High systemic levels of oestrogens are cholestatic and primary biliary cholangitis (PBC)—which is characterized by hepatic ductular inflammation—is thought to be triggered by exposure to xenobiotics such as those around landfill sites. Xenoestrogens may be a component of this chemical trigger. We therefore hypothesized that xenoestrogens are present at higher levels in the proximity of landfill sites. To test this hypothesis, soil samples were collected, extracts prepared and biological oestrogenic activity examined using cell-based reporter gene assays. Extracts from several sample sites around a landfill site contained a chemical(s) which activated the human ERα in a dose-dependent manner. Extracts from 3 separate control sampling sites were absent of any detectable activity. The mouse ERα and 2 variant mouse ERβ cDNAs were cloned and extracts from sample sites around a landfill site also activated these receptors. One variant murine ERβ was constitutively active when expressed in cholangiocytes, was readily inactivated by ICI182780 and activated in a dose-responsive, ICI182780-inhibitable manner by oestrogen. However, when this receptor was activated by extracts from landfill site soils, ICI182780 failed to antagonize activation. ERβ was readily detectable in murine cholangiocytes and exposing mice acutely to a pooled ER activating soil extracts also gave rise to a mild cholestatic injury. These data indicate that the environment around landfill sites may contain higher levels of xenoestrogens; that these chemicals have “super-activating” characteristics with a variant ERβ and therefore these chemicals could be a component of a xenobiotic insult that triggers PBC. A variety of man-made chemicals released into the environment by anthropogenic activity  have been shown to act as endocrine disruptors and have been proposed to be responsible for a spectrum of adverse effects in wildlife and man, from malformations in the male genital tract; decreased sperm quality; neuroendocrinological, behavioural and metabolic effects to cancer . OestrogOestrogens are primarily produced in the ovaries or testes from testosterone and androstenedione  through metabolism by aromatase, and their major biological function in women is to regulate the variety of physiological changes associated with female reproduction  ; hepaticwww.nursa.org/; last accessed November 18, 2016) and the primary role of ERα is the regulation of sexual reproduction, as exemplified in ERα knock out mice—females are infertile and males have decreased fertility (www.nursa.org/). ERβ knockout mice develop relatively normally and have normal fertility (Two ER genes are present within the human genome - ERα (NR3A1) and ERβ (NR3A2) and their products are both activated by E2  is a chronic liver disease thought to be of an autoimmune aetiology due to the presence of antibodies to mitochondrial proteins in the majority of PBC patients . At presPrevious investigations have suggested that only ERα is expressed in hepatocytes whereas both ERα and ERβ are expressed in cholangiocytes and markedly upregulated in disease settings. It has been considered that through actions on both (ERα) and (ERβ) subtypes, oestrogens regulate the expression of growth factors and cytokines, thereby modulating the proliferative response of cholangiocytes to damage .Given the widespread concern regarding man-made xenoestrogen chemicals and adverse health effects, we hypothesized that the environment around landfill sites contains a variety of xenoestrogens and that chronic exposure may give rise to some of the effects associated with endocrine disruptors, such as cholestasis. To test this hypothesis, soil samples were taken from around a currently active peri-urban landfill site positioned upon an area of historic mining (and from 3 separate control sampling sites), chemicals were extracted into an ethanol solvent and the presence of oestrogenic chemicals tested in biological assays (human and mouse ER-luciferase reporter gene assays).In this article, we demonstrate that several landfill sample sites contained oestrogenic chemicals, and in contrast to E2, behaved as irreversible agonists of a murine ERβ variant in a pancreatohepatobiliary cell context. Acute exposure of mice to a pooled oestrogenic environmental sample also resulted in evidence of cholestasis.The mouse cholangiocyte cell line 603B was a gift from Dr Yedidya Saiman, Mount Sinai School of Medicine, New York. The mouse pancreatic ductal cell line LTPA was originally obtained from the American Type Culture Collection  and is an epithelial cell line derived from a spontaneous pancreatic adenocarcinoma taken from a 12-month old female Lt/Sv mouse. When injected subcutaneously into Swiss nu/nu mice, LTPA cells form ductular structures . Although these cells are not of hepatic origin, the pancreatic and biliary ducts are physiologically connected and have a shared developmental origin (Soil samples (of approximately 0–5 cm in depth) were collected around the perimeter (within 200 m) to an active landfill site (located within a peri-urban area) which does not have a license to accept hazardous materials. In addition, 3 control soil samples were similarly collected from 3 separate sites. One sample was obtained from the University farm in rural Northumberland at a site with controlled fertilizer regime for the last 130 years and the remaining 2 control samples were obtained from gardens in urban areas in the region. Extraneous vegetable matter and stones removed, manually homogenized and stored at 5°C until downstream use. 125 g soil was sonicated in 300mls methanol for 10 min, followed by addition of a further 100 ml of solvent and sonication for a further 10 min prior to filtration with 25 µm filters and collection of filtrate. Filtrates were evaporated in a rotary evaporator and then blown down to near dryness under a stream of nitrogen prior to addition of 30 ml of ethanol. The solvated extracted chemicals were separated from any precipitate and stored at −20°C.HindIII and EcoRI restriction sites for mERα and AflII and EcoRI sites for mERβ cDNA products. The PCR products were initially cloned into a pCR-blunt vector using the Zero Blunt PCR Cloning Kit (Life technologies) prior to sub-cloning into pcDNA3.1 restricted with the appropriate restriction enzymes. Constructs were screened for correct insertion by restriction digest analyses and sequence integrity by DNA sequencing.The mouse  mERα and mERβ variant 1 (m mERβv1) and variant 2 (mERβv2) cDNAs were amplified from mRNA isolated from mouse uterus and mouse ovary, respectively, using the following primers, mERacloning, US, 5′- CGCCGAATTCCACTTACCATGACCATG-3′ DS, 5′- CCTGGAAGCTTTCAGATCGTGTTGGGG-3′; mERßcloning, US, 5′- CCGTGAATTCCTGAGAGCATCATGTCCA-3′ DS, 5′- TCCGCCTTAAGCCTGGCCGTCACTG-3′ to give 2 in air.603B, MCF-7 and 293 cells were cultured in low glucose Dulbecco’s Modified Eagles Medium , supplemented with 10% (v/v) foetal bovine serum (Sigma) and 80 U/ml of penicillin and streptomycin. LTPA cells were cultured in the above medium further supplemented with 0.1 mM non-essential amino acids  and 1 mM sodium pyruvate . All cell lines were maintained in a humidified atmosphere at 37 °C in 5% COTotal RNA was purified using Trizol . RT-PCR was carried out as previously outlined (3-pGL3promoter (3-pGL3promoter in response to E2 treatment (data not included) and therefore the 3XERE TATA Luc previously shown to respond to mouse ERα activation  as previously described in 603B and LTPA cells were transiently transfected in 24-well plates with 0.25 μg total DNA per well ; an oestrogen-responsive reporter gene luciferase construct, either (ERE)2HPO4 12H2O, 0.44 mM KH2PO4, 5.6 mM glucose, 6 mM HEPES, 0.35 g/l NaHCO3 and 1 mM CaCl2) supplemented with 1 mg/ml collagenase type 1A (Sigma) and 80 μg/ml DNAse I (Sigma). After 30–45 min incubation at 37 °C, the digest was filtered through a 125 μm Nybolt mesh and the resultant cell suspension made up to 100 ml with HBSS+. Cells were pelleted by centrifugation at 600 g for 5 min at room temperature. This washing step was repeated twice and the final cell pellet re-suspended in 12 ml HBSS+. Mouse cholangiocytes were semi-purified by density gradient centrifugation as previously described using percoll density centrifugation  in PBS for 10 min followed by permeabilization with 0.2% (w/v) Triton in 1× PBS for 15 min. Non-specific protein binding was blocked by incubation with 5% goat serum in 1× PBS-T (PBS + 0.02% w/v Tween 20) for 20 min at room temperature. Cells were incubated with anti-cytokeratin 19 (CK19)  plus anti-ERα  or anti-ERβ  primary antibodies in blocking buffer at 4 °C overnight. Following three 10-min washes in PBS-T, cells were incubated with the appropriate secondary antibodies  in blocking buffer for 45 min in the dark. Cell nuclei were stained with DAPI prior to mounting using Fluoroshield mountant (Sigma).Total protein was isolated from cell and tissue samples using RIPA buffer , 30 min agitation at 4 °C. 4× LDS sample buffer (Life Technologies) and 5% v/v 2-mercaptoethanol was added to cell lysates, samples were boiled for 5 min at 95 °C and the genomic DNA sheared by sonication. Proteins were separated using pre-cast 4–12% Bis-Tris polyacrylamide gels  and then transferred to nitrocellulose membranes using the iBlot device (Life technologies). Membranes were probed using standard protocols with the following primary antibodies, Rabbit polyclonal antibodies to ERα  and ERβ . Anti-GAPDH antibody was purchased from Cell Signaling . Appropriate HRP-conjugated anti-IgG antibodies were used for detection using the ECL reagent . Protein bands were visualized using a Syngene G, Box gel documentation system.Male adult C57Bl/6 wild type mice were purchased from Charles River  and housed in accordance with UK Home Office regulations. Males were chosen over females because the female oestrus cycle results in more variable levels of endogenous oestrogens in test groups of animals, which would result in more mice per test group being required to assess any potential effects of a xenoestrogen. On a 3Rs basis, it was considered more ethical to test any potential effects of xenoestrogens on as few mice as possible, and so male mice were selected. Food and water for animals were available ad libitum and conditions were kept at 23 ± 1 °C on a 12-h light/12-h dark cycle at 47% humidity. Mice were exposed to combined and concentrated soil extracts which have been shown to exhibit oestrogenic activity in a reporter gene assay or to combined soil extracts from the 3 control sampling sites without any detectable oestrogenic activity by single intraperitoneal injection. Other groups received either ethinyloestradiol  at 0.5 mg/kg bodyweight in 90% olive oil/10% ethanol vehicle or vehicle alone. For preparation of the extracts, combined oestrogen positive or oestrogen negative soil ethanol extracts were dried down under a stream of nitrogen and re-suspended in 90% olive oil/10% ethanol (v/v) at half the original volume. All animals were dosed at 20 ml per kg bodyweight. Mice were terminated 24 h postinjection and their blood and livers were collected. Experiments were performed under a UK Home Office licence with Local Ethics Committee approval.Blood was collected at termination, serum prepared and clinical chemistry  determined as described Normalized luciferase reporter gene expression was increased by selected landfill site ethanol extracts in a dose–response manner  and was The mouse was considered the preferred animal model in which to study the potential effects of xenoestrogens . Since cholangiocytes on a background of disease express both ERs in rats and man , 2006, cThe mERα cDNA was cloned as outlined in the methods section and expressed in LTPA cells . 3-pGL3promoter reporter gene constitutively (likely due to oestrogens present in the cell media) since co-addition of the ER antagonist ICI182780 significantly reduced luciferase gene expression. However, addition of 10 nM E2 significantly further induced luciferase gene expression confirming that the mERβv1 construct produced functional protein and that the 603B cells expressed co-factors permissive for mERβ responsiveness. In contrast, mERβv2 appeared to be constitutively fully activated since the ER antagonist ICI182780 significantly reduced luciferase gene expression to levels also seen with ICI182780 treatment with mERβv1 and addition of 10 nM E2 did not result in further induced luciferase gene expression  mediated degradation of the protein  and E2 activation (10−12 M) of the hERα, these data suggest that the concentration of xenoestrogens in landfill site soil 3 was approximately 5 nM E2 equivalents .The data in this article provide proof of concept that it is possible to detect xenoestrogenic activity in soil extracts and that measurably high levels can be detected in landfill site soil samples compared with control site soil samples not in proximity to landfill sites and not expected to be contaminated with chemicals such as pesticides. The study has been performed in an historic mining area therefore, to gain access to the most recently deposited material, surface sediments were taken. To our knowledge, this approach has not been employed before. The xenoestrogens present in landfill site soils were shown to activate the hERα in a dose–response manner, an effect that was antagonized by the ER antagonist ICI182780. However, the chemical identity(ies) of the xenoestrogen(s) present in the soil around the landfill site were not investigated. Given the wide range of natural and man-made chemicals shown to activate ERs .In order to be in a position to test the potential toxicological effects of xenoestrogen in man, an animal model is required to take into account exposure routes, toxicokinetic and toxicodynamic effects. Given the abundance of transgenic variants available, the mouse as a species was selected for its potential in future mechanistic in vivo studies. Given the focus of this study on the liver and the known expression and function of ERα in hepatocytes, the hepatocyte would have been the preferred option for screening the oestrogenicity of chemicals . However, since hepatocytes do not undergo mitosis to any significant extent in vitro, transfection efficiencies with hepatocytes are often too low. To generate high signal to noise readouts that are appropriately controlled, and given the need to screen a large set of samples with appropriate replication , the use of mouse hepatocytes in these studies was not a realistic option. Given the apparent role of ERs in human cholangiocyte biology, pancreatohepatobiliary ductal cell lines were selected for screening studies. The availability of pancreatohepatobiliary ductal cell lines in general is limited and in our hands, mERα was not functionally efficiently activated by any oestrogen in the cholangiocyte 603B cells, only in the pancreatic ductal LTPA cell line. The converse was the case for mERβ variants. The reason(s) for this is not clear but may be associated with a divergence between these cell types and the presence of factors required for mER transcriptional function. There may also be limited comparability between human and murine cholangiocyte ER expression which may impact on the relevance of observations in mice for man. However, should this difference in murine ERα and ERβ expression extend to human pancreatohepatobiliary tissues, it would suggest that hERβ transcriptional function predominates in cholangiocytes . The data from these current studies indicate however, that the landfill site soil extracts containing xenoestrogen(s) activate the mERα with similar potency to the hERα and therefore the mouse may be a good model for examining potential endocrine disruptor properties of these extracts In terms of the ERβ, the mouse has 2 variant transcripts which generate distinct mERβ proteins. Both transcripts were present in mouse ovary. The mERβv1 protein was transcriptionally activated by landfill site soil extracts containing xenoestrogen(s) with similar potency to the hERα and mERα. However, the mERβv2 protein was near constitutively transcriptionally fully activated when expressed in 603B cells and required inactivation by pre-treatment with ICI18270 and wash removal for dose–response activation by E2 or EE. To our knowledge, this has not been previously described, and may be a unique feature of the context in which the receptor is expressed . Furthermore, of potential significant toxicological significance, this receptor retains its sensitivity to antagonism by ICI182780 when activated by E2 or EE, but not by landfill site soil-based xenoestrogens. Assuming that the xenoeostrogen(s) interacts similarly with the ligand binding region of the mERβv2, this observation suggests that the(ir) rate of dissociation is significantly reduced compared with E2. Alternatively, the xenoestrogen(s) may interact and activate the receptor via a different mechanism (such as through a different binding site), rendering the receptor insensitive to ICI182780 antagonism.Multiple variant hERβ variant proteins generated from the hERβ gene have been described in man  indicateThe functional role of ERβ in cholangiocytes under normal conditions is likely to be negligible since its expression is low. However, in clinical disease situations, it has been reported that ERβ expression is markedly up-regulated  and/or infectious agent(s). There is an association between use of hormone replacement therapies and increased risk of having PBC  and was in part, funded by the National Institute for Health Research Health Protection Research Unit (NIHR HPRU) in Chemical and Radiation Threats and Hazards in partnership with Public Health England (PHE) and the MRC (in the form of an ITTP studentship to A.C.L.). NIHR HPRU funding was used for the part of the research that did not involve any live animals. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR, the Department of Health or Public Health England.Supplementary DataClick here for additional data file.


It is found that a phase transition from nonmagnetic to ferromagnetic ground state appears once above the critical hole density (~1014 cm−2). The spin magnetic moment can maintain a magnitude of 1.0 μB/hole with excellent stability of ferromagnetic state. Furthermore, we demonstrate that strain is very useful to modulate the DOS near the valence band, resulting in the reduction of the critical hole density to ~1013 cm−2 when the strain reaches 4% (6%) in SnS2 (SnSe2), which can be realized in common field effect transistors. Moreover, the phonon dispersion calculations for the strained SnX2 monolayers indicate that they can keep the dynamical stability under the hole doping. Therefore, the strain tunable magnetic transition in hole doped tin dichalcogenides indicates their potential promising applications in spintronic devices.By first-principles calculations, the magnetism of hole doped tin dichalcogenides SnX Moreover, these systems indicated obvious advantages in strong long-range exchange coupling interaction and no clustering of magnetic ions. To obtain 2D d0 ferromagnetic semiconductors, most studies have focused on inducing local magnetic moment by introducing nonmagnetic impurity atoms1516182021d0 semiconductors occurs when the relative gain in exchange interaction is larger than the loss in kinetic energy, i.e., when it satisfies the “Stoner Criterion”: D(EF)J > 1, where D(EF) is the density of states (DOS) at the Fermi level (EF), and J denotes the strength of the exchange interactionJ is large enough in elements N, O, P and S valence p orbitalsD(EF) becomes large enough to satisfy the Stoner Criterion, systems can be spin polarized. So the prime purpose of chemical doping and other methods is to increase the DOS at EF. However, these approaches would face serious challenges in experiments, for the structural disorder inherently always causes unnecessary complexity in the physical properties, even the concentration of dopants and vacancies is unfavorable for controlling.In addition to the magnetic elements doping in semiconductors, the type of EF locates near the top of valence band, the high DOS generally leads an electronic instability, resulting in phase transitions such as magnetism, superconductivity, and other phenomena. In semiconductor industry, carrier doping is considered as an effective approach to modulate EF, which performs obvious advantages including their completely free of structure disorder and remarkably simple to tune physical propertieset al.et al.1P3). In the previous work, we also explored the hole doping effect on magnetic properties in 2D graphene-like C2NIt is worth noting that some 2D semiconductors exist unusual band structures, one of which is the Mexican hat dispersions, such as gallium or indium monochalcogenides, often resulting in van Hove singularities (VHSs) with 2  have been widely studied for their excellent electrical, optical and magnetic properties, such as lithium ion batteries, photovoltaic devices, as well as field effect transistors3031322 nanostructures, such as manipulating SnSe2 armchair nanoribbons via edge hydrogenation22  monolayers. Results indicate that ferromagnetic ground state with outstanding stability of spin polarization can be obtained by hole doping. Particularly, by strain engineering, the critical hole density can be reduced to 1013 cm−2, which is an order of magnitude smaller than that of pristine structure. Therefore, SnX2 monolayers can be considered as a viable candidates for spintronic devices.In recent years, chemically stable and environmentally friendly semiconducting tin dichalcogenides SnX2 monolayers, density functional theory (DFT) calculations were performed using the Projector-Augmented Wave (PAW) pseudopotential implementation of the Vienna Ab Initio Simulation Package (VASP)3536k-point grid for all simulations. The convergence threshold was 1 × 10−5 eV for the electronic self-consistent field iterations. The atomic positions were optimized until the maximum Hellma-Feynman force on each atom was less than 10−2 eV Å−1. A vacuum spacing of 20 Å was placed to avoid the interactions between the monolayers and its periodic images. Moreover, to examine the dynamical stability of SnX2 monolayers with doping, the phonon dispersion was calculated by density functional perturbation theory in VASP.To study the electronic and magnetic properties of the SnX2  monolayers with the 1T structure are the hexagonal crystal structure of the CdI2-type, as shown in 2 monolayers consist of three atomic sublayers with the covalently bonded layer of X-Sn-X. The central Sn atom bonds to six nearest-neighbor X atoms located in the top and bottom sublayers. The structural stability of monolayer SnS2 has been theoretically predicted by Zhuang et al.2 were optimized, and atomic positions were relaxed to zero pressure following the convergence criteria. The optimized lattice constant of SnS2 and SnSe2 is 3.700 Å and 3.871 Å, which is in accordance with previous calculations by PBE formula3233SnX2 monolayers are displayed in M point and the valance band maximum (VBM) lies between Γ − M point, which means both the SnS2 and SnSe2 monolayers are indirect band gap semiconductors. The indirect band gap of SnS2 (~1.60 eV) is almost double of SnSe2 (~ 0.83 eV), which is consistent with previous reported value (1.57 eV for SnS2 and 0.79 eV for SnSe2)s, 5p, 4d-orbit partial DOS (PDOS) of Sn atom, and s, p-orbit PDOS of S (Se) atom are depicted in the right diagrams of p (Se-4p) states, besides, the CBM is co-contributed by Sn-5s and S (Se)-p states.The band structure and DOS of SnXEF would provide the possibility to develop a spontaneous ferromagnetism. We applied carrier doping to investigate the possible ferromagnetism in SnX2 monolayers. μB) per hole with the various hole density. At the nonmagnetic state, the magnetic moment is nearly zero. Once above the critical hole density, the local magnetic moment increases rapidly to a constant value with ~1.0 μB/hole, implying the transition from nonmagnetic to ferromagnetic ground state. The critical hole density (pc) of SnS2 and SnSe2 is about 2.2 × 1014 cm−2 and 3.2 × 1014 cm−2, respectively. In order to check the stability of ferromagnetic state, the spin polarization energy per hole, ΔEp, is discussed, which is defined by the energy difference between the spin-polarized state (Esp) and non-spin-polarized state (Enonsp) for each hole, i.e., ΔEp = (Esp − Enonsp)/number(hole). The hole density dependence of spin polarization energy per hole is also shown in Ep decreases monotonically with the increment of the hole density. At the hole density of 7.0 × 1014 cm−2, ΔEp is about −103 and −52 meV/hole for SnS2 and SnSe2, respectively, much higher than those in GaSe (−3 meV/hole)1P3 (−24 meV/hole)2 would be much more stable.It is well known that the high DOS around the 2 monolayers. At the first stage, although hole doping leads to the EF shift down to around the VBM, the spin up and spin down bands completely overlap, indicating the nonmagnetic structure. Once the hole density exceeds the critical value, the spin splitting appears around EF, resulting in the magnetic transition. When the hole density continue to increase, the spin up bands shift down, meanwhile, spin down bands shift up gradually. 2 in the spin polarized state with the hole density of 4.2 × 1014 cm−2 for SnS2 and 5.4 × 1014 cm−2 for SnSe2. Moreover, it is found that the spin down channel shows metallic characteristic, while the spin up channel shows a fascinate transition from metal to semiconductor, where the transition occurs at 2.5 × 1014 cm−2 for SnS2 and 5.4 × 1014 cm−2 for SnSe2, respectively. Thus, the half-metallic SnX2 monolayers can be modulated by hole doping. This half-metallic property would have good performance in transferring one particular spin oriented electrons such as in a spin filtering devices.We also studied the band structure and DOS of hole doped SnX2 monolayers, we notice that the magnetic moments are contributed mainly by the p states at the VBM. Like the previous study on d0 ferromagnetic semiconductors, such as ZnOxPy2 monolayers. The ferromagnetic characteristic would be achieved through a p-d hybridization-like p-p interaction between the p states of chalcogens. D(EF) and the energy difference ΔE of the two spin-type bands around the VBM by hole doping for monolayer SnX2. A large exchange-splitting of the two spin-type bands appears due to the strong exchange field in the ferromagnetic phase, and the ΔE increases with the increment of hole density when the D(EF) is large enough, which means a large strength of exchange interaction J in these systems. Consequently, the magnetism of SnX2 monolayers can be induced by hole doping.To understand the magnetization mechanism of hole doped SnX14 cm−2 in graphene and transitional metal dichalcogenides404113 cm−2 for conventional solid state gated voltage in FETs432 monolayers for their further practical applications.Currently, ultrahigh carrier density accumulation could be achievable using electric-field control in FETs in experiments, including conventional solid state gated voltage in FETs and electric double layer transistors (EDLTs) by using polymer electrolytes or ionic liquid as gated dielectrics. By using EDLTs, an accumulation of extremely high carrier density could up to 102 in the small region from −1.0 to −0.4 eV are inset in 14 cm−2) must be needed to obtain magnetic transition. Therefore, we devote to modulating the high DOS to the top of valence band. As we known, band structure is highly sensitive to the external conditions such as temperature, pressure or strain, especially in 2D layered structure. They often lead to dramatically changes about electronic, magnetic and optical properties. In particular, the strain engineering is commonly used to tune the band structure, due to the extraordinary break strength and structural stability in a wide range of strain45464748et al. recently reported the effects of in-plane biaxial strain on the electronic structure of single-layered SnS2The detail DOS of SnSe2 monolayers. 2 monolayers, in addition with unstrained states. It is clear that the DOS near the VBM increase with the increment of strain. When the strain reaches 4% for SnS2 and 6% for SnSe2, the DOS around the VBM are similar, and the VHSs occur, due to the Mexican hat-like band edges around Γ point. Once the EF shift down to the top of valence band, such unusual DOS would lead to the reduction of the hole density, and transitions to magnetism. So our next work studies the local magnetic moment and spin polarization energy dependent on the hole density under various strain strength.In this study, the biaxial tensile strain , with the increment of 2%, was applied for the SnX2 monolayers without hole doping, it is nonmagnetic at the ground state. The magnetic moment (~1.0 μB/hole) appears at the lower hole density in comparison to the unstrained SnX2. The inset map plotted relations between the critical hole density and the tensile strain. The critical hole densities reduce sharply in the small range of strain, i.e., 0–4% for SnS2 and 0–6% for SnSe2, and then maintain about 1.5 × 1013 cm−2 of SnS2 and 2.0 × 1013 cm−2 of SnSe2, respectively. These values are an order lower than that of unstrained structures, and could be realized in common FETs. Moreover, with the biaxial strain applied, the tendency of critical hole density is consist with that of DOS around the VBM described in D(EF) and ΔE for strained SnS2 (SnSe2) monolayers. It is clear that the SnX2 monolayers under biaxial strain are more favorable to achieve the spin splitting due to the greatly enhanced D(EF). Next, we check the stability of spin polarization of the strained structure, as shown in 2 monolayers, the lower spin polarization energy/hole can be obtained with the increment of strain, and finally the spin polarization energy nearly tend to the constant value. For example, at the hole density of 7.0 × 1014 cm−2 with 6% strain applied, values decrease monotonically to −180 meV of SnS2 and −160 meV of SnSe2, which is far more small than that of the unstrained structure. Therefore, by applying tensile strain, the magnetism of SnX2 monolayers would have promising applications for their achievable hole doping density and stable magnetic phase transition.The relations between the spin magnetic moment/hole and the hole density are shown in 2 in 1P32 and ≥6% for SnSe2), the critical hole density of SnX2 monolayers is smaller that of Si1P3 and GaSe. Furthermore, SnX2 monolayers perform better stability of spin polarization under both unstrained and strained state. Therefore, for 2D SnX2 monolayers, hole doping can induce stable spin polarization in a large range of hole density, and the critical hole density and spin polarization energy can be improved greatly by strain engineering.In addition, the critical hole density, magnetic moment and spin polarization energy are summarized for SnX2 monolayers is crucial for their applications on magnetism. So, we have also carried out the phonon dispersion calculations to study their dynamic stabilities. The phonon dispersions of SnS2 (SnSe2) monolayers under the tensile strain of 4% (6%) are shown in 2 (SnSe2) monolayers under tensile strain are stable. At 0.1 hole per unit cell doping, large enough to induce magnetism in SnS2 (SnSe2) monolayer with tensile strain of 4% (6%), there is still no imaginary frequency, except the frequency softening comparing to the SnS2 (SnSe2) monolayers without doping. Therefore, these results indicate that hole doping in SnX2 monolayers have negligible effect on their structural stabilities, and will provide feasible theoretical predictions for practical applications in spintronic devices.The stability of hole doped SnX2  monolayers are studied by DFT calculations. It is demonstrated that hole doping (~1014 cm−2) can drive a phase transition from nonmagnetic to ferromagnetic ground state. The electron spin magnetic moment can reach a constant magnitude (~1.0 μB/hole) with excellent stability of ferromagnetic state. Besides, the half-metallic characteristic can be modulated by hole doping. Furthermore, by introducing tensile biaxial strain to SnX2 monolayers, the critical hole density can reduce to ~1013 cm−2, which could be realized by conventional solid high dielectric FETs. The higher spin polarization energy indicates better stability in comparison with the pristine structure. In addition, the hole doped SnX2 monolayers still remains stable by phonon dispersion calculations. Therefore, investigation of the magnetism for hole doped SnX2 monolayers under the strain will provide an achievable idea for 2D functional materials in spintronics.In conclusion, electronic and magnetic properties of the SnXHow to cite this article: Xiang, H. et al. Strain tunable magnetism in SnX2  monolayers by hole doping. Sci. Rep.6, 39218; doi: 10.1038/srep39218 (2016).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


Samples requiring ≥ 3 arthrocentesis attempts had significantly higher PGE2 concentrations compared to samples requiring 1 or 2 attempts. Following correction for number of arthrocentesis attempts, PGE2 concentrations were significantly higher than baseline at 24 and 48 h in the bupivacaine group; however there were no significant differences between the bupivacaine and saline groups. In normal dogs, a single bupivacaine injection did not cause significant synovial inflammation, as measured by PGE2 concentrations, compared to saline controls. Future research should minimize aspiration attempts and include evaluation of the synovial response to bupivacaine in clinical cases with joint disease.There were no significant differences in synovial fluid prostaglandin E Bupivacaine is a local anesthetic that works directly on the neuronal cell membrane, blocking impulse conduction in nerve fibers. When administered intra-articularly, as part of a multimodal analgesic protocol, bupivacaine provides long-lasting local pain relief after joint surgery in people and animals.The safety of intra-articular bupivacaine came into question when clinical case reports concluded an association between intra-articular bupivacaine and glenohumeral chondrolysis, a rare complication following shoulder arthroscopy in people –3. Subse2 (PGE2). PGE2 is a marker of synovitis and stimulates expression of matrix metalloproteinases that play a major role in articular cartilage degeneration.The delay of several months between bupivacaine infusion and clinically evident chondrolysis suggests that chondrolysis may be a sequela to an extra-cartilaginous process (such as synovitis) that indirectly leads to chondrocyte toxicity or makes chondrocytes more vulnerable to subsequent insults. The idea that bupivacaine may cause acute synovitis is supported by studies that document signs of synovitis after local anesthetic injection in multiple species . Sustained synovitis can indirectly lead to cartilage degradation through the actions of enzymes, inflammatory mediators, and cytokines . An insu2 concentration compared to intra-articular saline injection.The objective of this study was to identify if there is a synovial response to a single intra-articular dose of bupivacaine in the normal canine stifle. We hypothesized that a single intra-articular bupivacaine injection would result in a detectable increase in synovial fluid PGEEight healthy adult male intact purpose bred Beagle dogs were purchased from Covance Inc.  and housed in our institution in a manner approved by the institutional animal care and use committee at Virginia Tech. The mean age of the dogs was 1.59 years (range 1.57–1.62) and the mean weight was 12.1 kg (range 10.1–13.4). Dogs were included if they were deemed healthy based on normal physical and orthopedic examinations. Dogs were heavily sedated with intravenous (IV) hydromorphone (0.05 mg/kg) and dexmedetomidine (5 mcg/kg). Bilateral orthogonal stifle radiographs were obtained and a radiologist reviewed all radiographic images to ensure there was no evidence of stifle osteoarthritis.Stifles were randomized using a random number generatorThe first 3 time points  were collected on the 1st day (Day 0). When necessary, dogs were administered additional sedation (dexmedetomidine 5 mcg/kg IV). A standard medial or lateral parapatellar arthrocentesis technique was used, based on dog positioning [2 quantified by competitive ELISAWithin 6 months of collection, samples were thawed on ice and PGE2 concentration 30 min following intra-articular injection of bupivacaine. A sample size of 6 stifles per group was estimated to achieve 83% power to detect a mean of paired differences of 480 pg/mL between groups with a significance level  of 0.05.Prior to the study, a power analysis was performed using data from an unpublished pilot study that measured the increase in PGEData were analyzed using commercial software.08 mL/kg  and fina2 concentrations were skewed and data are therefore summarized as median (range). A logarithmic transformation (base e) was applied to the concentrations before downstream data analysis. Effects of treatment and time were assessed using mixed-model repeated-measures ANOVA followed by Tukey’s procedure for multiple comparisons. Residuals were inspected to verify that the errors followed a normal distribution with constant variance. A post hoc analysis (mixed-model ANOVA) was performed to investigate the association between number of aspiration attempts required to collect an adequate sample and PGE2 concentration. This analysis revealed that ≥ 3 attempts resulted in significantly elevated PGE2 concentrations, and so a sensitivity re-assessment of the effects of treatment and time using data obtained after only 1 or 2 aspiration attempts was performed. Statistical significance was set at p < 0.05.Normal probability plots showed that PGE2 concentrations between the bupivacaine and saline groups (p = 0.229–0.898) or over time within each group  when all data were included in the analysis without consideration for number of aspiration attempts (Table No radiographic evidence of osteoarthritis was identified in any stifle in any dog. There were no significant differences in PGE2 concentrations varied significantly based on number of aspiration attempts (Table The number of aspiration attempts required to collect an adequate sample was 1 in the majority of cases 62/80), but was as high as 6 attempts in a single stifle at one time point (Dog 1 at T0). PGE/80, but 2 concentrations in the bupivacaine group increased significantly over time (p = 0.008). In the bupivacaine group, PGE2 concentrations in samples collected at T24 (p = 0.003) and T48 (p = 0.041) were significantly higher compared to T0. There were no changes in PGE2 concentrations in saline-treated joints over time (p = 0.207). There were no significant differences between saline- and bupivacaine-injected joints at any time point (p = 0.261–0.949).Based on the analysis of number of attempts, samples requiring ≥ 3 aspiration attempts were omitted and the data re-analyzed  or whether bupivacaine and saline stifles were analyzed separately .Finally, a post hoc analysis was performed to evaluate the effect of ≥ 3 attempts on all 2 concentrations compared to saline-injected controls regardless of number of aspiration attempts. Following re-analysis, PGE2 concentrations in the bupivacaine group increased significantly over time (p = 0.008). Measurement of synovial fluid PGE2 concentrations was complicated by the predictable increase in synovial fluid volume following injection, as well as the variable number of aspiration attempts for each joint. Our data indicate that synovial fluid PGE2 concentration is highly sensitive to number of aspiration attempts. This is an important consideration when performing arthrocentesis in clinical cases and in designing future studies.In normal canine stifles in our study, a single intra-articular injection of bupivacaine did not result in increased synovial fluid PGE2 following addition of IL-1β to the culture medium [2 from baseline within the bupivacaine group, suggesting an inflammatory response following injection. Confidence in this conclusion is weak considering the lack of significance between treatment and control groups.There is conflicting evidence regarding the expected inflammatory or anti-inflammatory effects of local anesthetics on the synovium. In an in vitro canine co-culture model of the synovial joint, 0.5% bupivacaine inhibited an expected rise in PGEe medium . Neurogee medium . These te medium –12. Simi2 concentrations in samples that required repeated arthrocentesis attempts (≥ 3 attempts in a single time point). Frequent needle penetration of the synovium is the most likely source of this inflammation rather than the treatment agent, because it occurred in both treatment groups. Increased PGE2 concentrations in synovial fluid following arthrocentesis is also documented in horses and as a result, those authors suggest separating repeated aspirations by 7 days to account for the inflammation created by arthrocentesis [An unexpected observation in our study was the significant increase in synovial fluid PGEcentesis . In our 2 concentrations would be decreased due to dilution by the larger relative fluid volume. In a previous study evaluating how quickly bupivacaine elutes from the stifle following intra-articular administration, a similar dilutional effect was noted following treatment injection, and our pre-injection fluid volume approximation was calculated based on those methods [We anticipated that the normal Beagle stifle would contain a small synovial fluid volume (1 mL fluid/12.5 kg dog). As a result, following aspiration at T0 and the subsequent infusion of the treatment volume (~ 2.5 mL), later PGE methods . Therefo methods .The effect of aspiration attempts influencing our data resulted in 11/80 samples being excluded from the final analysis. Dogs 1 and 5 were particularly difficult, contributing to 8/11 omitted samples. The difficulty associated with Dog 1 was attributed to the learning curve associated with the procedure while Dog 5 was attributed to his small size and resultant small initial synovial fluid volume.2 concentrations.A time- and dose-dependent effect of bupivacaine on chondrotoxicity in vitro is reported , 14–16; 2 concentrations and synovitis are a common feature of osteoarthritis and may either blunt the joint’s response to bupivacaine or prime the joint for an enhanced response to bupivacaine injection [An important limitation is the use of normal stifles; the results of our study may not accurately portray the response within a diseased joint (i.e. osteoarthritic joint), when bupivacaine is most likely to be used therapeutically. Elevated PGEnjection , 18. A s


Lithium cation is the charge carrier in lithium-ion battery. Electrolyte solution in lithium-ion battery is usually based on mixed solvents consisting of polar carbonates with different aliphatic chains. Despite various experimental evidences indicating that lithium ion forms a rigid and stable solvation sheath through electrostatic interactions with polar carbonates, both the lithium solvation structure and more importantly fluctuation dynamics and functional role of carbonate solvent molecules have not been fully elucidated yet with femtosecond vibrational spectroscopic methods. Here we investigate the ultrafast carbonate solvent exchange dynamics around lithium ions in electrolyte solutions with coherent two-dimensional infrared spectroscopy and find that the time constants of the formation and dissociation of lithium-ion···carbonate complex in solvation sheaths are on a picosecond timescale. We anticipate that such ultrafast microscopic fluxional processes in lithium-solvent complexes could provide an important clue to understanding macroscopic mobility of lithium cation in lithium-ion battery on a molecular level. The lithium solvation structure in the electrolyte solution for lithium-ion batteries has not been fully understood. Here, the authors show ultrafast fluxional exchange of carbonate solvent molecules in and out of lithium-ion solvation sheath utilizing coherent two-dimensional infrared spectroscopy. Typical electrolyte solutions used in LIB are composed of non-aqueous carbonate solvents that have certain advantages. For instance, the oxidation potential can be matched to the output voltage of LIB and carbonate solvents are also building blocks of solid-electrolyte interphase78910Electrolyte solvent capable of dissolving lithium salt is an integral part of Li-ion battery (LIB) that has become an indispensable power source for current mobile ecosystem and electrical vehicles123+ conductivity, mixed solvents have often been used not only because they increase the solubility of lithium salt but also because they serve as excellent media facilitating fast transport of Li-ion+ transport. To enhance Li+ ion mobility as well as to improve low-temperature performance, linear alkyl carbonates like diethyl carbonate (DEC) and dimethyl carbonate (DMC) are added to the LIB electrolyte solutions as co-solvents. It has long been believed that such low viscosity co-solvents act as a medium for transporting the sheaths of Li+ ion-solvent complex6, is dissolved in organic carbonate mixed solvents consisting of equal volumetric amounts of DEC, DMC and EC. Li+ is believed to be preferentially solvated by EC, and DEC and/or DMC provides the medium for large Li–EC complexes to flow between electrodes during charging and discharging processes. However, to our surprise, detailed microscopic solvation structures and ultrafast solvation dynamics around Li+ in such electrolyte solutions have not been fully understood and even they are still the key subjects under intense scholarly debate.To achieve high Li+ would, without a doubt, help one to better understand the working principles of LIB and to eventually design an improved LIB. Most of the previous studies primarily focused on the equilibrium structure of the first solvation shell of Li+, which has often been referred to as solvation sheath due to its rigidity13141516+ in mixed solvent electrolyte solutions18et al.17O NMR experiments show that Li+ makes a direct electrostatic interaction with carbonyl oxygen atoms of surrounding carbonates. Even in the case that Li+ is preferentially solvated by EC or PC, the participation of co-solvent molecules, such as DEC and DMC, in the solvation sheath is possible not only because of their high-mole fractions but also because of extra entropic gain. Therefore, it is by now clear that such co-solvents could potentially play a dual-role of low-viscous medium and solvating molecule. Co-solvents like DEC and DMC would add an extra flexibility to solvation sheath, because they comparatively weakly interact with Li+ and have many conformational degrees of freedom+ solvation sheath has been reported due to their ultrafast nature and experimental difficulties in finding an appropriate spectroscopic probe.Therefore, elucidating the molecular solvation structure of organic carbonates around Li+ ion. Here considering the carbonyl stretching vibration of DEC as an infrared probe, we carried out Fourier transform infrared (FTIR), femtosecond infrared pump-probe and two-dimensional (2D) infrared studies of chemical exchange processes between DEC molecules with and without Coulombic interaction with Li+. One of the most difficult problems in the present linear and nonlinear infrared spectroscopic investigations is that the infrared probe, DEC, itself is the solvent too. Thus, with normal infrared cells with several micrometre thickness at best, the linear absorption is saturated and the nonlinear infrared signal fields are self-attenuated, which renders a difficulty in detecting weak 2D infrared signal. To overcome this, we prepared a home-built thin sample cell coated with SiO2, using radio frequency magnetron sputtering technique , O−C−O asymmetric stretching , and O−CH2−CH3 asymmetric stretching  vibrations. As LiPF6 concentration in the DEC solution increases, two distinctively new peaks at 1,715.4 and 1,305.6 cm−1 appear. The frequencies and spectral widths of the main peaks at 1,747 and 1,258.9 cm−1 do not change much on increasing lithium salt concentration. Therefore, the two new peaks at 1,715.4 and 1,305.6 cm−1 can be safely assigned to the C=O stretch and O−C−O asymmetric stretch modes of DEC molecules directly interacting with Li+.The infrared absorption spectra of the C=O stretch and O−C−O asymmetric stretch modes in LiPF+. They are two ester ether oxygen atoms and one carbonyl oxygen atom. Therefore, at least two structures in 17O NMR experiment provided evidence that the ester ether oxygen atoms do not strongly interact with Li-ion−1 and 1,235.5 cm−1, respectively  of C=O frequency and red shift (96.8 cm−1) of O−C−O asymmetric stretch frequency. These results can be understood by noting that the Li+ ion interacting with carbonyl oxygen atom makes the double bond character (or bond length) of C=O group decrease (increase), which concomitantly makes the C−O bond lengths short  DEC surrounded by other DECs, (ii) DEC interacting with free Li+ and (iii) DEC interacting with Li+ in the CIP state, Li+:PF6−. In addition, the existence of two different types of anions, either PF6− solvated by DECs or PF6− in CIP imposes yet another complexity to the interpretation of linear and nonlinear infrared spectra. However, because the negatively charged PF6− tends to be away from the carbonate part of DEC due to Coulomb repulsion and preferentially surrounded by the DEC ethyl groups6− does not strongly affect the DEC carbonyl stretch infrared spectrum.The number of solvent molecules in the first solvation shell of a solute molecule is an important quantity providing information on the size and structure of Li-ion solvation sheath. Often, a series of lithium salt concentration-dependent spectroscopic experiments has been used to determine the solvation number around a Li+:PF6− as compared to DEC···Li+, we further analysed density functional theory calculation results of DEC interacting with Li+:PF6−  of free DEC and the positive 0–1 peak (ground state |0> bleach and stimulated emission from |1> to |0>) of Li–DEC complex at around 1,720 cm−1. Another notable feature is the prolonged positive signal  is found to be much shorter than that of free DEC (2.1±0.1 ps). Detailed discussion on the origin of such increased vibrational relaxation rate of DEC upon its electrostatic interaction with Li6 DEC solution at different waiting times. Each 2D infrared spectrum was normalized to the largest positive 0–1 peak magnitude. At a very short waiting time of Tw=0.3 ps, there are only two peaks on the diagonal line (see the upper-left panel in ωt) axis are the excited state absorption contributions. Both positive and negative peaks are elongated along the diagonal line at short waiting time up to Tw=1.0 ps. However, after 2.0 ps, all peaks become round in shape due to the spectral diffusion process. Diagonally elongated peaks at short waiting times indicate structural heterogeneity, distribution of coordination number and ethyl side-chain conformational distribution +. We found that the C=O stretch frequency–frequency correlation decays on a few picosecond timescale, which could result from ultrafast internal rotations of the ethyl groups in DEC and hindered rotational motions of carbonates.−1, which is slightly broader than that of free DEC (FWHM=18.9 cm−1). However, the vibrational lifetime of Li–DEC is just half of that of the free DEC, which means that the lifetime broadening of Li–DEC is larger than that of free DEC. These observations indicate that the inhomogeneous linewidth of Li–DEC may be narrower than free DEC, which is consistent with the notion that DEC molecules in Li–DEC complexes have stable conformers and the side-chain structure can be correlated with its solvation propertyIn principle, the inhomogeneous structural distribution can be studied by analysing the centre or nodal line slope and/or diagonal ellipticities of 2D infrared peaks3233+ could be affected by the presence of co-solvents in the first solvation shell. In a recent 17O NMR experiment, NMR peak of carbonyl oxygen atom in EC or DMC shows a motional narrowing behaviour, indicating the existence of fast equilibrium dynamics between free carbonate and Li-complexed carbonate molecules in nanosecond or even shorter timescales5+ in LIB electrolyte solutions. To date, however, little has been understood about how fast the solvation dynamics and the structural fluctuation of solvation sheath.In a real LIB electrolyte solution, detailed solvation dynamics around LiTw>2 ps), one can clearly observe cross peaks  induced by conformational transitions between different solvation structures. Indeed, at longer waiting time n–1 and DEC···Li+:PF6−(DEC)n–2. However, DEC in DEC···Li+(DEC)n–1 and that in DEC···Li+:PF6−(DEC)n–2 are neither spectrally nor kinetically resolvable, due to the similarity in solvation structures. This led us to use a simple two-state exchange model between DEC and DEC···Li+(ligands)n–1, where ligand refers to either DEC or PF6− and n varies depending on lithium salt concentration and solvent composition. Here the chemical exchange dynamics is therefore the dynamical equilibrium between Li+(ligands)n and Li+(ligands)n–1+free DEC (The Li–DEC coordination breaking and forming dynamics are manifested in the free DEC .+(ligands)n–1 can be obtained from the infrared spectral peak analyses after extracting information on the corresponding transition dipole ratio from the LiPF6 concentration-dependent infrared data (+(ligands)n–1 is 1.7 times larger than that of free DEC, which is in excellent agreement with quantum chemistry calculation results (+(ligands)n–1 to DEC is found to be 0.138, that is, [DEC···Li+(ligands)n–1]/[free DEC]=0.138. From the peak volumes  1.0 M LiPF6 in DMC:PC (1:1 in volume percent) solution, (ii) 1.5 M LiPF6 in DMC:PC (1.5:1) solution and (iii) 2.0 M LiPF6 in DMC:PC (1.5:1) solution. Here it should be noted that we considered DMC:PC mixed solvents instead of DEC:PC mixed solvents because of the following reasons. First, since the C=O stretching vibrational lifetime of DMC is comparatively longer than that of DEC, we could observe measurable diagonal and cross peaks of free DMC and Li–DMC complex in our 2D infrared spectra at long waiting times. Second, we here show that the chemical exchanges observed in Li–DEC (single carbonate solvent) solution do occur in different mixed solvent systems too.Despite that the ultrafast chemical exchange dynamics between free DEC and Li–DEC complex was observed in pure DEC solutions, typical LIB's use mixed solvents instead. To elucidate the separate roles and solvation properties of co-solvents with DMC and PC and to investigate the possibility of DMC playing dual roles both as a solvent medium for mobile Li–PC(DMC) complexes and as a ligand participating in the first solvation shell around LinPCm complex with n DMC and m PC molecules in the first solvation shell and free DMC molecules even in the DMC and PC (1:1 in volume percent) mixed solvents with 1.0 M LiPF6 –(iii) can be found in Despite that the 2D infrared spectra appear to be spectrally congested, we could clearly observe real-time chemical exchange processes between Li–DMCiPF6 see . The 2D ωτ=1,725 cm−1 and ωt=1,760 cm−1 increases (see the 2D infrared spectra inside the red boxes in + in LIB electrolyte solutions is stronger than that between DMC (or DEC) and Li+, which is consistent with the notion that the PC molecule with large dipole moment compared to that of DEC molecule tends to make a stable and rigid solvation sheath around a lithium cation in LIB electrolyte solutions.As waiting time increases, the cross peak amplitude at 6/DMC:PC (1:1 in volume percent) solution, we fitted the 2D infrared peaks with a 2D Gaussian function  vibrational lifetimes, (iii) rotational relaxation times and (iv) survival probabilities of the two species within the waiting time. The cross peak intensity, denoted as |μLi–DMC|2|μDMC|2, (ii) vibrational lifetimes, (iii) rotational relaxation times and (iv) condition probability of finding free DMC after finite waiting time when it was initially in a Li–DMC complex form. Therefore, the ratio of the cross peak intensity to the square root of the product of the two diagonal peaks, that is, To approximately examine the timescale of chemical exchange processes between Li–DMC complex and free DMC in 1.0 M LiPFfunction , which a6/DEC solution shown in 6/DMC:PC (1:1) solution in 6/DMC:PC (1.5:1) and 2.0 M LiPF6/DMC:PC (1.5:1) solutions, the intensity ratios are found to be 0.69 and 0.71, respectively (6/DEC solution is quantitatively similar to that in mixed solvents consisting of DMC and PC.From the 2D Gaussian fit to the 2D infrared spectra (at 10 ps) for 1.0 M LiPFectively . Our obs+ and PF6− solvation shells and ion aggregates, we carried out classical MD simulations for 1.0 M LiPF6/DEC solution using the force field parameters for PF6− in refs + is represented by a yellow sphere and PF6− is by an octahedron. At this high lithium salt concentration, ions form not just CIPs but also large aggregates with a polydisperse distribution. 4 and NaBF4 show a strong propensity to form a large-scale spatially extended networks that are tightly intertwined with water H-bonding networks39+ and PF6− ions form three-dimensional ion networks in LIB electrolyte solutions could be a key to understand detailed mechanism of Li+ ion mobility, since Li+ can jump from one to the other neighbouring anions in the same or different ion networks in the presence of non-zero gradient of electric potentialTo examine the structures and dynamics of Li+ solvation sheath structures in LIB electrolyte solutions are for the first time observed here with employing chemical exchange 2D infrared spectroscopic method. Combining FTIR spectroscopy, infrared pump-probe measurement and quantum chemistry calculation results, we completely characterized the vibrational properties of free DEC and Li–DEC complexes in both pure DEC solvent and mixed solvents consisting of DEC and PC. Subsequent 2D infrared data analyses revealed the ultrafast nature of chemical exchange dynamics in the immediate vicinity of Li+ in DEC solution. Here one of the most difficult challenges was to prepare very thin solution samples because the infrared probes are the C=O stretch modes of DEC and PC molecules that are also solvents. Using RF magnetron sputtering technique, we could control the thickness of coated SiO2 layer on CaF2 window for linear and nonlinear infrared spectroscopic studies. The picosecond Li–DEC complex making and breaking dynamics in both pure DEC solution and DMC:PC mixed solution suggest that the macroscopic Li transport during both charging and discharging processes might be interrelated to microscopic solvation fluctuation and discrete transitions between different solvation states of Li+ in LIB electrolyte solutions. We anticipate that the present ultrafast chemical exchange dynamics should also play an important role in solvation and de-solvation of Li+ at the solid-electrolyte interface on electrodes because of the following reason. If the rigidity of each Li+ solvation sheath is very high, any de-solvation of Li+ on the anode surface may not be efficient, which potentially restricts the function of LIB due to lowering the rate of lithium deposition on the anode. On the other hand, if the Li+ solvation sheath is not sufficiently stable due to a low dissolving power of solvent, the rate of Li+ release from the cathode could be slow. Therefore, it is naturally expected that ideal LIB solvent molecules should interact with Li+ with a properly balanced strength. Overall, the unexpected ultrafast fluxional solvation processes around Li+ in the LIB electrolyte solution is turned out to be prerequisite for optimally achieving the functional role of carbonate solvent in LIB. We anticipate that a further 2D infrared investigation on the Li-solvent fluctuation dynamics during both charging and discharging processes would provide critical information on the fluctuation-dissipation relationship between equilibrium microscopic solvent fluctuation and non-equilibrium macroscopic charge transport phenomenon.Ultrafast fluxional changes of Li6, Battery grade, purity >99.99% trace metal basis) and DEC (purity >99%) was purchased from Sigma-Aldrich. DEC was dried using thermally activated 3 Å molecular sieves. The water content of the sample determined by Karl-Fisher method was less than 50 p.p.m. All solution electrolyte preparation and injection into the infrared cell holder was carried out in Ar-filled glove box . The low water content in sample for infrared absorption measurement was confirmed by examining the water O–H stretch band at around 3,500 cm−1 in the infrared spectra.Lithium hexafluorophosphate  magnetron sputtering system  or −45° (perpendicular) by a linear polarizer on a computer-controlled rotator, sent through a polarizer fixed to 0° into the spectrograph, and detected by a 32-element array detector.Experimental details of pump-probe and 2D infrared spectroscopy employed in the present study have been described elsewhere. Briefly, 800 nm pulses with duration of ∼100 fs and 1.0 mJ were generated from Ti:Sapphire oscillator and regenerative amplifier operating at 1 KHz, which were then used to produce near- infrared pulses at ∼1.4 and ∼1.9 μm. With AgGaS nonlinear optical crystal, the signal and idler pulses are mixed to generate mid-infrared pulses with ∼8 μJ per pulse and ∼100 fs duration centred at 2,070 cm6-DEC solutions at 1.0 M LiPF6 concentration, where 378 LiPF6 molecules were dissolved in 1,000 DEC molecules. The force field parameters developed by Lopes and Pádua406− ion and DEC molecule. respectively, where the corresponding restrained electrostatic potential charges were obtained by carrying out B3LYP/6-311++G calculations. The particle mesh Ewald method was used for long-range electrostatic interaction and the cutoff distance for nonbonding interaction was set to be 10 Å. The solution system was first energy-minimized with the steepest descent method and the conjugate gradient method before running equilibrium MD simulations. Subsequently, a constant N, p and T ensemble simulation at 1 atm and 298 K was carried out for 2 ns to adjust the solution density. An additional 10 ns N, V and T ensemble simulation at 298 K was performed to make each LiPF6-DEC solution system to reach its thermal equilibrium state. Finally, the production run was performed for 10 ns at constant N, V and T conditions, where the simulation time step was set to be 1 fs, and atomic coordinates were saved for every 1 ps.Molecular dynamic (MD) simulations were performed for LiPFThe data that support the findings of this study are available from the authors on reasonable request, see author contributions for specific data sets.How to cite this article: Lee, K.-K. et al. Ultrafast fluxional exchange dynamics in electrolyte solvation sheath of lithium ion battery. Nat. Commun.8, 14658 doi: 10.1038/ncomms14658 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Supplementary Figures 1-12, Supplementary Tables 1-2, Supplementary Notes 1-6 and Supplementary References


Remarkably, the gas coexists with both magnetism and strong Rashba spin–orbit coupling. However, both the origin of superconductivity and the nature of the transition to the normal state over the whole doping range remain elusive. Here we use resonant microwave transport to extract the superfluid stiffness and the superconducting gap energy of the LaAlO3/SrTiO3 interface as a function of carrier density. We show that the superconducting phase diagram of this system is controlled by the competition between electron pairing and phase coherence. The analysis of the superfluid density reveals that only a very small fraction of the electrons condenses into the superconducting state. We propose that this corresponds to the weak filling of high-energy dxz/dyz bands in the quantum well, more apt to host superconductivity.In LaAlO 3/SrTiO3 interface. Here, Singh et al. report superfluid stiffness and the superconducting gap energy at such interface as a function of carrier density.The nature of the doping dependent superconducting transition remains elusive for a two dimensional electron gas at the LaAlO It ends into a quantum critical point, where the Tc is reduced to zero, as carriers are removed from the interfacial quantum well4. Despite a few proposals7, the origin of this carrier density dependence, and in particular the non-monotonic suppression of Tc, remains unclear. To investigate this issue, one must consider the two fundamental energy scales associated with superconductivity. On the one hand, the gap energy Δ measures the pairing strength between electrons that form Cooper pairs. On the other hand, the superfluid stiffness Js determines the cost of a phase twist in the superconducting condensate. In conventional superconductors, well described by Bardeen–Cooper–Schrieffer (BCS) theory, Js is much higher than Δ and the superconducting transition is controlled by the breaking of Cooper pairs. However, when the stiffness is strongly reduced, phase fluctuations play a major role and the suppression of Tc can be dominated by the loss of phase coherence8. Tunneling experiments in the low doping regime of LaAlO3/SrTiO3 interfaces evidenced the presence of a pseudogap in the density of states above Tc9. This can be interpreted as the signature of pairing surviving above Tc while superconducting coherence is destroyed by strong phase fluctuations, enhanced by a low superfluid stiffness10. Superconductor-to-insulator quantum phase transitions driven by gate voltage1 or magnetic field11 also highlighted the predominant role of phase fluctuations in the suppression of Tc.The superconducting phase diagram of LaAlO3/SrTiO3 band structure. Under strong quantum confinement, the degeneracy of the t2g bands of SrTiO3  is lifted, generating a rich and complex band structure13. The emergence of superconductivity for a given carrier density suggests that it could be intrinsically related to orbital occupancy in the interfacial quantum well. Experiments performed on (110)-oriented LaAlO3/SrTiO3 interfaces, for which the ordering of the t2g bands is reversed from that of the conventional (001) orientation, revealed that superconductivity behaves differently14. Instead of following the usual dome shape and disappearing at low doping, Tc is only weakly affected by gating over a wide range of carrier density. This shows the important role of orbitals ordering and also suggests that only some specific bands could host superconductivity15. In particular, it has been emphasized that the dxz/dyz band lying at high energy in the quantum well could play an important role because of its large density of states7.The two-dimensional (2D) superfluid density derived from the stiffness 3/SrTiO3 interfaces. This allows us to directly extract the evolution of the superfluid stiffness in the phase diagram that we also convert into a gap energy through BCS theory in the dirty limit. Both energy scales are compared with theoretical predictions. The superfluid density ns deduced from Js is found to be close to the carrier density of the dxz/dyz band extracted from multiband Hall effect measurements, highlighting the key role of this band in the emergence of superconductivity.Here we use resonant microwave transport to measure the complex conductivity of the superconducting (001)-oriented LaAlOJs is usually assessed either from penetration depth measurements17 or from dynamic transport measurements19. This latter method was adapted in this work for the specific case of LaAlO3/SrTiO3 samples. While superconductors have an infinite dc conductivity, they exhibit a finite complex conductivity σ(ω) at non-zero frequency, which in 2D translates into a sheet conductance G(ω) = G1(ω) − iG2(ω). The real part G1(ω) accounts for the transport of unpaired electrons existing at T ≠ 0 and ω ≠ 0, and the imaginary part G2(ω) accounts for the transport of Cooper pairs21. In the low-frequency limit G2(ω) = Lk is the kinetic inductance of the superconductor due to the inertia of Cooper pairs22. The superfluid stiffness is directly related to the inductive response of the condensate through the relation In superconducting thin films, 3 epitaxial layers were grown on 3 × 3 mm2 TiO2-terminated (001) SrTiO3 single crystals by pulsed laser deposition (see Methods section). After the growth, a weakly conducting metallic back-gate of resistance ~100 kΩ is deposited on the backside of the 200-μm- thick substrate. Figure 24. The LaAlO3/SrTiO3 heterostructure is inserted in a microwave circuit board, between the central strip of a coplanar waveguide guide (CPW) transmission line and its ground. It is embedded into an RLC resonant circuit whose inductor L1 and resistor R1 are surface mounted microwave devices (SMDs), and whose capacitor CSTO is due to the substrate in parallel with the 2D electron gas (2-DEG)  between the two ports is measured with a vector network analyzer. Standard microwave network analysis relates the reflection coefficient of the sample circuit Γ(ω) to S21(ω) through complex error coefficients, which are determined by a calibration procedure 25Ain and Aout are the complex amplitudes of incident and reflected waves, and Z0 = 50 Ω is the characteristic impedance of the CPW transmission line. A reflection measurement gives therefore a direct access to the load impedance ZL(ω) or equivalently its admittance GL(ω) = 1/ZL(ω), commonly called complex conductance. In the present case, ZL(ω) is the impedance of the RLC circuit represented in Fig. ω0 in the superconducting state is directly related to the kinetic inductance of the 2-DEG. Measuring ω0 as a function of gate voltage provides therefore a very direct method to determine the superfluid stiffness in the phase diagram. In addition, the setup of Fig. L1 and R1, allows measuring both the dc and ac microwave transport properties of the 2-DEG at the same time.In this study, 8-uc-thick LaAlOinated 00 SrTiO3 sEG) Fig. . Becauseinated 00 SrTiO3 sinated 00 SrTiO3 sinated 00 SrTiO3 s26. In the limit τ is the elastic scattering time) and for temperatures higher than Tc, the 2-DEG behaves as a metal whose Drude conductance is simply the inverse of the dc resistance  along with a 2π phase shift  ). The superconducting transition observed in dc resistance for positive gate voltages, VG, coincides with a shift of ω0 towards high frequency  coefficient , the resonance frequency remains unchanged as CSTO has no temperature dependence in the range of interest . On the same logarithmic scale, we also show the gate dependence of the superconducting Tc defined as the temperature where Rdc = 0 Ω. The accuracy in the determination of the superfluid stiffness is limited by the uncertainty on the exact value of the circuit inductance L1 and the contribution of the sample geometrical inductance. The total error, corresponding to the gray outline on Fig. In the following, we determine the gate dependence of the important energy scales in superconducting LaAlOentclass1pt{minimaτ is much shorter than the superconducting gap Δ(T = 0) (−3). Within this limit and for 28:Rn = Rdc(450 mK) is the normal state resistance  in the overdoped (OD) regime defined by VG > kBTc in Eq.  regime, corresponding to VG < Tc and JBCS evolve smoothly before vanishing only when approaching closely the quantum critical point where Tc ≃ 0 K (VG = 4 V). This indicates that the loss of phase coherence of the superconducting condensate is stronger than what expected taking into account conventional scattering by defects, as encoded in Eq. , in the UD regime, the charge carrier depletion makes the array more dilute. In this case, the system can maintain a rather high Tc (Rdc = 0 Ω) as long as the dc current can follow a percolating path through islands. However, the macroscopic stiffness Tc.entclass1pt{minimac in Eq. . In thisentclass1pt{minimaVG , the Fermi energy should enter into the dxz/dyz bands. Multiband transport in LaAlO3/SrTiO3 and LaTiO3/SrTiO3 interfaces has been observed experimentally in various magneto-transport experiments including quantum oscillations40, magneto-conductance41, and Hall effect44. Yang et al. 39 recently showed that, in addition to a majority of low-mobility carriers (LMCs), a small amount of high-mobility carriers (HMCs) is also present, with an effective mass close to the mxz/yz one. Despite a band mass substantially higher than the mxy one, these carriers acquire a high mobility since dxz/yz orbitals extend deeper in SrTiO3 where they recover bulk-like properties, including reduced scattering, higher dielectric constant and better screening. In Hall effect measurements, the Hall voltage is linear in magnetic field B in the low doping regime corresponding to one-band transport, but this is not the case at high doping because of the contribution of a new type of carriers (the HMC)3. We performed a two-band analysis of the Hall effect data combined with gate capacitance measurements to determine the contribution of the two populations of carriers to the total density ntot 3. The first clear signature of multiband transport is seen when the Hall carrier density nHall, measured in the limit B → 0, drops with VG instead of following the charging curve of the gate capacitance  which corresponds to the upper limit of the doping range commonly observed in LaAlO3/SrTiO3 interfaces. The fact that nHM. To clarify this situation, it is needed to go beyond single-band superconductor models that cannot account correctly for the unusual t2g-based interfacial band structure of LaAlO3/SrTiO3 interfaces. Further investigations of recent experimental45 and theoretical46 developments on superconductors having two dissimilar bands  should provide the starting framework to address this question.Bert et al.entclass1pt{minimaJs of LaAlO3/SrTiO3 interfaces by implementing a resonant microwave transport experiment. Whereas a good agreement with the BCS theory is observed at high carrier doping, we find that the suppression of Tc at low doping is controlled by the loss of macroscopic phase coherence instead of electron pairing strength as in standard BCS theory. The corresponding superfluid density represents only a small fraction of the total electrons density. We emphasize here that the monotonic raise of Tc in the OD region of the phase diagram cannot be attributed to a loss of superfluid density. The gate dependence of dxz/dyz bands taking place at higher energy in the quantum well. In addition to having a larger density of states, these dxz/dyz bands also extends much deeper in the substrate due to their out-of-plane mass. Away from the interface, the dielectric constant, which is most probably a fundamental ingredient for electron pairing47, is less affected by the interfacial electric field and therefore closer to its nominal value. These delocalized electrons therefore recover properties similar to the ones found in bulk SrTiO3, including BCS superconductivity48. Our finding is consistent with the observation of a gate-independent superconductivity in (110)-oriented LaAlO3/SrTiO3 interfaces for which the dxz/dyz bands have a lower energy than the dxy subbands and are therefore always filled14.In summary, we have measured the superfluid stiffness 3 epitaxial layers grown on 3 × 3 mm2 TiO2-terminated (001) SrTiO3 single crystals by pulsed laser deposition. The substrates were treated with buffered hydrofluoric acid to expose TiO2-terminated surface. Before deposition, the substrate was heated to 830 °C for 1 h in an oxygen pressure of 7.4 × 10−2 mbar. The thin film was deposited at 800 °C in an oxygen partial pressure of 1 × 10−4 mbar. The LaAlO3 target was ablated with a KrF excimer laser at a rate of 1 Hz with an energy density of 0.56–0.65 J cm−2. The film growth mode and thickness were monitored using reflection high-energy electron diffraction  during deposition. After the growth, a weakly conducting metallic back-gate of resistance ~100 kΩ (to avoid microwave shortcut of the 2-DEG) is deposited on the backside of the 200-μm-thick SrTiO3 substrate.In this study, we used 8-uc-thick LaAlOJs can be extracted directly from the raw measurements of S21 in most of the regions of the phase diagram /(ZL + Z0) of the sample circuit. This procedure also suppresses parasitic signals mainly due to wave interferences in the microwave setup, and improves the precision on the measurement as illustrated in Supplementary Fig. S21(ω) and the reflection coefficient of the sample circuit Γ(ω) is given byα′ = αβ, γ, and δ are three error complex coefficients. They can be determined using three known values of Γ = (ZL − Z0)/(ZL + Z0) which are obtained by imposing three different impedances ZL. It is customary to use an open, a short, and a matched load as standard impedances to calibrate microwave setup. However, such method is neither adapted to our very low temperature experiment nor to our sample circuit geometry. Instead, our setup was calibrated by directly varying the impedance ZL of the sample circuit with gate value. The main advantage of this method is that it fully takes into account the local microwave environment of the sample. The gate controls both the normal resistance of the 2-DEG whose value can be measured in dc and CSTO which can be extracted from ω0. In practice, we choose a set of three gate values which correspond to well-separated resonance frequencies. Other sets allow the accuracy of the calibration to be checked. Supplementary Fig. S21 and the corresponding calibrated Γ coefficient both in the normal state and the superconducting state for VG = 50 V.In this experiment, the resonance frequency shift and correspondingly 3 substrate can be retrieved from the value of CSTO plotted in Fig. -μm-thick 3 × 3 SrTiO3 substrate covered by two 100-μm-wide Au/Ti strips as represented in Supplementary Fig. CSTO − Cpara) measured experimentally is shown in Supplementary Fig. G = 0 V, 27.The dielectric constant of the SrTiOntot with VG is obtained by integrating the gate capacitance C(VG), measured by standard lock-in technique, over the gate voltage rangeA is the area of the sample and ntot(VG = −36 V) is matched to nHall since in this low doping regime the Hall effect is linear in magnetic field (single-band transport). In the multiband transport regime corresponding to VG > 0, the Hall resistance has been fitted with a two-band modeln1 and n2 are the 2D electron densities and, μ1 and μ2 the corresponding mobilities. Fits are performed with the two constraints: ntot = n1 + n2 and 1/Rdc = e(n1μ1 + n2μ2). The two populations of electrons are then identified as the LMC and the HMC.The dependence of the total carrier density All data that support the findings of this study are available from the corresponding authors upon request.Supplementary InformationPeer Review File


Roseburia but decreasing Faecalibacterium levels in the cecum relative to controls. Immunoglobulin A, interleukin 1β, and tumor necrosis factor α levels were also elevated in the intestine of these animals, corresponding to noise-induced abnormalities in glucose regulation and insulin sensitivity. These results suggest that lifelong environmental noise exposure could have cumulative effects on diabetes onset and development resulting from alterations in gut microbiota composition and intestinal inflammation.Chronic noise exposure has been implicated in increased risk of diabetes. However, there is limited experimental evidence of the mechanisms linking chronic noise stress and glucose metabolism. We addressed this in the present study by examining glucose metabolism, immune response, and changes in gut microbiota/host inflammatory homeostasis in rats exposed to noise for 30 consecutive days. Chronic noise exposure increased blood glucose and corticosterone levels for at least 14 days after cessation of noise. Stressed rats also exhibited elevated levels of glycogen and triglyceride in the liver and impaired hepatic insulin production via insulin-induced insulin receptor/insulin receptor substrate 1/glycogen synthase kinase 3β signalling, which persisted for 3–14 days after cessation of noise exposure. Chronic noise altered the percentage of Proteobacteria and Actinobacteria in the gut, increasing  The human population is increasingly being exposed to environmental noise from many sources including traffic, media, and household appliances. Long-term noise exposure is considered as a health hazard25Noise acts as a stressor that hyperactivates the sympathetic autonomic nervous system and activates the hypothalamic–pituitary–adrenal axis, which increases cortisol levels110111518We speculated that commensal microbiota play a role in noise stress-induced metabolic disorders. To test this hypothesis, we investigated the effects of chronic noise exposure on glucose regulation, microbiota composition, and inflammation in rats. Given the persistent effects of chronic noise on glucose homeostasisWe examined changes in body weight and measured blood glucose and CORT levels by enzyme-linked immunosorbent assay (ELISA) in rats exposed to noise for various periods. Noise exposure had no effect on body weight ; howeverThe effect of chronic noise exposure on insulin sensitivity was assessed by measuring blood plasma insulin, glycogen, and triglyceride (TG) levels by ELISA as well as liver insulin receptor (IR), IR substrate (IRS), and AKT and glycogen synthase kinase (GSK)-3β levels by western blotting. Exposure to noise increased insulin and TG concentrations, with corresponding reductions in liver glycogen that persisted for 0–3 days after noise cessation . IR and A total of six faecal samples (three each from control and noise-exposed rats on day 0 after exposure) were collected for 16S rRNA gene sequencing. Each sample has 1276 OTUs and an average of 41 847 sequences. Stress can alter the composition of gut microbiotaTo clarify the inflammatory events leading to metabolic dysfunction induced by chronic noise exposure, we measured immunoglobulin (Ig)A, interleukin (IL)-1β, and tumour necrosis factor (TNF)-α levels in the ileum by ELISA. Noise exposure induced increases in IgA, IL-1β, and TNF-α levels that persisted for at least 3 to 7 days after noise cessation . These dPrevious studies have reported that chronic noise exposure increases the risk of developing metabolic disorders such as diabetes and obesity568Exposure to road traffic noise is associated with a higher risk of incident diabetes141522Nighttime exposure to noise is associated with sleep disturbance and consequently, increased adiposity and risk of diabetes242526272830Psychological stress can interfere with carbohydrate metabolism and lead to insulin resistance33Escherichia, Salmonella, Helicobacter, and Yersinia genera. Noise exposure enriched Roseburia spp., which may be associated with weight loss and reduced glucose intoleranceFaecalibacterium spp., which are associated with Crohn’s disease, obesity, and asthmaHowever, a previous study strongly suggested that the persistent dysregulation of insulin sensitivity cannot be explained solely by abnormalities in blood glucocorticoid level and that other mechanisms are involved in noise-induced insulin resistanceInflammation is related to stress-induced metabolic dysfunction, and inflammatory mediators such as cytokines and chemokines may contribute to insulin resistance at the tissue level3945The above-mentioned previous study described the relationship between environmental noise exposure and risk of diabetes development in a laboratory settingOur results indicate that chronic noise exposure induces persistent abnormalities in glucose regulation accompanied by alterations in gut microbiota and host immune responses. Although the study examined a relatively small number of subjects, these findings nonetheless enhance our understanding of the etiological association between chronic noise exposure and metabolic dysregulation and suggest that exposure to environmental noise over a lifetime may have a cumulative effects on the development of metabolic syndromes.In total, 64 male Wistar rats  weighing 200–220 g used in this study and maintained in a room on a 12:12-h light/dark cycle (lights on from 06:00 to 18:00 h) with controlled ambient temperature (23 °C ± 2 °C) and humidity (50–70%) and free access to water and food. Animals were allowed to adapt to the laboratory environment for 5 days prior to the start of the experiment.The rats were randomly assigned to 0, 3, 7, and 14 day groups according to when the end-point evaluation was performed , and each group was further subdivided into noise-exposure and control groups; the former was exposed to 100 dB sound pressure level (SPL) white noise (4 h/day for 30 days from 8:00 to 12:00 h), whereas control rats were housed in similar cages but exposed to background noise (<40 dB SPL) from another chamber. At different time points  after the last noise exposure, rats were sacrificed by decapitation, and blood, liver, intestine, cecum, and caecal content samples were immediately collected for biochemical analyses and stored at −80 °C until use . At leasWhite noise was generated using a noise generator , amplified with a power amplifier , and delivered to a loudspeaker . Animals were exposed to noise in a reverberation chamber in wire-mesh cages placed at the centre of the sound field. A loudspeaker was suspended directly above the cages. Noise levels were measured with a 3560 C-size front end and 4938 1/4′′ pressure-field microphones (Brüel & Kjær Instruments) placed at the same level as the animals’ ears using linear weights. Variation in noise level was <2 dB within the space available to the animal. The main spectrum of the noise emitted from the speaker was in the range of 400–6300 Hz (measured using 1/3 octave bands).Blood was collected from the tail vein of each animal after an overnight (12 h) fast at each time point to determine blood glucose level using a Gold-Accu blood glucose test meter .Blood plasma CORT level, liver glycogen and TG levels, and TNF-α, IL-1β, and IgA levels in intestine homogenates were measured with ELISA kits  according to manufacturer’s instructions. Frozen rat liver and intestine samples were homogenised in ice-cold 1× phosphate-buffered saline  and total protein concentration was determined with the bicinchoninic acid assay . Glycogen and TG concentrations in the liver and inflammatory factor levels in the intestine were normalised to total liver or intestine protein concentrations, respectively, and the mean value of duplicate samples was taken as the final concentration for each animal.Western blotting was carried out as previously describedhttp://qiime.org/). Reads were first filtered by QIIME quality filters. Pick_de_novo_otus.py was used to select operational taxonomic units (OTUs) with a table. Sequences with ≥97% similarity were assigned to the same OTU. A representative sequence was selected for each OTU and the Ribosomal Database Project classifier was used to annotate taxonomic information for each representative sequenceTotal genomic DNA was extracted from samples using the cetyltrimethylammonium bromide/sodium dodecyl sulphate method. DNA concentration and purity were verified by 1% agarose gel electrophoresis. DNA was diluted to 1 ng/μl with sterile water and ~10 ng were used as a template to amplify the 16S rRNA gene in 30-μl reactions with 15 μl Phusion High-Fidelity PCR Master Mix  and 0.2 μM forward and reverse primers. Thermal cycling consisted of 98 °C for 1 min; 30 cycles of 98 °C for 10 s, 50 °C for 30 s, and 72 °C for 30 s; and 72 °C for 5 min. Equal volumes of 1× loading buffer (containing SYBR Green) and PCR product were mixed and resolved by 2% agarose gel electrophoresis. Samples between 400–450 bp were selected for further experiments. PCR products were mixed in equidensity ratios and purified using a GeneJET Gel Extraction kit . Sequencing libraries were generated using the TruSeq DNA PCR-Free Library Preparation kit  according to the manufacturer’s protocol, and index codes were added. The library quality was assessed with a Qubit@ 2.0 fluorometer (Thermo Scientific) and the Agilent Bioanalyser 2100 system. The library was sequenced on an Illumina HiSeq platform and 250-bp paired-end reads were generated. Sequences were analysed using Quantitative Insights Into Microbial Ecology (QIIME) software (10) for statistical analysis. Data obtained for the 16S rRNA gene in microbiota was assessed using the Wilcoxon rank-sum test. The Student t test was used to evaluate mean differences in all other data. P < 0.05 was considered statistically significant.Data presented in graphs indicate group mean ± standard deviation and were analysed using SPSS v.19.0 software . Normality of raw data and residuals was tested with the Shapiro-Wilk test. Because of skewed distributions, the values of CORT content, blood insulin, and the liver TG level were logarithmically transformed .Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.