging from 1.25 × 10–4 to 3.75 × 10–4 M to ensure pseudo-first order conditions. The processes were recorded by fluorescent emission in 0.7 cm optical length cuvettes each containing a total volume of 1 ml. The system, initially in equilibrium, was perturbed up to a final temperature of 25 °C, and the relaxation process was observed by fluorescent measurements. At least five replicates were performed for each composition probe-cation and the outliers were discarded. One monoexponential kinetic effect was observed in both cases. Examples of the recorded kinetic traces are provided in τ. The kinetic analysis yielded the apparent binding reaction between the probe (P) and the metal, M (Hg2+ or MeHg+).The kinetic study of the binding processes for BD116/Hgτ) on the reactants concentrations according to eqn (2) is shown in K = k1/k2.The linear dependence of the time constants  showed that the mechanism of PM formation was in accordance to eqn (1) in both systems. The values of k1, k2 and K of the BD116/Hg2+ and BD116/MeHg+ systems are shown in To a first approximation, the values of [P] and [M] are deduced from the thermodynamic values of the overall binding constant K, denote greater affinity of the BD116 probe with MeHg+ than with Hg2+. k1, and dissociation, k2, of the BD116/Hg2+ complex were 8 and 20 times larger, respectively, than those of the BD116/MeHg+ complex. That is, the S···MeHg+ binding dissociates 20 times slower than S···Hg2+, revealing that in the equilibrium the hydrophobic nature of the interaction prevails. The K values were calculated also by fluorescence titration, yielding very similar results  in CD3CN with Hg2+, in which a downfield chemical shift for both methyl groups of the dimethylthiophosphinoic group was recorded as Hg2+ was added, indicating that the coordination process took place by the close sulfur atom , so the process was not spontaneous. This result was in agreement with the experimental behaviour of the probes.To throw light to the fact that only probe BD116 was able to form stable complexes with MeHg2+ and MeHg+ was used for the chemical speciation of each analyte in aqueous test samples containing mixtures of both cations, performed for the first time only with the help of fluorogenic probes by the standard addition methodology  × 10–6 M [Hg2+] by titration with BD119 and (8.1 ± 2.5) × 10–6 M [MeHg+] from the difference between titrations with BD116 and BD119. BD116 was also used for the titration of stored solutions of MeHg+, with the surprising conclusion that aqueous solutions are fairly stable but methanol–water solutions slowly degrade on time by precipitation, a result confirmed by ICP-MS analysis of supernatant  species in live cells by incubating human embryonic kidney cell line 293 (HEK293) cells with BD116 or BD119 solutions (100 μM in PBS–MeOH 80 : 20) for 1 h at 37 °C. Then the plates were washed three times with PBS and incubated with Hg2+ (100 or 300 μM) in PBS for 1 h and the fluorescent emission was measured by using λexc = 388 or 358 nm. Cells remained viable after incubation in the presence of the probes, which were permeable to the cellular membrane. Controls of cells without probe were measured as blanks and the relative intensity of intracellular Hg2+ fluorescence was compared. Probe BD116 showed low levels of background intracellular fluorescence in the absence of Hg2+ but the intracellular fluorescence dramatically increased with addition of Hg2+ up to 300 μM. The emission was higher in the nucleus than in the cytoplasm of cells, due to a higher accumulation of the probe in the nucleus , viscosity and differential scanning calorimetry techniques. For the BD116/ctDNA system (in the absence of MeHg+), Fig. S88A–D . The titration curves were analysed at 344 nm according to eqn (5).The unusual accumulation of the probe BD116 in the nucleus suggested that the probe could interact with DNA. A deeper study on the interaction of BD116 and ctDNA alone, or incubated with MeHgCDNA and CP are the total dye and polymer concentrations, respectively; ΔA = A – εPCP is the change of absorbance (A) during titration, and εP = ΔA°/CP, where A° denotes the initial absorbance of the dye solution; Δε = εPDNA – εP is the amplitude of the binding isotherm. A similar equation was used in fluorescence titration experiments, by changing ΔA by ΔΦ and Δε by Δφ in eqn (5). Fitting according to eqn (5) required an iterative procedure as Δε was not known. This was put equal to zero in the left term of the equation in a first approximation, and then calculated from the reciprocal slope of the plot; calculation was repeated until convergence was reached . A similar behavior was found when ctDNA was replaced by ctDNA incubated overnight with MeHg+ + reduces its value by a half due to the DNA/MeHg+ interaction. To verify the type of binding, viscometric, CD and DSC measurements were performed at constant ctDNA and varying the probe concentration. Fig. S90A and B . Fig. S90E and F  derivatives  = 43) 5, also used for bioconjugation,6, closely related to a commonly used starting material for water solubilizing dendrimers,3)4]+PF6– and trismethyl)amine  species in image microscopy. HEK293 cells were incubated with JG47 or JG45 solutions (100 μM in PBS with Ca2+ and Mg2+) for 1 h at 37 °C. Then the plates were washed three times with PBS and incubated with Hg2+ (100–500 μM HgClO4) or MeHg+ (100–400 μM MeHgCl) in PBS + Ca2+ + Mg2+ for 1 h and the fluorescent emission was measured by exciting at λexc = 388 nm. Cells remained viable after incubation in the presence of the probes, which were permeable to the cellular membrane. Controls of cells without probe were measured as blanks and the relative intensity of intracellular Hg2+ and MeHg+ fluorescence were compared. Probes JG47 or JG45 showed low levels of background intracellular fluorescence in the absence of Hg2+ or MeHg+. There was very little change in the intracellular fluorescence when JG47 was tested in the presence of Hg2+ or MeHg+. The change in fluorescence was also very small for JG45 and Hg2+. However, the intracellular fluorescence dramatically increased by addition of MeHg+ up to 400 μM to the HEK293 cells incubated with JG45. The emission was observed almost exclusively in the nucleus of cells  species.In summary, we have prepared new fluorogenic probes that interact in different ways with two closely related cations of high environmental concern, HgSupplementary informationClick here for additional data file.


This compound undergoes a first order magnetic transition from a high temperature incommensurate phase to a low temperature commensurate one. Our investigation reveals that although these two phases have very different magnetic structures, the spin excitations are quite similar indicating a fragile low temperature ground state with respect to the high temperature one. Such a rare scenario is argued to be a manifestation of the competition between the exchange interaction and 4f magnetic anisotropy present in the system. It is concluded that the magnetic structure, hence the ferroelectricity, can be finely tuned depending on the anisotropy of the rare earth.In solid state science, multifunctional materials and especially multiferroics have attracted a great deal of attention, as they open the possibility for next generation spintronic and data storage devices. Interestingly, while many of them host coexisting 3d and 4f elements, the role of the coupling between these two magnetic entities has remained elusive. By means of single crystal neutron diffraction and inelastic neutron scattering experiments we shed light on this issue in the particular case of the multiferroic oxide DyMn The associated physics is undoubtedly challenging because of the coupled spin, charge, and lattice degrees of freedom. Particularly, the issue of determining the actual role of spin in the emergence of ferroelectric state has been a very crucial topic of research in recent years. Different mechanisms have been proposed, like the inverse Dzyaloshinskii-Moriya (DM) interaction model2, the exchange-striction mechanism1 and the spin dependent metal-ligand hybridization model6 among others, but no universal model could be implemented to the entire family. It remains that the magnetic properties of many of such magnetic ferroelectrics are driven by 4f rare earths and 3d transition metal ions. There is thus some urgency in understanding the role of the 3d-4f coupling. Noting the fact that 4f elements are also well known for their exotic electric properties, for their usage in various laser based applications and also as permanent magnets (NdFeB and SmCo for instance), understanding the coupling between the two magnetic species would significantly benefit the wing of material engineering as far as the technological implementation is concerned.Magnetic ferroelectrics, where the onset of ferroelectricity essentially requires the spins to be ordered, are a fascinating class of materials2O5, a spin induced multiferroic system of the RMn2O5  family showing large spontaneous electric polarization and colossal magnetoelectric effect, one of the strongests in the entire family14. With the coexistence of 4f and 3d metals, this composition appears to be an ideal seedbed to investigate the 3d-4f coupling in a very prominent way.In this report, we have chosen to study DyMn2O5 is mainly characterized by two transitions. Below T1 = 42 K, it undergoes a magnetic phase (HT phase) transition with a propagation vector k1 = (1/2 − δ(T), 0, 1/4 − ε(T))15. Ferroelectric order arises below 40 K which is very close to the HT transition. With lowering temperature, a first order phase transition occurs at T2 = 8 K, from the HT phase to a low temperature commensurate magnetic phase (LT) with k2 = . This transition is believed to be associated with the ordering of Dy3+ moments. In few literatures, the existence of a lock in transition around 18 K was also reported12. Although very weak, the HT phase persists below T2 and coexists with the LT phase. In spite of a few previous attempts, revealing the magnetic structures of different phases has remained a dubious issue for this compound17. In this study, we conclusively determine the magnetic structure of both the HT and LT phases, by means of detail single crystal neutron diffraction experiments. In addition, inelastic neutron scattering investigations were carried out to characterize the spin dynamics. It is found that the spin excitations of the HT and LT phases are surprisingly very similar, showing especially a soft mode along  plane, loops of five Mn ions (three Mn3+ and two Mn4+) are formed with three inequivalent nearest neighbor antiferromagnetic (AFM) interactions: J3, and J4 couple the Mn3+ and Mn4+ ions while J5 couples the Mn3+  and J2 (via Mn3+ layers) interactions, as shown in Fig. 3+ and Mn4+ ions . Such lattices have become famous in the problem of tiling a plane with regular polygons, but also after the beautiful streets in the city of Cairo paved in this design.As claimed in a recent study, DyMnsee Fig. . In cont3. About 400 to 500 nuclear and/or magnetic Bragg reflection’s integrated intensities were estimated at 50 K, 25 K, 15 K, and 2 K by measuring rocking curves around each reflections. Figure ω values have practically identical peak widths as expected for long range magnetic order. The refinement of the angular positions of the 50 K (above the magnetic transition) nuclear reflections revealed that the lattice constants are quite close to the one reported at room temperature. These values also match well with the literature reported earlier using powder sample15. Refinements of the nuclear and magnetic structures at 2 K and 15 K were performed using the FullProf software package20. We used the formalism of Becker-Coppens to refine the extinction in the crystal. The pretty low values of the discrepancy factors ensure the quality of the refinement we achieved. The scale factors obtained from the refinements were used in the refinements of the magnetic structures at 15 K and 2 K.We performed single crystal neutron diffraction operated in four circles geometry using a crystal with size nearly 2.5 × 1.7 × 1 mmQ-scans were performed below 45 K. The satellites are only visible below 30 K due to a large background. Two magnetic phases are observed, in agreement with previous reports: above 8 K, an incommensurate phase (HT) with k1 = (1/2 − δ(T), 0, 1/4 − ε(T)) with δ(T) and ε(T) being very small and, below 8 K, a commensurate phase (LT) with propagation vector k2 = . Figure Q-scans carried out along ε(T) increases slightly up to 8 K where the HT-LT transition occurs. Although very weak, the signal of the HT phase persists down to 2 K (in agreement with previous reports)15. Figure To follow the temperature evolution of the magnetic propagation vectors, k2 = . Unfortunately, representation analysis was not of any use to find symmetry relations among moments and thus determine a model of the magnetic structure. Refinement of the LT phase turned out to be challenging in presence of ten uncorrelated magnetic sites corresponding to three magnetic sub-lattices (Dy3+ Mn3+ and Mn4+). It was found that using a toy model with magnetic symmetry Pa2b′21m′ helped to reduce the free parameters in some of the RMn2O5 systems with  propagation vector21. This magnetic space group is a derivative of the crystallographic group Pb21m which was proposed to be the space group in the emerged ferroelectric phase of RMn2O5 at low temperature8. We started the refinement using this model. To make the scenario simpler, we also incorporated few constraints based on literature available for RMn2O521. These constraints included working with a spin density wave (SDW) model, putting all the spins in the  plain. In addition, moments of similar type of ions were set to be equal. Testing with different possible orientations among the spins under the constraint relations mentioned above eventually steered us to the solution of the magnetic structure depicted in Fig. RF = 8.6%) ensures that the proposed model is reliable. The spins are found predominantly along the b direction, and arranged in a ferromagnetic fashion along the c axis  plane is the manifestation of the different frustrated interactions. The obtained structure is close to the one reported by Blake et al. using powder neutron diffraction data15.At 2 K, 500 magnetic Bragg reflections were collected associated with the propagation wave vector k2 = /2, 0, 0.q =  was not useful due to the lack of correlations among the moments. Attempting the LT phase structure in the 15 K (HT phase) data was unsuccessful. The model proposed by Wilkinson et al. corresponding to k2 reflections at 4 K did not appear to be suitable as well16. Unlike the situation at 2 K, usage of SDW configuration under Shubnikov group Pa2b′21m′ was a failure too, despite its flexibility. However, execution of simulated annealing (SA) analysis helped to have some ideas about the nature of possible couplings with an indication of spirality in the Mn sublattices. Based on that, tests with numerous numbers of possible configurations were made. Such an attempt revealed that incorporating spiral structure descriptions for Mn3+ and Mn4+ sub-lattices along c direction indeed improves the data fitting quality significantly. This spiral arrangement is described by the classical Fourier decomposition: ψ is a relative phase factor applicable to each site. We have used spherical polar coordinate system to represent the components of MR, θR, ϕR) and  where M, θ, ϕ denote the amplitude, polar angle, and azimuthal angle respectively).Similar to the situation observed in the LT phase, irreducible representation analysis with propagation vector 3+ sublattice deteriorates the quality of fitting. Instead, inclusion of Dy3+ spins following SDW approach helps to improve the refinement quality further. It is worth noting that such states where the amplitude of the rare earth ordered magnetic moment is modulated, is a common feature in the RMn2O5 family7. The refined structure at 15 K is shown in Fig. RF (15.7%) factor is a manifestation of much weaker reflections at 15 K. However, the x, y, z) and  symmetry operations, have been taken into account, and their volume fraction was refined to 52/48%. Note that δ and ε are neglected in this approach. The obtained structure differs from two of the possible models made by Johnstone et al. using resonant x-ray technique at 15 K, since there is no spiral description in their proposition17. It is also different from the model proposed by Ratcliff et al. at 22 K where only the Mn4+ spins show a spiral arrangement12. It is important to mention that attempts of using these previously proposed models were unsuccessful.However, usage of a spiral model for Dyent Fig. . Two mag3+ spins are characterized by a strong easy-axis anisotropy that tends to align them along the b direction.With these results in hand, we now propose a scenario to account for this sequence of magnetic structures. The main idea is that the Dyc direction, whereas, at 15 K, Mn4+ spins rotate by 45° from site to site around the c axis, and by 90° in the case of Mn3+ ions. Interestingly, Dy3+ spins do not prefer to be arranged in a spiral manner. The refined structure at 15 K corresponds to ↑↑↓↓ stacking along c of Dy3+ spins pointing along the b direction. This is a strong argument in favor of a significant anisotropy along that particular axis. It is worth noting that thermal variation of magnetization measurement performed along the three crystallographic directions also indicates a slightly higher magnetization along b (not shown). To figure out quantitatively the nature of the Dy3+ anisotropy, numerical ab-initio calculations (including 4f electronic correlation and spin-orbit interaction) have been performed, on an embedded DyO5 fragment. We used the CASSCF code of the MOLCAS package22 for the orbital and correlation calculations, the EPCISO code23 for the spin-orbit calculation and a home made code for the anisotropy and magnetic moments calculation from the ab-initio results. We find that the low energy crystal field states of the Dy3+ ions are formed by a doublet. The corresponding |↑〉 and |↓〉 states have an Ising like character and carry a moment μ = 〈↑|τ|↑〉 = −〈↓|τ|↓〉 = 8.5 μB which forms an angle ϕ = 77° away from the a axis (hence close to the b axis). This compares very well with the experimental values of 7.5 μB and 115°. The |↑〉 and |↓〉 states are also well separated from the next excited states, located at 16 meV.There are indeed some salient features which make the magnetic structure at 15 K rather different from the 2 K structure. At 2 K, all the spins are arranged ferromagnetically along the 3+ and Dy3+ spins are anti-parallel to each other along the c direction, suggesting that those spins are coupled by an AFM J6 interaction  stabilize a complex spiral arrangement of the Mn spins. Note that, according to literature, J5 is AFM and likely the strongest interaction at play. Along J4 bonds, Mn4+ and Mn3+ are also anti-parallel to each other, suggesting that J3 is eventually the most frustrated coupling. Indeed, the effects of the different J3 on the magnetic energy cancel each other for a Pbam space group. It results that J3 only acts through weak symmetry breaking, and thus its global effect remains very small independently to its absolute value. The easy axis anisotropy of the Dy3+ spins competes with this underlying spiral order and maintains the Dy3+ spins along b. To take advantage of J6, however, the Dy3+ spins adopt this peculiar ↑↑↓↓ configuration. This is indeed a favorable compromise since for the sites where Mn3+ is along b, the J6 exchange energy is fully satisfied, while for the sites where Mn3+ is along a, Dy3+ and Mn3+ spins are perpendicular, so that the J6 exchange energy is zero.Meanwhile, a closer look at the 2 K structure shows that Mnsee Fig. . The pro24 to determine the magnetic structure at a certain temperature by means of energy minimization. The magnetic interactions at play are the J1,…,5 exchange couplings and the Mn3+-Dy3+ Ising coupling J6. By studying rigorously over a wide range of values, it was found that a parameter set corresponding to J1 = −1.5 meV, J2 = 1.5 meV, J3 = 0 meV, J4 = 1.75 meV, J5 = 3.5 meV and J6 = 0.03 meV could successfully yield a spiral magnetic structure similar to what is observed at 15 K with a propagation vector  . This analysis leads to the following relation for the magnitude of the exchange parameters: 26.Mean field calculations are then carried out using the SpinWave softwareJ6 is essential to stabilize the structure similar to the 15 K one. We propose that since the ordered Dy3+ moment increases with decreasing temperature, the competition turns in favor of the J6 exchange energy. It finally overcomes J1,2, destabilizes the spiral order, and results in a structure where all Mn3+ and Dy3+ spins are anti-parallel. In this scenario, the magnetic phase transition is thus a manifestation of the competition among the exchange interactions and the anisotropy energy.Although small, the inclusion of To further investigate this scenario, inelastic neutron scattering measurements have been carried out both in the HT and LT phases.QBragg. This branch corresponds to the long wavelength precession of the spins around the average local magnetization. With this picture in mind, the vicinity of the HT phase Bragg peak, QBragg = , has been mapped out as a function of energy transfer ω and Q wave-vector. Because of the small size (~mm3) of the sample, successful executions of the experiments were extremely challenging. All the measurements were thus repeated on different spectrometers (see Methods) to verify the genuineness of the data. Figures Q along c* and , i.e. along a* respectively (raw data). The directions of these two cuts in reciprocal space are depicted by dotted lines on the sketch presented in Fig. 2O525 and TbMn2O526. The data show indeed a feature (black dotted line) stemming from h. We note that this is a bit off from the Bragg position, expected at ħω = 4 meV at 2O525 and TbMn2O526.In standard magnets, an acoustic spin wave branch stems from the magnetic Bragg peak located at c* in the LT phase at 2 K. Note that the wider ħΩ = 2 meV. Along , see Fig. h, 0, 0), however, the spin wave branch is again clearly visible in Fig. c* 43. This hybridization may be the physical origin of the peculiar spectrum observed in Fig. The inelastic neutron scattering experiments of Figs see Fig. : the latmentclass2pt{minim2O5 have been investigated by means of neutron diffraction and inelastic scattering. A first order magnetic phase transition is reported at 8 K. It separates a high temperature magnetic structure spiraling around c direction with a propagation vector c with a propagation vector . Although the high temperature phase persists to exist down to 2 K, the magnetic Bragg reflections become extremely weaker compared to the actual low temperature phase ones. Surprisingly, the nature of spin excitations is practically identical above and below the transition temperature along c*. Based on both kinds of experiments, we propose that the physics at play is presumably associated to the competition between exchange interactions and the single ion anisotropy of the Dy3+ ion. More generally, these findings demonstrate that the magnetic structure, hence the ferroelectricity, by virtue of the magneto-electric coupling, strongly depends on the anisotropy of the rare earth. The 3d-4f coupling thus appears as one of the key ingredients in the physics of multiferroics.The magnetic structure and the spin dynamics in the multiferroic oxide DyMn2O5 were synthesized following the method described in ref.28. Single crystal neutron diffraction experiments were conducted on the D23 diffractometer, a CEA-CRG instrument installed at the ILL . The instrument was operated in a four circles geometry with an incident wave length of 1.280(1) Å. The sample (2.5 × 1.7 × 1 mm3) was attached to the cold finger of a displex refrigerator. Its crystalline quality was checked using the OrientExpress instrument installed at ILL.Single crystals of DyMnkf = 1.97 Å−1 and 1.8 Å−1 for 4F and IN12 respectively. To eliminate the higher order reflections, proper filtering was used. The sample was mounted to have access to mm3) of the sample, INS experiments were extremely challenging. They were thus repeated on 4F and IN12 to verify the genuineness of the data. We then pursued the idea that features being common to the two sets of experiments should be retained only. It could be noticed, fortunately, that the spectra are close to each other, giving good confidence that the features, yet weak, are real. Furthermore, in the present manuscript, we chose to show the raw data; for instance, no background has been subtracted. To identify the signal, we compared the data with the spectra published for similar materials, YMn2O5 and TbMn2O5 in refs25 and26 respectively. Indeed, we observe features clearly reminiscent of the spin wave branches reported in these two compounds. The spin wave energies were determined based on a simple fit assuming a standard Gaussian profile on top of a constant background. A flat feature at about 6.5 meV may also correspond to a signal (see Inelastic neutron scattering (INS) measurements were performed at the 4F and IN12 cold triple axes spectrometers installed at LLB and ILL respectively, on the very same crystal used for the diffraction measurements. The final wave vectors used were gnal see . Actuall