In immunoprecipitations, however, the homodimerization of MdmX could be observed only when the asparagine residue was replaced with cysteine in both RINGs. This result suggested that heterocomplexes consisting of one mutated MdmX RING with cysteine and one wild-type MdmX RING with asparagine might be less stable, despite being readily detectable in the cell-based assay. Moreover, Mdm2 C449N blocked Mdm2-MdmX heterodimerization but did not disrupt the ability of Mdm2 homodimer to promote p53 degradation, suggesting that the effect of the conserved cysteine and asparagine residues on dimerization was context-specific. Collectively, our results indicate that the effects of individual exchanges of conserved residues between Mdm2 and MdmX RING domains might be context-specific, supporting the hypothesis that Mdm2 RING homodimers and Mdm2-MdmX heterodimers may not be entirely structurally equivalent, despite their apparent similarity.Mdm2 and MdmX are related proteins serving in the form of the Mdm2 homodimer or Mdm2/MdmX heterodimer as an E3 ubiquitin ligase for the tumor suppressor p53. The dimerization is required for the E3 activity and is mediated by the conserved RING domains present in both proteins, but only the RING domain of Mdm2 can form homodimers efficiently. We performed a systematic mutational analysis of human Mdm2, exchanging parts of the RING with the corresponding MdmX sequence, to identify the molecular determinants of this difference. Mdm2 can also promote MdmX degradation, and we identified several mutations blocking it. They were located mainly at the Mdm2/E2 interface and did not disrupt the MdmX-Mdm2 interaction. Surprisingly, some mutations of the Mdm2/E2 interface inhibited MdmX degradation, which is mediated by the Mdm2/MdmX heterodimer, but did not affect p53 degradation, mediated by the Mdm2 homodimer. Only one mutant, replacing a conserved cysteine 449 with asparagine (C449N), disrupted the ability of Mdm2 to dimerize with MdmX. When we introduced the cysteine residue into the corresponding site in MdmX, the RING domain became capable of forming dimers with other MdmX molecules  Howeverelopment . Mdm2 anelopment . Both prelopment . Mdm2 caelopment .The N-terminal p53 binding domains and the RING domains of Mdm2 and MdmX are highly conserved in evolution , as is tin vivo (A notable feature of RING-type E3s is their tendency to form homodimers and heterodimers that seems to be the prerequisite for the E3 activity for many RING finger ubiquitin ligases . The RINin vivo . A preci2 in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal calf serum, 2 mM L-glutamine, 50 U/ml penicillin G, and 50 μg/ml streptomycin sulfate .Human U2OS and HEK293 cell lines were obtained from ECACC and DSMZ, respectively. Cells were cultured at 37°C / 5% COMammalian expression plasmids coding for wild-type Mdm2, Mdm2 mutant Mdm2∆9, lacking the conserved C-terminal tail required for RING dimerization, Mdm2 mutant C464A, disrupting the RING domain structure and function, Myc-tagged MdmX, FLAG-tagged human wild-type p53 (pcDNA3-FLAG-p53), and the Mdm2/MdmX chimera (MDM2/MDMXRFD) have been described previously . GFP-MdmU2OS cells in 60-mm dishes were transfected with 0.1 μg FLAG-p53 and 1.4 μg Mdm2 mutant plasmid constructs using Effectene transfection reagent according to guidelines of the manufacturer (Qiagen). Each transfection mixture also contained 0.2 μg pEGFP-N1 (Clontech) to control for transfection efficiency, and empty plasmid pcDNA3.1 (Invitrogen) was used to bring the total amount of transfected DNA to 1.7 μg. Cells were washed with cold PBS 30 h post-transfection and lysed with 0.25 ml of SDS sample buffer. Proteins were resolved by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by Western blotting with anti-Flag M2 , anti-Mdm2 Ab-1 , and anti-GFP 7.1/13.1  antibodies. In some experiments, p53 was also detected using the anti-p53 monoclonal antibody DO-1 .U2OS cells grown in 60-mm dishes were transfected with 1 μg Myc-MdmX and 6 μg wild-type Mdm2 or Mdm2 mutant plasmid constructs using Lipofectamine 2000 transfection reagent (Invitrogen) and DMEM was changed 4 h later. Each transfection mixture also contained 0.1 μg pEGFP-N1 as a control for transfection efficiency. The empty plasmid pcDNA3.1 was used to bring the total amount of transfected DNA to 7.1 μg in cells transfected with MdmX expression plasmid alone. Cells were cultured for 30 h, washed with cold PBS, and lysed with 0.3 ml of SDS-PAGE sample buffer. Proteins were resolved by SDS-PAGE and analyzed by Western blotting with anti-Myc 9E10 , anti-Mdm2 Ab-1 , and anti-GFP 7.1/13.1  antibodies.™594-conjugated secondary antibodies (Jackson ImmunoResearch) in the blocking solution containing 1 μg/ml DAPI (Sigma-Aldrich). Cells were washed with PBS and mounted with VECTASHIELD HardSet Antifade Mounting Medium (Vector Laboratories). Images were taken with the FluoView 500 confocal laser scanning fluorescence microscopes (Olympus).U2OS cells grown on coverslips were transfected with Myc-tagged MdmX or GFP-tagged MdmX (0.3 μg), and wild-type Mdm2 expression plasmids or the Mdm2/MdmX chimeric construct (1.2 μg) using Effectene transfection reagent (Qiagen) and the culture medium was changed 4 h later. The empty plasmid pcDNA3.1 was used to bring the total amount of transfected DNA to 1.5 μg in cells transfected with MdmX expression plasmid alone. Twenty-four hours post-transfection, cells were treated with 15 μM MG132 (Sigma-Aldrich) in DMEM for 3 h, washed with PBS, and fixed in 4% paraformaldehyde in PBS for 10 min at room temperature. After fixation, cells were washed with PBS and permeabilized with PBS containing 0.2% Triton X-100 (Sigma-Aldrich) for 5 min. Cells were blocked with PBS containing 0.5% bovine serum albumin at room temperature for 30 min and then incubated for 2 h at room temperature with anti-Mdm2 mouse monoclonal antibody IF2  in the blocking solution or the anti-Mdm2 antibody together with anti-c-Myc rabbit polyclonal antibody . Cells were washed with PBS and incubated for 1 h at room temperature with FITC- or DyLightHEK293 cells were transfected with 6 μg DNA per 100-mm plate using Lipofectamine 2000 reagent (Invitrogen). Cells were treated with a proteasome inhibitor (10 μM MG132 in cell culture medium) 24–36 h post-transfection, 4 h later washed with PBS, and lysed in Triton X-100 lysis buffer  containing protease inhibitors . Lysates were pre-cleared with 50 μl of protein G-Sepharose (Millipore). Immunoprecipitations were performed at 4°C with 1–2 μg of an antibody bound to 50 μl of protein G-Sepharose for 1 h. Mdm2 was immunoprecipitated with anti-Mdm2 antibody IF2  and Myc-MdmX with anti-Myc antibody 9E10 . Immunoprecipitated proteins were washed with lysis buffer and resuspended in SDS-PAGE sample buffer. Proteins from whole-cell extracts and immunoprecipitations were resolved by SDS-PAGE and analyzed by Western blotting with anti-Mdm2 Ab-1  and anti-Myc 9E10  or anti-Myc A-14 .U2OS cells grown in 60-mm dishes were transiently transfected with FLAG-p53 (0.5 μg), hemagglutinin (HA)-ubiquitin (0.5 μg), and Mdm2 plasmid constructs (5 μg) using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s recommendations. Empty plasmid pcDNA3.1 (Invitrogen) was used to bring the total amount of transfected DNA to 6 μg. Cells were treated 24 h post-transfection with 15 μM MG132 for 3 h, washed with PBS, and lysed in 0.3 ml 0.5% SDS. Lysates were boiled for 5 min, vortexed, cooled down to room temperature, and diluted with 1 ml of Triton X-100 lysis buffer. p53 was immunoprecipitated using 1 μg of anti-p53 monoclonal antibody DO-1 on a rotating wheel at 4°C. After 1 h of incubation, 20 μl of protein G-Sepharose beads (Sigma-Aldrich) were added for 45 min. The immunoprecipitates were washed three times with lysis buffer and analyzed by Western blotting. Ubiquitylated p53 was detected using anti-HA-Peroxidase antibody ; FLAG-p53 and MDM2 levels in the input were detected using anti-FLAG antibody M2 (Sigma-Aldrich), anti-Mdm2 Ab-1 (Merck).http://decibel.fi.muni.cz/cozoid/). The 2VJF, 2HDP, and 5MNJ structural data have been used for the Mdm2/MdmX analysis and visualization. Structures were visualized and simulated using the PyMOL tool .The Mdm2 contact residues mediating its binding to the MdmX and UbcH5b partners have been identified using COZOID (COntact ZOne IDentifier) tool . This protein retains most of the Mdm2 sequence  but the C-terminal portion of Mdm2 including the RING domain was exchanged with the MdmX sequence [Interestingly, in immunoprecipitations, the interaction of two MdmX RINGs was observed only when the cysteine residue was introduced into both of them , suggestvia its C-terminal tail -ubiquitin complex suggested that despite their apparent structural similarity, the Mdm2 homodimer and Mdm2-MdmX heterodimer have differing abilities to transfer Ub due to their abilities to interact with E2~Ub complexes (~ indicates thioester bond). While Mdm2 RING could bind both UbcH5b and ubiquitin, MdmX did not bind the E2 enzyme and contacted only ubiquitin, bcH5b~Ub .In the present study, we used PCR-mediated site-directed mutagenesis of the Mdm2 RING domain in the context of full-length Mdm2 to identify residues that could be responsible for the apparent lack of the capacity to homodimerize and low intrinsic E3 activity of the MdmX RING. We mutated parts of the Mdm2 RING into the corresponding MdmX residues and observed a significant drop in the activity toward MdmX in mutants affecting E436, VI439–440, QG442–443, C449, T463, and RQP479–481. Of these mutants, 436K, 439–440SL, 442-442EK, and 479–481KKE could be inactive because they lost at least one residue predicted to participate in the interaction with E2. Surprisingly, however, mutants 439–440SL and 442-442EK were still capable of targeting p53 for degradation, indicating that they remained active in the form of the Mdm2-Mdm2 homodimer. Such a result might suggest that a different set of residues at the Mdm2/E2 interface might be required for E2 binding in Mdm2 homodimers and Mdm2-MdmX heterodimers. Importantly, our mutant 479–481KKE was inactive both in the form of heterodimer and homodimer, confirming the critical contribution of R479 to the ubiquitin ligase activity of Mdm2 .in vivo. When cysteine was introduced into the corresponding position in the MdmX RING, the mutant MdmX protein gained the ability to form homodimers. Our data are consistent with earlier studies in which the RING domain of MdmX was mutated to contain selected Mdm2 residues. Iyappan et al. turned MdmX into an active ubiquitin ligase by mutating N448 to cysteine and replacing residues 465–480 with the corresponding Mdm2 sequence (in vitro. They found that substitution of N448 for cysteine and K478 for arginine granted the MdmX RING domain the E3 activity. Based on subsequent structural analysis, the authors reasoned that C449 of Mdm2 is critical for the stability of the RING dimer structure, while R479 could play a role in recruiting and activating the ubiquitin E2 conjugating enzyme (Our study also identified the conserved cysteine 449 of Mdm2 and asparagine 448 of MdmX located at the RING-RING interface as critical determinants of the ability of Mdm proteins to form dimers sequence . Egorovag enzyme . The crig enzyme .in vivo, in line with the results of structural analyses indicating that cysteine and asparagine residues contact in the Mdm2-MdmX heterodimer, but two asparagine residues would clash, preventing MdmX homodimerization. However, in immunoprecipitations, we could detect MdmX homodimerization only when N448 was mutated to cysteine in both MdmX RING domains, suggesting that the context of the RING domain also plays a role. Moreover, the interaction between Mdm2 and MdmX RING domains also seemed unstable when the two critical residues were swapped, as we could not detect the binding of Mdm2 449N to MdmX 448C in the immunoprecipitation assay, despite the predicted compatibility of the two residues. Last but not least, the Mdm2 449N mutant still served as E3 ubiquitin ligase targeting p53 for degradation, again suggesting that the asparagine and cysteine residues had a differential impact on Mdm RING domain function depending on the structural context.Interestingly, we found that only one RING with the N448C exchange was required for MdmX RING homodimerization It seems that the tight mutual regulation of Mdm2, MdmX, and p53 activity in cells is enabled by many individual differences between the RINGs of Mdm2 and MdmX that participate in the control of RING dimerization and E2 binding.PK, IS, and AV performed experiments and prepared the figures. BV, PM, and JP analyzed the results and prepared the figures. SU conceived the study, performed experiments, analyzed the results, and wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Fagus sylvatica) and gray poplar (Populus x canescens) were exposed under controlled conditions to 33P-ATP and/or 13C/15N labeled ATP in the presence and absence of the acid phosphatase inhibitor MoO42-. Accumulation of the respective label in the roots was used to calculate 33P, 13C and 15N uptake rates in ATP equivalents for comparison reason. The present data shown that a significant part of ATP was cleaved outside the roots before phosphate (Pi) was taken up. Furthermore, nucleotide uptake seems more reasonable after cleavage of at least one Pi unit as ADP, AMP and/or as the nucleoside adenosine. Similar results were obtained when still attached mycorrhizal roots of adult beech trees and their natural regeneration of two forest stands were exposed to ATP in the presence or absence of MoO42-. Cleavage of Pi from ATP by enzymes commonly present in the rhizosphere, such as extracellular acid phosphatases, ecto-apyrase and/or nucleotidases, prior ADP/AMP/adenosine uptake is highly probable but depended on the soil type and the pH of the soil solution. Although uptake of ATP/ADP/AMP cannot be excluded, uptake of the nucleoside adenosine without breakdown into its constituents ribose and adenine is highly evident. Based on the 33P, 13C, and 15N uptake rates calculated as equivalents of ATP the ‘pro and contra’ for the uptake of nucleotides and nucleosides is discussed.The present study elucidated whether roots of temperate forest trees can take up organic phosphorus in the form of ATP. Detached non-mycorrhizal roots of beech  because at least the nucleoside adenosine as N source is taken up without cleavage into its constituents ribose and adenine.Roots take up phosphorus from ATP as P Different to nitrogen (N) and sulfur (S), which are acquired by plant roots from the soil ms e.g.,  and fromes e.g., , P is exes e.g.,  further es e.g., . P inputes e.g.,  and a chg., org)  further g., org) , 2015a,bmitation . Mycorrhmitation . Increasectively .i) is adsorbed to Fe and Al oxyhydroxides and, hence, is not available for plant uptake (org (i) solubilization from chelated aluminum- and iron-P   .org by plant roots. However, the preferential Porg compound(s) used in Pi release  by acid and alkaline phosphatases, the significance of Pi release versus direct Porg uptake, and the interaction/competition between Pi and Porg for Pi uptake by the roots have not been established. Such interactions were found for the inorganic and organic N uptake by the roots of woody plants  the most important climax tree species of Central European temperate forests and poplar (Populus x canescens) a continuously, fast growing tree species of floodplains  of the INRA clone 717 1B4) were micropropagated 2 × 4 H2O, 0.3 mM MgSO4 × 7 H2O, 1.5 mM MgCl2 × 6 H2O, 0.25 mM KH2PO4, 2.3 μM MnCl2 × 4 H2O, 2 μM H3BO3, 0.08 μM CuCl2 × 4 H2O, 0.2 μM ZnCl2, 0.2 μM Na2MoO4 × 2 H2O, 0.04 μM CoCl2 × 6 H2O, 22.5 μM Na2-EDTA, 22.5 μM FeCl2 and was adjusted to pH 5.5. If necessary poplar plants were provided with distilled water.Poplar cuttings  were used.Beech seedlings were cultivated from beech nuts collected in 2011 from the Conventwald forest stand  [7.960 East; 48°02′ North (Google earth); 33P-Pi , roots were excised from P. x canescens plants, which were 14 to 18-weeks old and/or 0.7–1 m in height (i) supplemented with 2 mM MES buffer and adjusted to pH 5.0. In case of 33P-ATP treatments, the respective (pre-) incubation solutions in compartment A did not contain phosphate and molybdate but ATP. Beech roots were pre-incubated in the beech fertilization solution supplemented with 2 mM MES buffer adjusted to pH 5.0. The pH dependency of Pi uptake was analyzed with excised poplar roots over a range of pH 3.5 to pH 7  or with 0.169 mM 33P-ATP (5.3∗107 to 1.2∗107 Bq mmol-1 ATP). 33P-ATP was applied either as γ33P-ATP or as α33P-ATP   were diluted to 10 atom% or 14 atom% and were adjusted to the final concentration of 0.169 mM ATP and CTP. The soil microbial ATP concentration of active and dead microorganism, which are constituents of the rhizosphere, ranged from <1.2 μg g-1 soil  to >2 μg g-1 soil  . To simulate soil temperature, incubation chambers were placed on aluminum plates cooled down to 15°C for 4 h of incubation. Uptake of ATP or CTP was terminated by washing roots 3-times with the respective unlabeled solution. Root sections in the incubation compartment (compartment A) were separated from the root sections located in compartment B and C. Oven dried homogenized root samples were subjected to IRMS analysis for the determination of 13C and 15N accumulation.For uptake experiments with stable isotope labeled ATP/CTP , excisedAldrich)  and CTP biomass) . The ATPular ATP . Roots w13C and 15N uptake rates calculated as ATP equivalents in experiments under controlled conditions were similar to 13C and 15N uptake rates equivalent to ATP in the field, ATP uptake experiments were performed in September 2017 at two field sites, namely the acidic Conventwald (Con) and calcareous Tuttlingen (Tut) forest stands. The soils of these forests differ in their properties (silicate versus limestone bedrock) . Fine roots were cut from the trees after 4 h of incubation, rinsed with distilled water to remove adherent ATP-13C1015N5, dried in an oven  for at least 2 days and homogenized using mortar and pestle.To test if bedrock)  with the13C and 15N incorporation into root sections after ATP-13C1015N5 and CTP-13C915N3 exposure were determined in over dried powdered root samples of 0.1–2.0 mg aliquots filled into tin capsules  coupled via a Conflo II interface to an isotope ratio mass spectrometer . Alternatively, samples were analyzed with an elemental analyzer NA 1108, Fisons-Instruments, Rodano, Milan, Italy and a mass spectrometer  coupled by a ConFlo III interface  (13 CPDB = -26.39‰) and USGS 41 (glutamic acid δ13 CPDB = 37.63‰) for δ13C, and USGS 40 (glutamic acid δ15 Nair = -4.5‰) and USGS 41 (glutamic acid δ15 Nair = 47.600‰) for δ15 N. Standards were analyzed after every tenth sample to account for potential instrument drift over time as described by 15N and δ13C was used to calculate N and C uptake rates in equivalents of ATP and CTP  . A worki and CTP .33P as well as of 13C and 15N from differently labeled ATP/CTP was calculated from 33P, 15N and 13C incorporation as equivalents of ATP. This standardized calculation allows direct comparison between treatments and uncovers differences between the differently labeled ATP (®9.1 (OriginLab Corporation1). Normal distribution of the data was tested with the Shapiro–Wilk and Kolmogorov–Smirnov test; all data showed normal distribution at least by the Kolmogorov–Smirnov test. One Way ANOVA was applied followed by the Bonferroni and Tukey test with p < 0.5. Data are presented as single values (left to the box plots) and as box-plots showing the median (black line), the mean (open square), and the 25 and 75 percentile. Minimum and maximum values are given as error bars, whereas outliers (1%) are presented as stars.For comparison, uptake of eled ATP . Statisti uptake rates of excised roots calculated from 33P-Pi application  compared to growth at 0.05 mM Pi . The maximum Pi uptake rate was lower during growth at 0.25 mM Pi (271 ± 31 nmol g-1 fw h-1) compared to the growth at 0.05 mM Pi (367 ± 50 nmol g-1 fw h-1). Excised roots from beech seedlings cultivated with 0.02 mM Pi showed remarkably lower Km (39 ± 18 μM) and vmax values (178 ± 21 nmol g-1 fw h-1). The tripartite incubation chamber allowed calculation of the Pi that has been loaded into the xylem . In contrast, 33P-Pi uptake of excised beech roots significantly declined in the presence of ATP .e of ATP . Applicafrom ATP . By appl present . Xylem le of ATP . However33P from γ33P-ATP by excised poplar roots was determined as 33P incorporation and calculated as ATP equivalents (approximately 83 ± 27 nmol ATP g-1 fw h-1)  . 33P frof MoO42- . Xylem li cleavage for the uptake of 33P from 33P labeled ATP was tested by comparing 33P uptake from γ33P-ATP and α33P-ATP. Assuming that 33P prior its uptake must be cleaved from ATP by phosphatases, 33P uptake should be lower when the α-P instead of the end standing γ-P was labeled as 33P. Indeed, 33P uptake was significantly lower when the α-P instead of the γ33P in the ATP was labeled .Another approach to test the importance of P labeled . Hence, α33P-ATP . The xyl13C/15N labeled ATP was applied as another approach to investigate ATP uptake. In the 13C/15N labeled ATP, ribose was labeled only with 13C whereas adenine was labeled by both, 13C and 15N. In order to compare 13C and 15N uptake rates both were calculated as ATP equivalents, i.e., five 15N correspond for one ATP, while ten 13C are equivalent to one ATP. Incubation with doubled labeled ATP at 15°C, applied to simulate soil temperature in forest stands, resulted in similar 13C and 15N uptake rates equivalent to ATP and were not affected by the acid phosphatase inhibitor MoO42-  when α33P-ATP and of 1.71 ± 0.90  when γ33P-ATP was applied. These results support the view of an uptake of AMP and the nucleoside adenosine by the roots.The correlation of approach  as a fureriments , 33P upt applied . The appnditions . Both, 1 applied . Correla5 ± 0.09 . 13C upt13C915N3 . Despite high variability, 13C uptake rates calculated as ATP  and as CTP equivalents  were similar. The same was found when the 15N uptake rates were calculated as ATP  and CTP equivalents . This result was irrespective of the addition of MoO42- as acid phosphatase inhibitor or the addition of Pi for competition  was applied as CTP-petition . The rel for CTP . These r33P, 13C, and 15N from labeled ATP was investigated with excised roots from F. sylvatica seedlings. In parallel experiments, γ33P-ATP and 13C/15N double-labeled ATP (10 atom% ATP-13C1015N5) was applied . 13C and 15N uptake rates of beech roots equivalent to ATP  amounted to one fourth of the 33P uptake equivalent to ATP. 13C and 15N uptake of beech roots as equivalent to ATP was also neither affected by Pi nor by the acid phosphatase inhibitor MoO42-. The relationship between the 13C and 15N uptake equivalent to ATP showed a strong correlation of 1.004 ± 0.182  by phosphatases . The acid phosphatase inhibitor MoO42- did not affect 33P uptake calculated as ATP equivalents when γ33P-ATP was applied to excised non-mycorrhizal poplar or beech roots. This result indicates uptake of the intact γ33P-ATP molecule. If this assumption is correct, the labeling position of the 33P should not affect 33P uptake. However, when the αP of ATP was labeled, 33P uptake calculated as ATP equivalents was lower compared to the 33P uptake from γ33P-ATP. Both, phosphatases and ecto-apyrases can cleave the γPi and βPi unit from ATP, thereby contributing to the control of extracellular ATP abundance  The soils differ in pH, ranging from 5.7 to 7.5 for the calcareous Tut site and from 3.6 to 4.3 for the silicate Con forest, as well as in plant available soil Pi (Tut: 0.03 ± 0.01 μmol L-1 and Con: 0.23 ± 0.18 μmol L-1) (for detailed soil description see i cleavage from ATP depending on environmental conditions such as soil Pi, pH, microbial activity and the season. The lower plant available nitrogen and phosphorus in the soil of the Tut compared to the Con forest (13C and 15N uptake from ATP of beech offspring in the present study. Therefore, it is concluded that the processes described above are highly significant in determining the nutrient availability in forest soils.Under field conditions, the uptake of zosphere . Differen forest  coincide13C/15N-ATP uptake. FN performed ATP uptake experiments in the field.CH and HR designed the research project. CH wrote the manuscript and supervised all experiments. US performed most of the experiments. NT performed experiments on the temperature influence on The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Oxide perovskite materials have a long history of being investigated for thermoelectric applications. Compared to the state-of-the-art tin and lead chalcogenides, these perovskite compounds have advantages of low toxicity, eco-friendliness, and high elemental abundance. However, because of low electrical conductivity and high thermal conductivity, the total thermoelectric performance of oxide perovskites is relatively poor. Variety of methods were used to enhance the TE properties of oxide perovskite materials, such as doping, inducing oxygen vacancy, embedding crystal imperfection, and so on. Recently, hybrid perovskite materials started to draw attention for thermoelectric application. Due to the low thermal conductivity and high Seebeck coefficient feature of hybrid perovskites materials, they can be promising thermoelectric materials and hold the potential for the application of wearable energy generators and cooling devices. This mini-review will build a bridge between oxide perovskites and burgeoning hybrid halide perovskites in the research of thermoelectric properties with an aim to further enhance the relevant performance of perovskite-type materials. S2σ is defined as the thermoelectric power factor. The thermoelectric energy conversion efficiency and the maximum cooling temperature for Peltier devices are dependent on the value of ZT 3, includuding In , and raruding In ,95,96,975MnO2.98 . The electively) . The elet 1000 K . A ZT va3, including Mo . Ad. Ad3NH3Pectively . MoreoveExperimentally, the electrical conductivity of hybrid perovskite material was improved by photo or chemical induced doping ,160.3NH3MI3  samples were investigated. The electrical conductivity of CH3NH3PbI3 increased from 10−7 to 10−5 S·cm−1 with a carrier concentration of about 1014 cm−3 from light doping, when changed from dark to light intensities of 220 mW·cm−2. The S at room temperature (295 K) decreased upon illumination, from Sdark = 820 μV·K−1 to Slight = 540 μV·K−1. Additionally, the electrical conductivity of 10−3 S·cm−1 was achieved for CH3NH3SnI3 through impurity doping. The largest ZT values obtained was 0.008 at 295 K for CH3NH3SnI3 , resulting in vacancies of X−  and impurity doping on thermoelectric properties of CH3NH3SnI3  160]. A. A3NH3MI−1·K−2 by self-doping strategies [In short, chemical doping is more effective than light doping strategy in hybrid perovskite materials ,160. Therategies . Hybrid 18SbTe20 (LAST) [The ZT values of the oxide and hybrid perovskite materials from reported experimental data were summarized in 0 (LAST) , and SiG0 (LAST) ), the ZT3) [3) [3) [Oxide perovskites were applied as TE materials in a wide range of temperature (from 100 K–1400 K). The highest ZT value of oxide perovskite materials obtained experimentally so far is ~0.4. The reason for the low ZT value is that oxide perovskite materials have low electrical conductivity and high thermal conductivity. Doping is usually used as a strategy to improve the TE properties, which would normally increase the carrier concentration of materials, thus enhancing the electrical conductivity. In general, A-site doping  ,65,66,673) ,69,76 in3) ,73. On t3) ,107 and 3)  are typi3) [3) ,107,126. [3) [3) . Moreove [3) [3) . Further [3) [3) ,57,58,59 [3) [3) ,63,64, o [3) [3) , but it −1 for CH3NH3PbI3 at 295 K) [−1·K−1 for CH3NH3PbI3 at 295 K) [6H4NH2CuBr2I, a part of the Cu2+ sites could be replaced by Cu+, then the negatively charged Cu(+)’Cu(2+) defects would form in C6H4NH2CuBr2I, resulting in a carrier concentration of ~8.7 × 1020 cm−3 [Hybrid perovskite materials started to draw attention for TE application in recent years. It can be a promising TE because of high Seebeck coefficient (700 μV·Kt 295 K)  and low t 295 K) . To imprt 295 K) ,160. For020 cm−3 . The TE 020 cm−3 , but the020 cm−3 . Accordi020 cm−3 ,161,162


Flame-made TiO2 nanoparticles with tunable polymorphs, including the metastable TiO2-II phase, were prepared and a phase formation mechanism was proposed. 2-type TiO2 or TiO2-II is prepared via a single-step synthesis using a laminar premixed stagnation flame. Three other TiO2 polymorphs, namely anatase, rutile and TiO2-B phases, can also be obtained by tuning the oxygen/fuel ratio. TiO2-II is observed as a mixture with rutile under oxygen-lean flame conditions. To the best of our knowledge, this is the first time that this phase has been identified in flame-synthesised TiO2. The formation of TiO2-II in an atmospheric pressure flame cannot be explained thermodynamically and is hypothesised to be kinetically driven through the oxidation and solid-state transformation of a sub-oxide TiOx2– intermediate. In this scenario, rutile is nucleated from the metastable TiO2-II phase instead of directly from a molten/amorphous state. Mixtures containing three-phase heterojunctions of anatase, rutile, and TiO2-II nanoparticles as prepared here in slightly oxygen-lean flames might be important in photocatalysis due to enhanced electron–hole separation.A metastable “high-pressure” phase known as α-PbO These structural properties in turn control the catalytic activity of the formed TiO2-NPs.2 photocatalysts can be greatly enhanced by making use of anatase/rutile heterojunctions when compared to either pristine phase.2-B2-NPs can be controlled by changing the oxygen/fuel equivalence ratio,2 precursor loading,17A key attraction of flame-made TiO2 polymorphs have been identified, namely particle size, oxidising environment, and temperature. Zhang and Banfieldet al.2 phase transformation. For example, Mao et al.Three important factors affecting the stability of various TiO2 can exist in other, less-studied, polymorphic forms such as TiO2-II (columbite) and TiO2-H (hollandite). TiO2-II, an orthorhombic high-pressure phase of TiO2 isostructural with α-PbO2, is of particular interest in this work. Although pure TiO2-II is only thermodynamically stable at high pressure conditions, experiments and first-principles studies have shown that it can be retained at ambient pressure as a metastable phase.2-II has been found in nature as a mixture with rutile in ultra-high pressure metamorphic minerals.et al.2-II could be stabilised at a three-phase anatase/TiO2-II/rutile junction. They predicted that such a three-phase junction will lead to a synergistic effect in mixed-phase TiO2 catalysts to enhance the electron–hole separation in photocatalysis. However, the role of TiO2-II as a photocatalyst is still inconclusive as the properties are strongly dependent on the synthesis routes used.In addition to the structures mentioned above, crystalline TiO2-II phase at atmospheric pressure via stagnation flame synthesis in addition to the commonly observed phases anatase and rutile, as well as the previously reported metastable phase TiO2-B. The relative composition of these phases is strongly dependent on the oxygen/fuel ratio in the synthesis. The formation of metastable phases and their dependence on the oxidising environment give new insights into the phase formation and transformation mechanisms of TiO2-NPs in flames. The role of phase composition in the TiO2 photocatalytic activity is discussed further by Wu et al.27Herein we demonstrate, for the first time, the formation of the TiO22.12 nanoparticles (NPs) in this study were prepared with a premixed flame stabilised on a stagnation surface. A similar setup has been described in more detail elsewhere.–1 at 150 °C. The nozzle shape induced a flat plug flow of premixed gas that impinged on a stagnation surface. Titanium tetraisopropoxide  was injected into the unburned gas mixture with a syringe pump at 8 ml h–1. The gas line, precursor line, and burner surface were heated to 150 °C to prevent TTIP condensation. During the experiment, the undoped flame was first stabilised for 15 minutes before TTIP was injected for 4 minutes. A shroud flow of 20 slpm N2 gas was used to stabilise the jet flow.The TiO2 sample. The first one was a rotating (300 rpm), circular stainless steel plate with its rotational axis located 10 cm from the burner centerline . The 2θ scan range was 20–90° with a step size of 0.02° and 3 s per step. Zero-background silicon sample holders were used with powder samples pressed to create a dense film.Powder X-ray diffraction (XRD) patterns were recorded with a D8 Advance diffractometer (Bruker) with Cu Kα source . The photoelectrons were collected at an electron take-off angle of 90°. The binding energy shift was corrected by setting the C–C binding energy to 284.8 eV.X-ray photoelectron spectra (XPS) were recorded using a Kratos AXIS Ultra photoelectron spectrometer  fitted with a monochromatic Al K2 suspension in ethanol on TEM grids followed by air-drying.Transmission electron microscopy (TEM) images and selected area electron diffraction (SAED) patterns were acquired with a JEM-2100F FETEM (JEOL Ltd.) with 200 kV accelerating voltage. The TEM samples were prepared by applying a few drops of TiO2.3The XRD patterns were simulated with BRASSkinetics®kinetics® to estimate the flame adiabatic temperature , stoichiometric (φ = 1.00), and fuel-rich (φ = 1.67). 2-B and TiO2-II crystal phases, respectively.The TiOφ = 0.50, e.g., A(1 0 1) at 25.3° and A(2 0 0) at 48.1°. The presence of TiO2-B (ICSD no. 41056) can be identified from the smaller peaks at 2θ = 27–34°, i.e. B(0 0 2), B(1 1 1), B(3 1 0). The simulated XRD pattern with a 100 : 30 intensity ratio of anatase to TiO2-B correctly predicts all the main peaks observed in the experimental pattern. The simulated 2θ-dependent peak broadening due to the crystal size is insufficient to reproduce the broad peaks observed at high angles, e.g., A(2 1 5) and A(2 2 4), suggesting additional peak broadening due to micro-strain.In the lean flame .In the stoichiometric flame  planes of the TiO2-II polymorph (ICSD no. 158778). It is noted that the peak at 25.5° can either be ascribed to A(1 0 1) or II(1 1 0) but the lack of the A(2 0 0) peak at 48° suggests that the latter is the case. In addition, rutile can be identified from the R(1 1 0) peak at 27.5°. Comparison between the simulated and measured XRD patterns shows slightly broader experimental peaks at low 2θ, indicating that the measured crystals are smaller than the 9 nm assumed for the simulated XRD. The difference in peak broadening increases with 2θ, suggesting the presence of additional micro-strain. Furthermore, it can be observed that some peaks such as II(1 1 2) at 44.5° and II(1 1 3) at 62.5° are significantly smaller and/or broader than expected, most likely due to anisotropy in the crystals. It is interesting to note that similar XRD patterns with strong anisotropy in size and strain-induced broadening were observed in rutile and TiO2-II formed through high-energy milling experiments.In the rich flame  planes confirms the presence of the TiO2-B phase  patterns of the agglomerated particles  are cons-B phase .2-B and TiO2-II phases as discussed previously. The lattice spacings measured from the HRTEM images agree with those from the ICSD data to within 5% accuracy (a reasonable uncertainty expected from TEM2-B can be readily identified by the large spacing of B(0 0 1) planes as shown in d = 6.32 Å, ref. dB(001) = 6.24 Å). In d = 2.86 Å, ref. dII(1 1 1[combining macron]) = 2.85 Å) and II(0 1 0)  = 5.50 Å) are marked. The measured interplanar angle is 60.5° (ref. αII(1 1 1[combining macron])/(0 1 0) = 58.8°).φ = 1.67), explaining the anisotropy observed in the powder XRD patterns (While patterns . Unfortu3.3φ < 1.0), stoichiometric (φ = 1), and fuel-rich (φ > 1.0) flames were measured to elucidate the effect of fuel/oxygen equivalence ratio φ on the formed TiO2 polymorphs  with a small amount of rutile. McCormick et al.φ = 0.36 flame followed by annealing at 773 K. This discrepancy likely arises from varying stagnation surface temperature in these studies, leading to different quenching rates compared to this work. For example, Riad et al.2-B phase (27% as a mixture with anatase and amorphous phases) in samples prepared through flame spray pyrolysis (FSP) synthesis. In their study, TiO2-B was preferentially formed under oxygen-rich conditions, consistent with the observations made in this work. As increasing oxygen content leads to shorter residence time in the FSP, it is suggested that the short residence time is responsible for the formation of TiO2-B.Additional XRD patterns of samples prepared in fuel-lean (lymorphs . In the to 400 K . The parφ = 0.90–1.00), anatase and rutile are the two main phases observed. The rutile content increases with increasing equivalence ratio as is evident from the R(1 1 0) peak at 27.5°. An anatase/rutile transition near the stoichiometric point was also observed by Liu et al.et al.10Near the stoichiometric point φ = 0.0–1.00, aφ = 1.00–1.30), the XRD results indicate that the anatase content decreases (i.e. A(2 0 0) peak at 48.1°) while TiO2-II is formed. It is noted that the XRD results obtained by Liu et al.φ = 1.15 and 1.33 also showed a peak at 2θ = 31.5° but it was attributed to the presence of impurity (Ti3O5). As discussed previously, it is suggested that this peak originates from the II(1 1 1) reflection instead. It should also be noted that Liu et al.θ = 31.5° peak in the present study is likely caused by differences in the synthesis conditions such as the deposition time, actual gas flow rates, stagnation surface temperature, or burner nozzle diameter.As the equivalence ratio is increased further , soot is formed together with TiO2 causing the obtained powder to be coloured grey-black. Based on the XRD pattern  peak at 48.1°.For the very-fuel-rich flame condition , the calculated adiabatic flame temperature is 2400–2500 K (2 approx. 2100 K). The particles grow in size through surface growth and coalescence until they approach the stagnation surface where they are rapidly cooled and solidify  and a subsequent solid-state transformation to rutile. Both scenarios are considered here and will be discussed below.In the rich flames where TiO0–2500 K . At thisy Fig. S2. The pre2-II but this typically requires high-pressure conditions of up to 5–9 GPa.2-II in the present study was formed in an atmospheric pressure flame, a solid-state transformation of rutile to TiO2-II is considered unlikely.Numerous studies have documented solid-state transformations of rutile, anatase, or brookite to TiO2-II formation with subsequent phase transformation to rutile, additional XRD patterns ,i.e. oxygen-lean) flames. A preferred growth orientation for TiO2-II was also observed by Grey et al.2 sub-oxide (with composition close to Ti3O5) with boiling sulphuric acid. They proposed that TiO2-II was formed through a solid state transformation of an α-Ti3O5 due to a small long-range misfit between the atomic arrangements of α(1 1 0) and II(1 0 1) layers. Therefore, the results from Aarik et al.et al.3O5 sub-oxide in the formation of TiO2-II crystals. Such sub-oxide species can potentially be formed in the gas phase through clustering of species such as Ti and TiO which have recently been identified as important products in the TTIP decomposition.3O5 and Ti5O7 have been reported to form during plasma synthesis of TiO2 from TiC oxidation.2. With high cooling rates, it is possible that the sub-oxide clusters solidify prior to complete oxidation. In this case, diffusion of lattice oxygen can occur to further oxidise the TiO2 bulk, kinetically favouring the formation of TiO2-II over rutile through a mechanism similar to that described by Grey et al.,43i.e. solid-state transformation driven by the close structural match between (1 1 0) α-Ti3O5 and (1 0 1) TiO2-II planes.A comparison to other ambient pressure TiO2-II through the diffusion of lattice oxygen, some residue of Ti3+ could be expected. Such oxygen deficient titania was reported to be blue in colour,3+ or oxygen vacancies in the particle core does not contradict the absence of a Ti3+ peak in the surface sensitive XPS spectra  flames was elucidated based on the phase composition as a function of collection time and previous reports of TiO2-II formation. It is proposed that rutile is formed through a solid-state transformation of TiO2-II. The TiO2-II formation hereby likely involves some titania sub-oxide intermediate that is subsequently oxidised to stoichiometric TiO2-II. The formation of and transformation between the four different TiO2 polymorphs cannot be explained by the current understanding of TiO2 phase formation and transformation mechanism, and thus requires re-evaluation of the current working hypotheses.Notably, this is the first time that TiOThe authors declare no conflicts of interest.Supplementary informationClick here for additional data file.


Glycine max (L.) Merill). Flowering Locus T (FT) family genes function as key flowering integrators, with flowering-promoting members GmFT2a/GmFT5a and flowering-inhibiting members GmFT4/GmFT1a antagonistically regulating vegetative and reproductive growth. However, to date, the relations between natural variations of FT family genes and the diversity of flowering time and maturity in soybean are not clear. Therefore, we conducted this study to discover natural variations in FT family genes in association with flowering time and maturity.Flowering time and maturity are among the most important adaptive traits in soybean . They were diversified at the genome sequence polymorphism level. GmFT3b and GmFT5b might have experienced breeding selection in the process of soybean domestication and breeding. Haplotype analysis showed that a total of 17 haplotypes had correlative relationships with flowering time and maturity among the 10 FT genes, namely, 1a-H3, 1b-H1, 1b-H6, 1b-H7, 2a-H1, 2a-H3, 2a-H4, 2a-H9, 2b-H3, 2b-H4, 2b-H6, 2b-H7, 3b-H4, 5a-H1, 5a-H2, 5a-H4 and 5b-H1. Based on the association analysis, 38 polymorphic sites had a significant association with flowering time at the level of p < 0.01.Ten FT family genes, which might be involved in soybean adaptation to different environments and have an influence on diverse flowering time and maturity. This study will facilitate the understanding of the roles of FTs in flowering and maturity.Some natural variations exist within the 10 The online version of this article (10.1186/s12864-019-5577-5) contains supplementary material, which is available to authorized users. Soybeans are planted within a wide range of latitudes across the world resulting from the rich diversity of varieties in flowering time and maturity. In North America, soybeans are classified into 13 maturity groups: MG000 to MGX in the ascending order of maturity . MG0000,E1-E10 and J E1-E10 an E1-E10 an [E4 [E9 , 23 and 4 [E9 [J , 25, havlowering , 23, 26.FT, which encodes a putative florigen, is a key integrator gene in the regulation of flowering in Arabidopsis [FT-like genes have been identified in soybean among which GmFT2a and GmFT5a are confirmed as flowering promoters; whereas GmFT1a is divergent as a floral and maturity inhibitor [GmFT2a and GmFT5a coordinately control flowering as flowering integrators and can be regulated by E1, E2, E3 and E4 [FT family genes including GmFT2b, GmFT3a, GmFT3b and GmFT5b promoted flowering [GmFT4 delayed flowering [E1, GmFT1a and GmFT4 expression was up regulated, whereas the expression of GmFT2a and GmFT5a was suppressed [GmFT2c was recently found inserted in a putative transponson in the third intron, and GmFT2d was determined structurally rearranged with some remnant in the genome, indicating that GmFT2 subclades  have different evolutionary trajectories [bidopsis , and thebidopsis , 29. Tennhibitor , 30, 31.3 and E4 , 26, 32.lowering , 34, whelowering . In tranppressed . Currentectories .FT expression in response to diverse environmental signals, which enable flowering and other developmental responses to be seasonally timed [FT promoter sequences are confirmed responsible for diverse flowering time in Arabidopsis and also in rice [GmFT2a in soybean has high polymorphism, and some SNPs in the promoter region are associated with flowering time and photoperiod sensitivity [GmFT2a has a recessive allele for delayed flowering, which contains a Ty1/copia-like retrotransposon SORE-1 inserted in the first intron that attenuates GmFT2a expression by its allele-specific transcriptional repression [FT homologs have functional divergence in soybean [FT genes play roles that influence the flowering and maturation time through an interactive balance when receiving different environmental signals. Therefore, whether their natural variations are linked to flowering time and maturity requires investigation.In plants, multiple regulation pathways regulate ly timed . Differe in rice , 39. Thesitivity . GmFT2a pression . Critica soybean , 31, 32,FT family genes in all varieties were analyzed. Further analysis of FT haplotypes and their natural variations associated with flowering time and maturity was conducted. The results suggested that some natural variations of the 10 soybean FT genes existed in soybeans of different MGs and that these variations were related to soybean flowering time and maturity. Our findings will elucidate the roles of soybean FT family genes in flowering and maturity.In this study, 127 soybean varieties covering all known 14 maturity groups (MG) from MG0000 to MGX were selected and grown for phenotypic and genotypic identification. The sequence polymorphisms of 10 soybean Glycine max) varieties covering all known 14 maturity groups were collected for the experiment  method . SeventeFT family genes in soybean reference genome in the Phytozome database  were used as references. We chose polymorphic sites with minor allele frequency above 4 varieties and imported them into Tassel 5 to analyze the linkage disequilibrium (LD) [FT family genes were analyzed for association with flowering time and maturity. For those polymorphic sites that only appeared in these haplotypes with relation to flowering time and maturity, we defined them as candidate polymorphic sites  [p < 0.01 is selected as in our previous study on GmFT2a promoter variations [FT family genes is listed in Additional file We used Seqman 1.0 for sequence assembly . The seqium (LD) . We condesidual) , and theriations . The polExcept for some late-maturing varieties that did not reach R7 before the end of the experiment, the flowering time Ve-R1) varied from 22.2 to 120.5 days, with a span of 98.3 days, and the maturity (Ve-R7) varied from 61.4 to 155.7 days, with a span of 94.3 days  to 129 (GmFT1a) (Table w of nucleotide diversity parameters extended from 0.00039 (GmFT3a) to 0.01102 (GmFT1b) and from 0.00080 (GmFT3a) to 0.00787 (GmFT1b), respectively , including 105 SNPs and 24 Indels  was 0.00521, lower than that of GmFT1b (π = 0.01102) and GmFT2b (π = 0.00588)  reached the peak of 0.01102 among the 10 soybean FT family genes, whereas GmFT3a had the lowest π of 0.00039, with only 11 polymorphic sites . GmFT1b had a comparatively high π of SNPs of 0.00845; nevertheless, π of SNPs of GmFT4 was much lower at 0.00009 , except that the SNPs of GmFT3b exhibited a significant level (p < 0.01)  has a large contribution to haplotype analysis and eases the identification of some SNPs and Indels in relation to phenotypes. Soybean 1b Figs.  and 5. Ged Figs.  and 12. tes Fig. .Fig. 4GmFT family genes in the 127 varieties used in this study. Haplotypes of the 10 soybean FT family genes, variation types and polymorphic sites used for composing the haplotype are listed in Additional file GmFT3a, GmFT4 and GmFT6 did not exhibit any apparent associations , GmFT4 (θw = 0.00029), GmFT5a (θw = 0.00084) and GmFT6 (θw = 0.0008) should be conserved after strong selection. GmFT1a (θw = 0.0035), GmFT1b (θw = 0.00569), GmFT2a (θw = 0.00133), GmFT2b (θw = 0.00253), GmFT3b (θw = 0.00169) and GmFT1b (θw = 0.00144) might have experienced weak selection pressure and had high polymorphism in long-term evolution. This finding also reflected that the selected population had a wide genetic base. Because the ten FT family genes are diverse in the control of flowering and maturity, these genes could play concerto roles to elaborately regulate flowering and maturing and help soybean to adapt to greatly diverse environments and breeding selections. Neutral test indicated that GmFT3b and GmFT5b had some selected signals and might have experienced breeding selection. The polymorphic sites of GmFT3b and GmFT5b had a high proportion of strong LD ; GmFT2a and GmFT2b, which are located in Chr16, had the closest evolutionary relationship (34 kb apart); and GmFT3b and GmFT5b, which are located in Chr19, were only 16 kb apart. In the long process of natural selection and domestication, these short distances might result in linkage disequilibrium. As a result of certain selection effects, some polymorphic sites might have been inherited together in the adaptation to some environments.Haplotype combinations of soybean p < 0.01. These polymorphic sites may be subjected to natural and artificial selection in adaptation to diverse environments and have effects on pre- and postflowering photoperiod responses. An 8 bp fragment insertion in Intron3, close to exon4, appeared in 1b-H1 and 1b-H7 collectively and might have an effect on flowering time. This insertion might regulate GmFT1b expression directly or indirectly through changing the alternative splicing of GmFT1b. GmFT2a and GmFT5a coordinately control flowering and enable the adaptation of soybean to a wide range of photoperiodic environments [GmFT2a, namely, s4645, appeared in 2a-H9 and was distributed in the varieties with their maturity groups belonging to MGIX and MGX. The change at the site resulted in an amino acid substitution from nonpolar G169 to acidic D169. Notably, the flowering inhibitor GmFT1a has an amino acid of D169 in the same location, indicating that this amino acid substitution might have an important role in the function of GmFT2a. A 49 bp fragment deleted in the 3′ UTR appeared in 5a-H4 and 5a-H6 and was distributed in the varieties of MG0000, MG000 and MG00. They may affect the function of GmFT2a and GmFT5a in regulating soybean flowering.We chose polymorphic sites that only existed in the haplotypes with relations to flowering time and maturity. Further association analysis was conducted with GLM, and 38 polymorphic sites showed a significant association with flowering time at the level of ronments . A nonsyFT family genes exhibited variation in the aspect of genome sequence polymorphism. GmFT3b and GmFT5b had some selected signals and might have experienced breeding selection in the process of natural selection and domestication. By analyzing the haplotypes, we found that 1a-H3, 1b-H1, 1b-H6, 1b-H7, 2a-H1, 2a-H3, 2a-H4, 2a-H9, 2b-H3, 2b-H4, 2b-H6, 2b-H7, 3b-H4, 5a-H1, 5a-H2, 5a-H4 and 5b-H1 showed correlative relationships with flowering time and maturity; whereas GmFT3a, GmFT4 and GmFT6 did not have any apparent connections. Thirty-eight polymorphic sites showed a significant association with flowering time at the level of p < 0.01.Soybean varieties from the earliest maturing MG0000 to the latest maturing MGX showed high diversity in flowering time and maturity. The 10 soybean Additional file 1:Table S1. Varieties and their respective maturity group and origin. (DOCX 17 kb)Additional file 2:Table S2. Sequence of primers for the 10 FT family genes in soybean. (DOCX 15 kb)Additional file 3:Table S3. PCR reaction conditions for different templates of the FT family genes in soybean. (DOCX 13 kb)Additional file 4:Table S4. General linear model association of polymorphic sites of soybean FT family genes with relation to flowering time and growth duration. (DOCX 23 kb)Additional file 5:Table S5. Polymorphic sites of the 10 soybean FT family genes from the present study. (DOCX 56 kb)Additional file 6:Table S6. Flowering time (Ve-R1) and growth duration (Ve-R7 and Ve-R8) data in Beijing, China, in 2015. (DOCX 21 kb)Additional file 7:Table S7. Haplotypes of the 10 soybean FT family genes in 127 varieties covering 14 maturity groups. (DOCX 38 kb)Additional file 8:Table S8. Polymorphic sites used for defining the haplotypes of the 10 soybean FT family genes. (DOCX 14 kb)Additional file 9:Table S9. Polymorphic site features used for defining the haplotypes of the 10 soybean FT family genes. (DOCX 16 kb)Additional file 10:Table S10. Different types of soybean FT proteins and their haplotype distribution. (DOCX 18 kb)


Engineering of fragment crystallizable (Fc) domains of therapeutic immunoglobulin (IgG) antibodies to eliminate their immune effector functions while retaining other Fc characteristics has numerous applications, including blocking antigens on Fc gamma (Fcγ) receptor-expressing immune cells. We previously reported on a human IgG2 variant termed IgG2σ with barely detectable activity in antibody-dependent cellular cytotoxicity, phagocytosis, complement activity, and Fcγ receptor binding assays. Here, we extend that work to IgG1 and IgG4 antibodies, alternative subtypes which may offer advantages over IgG2 antibodies. In several in vitro and in vivo assays, the IgG1σ and IgG4σ variants showed equal or even lower Fc-related activities than the corresponding IgG2σ variant. In particular, IgG1σ and IgG4σ variants demonstrate complete lack of effector function as measured by antibody-dependent cellular cytotoxicity, complement-dependent cytotoxicity, antibody-dependent cellular phagocytosis, and in vivo T-cell activation. The IgG1σ and IgG4σ variants showed acceptable solubility and stability, and typical human IgG1 pharmacokinetic profiles in human FcRn-transgenic mice and cynomolgus monkeys. In silico T-cell epitope analyses predict a lack of immunogenicity in humans. Finally, crystal structures and simulations of the IgG1σ and IgG4σ Fc domains can explain the lack of Fc-mediated immune functions. These variants show promise for use in those therapeutic antibodies and Fc fusions for which the Fc domain should be immunologically “silent”. The biological functionality of therapeutic antibodies depends on the interactions of two regions of the protein with components of its external environment: the antigen binding region (Fab) interacting with an antigen, and the fragment crystallizable (Fc) region interacting with components of the immune system. The Fc region of the immunoglobulin (IgG) antibody, which is the focus of this study, can have interactions with Fc gamma receptors (FcγR) and first subcomponent of the C1 complex (C1q) that mediate antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), antibody-dependent cellular phagocytosis (ADCP), induction of secretion of mediators, endocytosis of opsonized particles, as well as modulation of tissue and serum half-life through interaction with the neonatal Fc receptor (FcRn) . NumerouFc-mediated effector functions are best avoided for some applications such as systemic neutralization of cytokines, targeting cell surface antigens on immune cells, or when engineering bispecific molecules to bring target diseased cells within proximity of effector immune cells to provide a more specific immune receptor engagement ,8,9. In 2 fragments can be generated; however, such molecules have shorter half-lives in patient sera. Chemical modifications can extend the half-life of such molecules, but can also bring potential risks with toxicities [When an antibody with no effector function is required, there are different approaches one may take to generate a molecule with the desired properties. Unfortunately, results of some strategies often come with liabilities to the molecular profile. For instance, Fab or F(ab′)xicities . Anotherxicities ,14,15,16xicities .An example of this approach is illustrated in the first marketed therapeutic antibody  in which two mutations in the lower hinge  were introduced to mitigate the induction of cytokine storm ,19. AlsoH2) domain [Although huIgG4 has weak binding affinity to most FcγRs except for the high affinity receptor FcγRI, it does retain the ability to induce phagocytosis by macrophages  and possibly activate monocytes when in an immune complex due to activating FcγRs on specific immune cells. Recent additional approaches to generate antibodies with no effector function have included disruption of proline sandwich motifs , and inc) domain . BecauseWe describe here the functional and structural characteristics of three novel silent Fc designs: huIgG1 sigma (IgG1σ), which is a variant of huIgG1, and huIgG4 sigma1 (IgG4σ1) and huIgG4 sigma2 (IgG4σ2), which are variants of huIgG4. The effector functions of these silent Fc variants are compared to those of previously described constructs such as huIgG1 L234A/L235A (AA) , huIgG4 H2 regions [Silent human IgG Fcs were designed by uniquely combining the sequence from previously characterized variants having mutations in the hinge and C regions . Mutatio regions ,38.H2 domains  antibodies are applied as serial dilutions. There is a reduction in  signal when competition takes place. To provide an avidity component comparable to that created by multiple antibodies bound to a cell surface, the test antibodies were cross-linked with anti-F(ab′)Competitive binding analyses of Ab1 (anti-tumor necrosis factor (TNF)α huIgG1) samples to the different FcγRs are shown in When the Fc region of an antibody binds to FcγR on immune cells, cytotoxic factors are released causing ADCC and death of target cells, whose membrane-surface antigens have been bound by the specific antibody. To compare cytotoxic potential of the silent Fc variants, K2 cells  expressing the Δ1–12 variant of human TNFα were used as target cells and human peripheral mononuclear cells (PBMCs) were the immune effector cells in ADCC assays . Figure Binding of the Fc region to FcγRs on certain immune cells such as macrophages can cause cell death by phagocytosis. ADCP relies on macrophages to bind and devour target cells following antibody binding. To further evaluate the lack of effector function with the silent Fc variants, ADCP flow cytometric assays were performed using macrophages derived from primary human monocytes as effector cells and K2 cells as target cells. Macrophages and K2 cells were labeled with separate fluorescent dyes and co-cultured for 5 h in the presence of different concentrations of test antibody. Quantitation of antibody-opsonized target cells was measured by dual-label flow cytometry. Representative data from two experiments are shown in Complement-dependent cell killing occurs when the Fc portion recruits serum complement proteins to the cell bound by the specific antibody; leading to induction of a membrane attack complex and target cell lysis. To determine whether the Fc variants have complement activation capability, CDC assays were performed using K2 target cells, rabbit complement and a panel of Ab1 huIgG Fc variants. As shown in Interaction with neonatal Fc receptor (FcRn) is a critical factor that contributes to sustaining circulating antibody half-life , thus beIn vivo, the Fc and FcγR interaction can be evaluated and detected by T cell activation . Human FAn initial study was done to confirm that T cell activation in the FcγR-hu mice is due to FcγR-Fc binding. Three strains of mice with different gene dosages were used: the 5-transgene FcγR-humanized mice, mice hemizygous for the 5 transgenes, and FcRα chain null mice (FcγR knockouts). Ab4 huIgG1 and Ab4 huIgG1σ were injected into the intraperitoneal cavity . Approximately 24 h later, mice were euthanized and their spleens removed for T cell activation analyses by flow cytometry. Results demonstrated the dependence on the presence of human FcγR, since very little or no T cell activation using huIgG1 was detected in the null mice, an intermediate level of activation was detected in hemizygous mice, and the most robust activation was noted in the homozygous mice A. Low to2 fragment and a non-T cell binding Ab1 huIgG1. Splenocytes from wild-type IgG (Ab4 huIgG1), low fucose IgG1 (Ab4 huG1 LoF with high affinity binding to CD16), and phorbol myristate acetate treated mice, collectively showed a robust CD69 signal  compared to un-activated splenocytes (25%) from phosphate- buffered saline (PBS) treated mice. Ab4 F(ab′)2 gave a substantial unexpected signal (46%) which suggested that T cells may have a low level of activation merely by bivalent CD3 binding without FcγR engagement. Ab4 huIgG1 induced a higher CD69 activation level than Ab1 huIgG1 WT because it has both CD3 and FcγR engagement. The dashed line in Comparison of CD69 activation results (expressed as a percentage of CD3ε cells) are shown in 2 caused a higher CD25 activation than expected (such as in the case of CD69 activation) which may be due to bivalent CD3 binding, clustering, and signaling. Ab4 huIgG1σ, Ab4 huIgG2σ, Ab4 huG4σ1 and Ab4 huG4σ2 induced minimal CD25 activation. These in vivo CD69 and CD25 results indicated that binding of IgG1σ, IgG2σ, IgG4σ1 and IgG4σ2 variants did not activate T cells compared to T cells from mice treated with huIgG WT or huIgG4 PAA.CD25 activation levels of splenic T cells (expressed as a percent of CD8+ cells) from mice treated with the silent Fc variants are shown in The B16 metastatic melanoma syngeneic model has been used to assess Fc and FcγR interactions. B16F10 mouse melanoma tumor cells can stimulate lung tumor metastasis, and murine TA99 antibody which targets the gp75 antigen on B16F10 cells, can inhibit tumor cell growth in mice ,45. AlsoThis B16 metastatic tumor model was used to test the effect of a huIgG1σ version of Ab5 (anti-grp75) on lung tumor metastasis in FcγR-hu mice. These mice treated with different versions of Ab5: huIgG1, huIgG1 LoF  and mIgG2a (mouse Ab as positive control) showed a significant reduction in lung tumor metastasis compared to the mice treated with PBS . Lung mePK studies were done to determine whether normal antibody half-life would be altered using antibodies with silent sigma Fc mutations compared to the wild-type huIgG. Human FcRn transgenic mice  were intravenously-injected once with 2 mg/kg doses of the test antibody. Blood was collected at various times up to 35 days post-injection and sera prepared by standard procedure. A Meso Scale Discovery (MSD) assay using anti-human IgG specific reagents was employed for quantitating human IgG in the mouse serum samples. Terminal half-life values were obtained using a one-compartment linear fit using Prism 6.0 software .1/2 = 5.5 ± 0.5 days) shows a 2-fold shorter half-life than the huIgG1 (t1/2 = 11.4 ± 1.7 days). However, PK values for the other Fc variants: huIgG1 AA (t1/2 = 7.5 ± 1.1 days), huIgG4 PAA (t1/2 = 7.8 ± 3.2 days), huIgG4σ2 (t1/2 = 7.9 ± 2.1 days), huIgG4σ1 (t1/2 = 8.9 ± 1.3 days), huIgG1σ (t1/2 = 9.6 days), huIgG2 (t1/2 = 11.2 ± 0.8 days) and huIgG1 FES (t1/2 = 9.7 ± 1.1 days) are not significantly different from that of huIgG1. These results indicate that the silent Fc mutations do not alter the PK compared to normal huIgG1, except for huIgG2σ which showed a reduction in half-life.HuIgG serum concentrations over time for the mice are shown in 1/2 = 11.7 ± 0.95 days) and its huIgG2σ counterpart (t1/2 = 10.3 ± 3.1 days); and for Ab2 × Ab5 bispecific (anti-RSV × anti-gp75) huIgG1σ (t1/2 = 5.6 ± 0.5 days) and its huIgG2σ counterpart (t1/2 = 5.2 ± 0.1 days) ) and glycoprotein gp120 of HIV envelope, huIgG1). Male cynomolgus monkeys  were intravenously injected once with 1.5 mg/kg of the test antibody. Blood samples were collected up to 21 days or 28 days, and PK analyzed as described. Although performed in separate studies, similar half-life values are observed for Ab3 huIgG1σ (t.1 days) B. Consid.1 days) , these mTM v2.7 human leukocyte antigen (HLA) class II-peptide binding prediction software  to predict potential immunogenicity in humans. These analyses generate IScores, which are weighted, population-relevant values that enable separation of individual 9-mer agretopes into groups by predicted immunogenic risk (PIR). Higher IScores indicate a higher PIR. Areas of sequence in the Fc variants which are identical to that of huIgG1 WT Fc sequence are excluded by the application of a tolerance threshold. IScores are averaged across all HLA-class II loci for each sequence of interest to create a single average IScore value. As shown in IScores of the tested variant sequences are like those of their respective wild-type isotype controls. The greatest deviation from wild-type Fc results is a reduction of 1.2% observed with huIgG4 PAA. However, differences observed between the tested variants are inconsequential because all produce average IScores of less than 5%. IScores of less than 10% are considered to indicate very low risk of immunogenicity with a prediction that less than 10% of the U.S. population would have at least one allele predicted to bind a 9-mer located within the tested sequence. A further breakdown of the IScore data into number of predicted agretopes, as well as number of low, medium and high PIR agretopes for each sequence of interest is shown in To assess the potential immunogenicity of huIgG1 and huIgG4 sigma variants, a T cell epitope analysis was done using the ImmunoFilterA panel of biophysical assays was conducted on Abs with Fc variations to assess potential problems in developability that might impact the development and commercial manufacturing of therapeutics. These studies included assessing antibody thermal stability, the stability of highly concentrated samples over time, and stability at low pH. The results are described in the sub-sections below.H2 domain) transitions, but huIgG1 FES, huIgG1σ, huIgG2σ, huIgG4 PAA, huIgG4σ1 and huIgG4σ2 show lower Tm1 transition values, suggesting reduced stability of its CH2 domain attributable to the presence of mutations  was used to assess the thermal stability of Ab1 Fc variants. A summary which includes the mid-point of denaturation (Tm) for the transitions is shown in utations . Ab1 huIutations . The Tm2nd 83 °C .Colloidal stability was evaluated over a temperature range by static light scattering (SLS) at two different wavelengths . The scaSamples were concentrated from their initial concentration of 1–2 mg/mL to 40–50 mg/mL and stored at 4 °C. To obtain a more complete picture of the stability of the Fc variants, protein melting points and aggregation onset temperature were measured for each by a combination of intrinsic fluorescence and static light scattering. h), percent polydispersity (% Pd) values, and percent mass comparison between the release lot and the low pH treated samples. All samples yielded a peak with an average radius of 5–7 nm, average % Pd of less than 15%, and an average % Mass of greater than 99%. These features are indicative of a mono-dispersed species for the antibody variants.To assess whether the Fc variants are stable after low pH treatment for viral load reduction in manufacturing, antibody samples were treated at pH 3.5 for 6 h and dialyzed back to pH 7.4 in PBS. Dynamic light scattering analyses were performed to quantitate presence of soluble aggregates and sub-visible particles. 12121, and had similar unit cell parameters. This isomorphism reflects a similarity in Fc packing within the crystal lattice where one intact Fc dimer is present in the asymmetric unit. In this crystal form, the A chain CH2 domain is less ordered as evidenced by the reduced quality of the electron density map and elevated temperature factors of the atoms. The reason for this is that the CH2 domains of the A and B chains have a difference in the crystal contacts. For the three Fc structures reported here and excluding the glycan contributions, the B chain has an average of 2082 ± 186 Å2 of buried surface area through interaction with symmetry related Fc molecules compared with only 1477 ± 181 Å2 of buried surface area for chain A. Consequently, for the three structures, the average B-factor calculated over residues 238–339 and glycan residues for chains B and A was 37.6 ± 4.5 Å2 and 54.1 ± 1.8 Å2, respectively.To assess the impact of the silencing mutations on the structure of the Fc, crystal structures of the Fc fragments of huIgG1σ, huIgG4σ1, and huIgG4σ2 were determined to resolutions of 1.90 Å, 1.90 Å, and 1.85 Å, respectively. Data collection and refinement statistics are summarized in The huIgG1σ Fc structure includes residues G236-S444 and A235-S444 for chains A and B, respectively, which are only those observed in the electron density map . The strThe structure of huIgG4σ1 Fc includes residues A237-L443 and G236-S444 for chains A and B, respectively, that were modeled into the visible electron density. For the huIgG4σ2 Fc structure, residues S238-L443 and E233-S444 were modeled for the respective chains . The lowPrevious crystal structures of huIgG1, huIgG2 and huIgG4 have shown two distinct conformations for the BC loop (residues 267 to 273) and the FG loop (residues 322 to 332) that seem to correlate with the ability to bind to FcγRs. Denoting the two conformations as ‘flipped-in’ and ‘flipped-out’, both loops are in the flipped-in conformation in structures of wild-type huIgG1 Fc (PDB 3AVE ) and huIα RMSD) to the flipped-in conformation for 57.1% of the simulation as compared to 44.4% in the case of huIgG1σ where the loop was found to be substantially more flexible  and huIflexible . NotablyIn addition to the FG loop, the simulations show that the BC loop is also more flexible in huIgG1σ than in the wild-type huIgG1 . In the HuIgG1 and huIgG1σ differ at position 268  in the BC loop, and at positions 330  and 331  in the FG loop. To investigate the impact of these mutations on the flexibility of the two loops, two additional simulations were performed after modelling the mutations in the huIgG1 wild-type crystal structure. One simulation (referred to as huIgG1-H268A) contained only the BC loop mutation, and the other (referred to as huIgG1-SS) contained only the two FG loop mutations. The flexibility of both loops in the huIgG1-H268A simulation was nearly identical to that in the wild-type simulation while the flexibility in huIgG1-SS simulation was closer to that in the huIgG1σ simulation . The mutMany therapeutic antibodies and Fc fusion proteins employ Fc activity as part of their mechanisms of action. Fc engagement with FcγR can activate myeloid cell and NK cell activity as well as the generation of reactive species that induce apoptosis and release of inflammatory cytokines which are important for eliminating unwanted target cells  ,53,54,55Several antibody panels  with Fc silent mutations were constructed, purified, and tested in vitro for Fc binding and function. The huIgG1σ, huIgG4σ1, and huIgG4σ2 antibodies tested as immune complexes had minimal binding to FcγRI, FcγRIIa, FcγRIIb, and FcγRIIIa, compared to huIgG1 WT using concentration ranges that could be found in clinical dosing. Since the TNFα on target cells and FcγRIII on effector cells are both multivalent, the engagement of IgG1 molecules to both cells would involve avidity. Thus, ADCC activity and the induction of immune effector function which depend on avidity and could occur at lower concentrations compared with monovalent antigen  . TherefoIn vivo studies emphasize again the lack of immune functionality with the huIgG sigma variants. Results using FcγR-hu mice demonstrated significantly lower levels of T cell dependent activation (CD69 and CD25 upregulation) with the sigma variants compared to huIgG1 AA, IgG1 FES, and huIgG4 PAA. In vivo half-life in a transgenic mouse model of human FcRn and in cynomolgus monkeys indicated that the silent mutations did not alter PK properties compared to normal huIgG. In addition, potential immunogenicity, evaluated in silico (for protein sequence “hot spots” that favor immune response initiation) predicts minimal immunogenic risk for these silent Fc variants. Although ex vivo immune responses measured by T cell proliferation and IL-2 secretion, were not tested here, the huIgG2σ (reported previously) shows relatively low risk of clinical immunogenicity as determined by comparing the frequency and magnitude of ex vivo T cell responses . The IgGH2 and hinge regions. However, there is little evidence that reduced thermal stability of these magnitudes translates into developability issues or in vivo stability issues. All Fc variants are stable for 4 weeks when concentrated (to 40–50 mg/ML), or subjected to low pH stress. Also, there are no additional post-translational modifications or significant changes in solution particle size associated with these Fc mutations. The impact of huIgG1σ, huIgG4σ1 and huIgG4σ2 mutations on biophysical properties was assessed for their effect on manufacturing. Thermal stress for all the sigma variants compared to their wild-type versions, suggests lower thermal stabilities in the CH2 domain FG loop between two tryptophan side chains in the Fc receptor is a conserved feature of huIgG Fc interaction with FcγRI , FcγRIIg., 4W4O ), and Fcg., 4W4O ). The reg., 4W4O ) as flipH2 domains from crystal structures of huIgG2 Fc and huIgG2σ Fc suggested that residue P271 in a flipped-out BC loop could clash with K326 in a flipped-in FG loop  mutations dramatically increase the flexibility of the FG loop even in the absence of the H268A mutation. Also, just the H268A mutation, in the absence of the FG loop mutations, only marginally increases the flexibility of the two loops relative to wild-type.It has been proposed that, in the case of huIgG2σ, the H268A mutation in the BC loop abolished an electrostatic interaction with E294 in the DE loop resulting in the observed flipped conformation of the BC loop which in turn triggered the flip of the FG loop [ FG loop . A simil FG loop . Davies PDB 4D2N , a strucThe results of our in vitro and in vivo studies demonstrated that huIgG4σ1 and huIgG4σ2 are more silent than huIgG4 PAA as discussed above. Given that all the mutations for these variants were localized to the lower hinge region  and thatIn summary, antibodies with huIgG1σ, huIgG4σ1, and huIgG4σ2 Fc regions have demonstrated minimal FcγR interactions by in vitro and in vivo methods. Immunogenicity, developability, and PK risks of these variants have been evaluated and determined to be comparable to that the huIgG1 WT  and huIgH2 domain [® technology  [Ab1 is a huIgG1 kappa antibody specific for tumor necrosis factor alpha (TNFα); Ab2 is a huIgG1 kappa monoclonal antibody specific for F glycoprotein of Respiratory Syncytial Virus (RSV); Ab3 is a huIgG1 bispecific antibody targeted against RSV and glycoprotein gp120 of the human immunodeficiency virus (HIV) envelope; Ab4 is an anti-mouse CD3ε-chain Ab, 145-2C11, and Ab5 is TA99, an anti-gp75 antibody which targets gp75 antigen on B16F10 melanoma cells . H435A i2 domain . All antDenmark) , and con2, in Iscove’s Modified Dulbecco’s Medium (IMDM) with GlutaMAX and 5% (v/v) heat-inactivated fetal bovine serum (FBS), 1× non-essential amino acids (NEAA), 1× sodium pyruvate, 0.5 μg/mL mycophenolic acid, 2.5 μg/mL hypoxanthine, and 50 μg/mL xanthine (MHX). Media components were purchased from Life Technologies  and the MHX components from Sigma .K2 cells are Sp2/0 mouse myeloma cells which express a mutant, transmembrane form of human TNFα ,63. K2 cv/v) FBS, 1× NEAA, and 1× sodium pyruvate.B16F10 mouse melanoma cell line was obtained from the American Tissue Culture Collection. Cells were cultured in RPMI 1640 supplemented with 10% (2 anti-huIgG F(ab′)2-specific fragment  in 1:1 molar ratio with the test huIgGs. Cross-linked test antibodies  were added to Corning white half-well 96-well assay plates  at the designated concentrations in competition with biotin-labeled huIgG Fc fragment (biot-Fc) at either 1 μg/mL  or 5 μg/mL (for FcγRIIIa assays). Biotinylated Fc fragment was used to prevent binding to the biot-Fc by the test article cross-linker described above. FcγRs as specified were added to a 200 ng/mL final concentration. Nickel chelate acceptor beads were added; followed by streptavidin donor beads. Plates were covered with foil adhesive plate sealers to protect from light, and placed on an orbital plate shaker with gentle shaking for 45 min at room temperature (RT). Subsequently, plates were read on the EnVision multi-label plate reader (Perkin-Elmer), and data plotted with GraphPad Prism v6.0 software .Binding of Ab1 variants to human FcγRs was assessed using an AlphaScreen  bead assay in a competition binding format. FcγRs were purchased from R&D Systems or Sino Biological Inc. . Since the outcome for huIgG with a silent Fc in a FcγR binding assay may be a negative result, assay sensitivity was increased by introducing avidity to the test samples via cross-linking. This was achieved using a goat F(ab′)w/v) Tween 20, and then incubated with blocking reagent  bovine serum albumin, 0.001% (w/v) Tween-20, pH 6.0, 10% (v/v) ChemiBlocker from Sigma-Aldrich, St. Louis, MO, USA. Plates were washed and serial dilutions of competitor test antibody in blocking reagent were added to plates in the presence of a fixed 4 μg/mL concentration of an indicator antibody (a biotinylated huIgG1). Plates were incubated at RT for 1 h, washed 3 times, and then incubated with a 1:10,000 dilution of HRP (Jackson ImmunoResearch Laboratories) at room temperature (RT) for 30 min to bind biotinylated antibody. Plates were washed and bound streptavidin-HRP was detected by adding TMB peroxidase substrate . Color development was stopped by addition of 0.5 M HCl. Optical densities were determined with a SpectraMax Plus384 plate reader  at 450 nm wavelength, and data plotted with GraphPad Prism v6.0 software.A competitive binding assay was used to assess relative affinities of different antibody samples to recombinant human FcRn (in-house expressed with transmembrane and cytoplasmic domains of FcRn replaced with a poly-histidine affinity tag). Ninety-six-well copper-coated plates (Thermo Scientific) were used to capture FcRn-His6 at 4 μg/mL in PBS, after which plates were washed with 0.15 M NaCl, 0.02%  for culturing overnight at 37 °C, 5% CO2.Peripheral mononuclear cells (PBMCs) were isolated from heparinized blood from in-house donors. Eighty (80) mL of blood from 2 donors were typically used per experiment. Blood was diluted 2-fold with PBS and 30 mL was layered over 15 mL of Ficoll-Paque  in a 50 mL conical centrifuge tube. Tubes were centrifuged at 400× ® EuTDA, PerkinElmer) for 25 min at 37 °C, washed 3 times in culture medium  heat-inactivated Fetal Bovine serum (FBS), 1× non-essential amino acids (NEAA), 1× sodium pyruvate, 1×, penicillin-streptomycin; all from Life Technologies and resuspended in culture medium. Target cells  were added to test antibody (100 μL) in 96-well U-bottom plates, then effector cells  were added. Plates were centrifuged at 200× g for 3 min, incubated at 37 °C for 2 h, and then centrifuged again at 200× g for 3 min. A total of 20 μL of supernatant was removed per well, and cell lysis was measured by the addition of 200 μL of the DELFIA Europium-based reagent (PerkinElmer). Fluorescence was measured using an Envision 2101 Reader (PerkinElmer). Data were normalized to maximal cytotoxicity with 0.7% (w/v) Triton X-100  or 10% (v/v) Lysis Buffer (DELFIA® PerkinElmer) and minimal lysis using target cells in the absence of any Ab. Samples were tested in duplicate. Percent specific lysis was calculated to be  divided by  × 100. Data were fit to a sigmoidal dose-response model using GraphPad Prism v6.0 software.K2 cells were used as target cells at a ratio of 50 effector cells per 1 target cell. Target cells were pre-labeled with BATDA (bis (acetoxymethyl) 2,2′:6′,2′′-terpyridine-6,′′-dicarboxylate, DELFIAv/v) heat-inactivated FBS media without phenol red were incubated for 5 h with a macrophage to K2 cell ratio of 1 effector to 1 target cell in the presence of test antibody. Two-color flow cytometry analyses were performed with a MACSQuant Flow Cytometer (Miltenyi) using optimal compensation in the B1 (PKH67) and B2 (PKH26) channels and gating on single cells. Dual-labeled cells (PKH26+/PKH67+) were considered to represent phagocytosis of K2 target cells by macrophages. Percent ADCP or phagocytosis of target cells was calculated to be 100× number of dual-labeled cells (macrophage + target) divided by the total number of target cells in the population (phagocytosed + non-phagocytosed) after >50,000 cell counts. The percent specific ADCP was obtained by subtracting from each sample the background value (macrophage + target incubated without Ab) [Monocytes were isolated from human PBMCs using a Monocyte Isolation Kit  and differentiated into macrophages for 1 week by culturing with 10 ng/mL recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) and 10 ng/mL recombinant human IL-4  in IMDM with Glutamax, 10% heat-inactivated FBS, 1× NEAA, 1× sodium pyruvate, 1× penicillin-streptomycin. Macrophages were labeled with PKH26  and K2 cells were labeled with PKH67 (Sigma). Labeled cells in IMDM-10%  heat-inactivated PBS, 1× NEAA, 1× sodium pyruvate, 1x penicillin-streptomycin. An additional 50 μL was added to the wells with or without test Abs and plates were incubated at 37 °C for 2 h. A total of 50 μL of 10% (w/v) rabbit complement  was added to the wells, and plates were incubated for 20 min at 37 °C. All samples were performed in triplicate. The plates were centrifuged at 200× g for 3 min, 50 μL of supernatant was removed to separate plates, and CDC was measured with a LDH cytotoxicity detection kit . Absorbance was measured using a Spectra Max Plus 384 (PerkinElmer). Data were fit to a sigmoidal dose-response model using GraphPad Prism v6.0 software. Maximal cytotoxicity was obtained with Triton X-100 (Sigma-Aldrich) and spontaneous release with cells and complement alone. Specific cell lysis was calculated as follows: Cytotoxicity (%) = 100 ×  of sample − OD of spontaneous release)/.K2 cells were used as target cells for CDC assays. A total of 50 μL of cells was added to wells of a 96-well plates for a final concentration of 8 × 10The FcγR-humanized (FcγR-hu) mice used in the T-cell activations studies express the different human FcγRs: CD16a (FcγRIIIa), CD16b (FcγRIIIb), CD32a (FcγRIIa), CD32b (FcγRIIb) and CD64 (FcγR1) and their endogenous mouse FcγRs have been inactivated . Three stmLDcrTg(FCGRT)32Dcr from The Jackson Laboratory) have their endogenous mouse FcRn α gene knocked out and are transgenic with the human FcRn α gene under the control of the native human gene promoter [Human FcRn transgenic animals (8–10 weeks old) used in PK studies were derived from C57BL/6 mice . Tg32 mipromoter ,66. The promoter . Tg32 heNaive cynomolgus monkeys used in the PK study at WuXi AppTec.,  were approximately 2 to 3.5 years old and weighed between 2.5 and 3.0 kg.2 asphyxiation and their spleens removed and placed into tubes containing cold RPMI-1640, 5% (v/v) heat-inactivated FBS, 1% (w/v) L-glutamine.FcγR-hu mice were used in the in vivo binding and T cell activation studies. The preliminary study was done with three strains of mice (8–10 weeks old): FcRα null females, FcγR-hu (hemi) females, and FcγR-hu homozygous (homo) females. Test antibody was injected into the intraperitoneal cavity of the mice at 0.5 mg/kg, 10 mL/kg. Approximately 24 h later, mice were euthanized by COMouse splenocytes were prepared from 3 to 6 mice as single-cell suspensions from each individual spleen on the day of harvest. They were washed with media, followed by anucleated red blood cell depletion using hypotonic RBC lysis solution (eBiosciences). Splenocytes were analyzed by flow cytometry for cell surface expression of CD25 and CD69 T cell activation markers. Cells were resuspended in staining buffer consisting of PBS for viability staining , washed, and then incubated with anti-CD16/32  to block nonspecific binding. Immunostaining was done in the presence of APC-CD25, FITC-CD8a, PE-CD4, PerCP-CD69, PE-Vio770-CD3ε  at 4 °C for 30 min protected from light, and followed by two washes.Cells were analyzed on the MACSQuant Analyzer (Miltenyi). Analyses of the multivariate data were performed using FlowJo v10 software . The percent of CD8+ and CD3+ cells that were also positive for CD25 (or CD69) expression were based on data collected with greater than 50,000 cells from each sample.5 cells/mouse, 0.2 mL of 1 × 106 cells/mL in PBS [FcγR-hu mice  were used for the syngeneic mouse lung tumor model. B16F10 tumor cells were intravenously injected into tail vein at 2 × 10L in PBS ,45. At 3L in PBS .Tumor index = lung weight × grade for animal tumor1.Less than 10 tumor foci2.10–100 tumor foci2.5.More than 100 foci, but countable3.One lobe of the lung is full of tumor4.Both lobes are full of tumor5.Lungs are full of tumor and tumor growing out into cavitiesGrading:2-anesthesized mice at indicated time points and terminal bleed was taken by cardiac puncture. After 30 min at RT, blood samples were centrifuged at 2500 rpm for 15 min and serum collected for analyses. All PK studies were approved by the Institutional Animal Care and Use Committee at Janssen Research & Development, LLC .For mouse antibody PK studies, female Tg32 hemi mice were injected with test antibody intravenously via tail vein at a dose of 2 mg/kg into 3 or 4 animals per group. Serial retro-orbital bleeds were obtained from CO2 goat anti-human IgG  in Starting Block T20 (Thermo Scientific) overnight at 4 °C and washed with Tris-buffered saline with 0.05% (w/v) Tween 20 (TBST from Sigma). Standards and serum samples were prepared in sample buffer (1% (w/v) bovine serum albumin in TBST and 20 mM EDTA) added to plates and incubated for 2 h at RT on a shaker. Plates were washed and incubated for 1 h with 1 μg/mL MSD-Sulfo (ruthenium)-labeled with a pan huIgG1 Ab, R10Z8E9. Plates were washed, 1× Read buffer T was added and plates were read on the MSD Sector Imager 6000 .An electrochemiluminescent immunoassay was used to measure human antibody concentration in mouse sera. Briefly, Streptavidin Gold multiarray 96-well plates 96-well plates  were coated with 50 μL/well of 1 μg/mL biotinylated F(ab′)1/2) calculations of the elimination phase for PK studies were determined using a 1-phase exponential decay model fitted by linear regression of natural log concentration versus time using Prism version 6.0 software. The least squares nonlinear decay model was weighted by 1/fitted concentration. Half-life calculations of the elimination phase were determined using the formula t1/2= −ln2/β, where β is the slope of the line fitted by the least square regression analysis starting after the first dose.Terminal half-life  using a similar MSD format. Protocols were reviewed and approved by WuXi AppTec Institutional Animal Care and Use Committee (IACUC) prior to procedures .TM, an HLA class II-peptide binding prediction tool to predict comparative immunogenicity in humans .The amino acid sequences for the different variants were analyzed in silico using ImmunoFilterexamples ,70,71). To assess whether antibodies with silent Fc regions would have good manufacturing properties, biophysical analytical tests were performed for thermal stability and solubility.DSC experiments were performed using a MicroCal Auto VP-capillary DSC system  in which temperature differences between the reference and sample cell were continuously measured and converted to power units. Samples were heated from 25 °C to 110 °C at a rate of 1 °C/min. A pre-scan time of 10 min and a filtering period of 10 s were used for each run. DSC measurements were made at sample concentrations of approximately 0.5 mg/mL in 1× PBS buffer in duplicate. Analysis of the resulting data was performed using MicroCal Origin 7 software .g using Amicon Ultra-15 centrifugal filter units with Ultracel-30 membranes (Sigma-Aldrich). Samples were inspected for signs of precipitation until volumes were reached for a concentration of 40–50 mg/mL. The sequence-predicted absorbance constants (A280/mg/mL) for each antibody were used to calculate sample concentrations at absorbance 280 nm.Antibody samples were concentrated by centrifugation at 2250× Particle size and size distributions were determined using DLS on a DynaPro Plate Reader  at 23 °C. For each analysis, 30 μL of each sample at 1 mg/mL was placed in a 384-well black polystyrene plate with a clear flat bottom . Triplicate measurements were performed for each sample with each measurement consisting of 20 runs.Tm was determined for each sample using intrinsic fluorescence with the Uncle instrument . Tagg was assessed by Static Light Scattering (SLS) to monitor protein aggregation using the same instrument. For the combined Tm and Tagg method, antibody was loaded and run with a thermal ramp from 15 to 95 °C; at a ramp rate of 0.3 °C/min.Samples were separated over a TOSOH TSKgel BioAssist G3SWxL column  that had been equilibrated with PBS supplemented with 500 mM NaCl, at a flow rate of 0.5 mL/min using an Agilent 1100-series HPLC . A target of 100–200 μg of total protein was injected per run. Peaks were monitored using absorbance at 280 nm. Data analysis of species found in each sample was performed using ChemStation software (Agilent Technologies).Exposure to low pH was performed for accelerated stability testing. Protein samples were prepared at a concentration of 1 mg/mL. Samples were dialyzed for 6 h into 0.05 M sodium acetate buffer, pH 3.5; then dialyzed for 16 h in 0.1 M PBS, pH 7.4, and stored at 4 °C prior to analyses.w/v) PEG 20,000, 0.1 M sodium acetate, pH 5.5  MPD). Prior to data collection, crystals were cryo-protected in reservoir supplemented with 20% (w/v) glycerol and flash frozen in liquid N2. X-ray diffraction data for huIgG1σ and huIgG4σ1 were collected on the IMCA-CAT beam line (17-ID) at the Advanced Photon Source (APS) at Argonne National Laboratory equipped with a DECTRIS Pilatus 6M pixel array detector. Diffraction data for huIgG4σ2 were collected by Shamrock Structures, LLC on the SER-CAT beam line (22-ID) at APS equipped with a Rayonix 300HS CCD detector. All data were processed with the program XDS [Recombinant huIgG1σ Fc and huIgG4σ2 Fc were transiently expressed in HEK 293 cells and purified by Protein A affinity chromatography at Sino Biological Inc. (China). Recombinant huIgG4σ1 Fc was transiently expressed in 293 Expi cells and purified in two steps using Protein A affinity chromatography and size exclusion chromatography (Superdex 200 PG) at Aldevron . Proteins were delivered in 20 mM Tris, 50 mM NaCl, pH 7.5 at concentrations ranging from 2 to 4 mg/mL. Fc molecules were further concentrated to 10–13 mg/mL prior to crystallization. Crystallization experiments employed the vapor-diffusion method. Crystallization drops were set up using a Mosquito liquid handling robot  in 96-well Corning 3550 (huIgG1σ and huIgG4σ2) or MRC 2 well (huIgG4σ1) crystallization plates. Diffraction quality crystal were obtained for all three Fc regions in 9–10%  [Initial phases were determined by the method of molecular replacement with the program Phaser  as implePDB 3AVE  (huIgG1 t (COOT)  and Phent (COOT) ,77 respeH2 and CH3 domains, and the glycans present in the corresponding crystal structures. The simulations were set up in the Maestro graphical user interface and run using the Desmond program (multisim version 3.8.5.19 and mmshare version 3.5) [H2 domain. Explicit solvent MD simulations were performed on intact Fc to investigate the flexibility of BC and FG loops. Two simulations were initiated from the available crystal structures: IgG1 WT using the crystal structure (PDB 3AVE) of IgG1 wild-type Fc and IgG1σ using the crystal structure described here. Additional two simulations were initiated after modelling mutations in the wild-type crystal structure: IgG1-H268A containing H268A mutation and IgG1-SS containing A330S and P331S mutations. The simulations included both chains of Cion 3.5) , both paion 3.5) . The sysion 3.5)  water moion 3.5)  was usedion 3.5)  (REST) MA requirement for truly silent human Fc designs is evident as more antibodies are being developed for immunotherapy. The Fc engineered human mutations described here as IgG1σ, IgG4σ1 and IgG4σ2 demonstrate equal or lower immune functionality than the corresponding IgG2σ design. Lack of immune effector function is demonstrated in vitro with FcγR binding, cytotoxicity assays, and in vivo with T cell activation and tumor inhibition studies. Crystal structures and simulations of these Fc variants reveal altered conformational preferences within the lower hinge and BC and FG loops relative to wild-type IgG, providing a structural rationalization for diminished Fc receptor engagement. Immunogenicity predictions, pharmacokinetic studies in mice and monkeys, and biophysical analyses also support these novel mutations as optimized silent Fc choices for the development of therapeutic antibodies.