Assessing the N2O emission from lucerne grasslands with nitrogen addition will aid in estimating the annual N2O emissions of such agriculture areas, particularly following summer rainfall events in light of precipitation variation associated with global change. Here, we measured soil N2O emissions, soil temperature and water content of lucerne grasslands with four levels of nitrogen addition over 25 days, which included 10 rainfall events. Results showed that nitrogen addition was observed to increase soil NO3−-N content, but not significantly improve dry matter yield, height or leaf area index. Nitrogen addition and rainfall significantly affected N2O emissions, while the response of N2O emissions to increasing nitrogen input was not linear. Relative soil gas diffusivity (Dp/Do) and water-filled pore space (WFPS) were good indicators of N2O diurnal dynamics, and Dp/Do was able to explain slightly more of the variation in N2O emissions than WFPS. Collectively, nitrogen addition did not affect lucerne dry matter yield in a short term, while it induced soil N2O emissions when rainfall events alter soil water content, and Dp/Do could be a better proxy for predicting N2O emissions in rainfed lucerne grasslands.Nitrous oxide (N Altho Plateau ,6. Howevnitrogen ,9,10,11.2O emissions in agricultural soils are produced from nitrification by ammonia-oxidizing bacteria (AOB) and archaea (AOA) under aerobic conditions, which convert ammonia, via nitrite (NO2−), to nitrate (NO3−); denitrification under anaerobic conditions sequentially reduces NO3− to dinitrogen (N2), and N2O is a by-product [2O emissions in semi-arid regions such as the Loess Plateau are often greater following summer rainfall events, especially with nitrogen fertiliser application [2O emissions with changes in soil water content [p/Do), defined as the ratio of the soil gas diffusion coefficient to the free air gas diffusion coefficient, can be an excellent factor to describe soil gas transport between the soil and the atmosphere [p/Do be a key predictor of N2O emissions even under varied soil matrix potential and bulk density levels [N-product ,2,3,4,5.-product ,13,14,15lication ,13,14,15 content ,17,18. H content ,19, whic content ,21. Relamosphere ,22. Dp/Dy levels ,26,27,282O emissions in lucerne grasslands under nitrogen addition during periods of high rainfall variability will aid us in estimating annual N2O emissions and increase our knowledge about N2O emissions in dryland forage production systems. Thus, the aims of the study were as follows: (1) to measure soil N2O emissions from lucerne grasslands with four levels of nitrogen addition over 25 days, which included 10 periods of rainfall; (2) to relate N2O emissions to changes in soil water content by WFPS and Dp/Do. We hypothesized that besides WFPS, Dp/Do is also a strong indicator of N2O emissions in rainfed lucerne grassland when rainfall events alter soil water content.Evaluating the effect of soil water content on NThis study was undertaken at the Loess Plateau Research Station of Lanzhou University , Lanzhou, China. The area is characterized by a cool continental climate, with mean annual precipitation of 548 mm and rainfall that is most abundant from July through September. The average annual temperature ranges from 8 °C to 10 °C. The mean temperatures in the warmest (July) and coldest (January) months are 21.3 °C and −5.3 °C, respectively. Soil temperature (0–100 mm) ranged from −1.6 °C (March) to 30.6 °C (August), and soil water content (0–100 mm) ranged from 7.8% (January) to 14.8% (August). The soil is classified as silty loam soil according to the FAO/UNESCO soil classification, and the soil characteristics, based on samples collected at the start of the measurement periods, are described in −1, and are referred to herein as N0, N50, N100 and N150. The fertiliser used was based on urea with 40% nitrogen content. Fertiliser applications occurred twice, on April 5 (recovery stage) and June 5 (after the first cutting) with 80% and 20% of the allotted fertiliser, respectively. This study was conducted from the 1st to 25th of August 2014 using lucerne crops, corresponding to when rainfall would be abundant, and the days of the experiment are thus referred to as Day 1 to Day 25.The experiment was conducted according to a randomized block design; each plot was 3 m wide × 3 m in length with three replicates. A 0.5-m-wide buffer zone was included between each plot. One of four nitrogen addition levels was applied to each plot, 0, 50, 100 and 150 kg N haMedicago sativa L. cv ‘Longdong’) were planted by drilling in 2009, with 300 mm row spacing and a sowing rate of 15 kg ha−1. The previous crop on the lucerne grasslands was maize (Zea mays). The Lucerne grasslands were rainfed, and 302 kg ha−1 of KH2PO4 was applied each year during the regreening stage. Lucerne was harvested twice at its flowering stage each year (June and July) as is typically done by growers in the region [−2.Lucerne grasslands  connected to three replicates in closed dynamic soil respiration chambers  placed in each plot. The slope of the change in N2O concentration of the chamber headspace over the measuring time was calculated to obtain the N2O flux  using Equation (1):Measurement of NA is the surface area (m2) of the chamber, V is the volume (L) of the chamber, M0 is the molecular weight of the N2O; t1 and t2 are the times in the beginning and ending of gas measurement, m1 and m2 are the masses of the N2O (mg) in the chamber at t1 and t2, respectively; C1 and C2 are the volume concentrations of N2O in the chamber at t1 and t2, respectively; and T1 and T2 are the air temperatures in the chamber at t1 and t2, respectively. The N2O flux was simultaneously computed with an in-built microcomputer in the N2O/CO near-infrared gas analyser.Here, 2O/CO near-infrared gas analyser is 1 ppb. Minimum detectable N2O fluxes for the N2O/CO analyser were determined by using the linear regression method described by Parkin et al. (2012) [2O m−2 h−1. Detection limits were used to calibrate flux data, and fluxes below the detection limit were assigned a value of zero, in total, 5% of N2O fluxes were assigned as 0.The measurement accuracy of the N. (2012) . The min2O/CO analyser for N2O flux recording. The daily dynamics of N2O emissions (over 24 h) were monitored overall, and each plot was measured 24 times during each 24-h cycle. At the same time, chamber temperature was measured with sensors installed inside of the chamber lid.Each chamber was fitted with a small fan mounted inside the lid to maintain well-mixed conditions. Three replicate chambers were used for each plot, randomly distributed in each plot and pressed 50 mm into the soil. Flux data were logged and calculated by a Soil Flux System . The Soil Flux System was connected to a DLT-100 Nv) readings were measured by probes close to each chamber, installed at a 50 mm depth. Soil samples, at depths of 0–100, 100–200 and 200–300 mm, were taken at five sites near each chamber with a 50-mm-diameter gouge auger. Soil samples were placed in airtight plastic bags, transported to the laboratory and stored temporarily at 4 °C in the dark. The fresh soil was analysed for nitrate nitrogen (NO3−-N). Soil organic carbon (SOC) and total nitrogen (TN) were analysed using air-dried soil.During each gas sampling period, hourly soil temperature and soil volumetric water content . NO3−-N was extracted with 1 M KCl and measured with a colorimetric continuous flow analyzer .SOC was determined using the potassium dichromate/sulfuric acid mixture titration method, and TN was measured by the Kjeldahl method (Kjeltec−3) of the soil at a 0–100 mm depth using a ring  was computed as the ratio of the mass of dry soil (g) to the volume of the sample (m3) [v [p/Do [Bulk density (Mg mple (m3) . The ave (m3) [v . Water-i[v [p/Do .−1) was measured after oven drying for 48 h at 65 °C.Samples were collected along two 0.5 m transects, which were made at each plot before (16 July) and at the end of the experiment (23 August) when stubble height was 50 mm. The dry matter yield  was determined by an AccuPAR/LAI canopy analyzer  with four replicates per treatment.3−-N, soil water content and temperature data among the nitrogen fertiliser addition treatments for each period. Comparisons of soil N2O emissions were made using the least significant difference test (LSD) at 5%. After confirming the normality of the data, interactions between nitrogen addition and rainfall to N2O emissions were analysed using two-way ANOVA. A general exponential model was used to determine soil water content effects on N2O emissions. All statistical analyses were performed using Genstat .Analysis of variance (ANOVA) was utilized to determine significant differences in soil NOv (c) of four treatments at a depth of 0–100 mm during the 25 days. There were no significant differences in mean chamber temperature among the treatments. Mean chamber temperature under N0, N50, N100 and N150 decreased significantly, by about 38%, 40%, 39% and 39% (p < 0.05), respectively, from Day 1 to 8 after three rainfall events, all exceeding 6.0 mm, respectively. The biggest decrease in chamber temperature occurred when the precipitation reached 32.6 mm and the temperature declined about 23% from Day 5 to 6 (p < 0.05).p < 0.05).Differences among mean soil temperatures of the four treatments at a depth of 0–100 mm were lower than those among chamber temperatures, and the trends in soil temperatures were similar to those of chamber temperatures a,b. The v of the four treatments at a depth of 0–100 mm was from 8.22% to 8.54% between Day 1 and 5. The largest θv value occurred after a 32.6 mm rainfall event, and the content increased by about 78% from Day 5 to 6 (p < 0.05). The range of mean θv values for each treatment was from 11.22% to 14.77% between Day 6 and 25.The range of θ3−-N contents were lower than 8.34 mg kg−1 before the first fertiliser application ; The 0–100 mm soil NO3−-N content under the N50, N100 and N150 treatments was 141%, 151% and 194% higher, respectively than that under N0 (p < 0.05). The 200–300 mm soil NO3−-N content under the N50, N100 and N150 treatments were significantly higher than that under N0 (p < 0.05). Soil NO3−-N contents in the 0–100 mm soil layer for the N0, N50, N100 and N150 treatments were 7.26, 11.91, 13.18 and 15.93 mg kg−1, respectively, at the end of the experiment , 0.0089 ± 0.0032, 0.0088 ± 0.0033 and 0.0206 ± 0.0089 mg m−2 h−1, respectively . Daily N2O emissions were low under the N0, N50 and N100 treatments, but emissions under each treatment increased sharply on Day 6 following a 32.6 mm rainfall event, with emissions of 0.0187, 0.0127, 0.0123 and 0.0409 mg m−2 h−1, respectively. Emissions under the N0 treatment increased continually from Day 3 to 8, which included four periods of precipitation, ranging from 0.0066 to 0.0626 mg m−2 h−1. Emissions increased sharply from 0.0102 to 0.034 mg m−2 h−1 between Day 15 and 17 following a 32.6 mm rainfall event under the N150 treatment and increased again on Day 21 and 22, ranging from 0.0261 to 0.0374 mg m−2 h−1. While emissions increased slightly from 0.0051 to 0.0087, 0.0030 to 0.0058, 0.0028 to 0.0067 and 0.0081 to 0.0097 mg m−2 h−1 between Day 4 and Day 5 following a 6.3 mm rainfall event under N0, N50, N100 and N150, respectively. Emissions decreased under the N50, N100 and N150 treatments on Day 7, following a 32.6 mm rainfall event.Mean Nectively . Nitroge2O emissions were significantly affected by the nitrogen addition amount (p < 0.001) and rainfall .N2O emissions after precipitation was different under each treatment, especially on Day 4 when the first rain occurred, after a more than a 30-day dry period, and on Day 6, when the highest rainfall occurred. We chose Day 2 as a representative dry period. The daily variability of N2O emissions was highest for N150 on Day 2, with a minimum of 0.0001 mg m−2 h−1 and a maximum of 0.0394 mg m−2 h−1 , who suggested that modest nitrogen addition would have little impact on N2O emissions. Additionally, legume–Rhizobium symbioses could produce N2O as a result of rhizobia denitrification [2O emission via denitrification.Although Naddition , the rel obvious . Topsoile of N2O , did notdentical , and at ined too c. More Nntration b,c. Normfication , but rhification  and, thu2O emissions by influencing metabolic activities of nitrifiers or denitrifiers and substrate availability [2O dynamics, such as those observed on Day 6  that affect N2O emission, and further study is warranted.Soil temperature affects Nlability . Differeon Day 6 c. Howevey stable b. Soil waeration . Change 6, Day 8  and Day missions . Thus, lll event b. N2O emp/Do, mean N2O flux increased significantly  flux and Dp/Do was stronger than that between Dp/Do and N2O flux. Balaine (25) found that Dp/Do reflects both N2O and N2 emissions as well. Because our study did not measure N2 flux, Dp/Do may have appeared superior to WFPS in explaining N2O emissions of lucerne grassland, which also demonstrates the importance of measuring total nitrogen (N2O + N2) flux. Nitrification might be the largest source of N2O emissions in semi-arid regions, where soils are rarely under anaerobic conditions that promote denitrification [v values in the current study was 8.22–14.77%  observed that the relationship between total nitrogen , which was also consistent with Dp/Do being just slightly better than WFPS in explaining the variation in N2O emissions (Owens (39) found that values of D to 0.15 b, and lomissions c,d.2O emissions occurred unexpectedly in the study  suggested that negative N2O fluxes would not be a common phenomenon. Particularly, the study was performed over a time period when N2O uptake occurred unexpectedly.Negative Nhe study . Negativactivity ,34. Bart2O emissions, while the N2O emission response to increasing nitrogen input was not linear. Soil Dp/Do and WFPS were good indicators of N2O diurnal dynamics, and Dp/Do was able to explain slightly more of the variation in N2O emissions than WFPS, even when θv was less than 20 m−3. Thus, more research should be focused on Dp/Do, which could be a better proxy for N2O emissions, even when both rainfall and the addition of nitrogen strongly shape variability in N2O emissions.Nitrogen addition and rainfall significantly affected N


TL1A (TNFSF15) is a TNF superfamily ligand which can bind the TNFRSF member death receptor 3 (DR3) on T cells and the soluble decoy receptor DcR3. Engagement of DR3 on CD4+ or CD8+ effector T cells by TL1A induces downstream signaling, leading to proliferation and an increase in secretion of inflammatory cytokines. We designed a stable recombinant TL1A molecule that (1) displays high monodispersity and stability, (2) displays the ability to activate T cells in vitro and in vivo, and (3) lacks binding to DcR3 while retaining functional activity via DR3. Together these results suggest the TL1A ligand can be amenable to therapeutic development on its own or paired with a tumor-targeting moiety. The best characterized immune checkpoint modulatory axis is that of programmed death 1: ligand (PD-1: PD-L1)2. Indeed, antibodies that block the PD-1: PD-L1 interaction have shown marked clinical success in subsets of patients highlighting both the promise of immune checkpoint blockade therapy and the need for therapeutic molecules which can modulate other immune checkpoint axes3.Cytotoxic (CD8+) T cells are part of a potent adaptive immune system to eliminate tumor cells by recognition of tumor peptides presented on major histocompatibility (MHC) class I molecules on tumor cells via their T cell receptors (TCRs). However, tumor infiltrating lymphocytes (TILs) can fail to suppress tumor growth due to a variety of immunosuppressive mechanisms exploited by tumor cells. Three hallmarks of T cell exhaustion are upregulation of immune checkpoint receptors, lower inflammatory cytokine secretion, and altered gene expression profiles4. First, the T cell receptor (TCR) complex recognizes its cognate peptide MHC complex. Second, a host of co-signaling receptors determine whether the T cell becomes activated or suppressed. The co-stimulatory receptor CD28 co-localizes with the TCR to the central super-molecular activating center (cSMAC) while other immunomodulatory receptors may localize to the peripheral or distal super-molecular activating centers (pSMAC or dSMAC) to guide signaling for T cell activation or differentiation into regulatory T cells (Tregs)5.T cell activation occurs via a two-signal mechanism6. Additionally, TL1A can also bind to the soluble decoy receptor DcR3 (TNFRSF6B) at higher affinity than to DR3 thereby preventing TL1A from stimulating T cells8. DcR3 is structurally homologous to DR3 but shares only ~ 20% sequence identity with DR3. DcR3 can therefore serve as a sink to prevent TL1A from activating T cells. DcR3 is up-regulated in a variety of tumors and is associated with metastasis10. DcR3 displays promiscuity in its abilities to bind to three TNFSF ligands: TL1A, Fas ligand (FasL), and LIGHT , a receptor expressed on T cells). These interactions may sequester TL1A from engaging DR3 thereby down-regulating cell immune response to tumors11.One co-stimulatory receptor, TNF-like factor 1A (TL1A) protein belongs to the TNF family and is expressed on a variety of cell types as a type II single-pass transmembrane protein which can be cleaved from the cell surface. Soluble or membrane-bound TL1A binds to the co-stimulatory death receptor 3 (DR3) on T cells or NK cells12. Interaction between TL1A and DR3 contributes to T cell activation, leading to increased inflammatory cytokine production and T cell proliferation. Thus, the potential of employing such interaction to overcome T cell exhaustion in solid tumors makes DR3 an attractive immune checkpoint target. Nonetheless, significant challenges to therapeutic targeting of the TL1A:DR3 axis remain. The 3:3 stoichiometry of trimeric TL1A and DR3 binding is finely tuned suggesting that a ligand-based approach may be more amenable to T cell activation compared to bivalent antibody-based approaches. However, the high levels of DcR3 require either higher dosing regimens or engineering to eliminate this interaction as a sink in tumor micro-environment.TL1A blocking antibodies have been used in the clinic to treat ulcerative colitis and other inflammatory conditions13. Additionally, the crystal structure of TL1A bound to DcR3 showed the molecular basis for these interactions14. The close structural homology but low sequence identity of DR3 and DcR3 suggest that selective mutations could influence specific binding to DR3 over DcR3.Here, we sought to engineer a recombinant TL1A ligand that would be suitable for therapeutic development for T cell activation. The desired engineered TL1A therapeutic would satisfy 3 criteria: (1) specific binding to DR3 but not DcR3; (2) display a long serum half-life; and (3) demonstrate structural stability. TL1A ligand has been engineered to modulate affinity for DcR315. We determined the minimal sufficient fragment of TL1A suitable to form a stable trimer capable of binding DR3. A structure and model-guided strategy was used to design and engineer TL1A mutations, leading to a molecule that retained DR3 binding with affinity similar to wild-type TL1A but displayed no measurable binding to DcR3. This Fc-TL1A fusion displayed T cell co-stimulation in in vitro and in vivo models.We tested several molecular architectures of TL1A, including single-chain (sc) variants, in which three TL1A monomers were fused into a single polypeptide. TL1A ligands were tested as fusion proteins fused to either human serum albumin (HSA) or to an Fc domain, both of which have been demonstrated to increase the serum half-life of biologics1, we used a scTNFα molecule as a comparison for protein monodispersity. After expression and affinity capture, proteins were purified by preparative gel filtration, and the relative population of target species of each molecule was quantified . When comparing the scTL1A constructs; His-scTL1A, Fc-scTL1A, and HSA-scTL1A; each was > 70% target species with Fc- and HSA-scTL1A displaying ~ 80% target oligomer. We further assessed the molecular weights of each species of the Fc-scTL1A by size-exclusion chromatography multi-angle light scattering (SEC-MALS)  would display an increase binding avidity compared to molecules having a single TL1A trimer   and favorable monodispersity.We next determined whether the TL1A constructs could co-stimulate T cells, leading to cytokine production using a T cell activation assay. In the absence of aCD3 antibody, the TL1A molecules could not stimulate T cells, as expected due to the lack of TCR stimulation Fig. E,F. In t16. C57BL/6 mice were injected with equal molarity of Fc-TL1A, Fc-scTL1A or Fc-His-scTL1A intraperitoneally, and anti-CD3 Ab intravenously 13, and our ELISA and SPR-based binding analysis confirmed that the C162S, C202S TL1A variant could bind only DcR3 but not DR3  which showed modestly decreased DR3 binding, the other 3 combination mutants maintained similar binding to DR3 compared to wild-type TL1A  are prevented from acting against tumor cells15. We tested several architectures of TL1A ligand, including single-chain versions of the trimeric ligand, in which each domain was linked by a Gly-Ser linker, as well as both human serum albumin- and Fc-fusions. A version of TL1A in which the ligand was formatted as a single-chain and was fused onto the C-terminus of the Fc showed the highest monodispersity upon purification. Surprisingly, molecules in which TL1A was fused onto the N-terminus of the Fc failed to show binding to receptors, although the mechanism preventing binding was unknown. TL1A contains two cysteines  which form an intra-molecular disulfide bond and stabilize a loop which is critical for binding DR3  compared to antibodies (~ 3 weeks). Fusion to either an Fc or serum albumin, both of which display long serum half-lives due to their abilities to undergo FcRn-mediated recycling have been successful methods to extend the half-life of protein therapeuticseines C16, C202 wh13. Our studies suggested that mutations in TL1A which disrupt multiple interactions with DcR3 were needed to achieve full abrogation of binding  have been generated as bispecific antibodies which can bind both co-stimulatory receptors and to a tumor antigen. One study showed that an anti-EGFR × LIGHT-Fc BsAb could target tumors and activate LTβR signaling to recruit TILS and showed anti-tumor efficacy20. Results from this work suggest that targeting co-stimulatory activity to a tumor has therapeutic promise. DcR3 is upregulated in a number of solid tumor types, particularly colon, pancreatic, and stomach cancers21, suggesting that DcR3 in these tumors may be a mechanism of T cell inhibition and that T cells within these tumors could be stimulated by a TL1A ligand which can overcome the DcR3-mediated sink. Bispecific antibodies which can deliver the Fc-scTL1A-AKALFF specifically to these tumors may offer similar anti-tumor activity with limited risk of systemic T cell activation. The Fc-scTL1A-AKALFF variant developed in this work was designed to function as a module which can be joined with a tumor-targeting moiety to specifically activate T cells in the tumor micro-environment, and its favorable monodispersity, stability, and activity suggest it will mediate anti-tumor activity.A particular challenge for TNFSF-targeting therapeutics is tumor targeting. Systemic T cell activation is associated with the potential for rapid and severe toxicity associated with cytokine release via off-tumor T cell activation and the potential for reactivation-induced T cell death, wherein tumor-infiltrating T cells (TILS) undergo apoptosis in response to over-activation by bispecific T cell engagers  and expressed according to the manufacturer’s protocol. Proteins were purified by either Ni–NTA affinity chromatography (His-TL1A and HSA-TL1A) or by mAbSelect SuRe  (Fc-TL1A) according to the manufacturer’s protocol. Preparative gel-filtration was performed using a Sepax SRT-C SEC-300 column . Gel-filtration was performed in buffer consisting of 20 mM sodium phosphate, pH 6.8.SEC separations were performed on a Waters Acquity UPLC system consisting of an H-class quaternary pump, FTN autosampler, heated column compartment and TUV detector. Samples were maintained at 4 °C in an autosampler until analysis. Aliquots (5 µg) of the samples were analyzed on a Waters BEH SEC column  maintained at 25 °C. The mobile phase was PBS, pH 7.4 at a flow rate of 300 µL/min, and UV absorbance was monitored at 280 nm. The entire flow from the TUV detector was directed to a Wyatt µDAWN detector equipped with additional DLS channel at a scattering angle of 135° and a serially connected Wyatt Optilab T-rEX refractive index detector. The LC was operated under control of MassLynx V4.2 SCN1007 software, while Wyatt components were managed with Astra V7.3.2. UV data was acquired by Astra via auxiliary input of 0.2 AU/V signal. All data was processed with Astra V7.3.2.U-PLEX TH1 TH2 cytokine (cat # K15010B-2) plates were coated with 25 ml of cell culture supernatant overnight at 4 °C. The plates were washed 3X with PBS with 0.05% Tween 20 (PBST). Anti-human Fc detection reagent at 2 mg/ml final concentration was applied for 1 h at RT with shaking. The plates were washed 3X with PBST. 150 ml of 2X read buffer was added to the plates, and data was collected on an MSD instrument. Raw data was processed in MSD Discovery Workbench and imported into GraphPad Prism 8 software. Data were analyzed and plotted based on the results of three independent experiments, and statistics were generated using GraphPad Prism 8.All ELISA-based measurements were collected in triplicate, and error values report the standard error among measurements. Recombinant DR3-Fc  was non-specifically immobilized onto 96-well White Maxisorp plates  through dilution to 10 mg/mL . Plates were blocked for 1 h at room temperature with Casein buffer, 250 ml/well. TL1A was added at the indicated concentrations at 50 mL/well diluted in StartingBlock PBS, at 25 mg/mL*, diluting 3 × over each well on a 96-well White Maxisorp plate  and incubated overnight at 4 °C. Note that TL1A variant concentrations were normalized to moles of TL1A trimer/molecule. For example, 1 molecule of His-TL1A contains 1 TL1A trimer, and thus 25 mg/mL of TL1W2 contains 376 nM TL1A trimers. Conversely, TL1W14 contains 2 TL1A trimers per molecule, and has a different molecular weight. Thus, 25 mg/mL TL1W14 = 290 nM. Therefore, 376 nM is equivalent to 32.4 mg/mL. Plates were then washed 3 × with TBST. Bound TL1A was detected by addition of 100 µl/well polyclonal rabbit anti-human TL1A-biotin antibody, diluted 1:1,000 in Starting Block PBS and incubated for 1 h at room temperature with shaking ~ 150 rpm. Anti-TL1A was detected by addition of 100 ml/well streptavidin-HRP conjugate, diluted 1:10,000 in Starting Block PBS and incubated for 1 h at room temperature with shaking ~ 150 rpm. Plates were washed 3 × with TBST. Detection was carried out by addition of 100 µl/well POD Chemiluminescence substrate  immediately prior to reading plates. Luminescence was detected using a Wallac Envision plate reader.96-well U-bottom tissue culture plates  were coated with 0.1 or 0.01 mg/mL of anti-CD3  in PBS overnight at 4 °C. Plates were then washed three times with complete RPMI media (RPMI + 10% FBS). Pan T cells were isolated from peripheral blood mononuclear cells (PBMCs) using negative selection . 30,000 pan T cells were plated in each well of anti-CD3 pre-coated 96-well U-bottom plates. Engineered TL1A ligands and/or 0.1 mg/mL of anti-CD28  were added to appropriate wells. To assess DcR3-mediated inhibition on T cell activation, recombinant DcR3 protein (R&D cat# 142-DC-100) was added to a final concentration of 30 nM to anti-CD3 stimulated T cells with or without TL1A ligands.TL1A constructs were evaluated for the ability to co-stimulate T cells to produce cytokine in an in vivo mouse model. Suboptimal dose of anti-mouse CD3 antibody (2 mg/mouse) 2 fragment; cat. # BE0001-1FAB) was injected intravenously into C57BL/6 mice. Fc-TL1A W3 or Fc-scTL1A W14 (30 µg per mouse) was injected intraperitoneally. Fc-TL1A W3 (100 µg per mouse) was injected intraperitoneally into mice in the absence of anti-mouse CD3 stimulation. Mice were bled 6 h later. Sera were collected and frozen. Frozen sera were thawed and diluted 1:10 for mouse IFNg ELISA . Briefly, Nunc MaxiSorp 96-well plates were pre-coated with capture antibody overnight at 4 °C. Plates were washed and then blocked with ELISA diluent for 1 h at room temperature. Plates were washed with Wash Buffer. Diluted samples were loaded into plates and incubated for 2 h at room temperature. Plates were washed with Wash Buffer 3 to 5 times. Diluted detection antibody was added to each well, incubated for 1 h at room temperature, and washed. Diluted Streptavidin-HRP detection reagent was added, incubated for 30 min at room temperature, and washed. TMB substrate solution was added and incubated at room temperature for 15 min. Stop solution was added to each well. Plates were read in a SpectraMax ELISA plate reader.14.Models of the TL1A-DR3 complex were generated using the protein modeling component of MOE . The sequence of DR3 (Uniprot ID O95407) and the structure of TL1A bound to DcR3 (PDB ID 3K51) were used as inputs22, used to calculate the distance distribution function P(r), and DAMMIF23, used in automated bead modelling for shape determination. To generate the models of Fc-scTL1A, 2-fold symmetry constraints were imposed, which produces more reliable envelopes by reducing noise24. For each sample, 5 independent shape models calculated with DAMMIF were averaged using DAMAVER to produce the final ab initio envelopes. Surface rendering was done with Chimera25.Scattering data collection was carried out on an in-house x-ray source at 20 °C. Fc-scTL1A samples were analyzed in 20 mM HEPES HCl pH 6.5, 100 mM NaCl. To limit radiation damage, the samples were continuously oscillated inside the cuvette during the exposures. Independent measurements were collected from each sample at different concentrations , checking the linear dependence of the scattering intensity at zero angle as a function of concentration to ensure lack of aggregation . Kinetic analysis was done by grouping the kon, koff and RUmax, and holding the RI constant and equal to zero. The Chi2 value was used to assess the quality of the fit. Data were evaluated only qualitatively.Goat anti-human-Fc was immobilized at 30 mg/ml, Acetate, pH 5.0 on L1-L6. DR3, DcR3 were immobilized at 5 or 1 mg/mL. The desired absolute ligand levels for either indirect capture format or immobilization formats are the range that can produce final analyte binding signals between 50~200 RUs. BSA was used as a non-binding analyte control. Experiments were performed at 25 °C, with PBST  Tween) as the running buffer. A kThe datasets used and/or analysed during the current study available from the corresponding author on reasonable request.Supplementary Information.


The dual-luciferase reporter system results indicated that FEZ1, SCOC, ULK1 and NBR1 were all miR-129-5p target genes. Furthermore, immunoprecipitation assay revealed that SCOC, ULK1 and NBR1 could directly bind to the FEZ1 protein. The experiments at an animal level demonstrated that miR-129-5p could effectively alleviate the behavioral indicators of PSD model mice. Taken together, this study testified that SCOC/ULK1/NBR1 proteins could directly bind to FEZ1 to form protein complex, and all of the four proteins FEZ1/SCOC/ULK1/NBR1 were miR-129-5p target genes. miR-129-5p overexpression could effectively restore the behavioral characteristics of model mice, and reduce the autophagy-related proteins FEZ1/SCOC/ULK1/NBR1.Post-stroke depression (PSD) seriously affects the normal life of patients. Based on the previous sequencing results, this study selected miR-129-5p as the research object, which was significantly reduced in the PSD model by screening. To clarify the regulatory role of miR-129-5p, this study overexpressed and interfered with miR-129-5p in neuronal cells cultured in vitro, tested its effect on neuronal cell autophagy, and determined expressions of fasciculation and elongation protein zeta-1 (FEZ1), short coiled-coil protein (SCOC), unc-51 like autophagy activating kinase 1 (ULK1) and autophagy cargo receptor (NBR1) autophagy-related proteins. The dual-luciferase reporter system and immunoprecipitation were applied to detect the molecular regulatory mechanism of miR-129-5 and FEZ1, SCOC, ULK1 and NBR1. Findings of the present study revealed that the autophagy of neuronal cells was markedly decreased by overexpressing miR-129-5p (  SCOC/ULK1/NBR1 proteins could bind to FEZ1 to form protein complex.FEZ1/SCOC/ULK1/NBR1 were miR-129-5p target genes.Overexpression of miR-129-5p could reduce the autophagy-related proteins FEZ1/SCOC/ULK1/NBR1.1Stroke is currently considered to be mostly responsible for mortality and severe long-term disability worldwide. It has emerged as a serious public health problem . Post-stCurrent PSD research has found that acute inflammation occurs after a stroke, leading to the release of glucocorticoid . These pThe pathophysiological mechanism of PSD is unclear. Although the etiology of PSD is thought to be multifactorial both psychosocially and biologically. Multiple studies have shown that PSD may be caused by post-stroke cognitive impairment, physical disability or psychological stress caused by social isolation. More evidence has shown that multiple biological factors, such as the location of stroke, genetic susceptibility and cortex–striatum–globus pallidus–thalamic cortex projection or destruction of neurotransmitter projection pathways, and reduced neurotrophic factors or inflammation, all are associated with PSD occurrence . As the Clinically, focal brain injury is found to be likely to induce delayed cognitive ,24and deSome researchers have reported that miR-129-5p mediates LINC00574 and reduces the expression of UDP glucuronyl transferase (UGT2B15) in HepaRG cells through LINC00574 . MeanwhiFrom these evidences, the role of FEZ1 in mental illness and emotional cognitive impairment cannot be ignored. Despite its importance, the basic mechanism, especially its internal relationship and dynamic expression changes in post-stroke depression, is still not fully elucidated. To solve this problem, we established a post-stroke depression model by injecting endothelin (ET)-1 into the left medial prefrontal cortex of mice. We then performed behavioral assessments on these mice. Next, the FEZ1 protein of the hippocampus of this model and the changes of the three autophagy proteins that interacted with FEZ1, SCOC, ULK1, and NBR1 were evaluated by Western blot and qPCR analysis. And after regulating the changes of miR-129-5p in in vitro cell experiments, the changes of FEZ1, SCOC, ULK1, and NBR1 proteins were observed. Meanwhile, the miR-129-5p gene expression was regulated in the PSD model group, and the behavioral changes of the mice were visualized.We hypothesized that the overexpression of miR-129-5p could restore the behavioral characteristics of model mice via reduce the autophagy-related proteins FEZ1/SCOC/ULK1/NBR1. To prove this hypothesis, HT22 cells were used as materials in vitro and behavioral testing of model mice was performed. Our results demonstrated that miR-129-5p had an important effect on neuronal cell autophagy, which its overexpression could decrease the expression of the autophagy-related proteins FEZ1/SCOC/ULK1/NBR1.22.15/well. Lentifusion Max and transfection diluent were used to transfect the lentivirus into the cells. After 48 h, stable transfected strains were selected with 5 μg/mL adenine [Neuronal cells (HT22) were obtained from American Type Culture Collection (ATCC), and cultured in 10% FBS and 1% penicillin–streptomycin in a complete cultural medium. miR-129-5p overexpression lentivirus and interference lentivirus were packaged by Chongqing Biomedicine Biotechnology Co., Ltd. 24 h prior to transfecting lentivirus, HT22 cells were inoculated into a 24-well plate at 1 × 10 adenine . This re2.2Trizol method was adopted for total cell RNA extraction, and T5 Fast qPCR kit  was used for qPCR test. The reaction system was: 2 × T5 Fast qPCR Mix, 10.0 μL; 10 μM Primer F, 0.8 μL; 10 μM Primer R, 0.8 μL; 50 × ROX Reference Dye II, 0.4 μL; template DNA, 0.5 μL; and ddH2O, 7.5 μL. The reaction conditions were set at 95°C, 30 s; 95°C, 5 s; 55°C, 30 s; 72°C, 30 s, 40 cycles of reaction. The primer sequences are shown in 2.35 cells/well. The cell confluence rate of the cells on the second day was 50%~70%. The next day, the original medium was refreshed using 250 μL medium with the corresponding virus, and incubated for 2 h in an incubator. After 2 h, the medium with virus was changed using 500 μL of fresh medium. 48 h after infection, they were treated with amino acid deprivation for 2 h, followed by two cycles of washing with PBS and 4% paraformaldehyde fixation for 20 min, and photographed after loading for analysis [mRFP-GFP-LC3, autophagy double-labeled adenovirus, was bought from Hanbio Biotechnology Co. Ltd. 24 h before the lentivirus transfection, and the neuronal cells were subcultured in a 24-well plate pre-plated with slides at 1 × 10analysis .2.4The protein was lysed using RIPA lysis buffer, and BCA method was adopted to determine the protein concentration. The precast gel was removed from the refrigerator at 4°C and put into the electrophoresis tank. Subsequently, 500 μg of total protein was added to each sample and mixed by the additional of 5× SDS loading buffer at 4:1 ratio. After mixing, the protein concentration was set at 3.3 μg/μL. The metal bath was heated at 100°C for 6 min to denature the protein. The denatured total protein was taken 60 μg for loading. After running at 80 V through the concentrated glue, the voltage was adjusted to 120 V, and waited until the bromophenol blue reached the bottom of the rubber sheet without spilt out. After transferring the membrane, 5% skimmed milk blocking solution was supplied for blocking at room temperature for 1 h. Primary antibody was added for incubation overnight at 4°C, washed 3 times with TBST, 10 min each time, added secondary antibody and incubated at room temperature for 1 h, washed with TBST 3 times, 10 min each time, and exposed using ECL exposure liquid. The antibodies used were: FEZ1 , 1:500; SCOC , 1:500; ULK1 , 1:500; NBR1 , 1:500; GAPDH , 1:2000; and secondary antibody , 1:1000.2.5Following three cycles of washing with PBS, 5 min each time, 0.5% TritonX-100 was added dropwise to the slides to punch holes at room temperature for 10 min. PBS was supplemented to immerse the 2.6The vectors of dual-luciferase reporter system were constructed by Chongqing Biomedicine Biotechnology Co., Ltd. The plasmids were co-transfected into the cells, added with 50 μL culture medium free from antibiotics and serum, added 1 μg (mixed in each group) plasmid DNA, and mixed gently using a pipette; then added 1.6 μL Nanofusion transfection reagent, mixed gently using a pipette, let stand 5–20 min at room temperature, and added the cells in a 12-well plate. Distilled water was added to dilute 5*PLB (Passive Lysis Buffer) to 1*PLB, added 500 μL per well of a 12-well plate, break up the cells using a pipette, placed on a shaker at room temperature and vibrated slowly for 15 min. The lysate was aspirated into a 1.5 mL centrifuge tube for centrifugation at 4°C 12,000 rpm for 10 min, and the supernatant was transferred to a new tube. By adding 100 μL of Luciferase Assay Reagent II (LAR II)  working solution to the 96-well plate, 20 μL of cell lysate was supplied, mixed 2–3 times using a pipette, measured and recorded the value of Firefly luciferase, which was the luminescence value of the reporter gene. Ultimately, 100 μL Stop & Glo® Reagent  was added, mixed 2–3 times using a pipette, measured and recorded the Renilla luciferase value, which was the internal reference value.2.7Pre-chilled RIPA buffer was added to the cultured cells to lyse proteins, and maintained on ice for 30 min. After being scraped from the culture dish using a pre-cooled scraper, the cell suspension was transferred to a 1.5 EP tube and centrifuged at 12,000 rpm for 10 min at 4°C. The supernatant was immediately transferred to a new centrifuge tube. Protein G/A agarose was prepared initially. Beads were washed with PBS twice. PBS was used to prepare a medium of 50% concentration. Of each 1 mL of total protein, 100 μL Protein G/A Sepharose beads (50%) was added vibrated for 10 min at 4°C to remove nonspecific contaminants and reduce background, centrifuged at 12,000 rpm 4°C for 15 min, and transferred the supernatant to a new centrifuge tube. The protein concentration was detected using BCA methods. Of 5 μL FEZ1 IP antibody was added to 500 μL of total protein. A mixture of antigen–antibody was vibrated slowly overnight at 4°C and its complex was captured following the addition of 50 μL Protein G/A agarose beads, and vibrated mildly at 4°C for 3 h, and centrifuged briefly at 2000 rpm for 5 s. The agarose beads-antigen-antibody complex was collected, followed by the removal of the supernatant, washed 3 times with pre-cooled RIPA buffer, and completely aspirated the supernatant. The complex was added with 60 μL 1× loading buffer following mild vibration . The loa2.8The animal keeping and PSD model establishment were conducted as our previous study . The micElevated plus maze test: the experimental animals were placed into the central area of the maze, with their heads toward the open arm. Each experimental animal should be placed at the same site thereafter. At the same time, the camera monitor was turned on to record the movement of the experimental animals within 5 min, and the corresponding data were recorded. During the experiment, the experimenter should be at a distance of 1 m away from the maze, and keep the experimental environment quiet free from external interference.Open field test: the experimental animals were placed into the central area of the open field and started to record the movement of the experimental animal within 5 min, and noted the corresponding data. During the experiment, the experimenter should be at a distance of 1 meter from the open field, and keep the experimental environment quiet without external interference.Tail suspension test: the tail of the mouse was fixed at the 1/3 part with tape and hang on a bracket. Its head was kept 15 cm away from the table, and a camera was used for photograph. The background of the camera was in obvious contrast with the fur color of the mouse. The C57 mouse used a white background and the timing was terminated after 6 min.2.9The paraffin sections of the tissues at 5-μm thickness were baked overnight at 37°C, deparaffinized and incubated using 3% hydrogen peroxide for 15 min. The sections were microwaved to heat the tissue antigens, and then cooled at room temperature for 30 min. The tissue was incubated with mouse FEZ1 (1:50), SCOC (1:50), ULK1 (1:50), and NBR1 (1:100) antibodies, and all of which were obtained from Abclonal company in China. Finally, the tissue was incubated with a biotin-labeled secondary antibody  to observe protein expression.2.10P less than 0.05 were considered the difference was statistically significant.All data were expressed as mean ± standard deviation. One-way analysis of variance was adopted and least significant difference test was performed for analysis. All statistical analyses were conducted via SPSS  software. The values of 3The expression of miR-129-5p was significantly reduced in the PSD model by screening. We hypothesized that the overexpression of miR-129-5p could restore the behavioral characteristics of model mice via reducing the autophagy-related proteins FEZ1/SCOC/ULK1/NBR1. To clarify the regulatory mechanism, transfection of neuronal cells with miR-129-5p overexpression lentivirus and interference lentivirus were performed in this study. It was proved that the FEZ1/SCOC/ULK1/NBR1 were related to PSD, which changes were regulated by miR-129-5p-mediated pathway.3.1p < 0.05)  , indicat3.2p < 0.05) (p < 0.05) (p < 0.05) (p < 0.05)  . After i < 0.05) . Further < 0.05) . Whereas < 0.05) . Similar < 0.05) . The pre3.3In our previous studies, we predicted that FEZ1, SCOC, ULK1 and NBR1 might be miR-129-5p target genes, and PPI predicted FEZ1 and SCOC, ULK1 and NBR1 interacted with each other. Therefore, in this study, we used dual-luciferase reporter system detection and immunoprecipitation test to verify their mutual regulation relationship. The results are shown in 3.4The elevated plus maze test revealed that the times of open arm entry of mice in normal group, model group, model + control virus group and model + overexpressing miR-129-5p virus group were 3.33 ± 2.16, 1.00 ± 1.26, 1.83 ± 1.17 and 2.67 ± 1.03 ; the timThe results of open field tests indicated that the times of entry to center in normal group, model group, model + control virus group and model + overexpressing miR-129-5p virus group were 6.66 ± 1.86, 3.67 ± 3.20, 5.67 ± 4.22 and 9.33 ± 6.09 ; the traThe tail suspension test revealed that inactive incubation period of mice in normal group, model group, model + control virus group and model + overexpressing miR-129-5p virus group were 0.35 ± 0.54 s, 0.167 ± 0.41 s, 0.33 ± 0.82 s and 1.83 ± 4.49 s, respectively ; the inaThough the results of the elevated plus maze, open field, and tail suspension test did not present significant changes in the detection indicators, clearly all indicators had a recovery trend. It showed that miR-129-5p could effectively alleviate the behavioral indicators of post-stroke depression model mice.3.5p < 0.05) (p < 0.05)  . Meanwhi < 0.05) . Finally < 0.05) .Figure 4Post-stroke depression seriously affects the normal life of patients, and research on its pathogenesis and treatment methods is urgently needed. Based on the previous sequencing results, the present study screened miR-129-5p, which was greatly reduced in the PSD model, as the research object, and clarified its regulatory function. Consequently, overexpression of miR-129-5p could effectively restore the behavioral characteristics of model mice, and reduce the autophagy-related protein FEZ1/SCOC/ULK1/NBR1. This study found that SCOC/ULK1/NBR1 protein could directly bind to FEZ1 to form a protein complex, and all the four proteins of FEZ1/SCOC/ULK1/NBR1 were miR-129-5p target genes. miR-129-5p targeting FEZ1/SCOC/ULK1/NBR1 protein complex might provide new ideas for the research and treatment of PSD.FEZ1 has been identified as an interaction partner of the disrupted-in schizophrenia 1 (DISC1). The latter also links to the cerebral cortex development. The growth cones of cultured hippocampal neurons contain FEZ1 and DISC1, and both can interact with F-actin. Furthermore, the synergistic effect between FEZ1 and DISC1 promotes the differentiation of PC12 cell neurons . TherefoAutophagy is intimately correlated with multiple individual disorders in the nervous system as it is of vital importance for the digestion and removal of residual organelles, injured proteins and cytoplasm ,42. AutoSCOC represents an effector of GTPase Arl1 of the Golgi apparatus. It has been lately recognized to play a role in positively regulating autophagy in genome-wide siRNA screening . The proNBR1 is known to be a widely expressed protein with high conservation in eukaryotes. NBR1 acts as a binding partner of autophagy-related protein 8 (ATG8) family proteins in mammalian cells. p62 and NBR1 can function in selective autophagy via binding of ubiquitin and autophagosome protein ,52. MeanRecent studies have bred FEZ1-deficient mice, which behaved abnormally, including excessive exercise phenotypes and increased ability to respond to psycho stimulants. Relevant abnormal mental behaviors are the result of changes in the dopamine release in the cerebral limbic pathways . As the Taken together, this research provides a foundation for the relationship between PSD and autophagy proteins. In recent years, increasing studies on the relationship between autophagy and depression have been carried out, but the research on the relationship between PSD and autophagy needs further and in-depth investigation to elucidate the pathway of PSD miR-129-5p gene. It can provide targets for clarifying the pathogenesis of PSD and offer reference for the treatment of PSD clinically.55.1In our previous study, we clarified that the differential expressions of microRNAs and mRNAs in a PSD model, found that a total of 21 microRNAs expression levels were substantially upregulated, and 32 microRNAs expression levels were markedly downregulated. Thus, we drew a conclusion: miR-129-5p and autophagy-related genes FEZ1, NBR1, ULK1, and SCOC could be used as targets for further research. Thus, we performed this study.5.2In this study, we established PSD model in C57BL/6 J mice, and used the dual luciferase reporter system and immunoprecipitation to detect the molecular regulatory mechanism of miR-129-5 and FEZ1, SCOC, ULK1 and NBR1. We found that SCOC/ULK1/NBR1 proteins could directly bind to FEZ1 to form protein complex, and all of the four proteins FEZ1/SCOC/ULK1/NBR1 were miR-129-5p target genes. These findings mean overexpression of miR-129-5p could effectively restore the behavioral characteristics of model mice and reduce the autophagy-related proteins FEZ1/SCOC/ULK1/NBR1. It might provide new ideas for the research and treatment of PSD.5.3This research provides a foundation for the relationship between PSD and autophagy proteins. It can provide targets for clarifying the pathogenesis of PSD and offer reference for the treatment of PSD clinically.5.4Recently more and more studies on the relationship between autophagy and depression have been carried out. Though our study provides the relations among PSD, miR-129-5p and autophagy-related protein, it is still unknown the pathway of PSD miR-129-5p gene, further and in-depth investigation needs to be elucidated. In the future, research of the mechanism of miR-129-5p gene mediated-pathway and studies of the autophagy as a potential pharmacological target for curing PSD should be carried on. There is still a lot of research to be done. Overall, this study revealed that SCOC/ULK1/NBR1 protein could directly bind to FEZ1 to form a protein complex, and the four proteins of FEZ1/SCOC/ULK1/NBR1 were all miR-129-5p target genes. Overexpression of miR-129-5p could effectively restore the behavioral characteristics of model mice, and simultaneously reduce the autophagy-related proteins FEZ1/SCOC/ULK1/NBR1.


Background: Night-migratory birds sense the Earth’s magnetic field by an unknown molecular mechanism. Theoretical and experimental evidence support the hypothesis that the light-induced formation of a radical-pair in European robin cryptochrome 4a (ErCry4a) is the primary signaling step in the retina of the bird. In the present work, we investigated a possible route of cryptochrome signaling involving the α-subunit of the cone-secific heterotrimeric G protein from European robin. Methods: Protein–protein interaction studies include surface plasmon resonance, pulldown affinity binding and Förster resonance energy transfer. Results: Surface plasmon resonance studies showed direct interaction, revealing high to moderate affinity for binding of non-myristoylated and myristoylated G protein to ErCry4a, respectively. Pulldown affinity experiments confirmed this complex formation in solution. We validated these in vitro data by monitoring the interaction between ErCry4a and G protein in a transiently transfected neuroretinal cell line using Förster resonance energy transfer. Conclusions: Our results suggest that ErCry4a and the G protein also interact in living cells and might constitute the first biochemical signaling step in radical-pair-based magnetoreception.  Sensing the Earth´s magnetic field is a widespread ability in the animal kingdom. Behavioral experiments and analyses of data from wild animals have shown that night-migratory songbirds, fish, amphibians, reptiles and insects orientate and navigate using magnetic sensing ,2,3,4,5.Cry proteins are flavoproteins harboring a flavin adenine dinucleotide (FAD) in a binding pocket situated in a conserved photolyase homology region. The FAD chromophore enables Cry to absorb blue light, which triggers the formation of a radical pair involving a series of three or four neighboring tryptophan (Trp) residues ,13. ErCry4a) can be purified with a bound FAD chromophore, exhibits a photo-induced electron transfer pathway leading to the formation of a [FAD●− TrpH●+] radical pair, and it is magnetically sensitive in vitro [ErCry4a than in chicken and pigeon Cry4 [ErCry4 expresses in the outer segments of double cones and long-wavelength single cones but not in other retinal cells [ErCry4 and iodopsin (long wavelength opsin) in long-wavelength single cones and in double cones [Thus far, six different Crys  have been identified to be expressed in the retina of different bird species, but only Cry1a and Cry4a are currently discussed as the primary magnetic field receptors. Cry1a is localized in UV-sensitive cones in the retina of European robins  and a liin vitro ,18,19. Feon Cry4 . These aal cells . In the le cones .ErCry4a by a yeast two-hybrid screening approach [GNAT2 coding for the α-subunit of a cone-specific heterotrimeric G protein, GNG10 coding for the γ-subunit of a cone-specific heterotrimeric G protein, LWS, also called iodopsin, coding for long-wavelength-sensitive opsin, KCNV2 coding for potassium voltage-gated channel subfamily V member 2, RBP1 coding for retinol-binding protein 1 and RGR coding for retinal G protein-coupled receptor. The identified α- and γ-subunit of the G protein are the cone-specific orthologues of the heterotrimeric G protein transducin (Gt) that mediates phototransduction in mammalian and other vertebrate rod and cone photoreceptor cells. The versatile role of heterotrimeric G proteins in signal transduction pathways makes them primary candidates to test for direct interaction with Cry4 variants. Could the G protein be the long-sought-after first interaction partner of Cry4a in a radical-pair-based magnetoreception signaling pathway?A further important step for validating that Cry4 variants indeed operate as magnetoreceptive molecules is to link them to signal transduction processes causing magnetic-field-sensitive changes in the membrane potential. Wu et al. (2020) identified six putative protein interaction partners of approach . All sixErCry4a directly interacts with the G protein α-subunit of European robin (ErGtα) on the molecular level. We expressed recombinant variants of both proteins, purified them and verified their functional state by independent functional assays. Using a biosensor approach employing surface plasmon resonance (SPR), we investigated the protein–protein interaction process and analyzed the kinetic parameter of the binding process, which was further corroborated by in vitro pulldown affinity assays. Using acceptor photobleaching Förster resonance energy transfer (FRET), we further validated that the interaction between ErCry4a and ErGtα also occurs in living cells in a neuroretinal bird cell line.The aim of the present study is to test whether ErGnat2  to create the pCold ErGnat2 construct. All primers for cloning steps are listed in the ErGnat2 in the pCold vector was performed, and the corresponding region of bovine inhibitory alpha subunit was amplified from the commercial pCS6 vector containing bGNA1i. The products were then assembled using the Gibson assembly to create the completed pCold ErGnat2 chimera. To create the pET21a ErGnat2 chimera vector, the chimera sequence was amplified from pCold and assembled using the Gibson assembly with NdeI and XhoI digested pET21a. To create the pACE SUMO ErGnat2 chimera vector, the chimera sequence was amplified and combined with the pACE SUMO vector using the Sequence and Ligation Independent Cloning (SLIC) technique. To remove 6 non-native amino acids from the coding sequence, PCR mutagenesis was performed. To create the chimera, a deletion mutagenesis of amino acids 220–298 of tα showed that expression in E. coli resulted in misfolded protein [ErGtα. However, it is common practice to construct, express and purify chimeric mammalian Gtα proteins for functional studies [tα/Giα is based on the coding sequence for the European robin gene ErGNAT2 [ErGtα and the Bovine taurus Giα1 . Primer pairs utilized and more details about the cloning steps can be found in Earlier work on mammalian G protein , and we  studies . The chi ErGNAT2  encodingrus Giα1 . The seqrus Giα1 . The iniE. coli cells (prepared in-house). Subsequently, the cells were cultured at 37 °C and 180 rpm in LB medium containing 10 g/L NaCl and 100 µg/L ampicillin . Upon reaching an OD600 of 0.6, the shaking was reduced to 160 rpm and the cells were cooled to 17 °C for 30 min. Next, the cells were induced by adding IPTG (Roth) to 10 µM, incubated for 20–24 h at 17 °C, and then harvested and stored at −20 °C. The plasmid was transformed into chemically competent BL21 2, 10 mM Imidazole, 10 mM ß-mercaptoethanol, Roche cOmplete™ EDTA-free Protease Inhibitor Cocktail) and lysed by sonication using a Bandelin GM 2200 ultrasonic generator with a UW 2200 converter and MS72 microtip at 45% power. Cell lysates were then centrifuged at 18,000 rpm  in a JA25.5 rotor at 4 °C for 60 min, and the supernatants were applied to equilibrated gravity flow columns containing 2 mL of Ni-NTA resin each. Each column was washed with 80 matrix volumes (160 mL) wash buffer , and then eluted twice using 4 mL of elution buffer . Cells from 3.5 L culture were resuspended in 50 mL Ni-NTA binding buffer  after adding Ulp1 protease (in-house) against at least 4 L of dialysis buffer  overnight. The digest was then applied to the same equilibrated gravity flow columns containing Ni-NTA matrix to remove digested tag sequences. For this, the columns were washed 3 times with 5 mL wash buffer and eluted with 5 mL elution buffer. The flow-through and wash fractions containing the Gtα/Giα chimera were pooled and concentrated using a Macrosep Advance 10k MWCO centrifugal filter to a concentration of up to 15 mg/mL. Concentrated fractions were applied to house-packed size exclusion chromatography column  pre-equilibrated with size exclusion chromatography buffer . The flow rate was maintained at 1 mL/min, and fractions containing the G-protein were identified with SDS Page, pooled, and concentrated and, then, flash frozen in liquid N2 in 50 or 100 µL aliquots and stored at −80 °C. Both elution fractions were dialyzed together (SERVAPORE. coli BL21 (Codon+) cells, harboring the pET21a Gtα/Giα chimera plasmid and pBB131 encoding yeast myristoyltransferase, were grown in 5 × 500 mL yeast tryptone (YT) media at 37 °C and 180 rpm in the presence of 100 µg/mL ampicillin (Roth) and 30 µg/mL kanamycin . At OD600 of 0.4 myristic acid  was added to a final concentration of 50 µg/mL, and the incubation was continued until an OD600 of 0.5–0.6 was reached. Then, the cultures were cooled down to 17 °C at 160 rpm for 30 min. IPTG (Roth) was added to a final concentration of 150 µM, and incubation was continued for 20–24 h at 17 °C and 160 rpm. Afterward, the cell pellets were harvested by centrifugation at 7000 rpm for 7 min at 4 °C. Each cell pellet was resuspended in 10 mL in Ni-NTA binding buffer  and a protease inhibitor cocktail (Roche cOmplete™ EDTA-free Protease Inhibitor Cocktail) was added . The cells were lysed by ultrasonication using a Bandelin GM 2200 ultrasonic generator with a UW 2200 converter and MS72 microtip at 45% power and centrifuged at 100,000× g for 30 min at 4 °C. The supernatant was used for further purification. An empty 20 mL column was packed with 2.5 mL Ni2+-nitrilotriacetic acid-agarose resin (HisPurTM Ni-NTA) and equilibrated with 2 × column volumes of Ni-NTA binding buffer. The supernatant was loaded on the Ni-NTA column by gravity flow and washed with 50 × matrix volume of Ni-NTA washing buffer . The protein was eluted with 2 × matrix volume of Ni-NTA elution buffer . Next, an anion exchange chromatography (AEC) was performed. The elution fraction from the NTA-affinity chromatography was diluted 1:3 in AEC buffer A (20 mM Tris-HCl pH 7.0 and 1 mM DTT). A HiTrapTM 5 mL Q Sepharose High Performance  column was equilibrated with 5% AEC buffer B  and 95% AEC buffer A. The protein sample was loaded on the column and washed with 5% AEC buffer B and a flow rate of 1 mL/min until a stable UV signal was reached. The protein was eluted with a salt gradient from 5 to 50% AEC buffer B over 112.5 mL at a flow rate of 1 mL/min. Fractions containing the Gtα/Giα chimera were pooled, and to remove all remaining contamination, a size exclusion chromatography (SEC) was performed. A Superdex 200 16/600 column with a volume of 120 mL was equilibrated using SPR buffer , and the pooled AEC fractions were concentrated using a Macrosep Advance 10k MWCO centrifugal filter. Using a flow rate of 0.5 mL/min, the sample was applied to the column, and the SEC run started. Fractionation was started after 0.4 CV using 3 mL fraction sizes. Desired fractions were pooled, concentrated, and flash frozen in liquid N2 for storage at −80 °C. Myristoylation was analyzed and verified by reversed phase analytical HPLC as described previously for myristoylated neuronal calcium sensor proteins [ErCry4-497 was produced by mutagenesis using forward and reverse primers as listed in the ErCry4a pCold plasmid as described [ErCRY4-497 was expressed and purified as ErCry4a, except that the expression time was extended from 22 to 44 h. Basic steps of cloning, expression and purification of ErCy4a have been described previously . The folescribed . ErCRY4-ErCry1a and ErCry1b, as well as the chimeric G-protein α-subunit Gtα/Giα were cloned into the pFastBacHT B vector  using BamHI and XhoI restriction sites and primers listed in ErCRY4 in the pFast vector was a gift from Joseph S. Takahashi .g and resuspended in 30 mL homogenization buffer  per 10 g cell pellet. Cells were lysed with a Potter-Elvehjem-homogenizer and clarified by centrifugation at 48,384× g. Clarified cell lysates were applied to Ni-NTA agarose columns , pre-equilibrated with homogenization buffer. Bound proteins were eluted with elution buffer . The proteins were diluted 1:10 in 20 mM Tris and further purified on anion-exchange 5 mL Hitrap Q columns , after equilibration with buffer A . Proteins were eluted with a gradient increasing NaCl to 0.5 M at a flow rate of 1 mL/min. While fractions containing ErCry4 were yellow in color, due to the absorption of bound FAD, those of ErCry1a and ErCry1b were colorless.Baculovirus was produced in SF9 cells (Thermo Fisher) using the Bac-to-Bac Baculovirus expression system (ThermoFisher), while Tni cells  were used for protein expression. Cells from 1 L culture were sedimented by centrifugation at 3745× tα/Giα chimera samples were thawed. Two reaction mixtures were prepared, both containing 50 mM Tris/HCl at pH 7.4, 50 mM NaCl, 2 mM MgCl2, 10 µM GDP, and 10 µM Gtα/Giα chimera. To one reaction, 50 µM AlCl3 and 10 mM NaF were added. The reactions were started by adding chymotrypsin (Roth), from a frozen stock solution containing 0.5 mg/mL chymotrypsin in 0.5 mM HCl and 50% glycerol, to 12.5 µg/mL and a final volume of 300 µL. Upon addition of protease, the reactions were incubated at 37 °C. Digestion was stopped after 0.5, 2, 5, 10, 20, 40, 60, and 90 min by removing 30 µL of the reaction mixture each, mixing with 10 µL of 4× Laemmli sample buffer, heating to 95 °C for 3 min, and storage at −20 °C. Additionally, a sample was prepared before the addition of protease. Analysis was performed by loading identical volumes of each sample onto 10% Bis-tris SDS-PAGE gels. Gels were stained using Coomassie Brilliant Blue R250 and decolorized using 10% acetic acid and 40% ethanol. Visualization was performed using an Azure c400 Gel Imaging System by Azure Biosystems, Dublin, CA, USA.The proteolytic digestion was performed under conditions such as those previously described ,26 for ttα/Giα sample was thawed and diluted in fluorescence buffer  at a final concentration of 1 µM. Trp fluorescence emission was recorded at 340 nm after excitation at 290 nm. Changes in emission rate were triggered by injection of 50 µM AlF4− (premix of 50 µM AlCl3 and 10 mM NaF). Recording time was 500 s.The intrinsic Trp fluorescence assay for measuring the activation-dependent conformational change in G protein α-subunits is a widely used assay to test their functional intactness. Details of the experimental setup are described . We recotα/Giα chimeraPurification of ErCry4 and G—Both ErCry4a and non-myristoylated Gtα/Giα chimera were expressed and purified essentially as described above, except that all steps were carried out under far-red light (750+ nm). For the Gtα/Giα chimera, concentrated fractions after affinity purification were not digested to remove the tag but directly purified by size exclusion chromatography using a Superdex200 increase 10/300 column pre-equilibrated with a running buffer . The flow rate was maintained at 0.17 mL/min, and fractions containing the G-protein were identified with SDS-PAGE and used immediately for pulldown experiments. For ErCry4a, after affinity purification, elution fractions were dialyzed together  after adding TEV protease (in-house) to an approximate molar ratio of 1:20 against at least 3 L of dialysis buffer  overnight. The digest was then applied to the gravity flow columns  equilibrated with dialysis buffer to remove His-tag and protease. The flow-through containing ErCry4a was concentrated using a Vivaspin Turbo 15 10k MWCO centrifugal filter and further purified by size exclusion chromatography using a Superdex200 increase 10/300 column  pre-equilibrated with a running buffer . The flow rate was maintained at 0.17 mL/min, and fractions containing ErCry4a were identified with SDS-PAGE and used immediately for pulldown experiments.Pulldown—All steps until SDS-PAGE gel electrophoresis were carried out under far-red light (750+ nm). For protein pulldown experiments, 1.47 nmol each of ErCry4a and His-Gtα/Giα were mixed and incubated overnight at 6 °C with gentle agitation. For the negative controls, His-Gtα/Giα was replaced with a running buffer. Next, 50 µL Super Ni-NTA agarose resin  was added, and the sample was incubated for an additional 1.5 h at 6 °C with gentle agitation. The slurry was transferred to Micro Bio-SpinTM Columns , washed five times with 1 mL of wash buffer , then incubated with 70 µL elution buffer  for 30 min on ice. For elution, columns were centrifuged for 1 min at 4 °C and 500 g. SDS-PAGE samples were prepared as follows. For the input controls, 2.5 µg of either ErCry4a or His-Gtα/Giα were mixed with SDS-sample buffer, boiled for 5 min at 95 °C, and then placed on ice until loaded on the gel. For the other samples, 20 µL of the elution fraction were mixed with 5 µL SDS-sample buffer, boiled for 5 min at 95 °C, then placed on ice until loaded on the gel. Samples were run on 4–20% Tris SDS-PAGE gradient gels. Gels were stained using Coomassie Brilliant Blue R250  imaging system.Densiometric analysis—Analysis was carried out in the Fiji software package using the SDS-PAGE analysis function allowing for the integration of grey values with background correction. For each experiment, the ErCry4a input band was quantified. Then the same region of interest was quantified for negative control and pulldown lanes. The bar diagram visualizes the average percentage of input ErCry4a recovered in negative control and pulldown, respectively, with error bars indicating the standard deviation. Individual experimental results are indicated by circles.2-groups. In preliminary tests, we compared different immobilization geometries, immobilization densities and regeneration protocols. Immobilization densities of myristoylated Gtα/Giα were 3.6–3.7 ng/mm2 and of non-myristoylated Gtα/Giα were 2.6–3.9 ng/mm2. Interaction processes were studied by injection of ErCry variants at different concentrations at a flow rate of either 5 or 20 µL/min. Some recordings were also performed at higher flow rates of 30 and 50 µL/min. SPR running buffer was 10 mM HEPES/NaOH, pH 7.4, 150 mM NaCl, 10 mM MgCl2, 0.005% Tween-20, 3.4 mM EDTA. Control surfaces were coated with Ulp1 (in-house made) at a density of 2.4–3.6 ng/mm2 or by an amino coupling activation/deactivation cycle (SPR recordings with ErCry1a). Surface plasmon resonance (SPR) measurements were performed on a Biacore 3000 . The general operation principle, including the immobilization procedures and quantitative data analysis, has been described before ,28. We uD values. SPR sensorgrams that did not show mono-exponential binding curves were evaluated by a two-state-reaction model according to A + B ↔ AB ↔AB* (* indicates a different protein conformation). The latter describes the binding process of A + B by a forward rate constant ka1 and a backward rate constant kd1 leading to complex formation AB and a conformational change of AB to AB* . We calculated the apparent KD from the ratio of kd1/ka1.For the evaluation of sensorgrams, we used nonlinear curve fitting implemented in the BIAevaluation software 4.1  by applying the global fitting approach. Sensorgrams obeying a mono-exponential Langmuir binding process (A + B ↔ AB) yielded association and dissociation rate constants and apparent KCloning of FRET constructs—cDNA production: RNA was extracted from the retina of one European robin, which was wild-caught in the vicinity of the university campus using mist nets. The animal was sacrificed by decapitation, and the eyes were immediately removed. The retina, free of vitreous, was shock-frozen in liquid nitrogen and stored at −80 °C until RNA extraction. RNA was extracted using the NucleoSpin RNA XS kit , and a cDNA library was generated using the Make Your Own “Mate & PlateTM” Library System .http://n2t.net/addgene (accessed on 26 February 2020): 74252; RRID: Addgene_74252) [ErCRY4 was generated by first linearizing pKanCMV-mClover3-mRuby3 with primers 1 and 2 .All FRET constructs were based on the pKan-CMV-mClover3-mRuby3 vector, a gift from Michael Lin  . Table 1 1 and 2  using PrErCRY4 by deletion of ErCRY4 and mRuby3 by using the Q5 site-directed mutagenesis kit (New England Biolabs) with primers 5 and 6, introducing a XhoI restriction site and a stop codon after the mClover3 plus linker. pKan-CMV-mClover3-ErCRY4-497 was generated by amplifying the ErCRY4 cDNA with primers 7 and 8 and In-Fusion recombination with pKan-CMV-mClover3 digested with AscI and XhoI. pKan-CMV-mRuby3 was generated by deletion of mClover3 from pKan-CMV-mClover3-mRuby3 using the Q5 site-directed mutagenesis kit  and primers 9 and 10. pKan-CMV-ErGNAT2-mRuby3, with GNAT2 being the corresponding gene to the wildtype ErGtα protein, was generated by first linearizing pKan-CMV-mClover3-mRuby3 with primers 11 and 12 using PrimeSTAR Polymerase, and then amplifying the GNAT2 gene with primers 13, introducing an AscI restriction site, and 14, introducing a XhoI restriction site. Both PCR products were then recombined using In-Fusion, resulting in the deletion of mClover3 and a fusion construct of GNAT2-mRuby3. The GNAT2 sequence was compared to the genome sequence (NCBI Sequence ID LR812129.1).pKan-CMV-mClover3 was generated from pKan-CMV-mClover3-Cell Culture and Expression—The QNR/K2 Neuroretina Quail cell line was purchased from ATCC (CRL-2533) and cultured in DMEM + GlutaMAX  supplemented with 10% fetal bovine serum (Gibco) at 39 °C and 5% CO2. QNR/K2 cells were (co-)transfected with the FRET constructs using Lipofectamine 2000 (Invitrogen) according to manufacturer’s instructions, after which cells were handled in dim red light until fixed. Forty-eight hours after transfection, cells were washed twice with PBS (Gibco), fixed in 4% (wt/v) paraformaldehyde for 30 min, and washed again twice with PBS and kept in PBS for imaging. Transfection, fixation and imaging were all performed in µ-Slides 8 Well with a #1.5 polymer coverslip bottom (Ibidi). Acceptor photobleaching FRET—Imaging of transiently transfected QNR/K2 cells was performed with an inverted Leica TCS SP5 II confocal microscope. A 63×/water objective with 1.2 numerical aperture was used . A white light laser was used as an excitation light source. The excitation wavelengths were set to 488 nm for mClover3 fluorescent protein and 560 nm for mRuby3. Photomultiplier tubes were used as detectors. The detection range was set to 500–550 nm for mClover3 emission and 570–644 nm for mRuby3 emission using AOTF . To determine the FRET efficiency, the acceptor photobleaching protocol was used. Acceptor photobleaching was performed with the FRET AB wizard in the LAS AF software on selected regions of interest (ROIs), generally one to two per cell in a region where both fluorophores showed expression. Acceptor bleaching was achieved by setting four parallel laser lines of white light laser  to 100% intensity. Donor bleaching was found to be negligible under these conditions. Prebleach and postbleach images were acquired. FRET for the ROIs was observed by an increase in donor (mClover3) fluorescence intensity following the acceptor (mRuby3) photobleaching. FRET efficiency was measured in percent and calculated automatically by Leica LAS AF software as (Dpost − Dpre)/Dpost, where Dpost (Dpre) is the fluorescence intensity of the donor after (before) photobleaching. For each condition, at least three individual experiments from freshly transfected cells were performed.Statistics—Since the FRET efficiency read-outs were reported as percentages, we opted for a binomial generalized linear model (GLM) followed by an analysis of variance, type III from the ‘car’ package in reference [eference  of the reference . ErGtα, we used purified recombinant ErCry4a that matches the in vitro requirements for a magnetic sensing protein [ErGtα, we constructed a Gtα/Giα chimera, in which a region of 79 amino acids was replaced by the corresponding region from an inhibitory bovine Gα-subunit. The inserted region is highly homologous to the ErGtα counterpart and differs only in 23 positions (of which ten are conservative replacements). The benefit is that the replacements facilitate heterologous expression and subsequent purification as a soluble protein [tα/Giα chimera was used as a surrogate for Gtα.For interaction studies with  protein . HoweverErCry4a and the Gtα/Giα chimera. We took into account that α-subunits of G proteins are myristoylated at the amino-terminus and expressed and purified myristoylated and non-myristoylated Gtα/Giα chimera. Both variants were immobilized by a standard SPR amine coupling procedure on a CM5 sensor chip surface [ErCry4a in the mobile phase that is flushed over the Gtα/Giα chimera coated chip surface. A control surface was coated with ubiquitin-like-protease 1 (Ulp1). Purified ErCry4a was injected and flushed over both surfaces (control and the Gtα/Giα chimera coated surface), and the control recordings were subtracted from the sample recordings. A positive increase in SPR resonance units (RU) indicated a binding process and injection of increasing ErCry4a concentrations resulted in sensorgrams of increasing amplitudes .Using surface plasmon resonance (SPR), we investigated the binding affinities and kinetic parameters of the interaction between  surface . In a seccessful A resulteErCry4a to non-myristoylated Gtα/Giα also occurred in a concentration-dependent manner (D = 35 ± 15 nM (n = 5). These results showed a moderate to high affinity of ErCry4 to myristoylated Gtα/Giα and a high affinity to non-myristoylated Gtα/Giα, indicating an impact of the myristoyl group for the interaction process .Binding of t manner B. By pertα/Giα to a C-terminally truncated variant of ErCry4a (ErCry4a-497) and determined an apparent KD of 0.2 ± 0.14 µM  analysis  and/or size exclusion chromatography (SEC) are functionally active by measuring their well-known conformational change in response to GDP/GTP exchange , supporting that the C-terminus is not involved in the interaction. The acceptor photobleaching technique tells us that ErGtα and ErCry4a and ErCry4a-497 are in close proximity to each other, indicating a protein–protein interaction.We observed a slightly higher false FRET background of mClover3-ErCry1a is the other Cry form currently discussed as a potential magnetoreceptive protein [ErCry1a and ErCry1b in our interaction analysis by testing for binding of these purified ErCry1 forms (the apo forms without any bound FAD) to myristoylated Gtα/Giα by SPR. Using the same experimental approach as for ErCry4a, ErCry1a and ErCry1b showed a concentration-dependent binding to myristoylated Gtα/Giα  confirmed this result and showed further that Gtα/Giα bound to ErCry4a-497 with similar affinity  compared to wildtype ErCry4a. For this, we do not have a clear interpretation but point out that purified ErCry4a-497 was less stable compared to the wildtype protein leaving some ambiguities about the quantitative kinetic analysis of this variant. However, similar FRET efficiencies between wildtype Gtα and ErCry4 either with or without the C-terminus in a transiently transfected bird neuroretinal cell line confirm the in vitro data and suggest that these two proteins also interact in living cells and that the C-terminal tail of ErCry4 is not involved in the interaction  also reported that the C-terminus of  process . Conducteraction .tα/Giα to the Cry forms ErCry1a and ErCry1b, which are also expressed in retinal cell types of migratory birds, pointing to putative roles in magnetic sensing [D values of 0.4 and 1.53 µM for ErCry1a and ErCry1b, respectively. These values are similar to those determined for ErCry4a and did not disclose a preference of Gtα/Giα for either of them. However, the shape of the SPR sensorgrams indicates differences in the association and dissociation rate constants, which was confirmed by nonlinear curve fitting , who found no evidence for GNAT2 (Gtα) interacting with ErCry1a [ErCry1b with Gtα was observed, but reporter gene expression signals indicating a binding process did not differ from control incubations. Thus, reduced stability of complexes involving ErCry1a and ErCry1b could account for the differences in the yeast two-hybrid experiments. Furthermore, binding of Gtα/Giα to different ErCry isoforms seems to be a new variation of a common theme since G proteins exhibit a multiplicity of G protein-coupling and targeting [Finally, we extended the interaction analysis of myristoylated G sensing ,16,45,46 ErCry1a . An appaargeting ,39. For argeting ,48, two ErCry4a and the α-subunit of a cone-specific G protein from European robin interact. We speculate that this interaction could be the first trigger step of biochemical signaling in radical-pair-based magnetoreception. This finding will open several routes of research addressing potential downstream signaling pathways, the sequence of individual binding steps, and the colocalization of signaling components in retinal cells.In summary, we provide strong experimental evidence from in vitro data and measurements in living cells showing that


Further optimization of functional properties for different types of applications can be achieved by modification of processing parameters or chemical composition. In the present work, the influence of pentavalent niobium substitution for trivalent ferric ions on the structure, microstructure and dielectric properties of 0.7BF–0.3BT ceramics was investigated systematically. Doping with niobium led to incremental reductions in grain size (from 7.0 to 1.3 µm) and suppression of long-range ferroelectric ordering. It was found that core-shell type microstructural features became more prominent as the Nb concentration increased, which were correlated with the formation of distinct peaks in the dielectric permittivity–temperature relationship, at ~470 and 600 °C, which were attributed to the BT-rich shell and BF-rich core regions, respectively. Nb-doping of BF–BT ceramics yielded reduced electronic conductivity and dielectric loss, improved electrical breakdown strength and enhanced dielectric energy storage characteristics. These effects are attributed to the charge compensation of pentavalent Nb donor defects by bismuth vacancies, which suppresses the formation of oxygen vacancies and the associated electron hole conduction mechanism. The relatively high recoverable energy density (Wrec = 2.01 J cm−3) and energy storage efficiency (η = 68%) of the 0.7BiFeO3–0.3BaTiO3 binary system were achieved at 75 °C under an electric field of 15 kV mm−1. This material demonstrates the greatest potential for applications in energy storage capacitors and temperature-stable dielectrics.BiFeO In recent decades, research on energy storage materials has generated significant interest from academia and industry. Dielectric materials have been intensively investigated due to their fast charge/discharge rate, good performance under pulsed current/voltage conditions, excellent thermodynamic and mechanical stability and long cycling lifespan ,2. In getotal), recoverable energy density (Wrec) and energy storage efficiency (η) are obtained by numerical integration of the polarization–electric field (P–E) loop according to the following formulae:m, Pr and E are the maximum polarization, remanent polarization and applied electric field, respectively. For future high-performance energy storage materials, the following three points need to be considered: high polarization difference (ΔP = Pm − Pr), high breakdown strength (BDS) and sustainable, environmentally friendly compositions.Generally, the total energy density (W3 (PZT), using various types of dopants and compositional adjustments to achieve antiferroelectric [rec value for O3 antiferroelectric ceramics was reported as 12.3 J cm−3 at an electric field of 42 kV mm−1 [3 (PLZST) relaxor thin films reached 31.2 J cm−3 at 200 kV mm−1 [Recently, lead-free ceramics have attracted increasing attention to replace conventional lead-based dielectric materials, although the latter have been shown to exhibit excellent energy-storage properties. There are already numerous reports of lead-based dielectrics based on PbOelectric ,5 or relelectric ,7. For e kV mm−1 . On the rec values have been reported in more environmentally-friendly, lead-free bulk ceramics, such as NaNbO3–(Bi0.5Na0.5)TiO3 (Wrec~12 J cm−3 at 68 kV mm−1). Significant random fields are thought to occur in the relaxor AFE matrix in such NaNbO3-based compositions, which suppress the growth of nanodomains and the phase transition process, resulting in outstanding energy-storage properties [3–BaTiO3–NaNbO3, the NaNbO3 substitution was found to improve the dielectric breakdown strength, resulting in Wrec~8 J cm−3 at 36 kV mm−1 [0.5Na0.5)NbO3–SrTiO3 ceramics, the grain size was reduced into the submicron range by the addition of SrTiO3, leading to high BDS and high Wrec (Wrec~4 J cm−3 at 40 kV mm−1) [0.03Ag0.91NbO3 the Sm dopant was found to stabilize antiferroelectricity and increase the degree of disorder, resulting in slimmer P–E loops and improved recoverable energy density (Wrec~5 J cm−3 at 29 kV mm−1) [Promising W kV mm−1 . In (K0.kV mm−1) , while fkV mm−1) .3 (BF) was recognized as an important component due to its “giant” spontaneous polarization (>100 μm cm−2) [r, BF-based compositions have the potential for a high polarization difference (ΔP), resulting in excellent energy storage performance. In particular, 0.7BF–0.3BT ceramics have been shown to exhibit high switchable polarization values (Pm~44 μm cm−2 and Pr~40 μm cm−2) due to the proximity of this composition to the morphotropic phase boundary (MPB) at 0.67BF–0.33BT [2O3 and the changes in oxidation state of Fe3+ and Fe2+ ions during sintering, is another problem that needs to be addressed [3, SrTiO3, La(Mg0.5Ti0.5)O3 and Ba(Mg1/3Nb2/3)O3 [The various materials mentioned above represent some examples of the numerous studies on dielectrics for energy storage. Among the environmentally friendly lead-free materials, BiFeOμm cm−2) ,13. By rF–0.33BT . Howeverddressed ,16. The Nb2/3)O3 ,19,20,215+ substitution for Fe3+ on the B-site can dramatically increase the electrical resistivity of BiFeO3-based ceramics [3-based ceramics were systematically investigated in recent years [rec, such as BiFeO3–BaTiO3–NaNbO3 [3–BaTiO3–Bi0.5Na0.5TiO3 [3–0.35BaTiO3 with 3 mol% Nb2O5 addition yielded an improved Wrec of 0.71 J cm−3 at 9 kV mm−1 [rec by modification of stoichiometry, selection of appropriate minor dopants and microstructural control.It was found that Nbceramics . Moreoveceramics . The strnt years ,25,26,273–NaNbO3  and BiFea0.5TiO3 . However kV mm−1 . The potrec (2.01 J cm−3) and energy storage efficiency η (68.1%) were achieved for 0.7BiFe0.98Nb0.02O3–0.3BaTiO3 ceramics at an electric field level of 15 kV/mm and temperature of 75 °C. In addition, both Wrec and η are significantly improved at elevated temperatures, which indicates that Nb-doped BF–BT ceramics have the potential to be applied as energy storage dielectrics at elevated temperatures without property deterioration. Donor doping with niobium leads to a significant reduction in electronic conductivity, improvement of electrical breakdown strength and enhanced dielectric energy storage properties for BF–BT ceramics. In this work, a sequence of lead-free Nb-doped BF–BT ceramics were synthesized by solid state reaction and conventional sintering. A relatively high W1–xNbxO3–0.3BaTiO3  ceramics were synthesized by the solid-state reaction method using Bi2O3 (99%), Fe2O3 (99%), BaCO3 (99%), TiO2 (99%) and Nb2O5 (99.99%) powders . The precursor powders were weighed out according to the required stoichiometry, followed by mixing and milling in propan-2-ol with yttria-stabilized zirconia media for 24 h and drying for 12 h at 90 °C. Afterwards, the powders were calcined at 800 °C for 4 h in air and milled again for 24 h. An addition of 2 wt% polyethylene glycol (PEG) lubricant was made into the dry calcined powders. Green pellets, 10 mm in diameter, were prepared by uniaxial pressing at a pressure of 100 MPa. The ceramic pellets were then sintered in a covered alumina crucible at 1010 °C for 3 h with heating and cooling rates of 5 °C/min. Conductive electrodes were applied using a silver paste , which was heated to 550 °C for 30 min for densification.0.7BiFeThe sintered ceramic pellets were ground using 800, 1200, 2400 and 4000 grit SiC paper followed by polishing procedures with 3, 1, 0.25 μm diamond paste and 0.04 μm OPS silica colloidal suspension. Examination of microstructure and micro-chemical elemental mapping of the polished cross-sections were conducted using a scanning electron microscope  equipped with an energy-dispersive X-ray spectroscopy (EDS) system. The average grain sizes were determined quantitatively from the SEM images by line intercept analysis using ImageJ software . X-ray diffraction (XRD) patterns of as-sintered samples were obtained using a PANalytical X’Pert Pro X-ray diffractometer  in the 2θ range from 10 to 100°, with a step size of 0.0167° and a counting time of 1.5 s for each step. TOPAS v5 software  was utilized for Rietveld refinement of the XRD data.total, Wrec and η were obtained by numerical integration of the P–E loop, according to Equations (1) to (3) above. Five specimens of each composition were tested to provide statistically averaged polarization values and energy storage parameters. Typical uncertainties in the polarization, energy storage density and efficiency values were estimated as 0.01 C m−2, 0.01 J cm−3 and 0.05%, respectively.Temperature-dependent dielectric measurements were conducted using an Agilent 4284A impedance analyzer  and a Carbolite CWF1200 horizontal tube furnace , using pure silver conductor wires for electrical contacts to the test specimens in the furnace. The dielectric constant and loss were determined at fixed frequencies of 1 kHz, 10 kHz and 100 kHz over the temperature range from 25 to 650 °C, using a single parallel capacitor–resistor network as the equivalent circuit element. Ferroelectric hysteresis measurements were conducted using a function generator  together with a Chevin Research HV amplifier. Four cycles of a sinusoidal electric field with a frequency of 2 Hz were applied to the samples. Polarization–electric field (P–E) loops were constructed from the recorded electric potential and induced current waveforms, according to the methods described by Stewart et al. . Wtotal,5Ti3FeO15 (ICSD_PDF#01-089-8545) [2O6 (ICSD_PDF#01-077-0541) [2O6 appears first, followed by Bi5Ti3FeO15, which may help to rebalance the stoichiometry of the perovskite solid solution. The {111} and {200} peak profiles, presented in 89-8545)  and BaNb77-0541)  were ide5+ (IR = 0.64 Å) to Nb4+ (IR = 0.68 Å) during sintering [4+ for Fe3+ (IR = 0.645 Å). Alternatively, the observed expansion of the lattice could possibly be caused by partial reduction of Fe3+ to Fe2+ ions, with an associated increase in ionic radius for Fe2+ (IR = 0.78 Å). The latter reaction can be represented by the following defect chemical equation, which involves charge compensation by the reduction of ferric to ferrous oxide in response to donor-type substitution of Nb5+ for Fe3+ in BiFeO3.The lattice parameters obtained by Rietveld refinement, summarized in intering , resulti3+ and/or Nb5+ ions in Nb-doped BF–BT ceramics during sintering.Further investigations are necessary to investigate the potential changes in the valence states of Fe5+ ions influences the lattice diffusion and increases the activation energy for grain boundary migration [The influence of Nb-doping on the microstructure of the 0.7BF–0.3BT–xNb ceramics is illustrated by the SEM micrographs shown in igration . Similarigration ,35.3- and BaTiO3-rich solid solutions. On the other hand, Nb and O appear to be distributed evenly within the characterized region. However, accurate determination of the distribution of Nb needs to be investigated further by other techniques due to the insensitivity of the EDS detector for this low dopant concentration. Bright features located at the triple points and along grain boundaries in the BSE images indicate the development of Bi-rich liquid phases during sintering, which may assist with densification. The existence of chemical heterogeneity in the form of core-shell type microstructural features has a significant impact on functional properties and also causes differences in crystal structure between the core and shell regions, as reported previously in BF–BT and other solid solutions such as 0.24BiFeO3–0.56K0.5Bi0.5TiO3–0.20PbTiO3 (BF–KBT–PT) [The occurrence of chemical heterogeneity is supported by EDS mapping of the elemental distribution in the 0.7BF–0.3BT–4Nb ceramic, as illustrated in –KBT–PT) . Similarr) and loss (tan δ) of the 0.7BF–0.3BT–xNb ceramics are shown in The temperature dependence of dielectric permittivity  and (6), respectively.Assuming that the conduction mechanisms in BF–BT are similar to that of BiFeO5+ substituted for Fe3+, provides a mechanism to suppress the formation of oxygen vacancies and therefore reoxidation cannot occur. In this case, the (relative) negative charge on a bismuth vacancy is compensated by the (relative) positive charge of the donor, rather than that of the oxygen vacancy, according to Equation (7).Incorporating a donor dopant, such as Nb2O3 may be volatilized without the formation of oxygen vacancies when three units of Nb2O5 are incorporated into solid solution, due to the necessary charge balance between the bismuth vacancies and the substituted Nb defects. Excessive Nb doping exceeds the solubility limit and leads to the formation of second phases, as noted in According to the stoichiometry of this equation, two molecules of Bi3-rich core regions, as identified in the microstructural observations reported above. A relatively stable dielectric plateau was formed between 150 and 500 °C for the Nb doped samples with x in the range of 2 to 4 at%, which is attributed mostly to the contribution from the shell regions. We suppose that incorporation of Nb leads to enhanced disorder of the local charge and lattice distortion, leading to nano-scale local random fields and the formation of polar nano regions (PNRs). The long-range ferroelectric order is suppressed, which induces relaxor-like behavior and the formation of the plateau region [The main dielectric peak at 470 °C was strongly suppressed by Nb-doping, while a second peak appeared at elevated temperatures around 600 °C b. This su region ,39.m) and remanent (Pr) polarization values reduced dramatically with increasing Nb concentration in the range from 0.5 to 2 at%, followed by a gradual decrease in Pm and slight variations in Pr from 3 to 5 at%, as shown in r was already reduced to a negligible level at 2% Nb, further increases in Nb concentration resulted in continued reductions in Pm, which led to progressive reductions in recoverable energy density, Wrec. It is considered that the long-range ferroelectric ordering was disrupted, along with an evolution from nonergodic to ergodic relaxor ferroelectric behavior with increasing Nb concentration, yielding more reversible polarization switching characteristics. It was observed that donor doping with Nb led to improved breakdown strength, which is attributed to the combined effects of a reduction in the free carrier concentration and reduced grain size.The polarization–electric field (P–E) loops of the BF–BT ceramics are shown in rec) and efficiency (η) are shown in rec (0.9 J cm−3) and η (69.6%) at room temperature are achieved for the 0.7BF–0.3BT ceramics doped with 2 at% Nb; this material demonstrates the greatest potential out of those investigated in the present study and may be suitable for applications in energy storage capacitors and temperature-stable dielectrics.The compositional dependencies of the recoverable energy density (Wm) exhibits little sign of saturation, as shown in m at high fields (above 12 kV/mm) is observed in the measurements conducted at 75 °C, indicating a tendency for saturation. The remanent polarization is also reduced significantly at 75 °C, in comparison with that measured at 25 °C. These observations indicate a more reversible field-induced switching of the PNRs at elevated temperatures, leading to enhanced Wrec and η values, which improved from 1.65 J cm−3 to 2.01 J cm−3 and from 58% to 68% at 15 kV/mm, as the temperature increased from 25 to 75 °C. A schematic illustration of the changes in the nano-polar ferroelectric response for the 0.7BF–0.3BT–2Nb at different temperatures is shown in rec = 2.01 J cm−3) and the energy storage efficiency (η = 68%) were achieved in the 0.7BF–0.3BT binary system. These results demonstrate that Nb-doped BF–BT ceramics are promising lead-free dielectrics for next-generation pulsed power capacitor applications.The present paper demonstrates that undoped 0.7BF–0.3BT ceramics exhibit a rhombohedral phase and well-defined ferroelectric domain structures. Nb-doping induces an apparently pseudo-cubic structure, accompanied by core-shell microstructural features, even at the lowest substitution level of 0.5 at%. The presence of bismuth and niobium rich second phases at high Nb concentrations indicates a solubility limit of approximately 2 at%. Distinct peaks in the dielectric permittivity-temperature relationships for Nb-doped 0.7BF–0.3BT ceramics at temperatures of 470 and 600 °C are attributed to dominant contributions from the shell and core regions, respectively. The relatively high recoverable energy density (W