Furthermore, TH287 was radiolabeled with carbon-11 for in vivo microPET studies. Saturation binding assays show that [3H]TH287 has a dissociation constant (Kd) of 1.97 ± 0.18 nM, Bmax of 2676 ± 122 fmol/mg protein for U251MG cells, and nH of 0.98 ± 0.02. Competitive binding assays show that TH287 (Ki: 3.04 ± 0.14 nM) has a higher affinity for MTH1 in U251MG cells compared to another well studied MTH1 inhibitor: (S)-crizotinib (Ki: 153.90 ± 20.48 nM). In vitro enzymatic assays show that TH287 has an IC50 of 2.2 nM in inhibiting MTH1 hydrolase activity and a Ki of 1.3 nM from kinetics assays, these results are consistent with our radioligand binding assays. Furthermore, MicroPET imaging shows that [11C]TH287 gets into the brain with rapid clearance from the brain, kidney, and heart. The results presented here indicate that radiolabeled TH287 has favorable properties to be a useful tool for measuring MTH1 in vitro and for further evaluation for in vivo PET imaging MTH1 of brain tumors and other central nervous system disorders.MTH1 (MutT homolog 1) or NUDT1 (Nudix Hydrolase 1), also known as oxidized purine nucleoside triphosphatase, has potential as a biomarker for monitoring cancer progression and quantifying target engagement for relevant therapies. In this study, we validate one MTH1 inhibitor TH287 as a PET MTH1 radiotracer. TH287 was radiolabeled with tritium and the binding of [ Improved cancer diagnosis and cancer-specific therapeutics are needed to reduce cancer mortality rates. Positron emission tomography (PET) can measure disease-based changes in cell function at the molecular level. PET combined with computed tomography (CT) or magnetic resonance imaging (MRI) provides a powerful hybrid of anatomical and functional assessments of cancer and other diseases.Dysfunctional redox regulation and increased reactive oxygen species (ROS) cause oxidative damage to nuclear and mitochondria DNA that contributes to the pathogenesis and progression of cancer and other diseases . Thus, iCancer cells require high ROS concentrations due to their high proliferation rate. In mitochondria, where ~90% of total oxygen consumption occurs, about 1–4% consumed oxygen molecules are partially reduced by electrons to generate ROS including superoxide, hydrogen peroxide, and hydroxyl radicals . ROS cauMisincorporation of oxidized nucleoside triphosphates into DNA/RNA during replication and transcription can cause mutations that may lead to carcinogenesis, senescence, apoptosis, or neurodegeneration ,11. MTH1Thus, monitoring MTH1 status in patients through in vivo imaging would facilitate the design of therapeutic strategies that target oxidative DNA damage involved diseases such as cancer. TH287  is among3H]TH287 to MTH1 in glioblastoma cells (U251MG) was evaluated in vitro using saturation binding, competitive binding, and enzymatic assays. Furthermore, TH287 was radiolabeled with carbon-11 to study whether [11C]TH287 is a suitable radiotracer for PET. The uptake and distribution of [11C]TH287 in different organs of mice were investigated with microPET imaging. This is the first study that explores the feasibility of developing a radiolabeled MTH1 inhibitor for use as an in vitro binding assay or in vivo PET imaging radiotracer.In the present study, the affinity of TH287 to MTH1 in live glioblastoma U251MG cells and in vivo was investigated using its radiolabeled form. TH287 was radiolabeled with tritium and the binding of [3H]TH287 with U251MG cells. The saturation curve, Scatchard plot, and Hill plot are shown in 3H]TH287 has a Kd value of 1.97 ± 0.18 nM, a maxB value of 2676.26 ± 122.38 fmol/mg protein, and a nH value of 0.98 ± 0.024 for U251MG cells. These values suggest TH287 is within the pharmaceutic limit to be adopted as a radiotracer for microPET imaging.To validate TH287 as a potential radiotracer for microPET imaging, its affinity to MTH1 was first characterized in live cells through saturation binding studies. The assays were carried out by incubating increasing concentrations of [3H]TH287 in the saturation binding assays, MTH1 was further knocked down through reverse transfection of MTH1 siRNA into U251MG cells. MTH1 knockdown completely abolished [3H]TH287 binding as shown by radioligand binding readings of MTH1 siRNA transfection groups similar to cells treated with 10 µM TH086, which is normally considered as non-specific binding, suggesting that [3H]TH287 binds specifically to MTH1 in U251MG cells instead of other targets -crizotinib shows a Ki of 153.90 ± 20.48 nM, indicating that its affinity is much lower than TH287. No binding was detected for Raclopride—a nonselective compound -crizotinib respectively.To further validate the potential of TH287 as a radiotracer for MTH1, full dose range competition binding assays were performed between a well-studied MTH1 inhibitor, (S)-crizotinib, and TH287 was synthesized and microPET imaging was performed in normal mice. In the microPET imaging of normal mice, summed (0–10 min) frames of the microPET images (11C]TH287 (~0.4 mCi) had high initial uptake and fast clearance of [11C]TH287 from the brain, heart, and kidney . Several existing MTH1 inhibitors besides TH287, including TH086 and (S)-crizotinib, were also tested in the study to validate TH287 as a specific MTH1 probe. TH287 has a higher affinity to MTH1 compared to all these two ligands. The cell viability studies showed that up to 30 µM TH287 exhibits minimal inhibition on U251MG cells while 30 µM (S)-crizotinib killed all the cells via an off-target other than MTH1. MicroPET imaging shows that TH287 penetrates the blood-brain barrier of mice with a fast clearance.In this study, we explored the feasibility of developing TH287 as a PET radiotracer for cancer by investigating the affinity of TH287 to MTH1 in live cells and its distribution and clearance in mice. TH287 exhibits a high affinity for MTH1 in the nanomolar range (IC50 = 0.043 nM) TH287. Competitive binding assays show that TH287 has a high binding affinity, with a Ki of 3.04 ± 0.14 nM while (S)-crizotinib shows a Ki of 153.90 ± 20.48 nM, indicating that its affinity is much lower than TH287. No binding was detected for Raclopride—a nonselective compound -crizotinib respectively.To further validate the potential of TH287 as a radiotracer for MTH1, full dose range competition binding assays were performed between a well-studied MTH1 inhibitor, (S)-crizotinib, and TH287 was customer synthesized by ViTrax  via direct catalytic tritium gas exchange, a radiochemical purity of >99% was determined by the high performance liquid chromatography (HPLC). Product identity was confirmed by Mass spectrometry and HPLC co-elution with Authentic Standard  and mobile phase B (acetonitrile). From 10% B to 90% B over 10 min at flow rate 1 mL/min.).Specific Activity was determined to be 31.2 Ci/mmol by Mass Spec. The location of the tritium label has not been determined, however, can be assumed to be located on the aromatic ring.Chemical reagents and the standard compounds (Raclopride and (S)-crizotinib) were purchased from Sigma-Aldrich  and Tocris . 5) is shown in 2 was synthesized by coupling dichlorophenyl boronic acid and 4,6-dichloropyrimidin-2-amine (1) with Pd(PPh3)4 as the catalyst. The amine group of 2 was then protected with a Boc group to give compound 3. The chloro group of compound 3 was substituted with an azide group to afford compound 4. the azide group of 4 was reduced to an amine group with PPh3 to give the precursor 5.The synthesis of the C-11 labeling precursor of TH287  downfield from tetramethylsilane (TMS). All coupling constants (J) are given in Hertz (Hz). Splitting patterns are typically described as follows: s, singlet; d, doublet; t, triplet; m, multiplet.All chemicals were obtained from standard commercial sources and used without further purification. All reactions were carried out by standard air-free and moisture-free techniques under an inert nitrogen atmosphere with dry solvents unless otherwise stated. Flash column chromatography was conducted using Scientific Adsorbents, Inc. silica gel, 60A, “40 Micron Flash” (32–63 µm). Melting points were determined by using the MEL-TEMP 3.0 apparatus and are uncorrected. Routine 4-Chloro-6-pyrimidin-2-amine (2) A solution of boronic acid  and 4,6-dichloropyrimidin-2-amine  in dioxane (30 mL) and water (10 mL) was added Pd(PPh3)4  and Na2CO3 . The mixture was heated at 90 °C for 4 h. At room temperature, the reaction mixture was diluted with ethyl acetate (100 mL), washed with water (50 mL × 2) and NaCl solution (75 mL), and dried over Na2SO4. After evaporation of the solvents, the crude product was purified by column chromatography with CH2Cl2/EtOAc (5:1) to afford 306 mg (37%) of 2 as a white solid, mp 210.6–211.7 °C. 1H NMR  δ 7.56 , 7.40 , 7.31 , 6.93 , 5.31 .tert-Butyl pyrimidin-2-yl)carbamate (3) A solution of 2  and di-tert-butyl dicarbonate  in THF (20 mL) was added N,N-dimethylpyridin-4-amine . The mixture was stirred at room temperature overnight. After evaporation of the THF, the crude product was purified by column chromatography with CH2Cl2 to afford 296 mg (79%) of 3 as a colorless oil. 1H NMR  δ 7.90 , 7.61 , 7.56 , 7.37 , 1.46 .tert-Butyl pyrimidin-2-yl)carbamate (4) A solution of 3  and NaN3  in DMF (10 mL) was heated at 90 °C for 24 h. At room temperature, the reaction mixture was diluted with ethyl acetate (75 mL), washed with water (50 mL × 2), and NaCl solution (50 mL), and dried over Na2SO4. After evaporation of the ethyl acetate, the crude product was purified by column chromatography with CH2Cl2 to afford 216 mg (85%) of 4 as a colorless oil. 1H NMR  δ 7.54 , 7.50 , 7.30 , 6.78 , 1.55 .tert-Butyl pyrimidin-2-yl)carbamate (5) A solution of 4  and Ph3P  in THF (10 mL) and water (1 mL) was heated to reflux for 4 h to complete the reaction. At room temperature, the reaction mixture was added ethyl acetate (50 mL), and then was washed with water (30 mL × 2) and NaCl solution (30 mL) and dried over Na2SO4. After evaporation of the ethyl acetate, the crude product was purified by column chromatography with CH2Cl2/EtOAc (1:1) to afford 116 mg (83%) of 5 as a colorless oil. 1H NMR  δ 7.65 , 7.50 , 7.41 , 7.26 , 6.36 , 5.39 , 1.51 .5 to prepare [11C]TH287 is shown in 11C]CH3I was produced from [11C]CO2 using a GE PETtrace MeI Microlab. [11C]CO2 was produced at the Cyclotron Facility of Washington University School of Medicine using a JSW BC-16/8 cyclotron by irradiating a gas target of 0.2% O2 in N2 for 15–30 min with a 40 µA beam of 16 MeV protons. [11C]CO2 was converted to [11C]CH3I by the GE PETtrace MeI Microlab using a nickel catalyst  in the presence of H2 at 360 °C and followed by a reaction with iodine in the gas phase at 690 °C. [11C]CH3I was delivered in the gas phase with helium approximately 12 min following the end of the bombardment.The C-11 radiolabeling of the precursor 5 (1.1 mg) was treated with TFA (300 µL) at 80 °C for 10 min, and then TFA was removed under a flow of nitrogen at 80 °C. The residual TFA was removed by the addition of acetonitrile (1 mL) and heating at 80 °C under a flow of nitrogen. This process was repeated two more times to obtain a white residue, which was transferred to the labeling vessel by DMF (2 × 100 µL). [11C]MeI was produced at the Washington University in Saint Louis Cyclotron facility. Before the trapping of [11C]MeI, NaH (1 mg) in DMF (100 µL) was added to the labeling vessel and the vessel was kept at 0 °C to trap [11C]MeI. After trapping of [11C]MeI, the reaction was heated at 90 °C for 6 min and then diluted with 1:1 HPLC mobile phase/water for HPLC purification . [11C]TH287 was collected at 15 min in 50 mL of water and extracted by a Waters C18 cartridge. The cartridge was rinsed with 10 mL of water and [11C]TH287 was eluted with ethanol and saline to achieve a final dose of 10% ethanol/saline. The radiochemical yield (decay corrected) was 5.9 ± 3.2% (n = 5), and the specific molar activity at the end of the bombardment was 2494 ± 1603 mCi/µmol (n = 5) and radiochemical purity was >99%. At this preliminary stage of our research, we have yet to optimize the labeling. We believe the low yield is due to the slow methylation reaction on the aromatic amine, being limited by the short half-life of carbon-11. The use of more reactive [C-11]methyl triflate may improve the yield under mild conditions.The boc-protected precursor 2 and 95% air at 37 °C. Short tandem repeat (STR) profiling was carried annually from Cell Line Authentication Service from the University of Arizona Genetics Core to ensure quality and integrity.U251MG cells (RRID: CVCL_0021)  were grown in RPMI 1640 medium  supplemented with 10% fetal bovine serum  and 1% penicillin/streptomycin . Cells were maintained in a humidified environment of 5% COReverse transfection of MTH1 siRNA was used to knockdown MTH1 expression and cells transfected with control siRNA served as the experimental control. A Lipofectamine RNAiMAX group only was also used as a control group. Briefly, 0.2 µL Lipofectamine RNAiMAX  mixed with 0.6 µL of 10 µM siRNA in 20 µL of Opti-MEM was added to each well of the 96-well plates. Then, 100 µL of the cell suspension containing 8000 cells was added to each well. The culture medium was changed after overnight incubation at 37 °C. 48 h after the transfection, saturation binding assays were performed on all experimental groups of cells.3H]TH287 in a total volume of 150 µL of media at 37 °C in 96 well stripwell tissue culture plates  for 30 min. The concentrations of the radioligand ranged from 0.5–15 nM. After incubation, each well was washed with 200 µL of ice-cold PBS and transferred to a vial with 2 mL of scintillation fluid. A liquid scintillation system AccuFLEX LSC-800  was used to quantify the bound radioactivity. Nonspecific binding was determined from cells treated with 10 µM TH086. Experiments were repeated three times.U251MG cells  were seeded 24 h before the assay. The cells were incubated with TH287 (1.97 ± 0.18 nM) were derived from U251MG experiments and Lt is the concentration of radioligand used in each assay.Data from the competitive binding experiments were modeled using nonlinear regression analysis to determine the concentration of inhibitor that inhibits 50% of the radioligand specific binding (equation :(3)Ki=ICn’H, defined as:Data from competitive radioligand binding studies were transformed to determine the pseudo-Hill coefficient, n’H, which is the negative of the Hill slope, was readily determined from the plot of IC50 values were determined by fitting a dose-response curve to the data points using nonlinear regression.The enzymatic hydrolysis of dGTP by purified human recombinant MTH1 to form dGMP and pyrophosphate was applied for screening purposes using a PPiLightTM inorganic pyrophosphate bioluminescent assay—according to published procedures . After s3 U251MG cells were plated into 96-well plates and treated with different concentrations  of TH287 or (S)-crizotinib at 37 °C for 72 h. After incubation with MTT for 4 h at 37 °C, the culture medium was aspirated and 200 µL DMSO was added to each well to dissolve the formazan crystals, then the absorbance was measured at 540 nm using a Victor 3 Microplate reader .MTT assays were used to test cell toxicity of compounds. 2 × 1011C]TH287 via the tail vein. The imaging sessions were 1 h dynamic scans using the MicroPET® Focus 220 and Inveon scanners . Acquired list mode data were histogrammed into a 3D set of sinograms and binned to the following time frames: 5 × 60 s, 5 × 2 min, and 9 × 5 min. Sinogram data was then processed using a filter back-projection algorithm with attenuation and scatter corrections. Regions of interest (ROI) were manually drawn on the brain, heart, and kidney of the mice using 0–10 min summarized images as references. The software Acquisition Sinogram Image PROcessing using IDL’s Virtual MachineTM (ASIPro VMTM)  was used to draw ROI and obtain the radioactivity uptake (nCi/c.c.) curve over the time course of the scan, and the uptake data were further normalized by the injected radioactivity dose to get %I.D./cc.The Washington University Animal Studies Committee approved all procedures used for the mouse experiments described in the present study under ACS# 20,170,165 (approved on 9/1/2017). Overall care of the animals was consistent with The Guide for the Care and Use of Laboratory Animals from the National Research Council and the USDA Animal Care Resource Guide. Four male athymic nude mice , purchased from Charles River Laboratories , were included for the preliminary microPET imaging evaluation of MTH1 in vivo. The mice were anesthetized with isoflurane and injected with ~400 µCi of [p-value of <0.05 was considered statistically significant. All data were reported as mean ± standard deviation if not otherwise noted.The data and statistical analysis comply with the recommendations on experimental design and analysis in pharmacology . Statist


The preferable non-invasive and real-time diagnosis in vivo is more accessible by PET scan using 16α-[18F]FES. The objective of this study was to develop a quick automatic method for synthesis of solvent-free 16α-[18F]FES using a CFN-MPS-200 synthesis system and compare the catalytic efficiency of two phase transfer catalysts, Kryptofix 222/K2CO3 (K222/K2CO3) and tetrabutylammonium hydrogen carbonate (TBA·HCO3). In this method, phase transfer catalysts K222/K2CO3 and TBA·HCO3 were used, respectively. The intermediate products were both hydrolyzed with hydrochloric acid and neutralized with sodium bicarbonate. The crude product was purified with semi-preparative HPLC, and the solvent was removed by rotary evaporation. The effects of radiofluorination temperature and time on the synthesis were also investigated. Radiochemical purity of solvent-free product was above 99% and the decay-corrected radiochemical yield of 16α-[18F]FES was obtained in 48.7 ± 0.95%  and 46.7 ± 0.77% . The solvent-free 16α-[18F]FES was studied in clinically diagnosed breast cancer patients, and FES-PET results were compared with pathology diagnosis results to validate the diagnosis value of 16α-[18F]FES. The new method was more reliable, efficient, and time-saving. There was no significant difference in catalytic activity between K222/K2CO3 and TBA·HCO3.Estrogen receptor (ER) expression level of human breast cancer often reflects the stage of disease and is usually monitored by immunohistochemical staining  An ideal non-invasive method monitoring real-time ER expression in the whole body is preferable in clinical practice. 16α-[18F]FES, an ER imaging biomarker, shows high binding affinity to ER. The utility of 16α-[18F]FES, especially in conjunction with [18F]fluorodeoxyglucose, is considered of important diagnostic value.Estrogen receptor (ER) expression level in breast cancer is known as a meaningful prognostic indicator. ER-positive breast cancer is considered less aggressive and more sensitive to appropriate hormone therapies , 2. The 18F]FES to meet clinical requirements. The first synthesis of 16α-[18F]FES was reported by Kiesewetter , Kryptofix 222 (K222), and 16α-[19F]FES reference standard were obtained from ABX . Cartridges were purchased from Waters . Before use, QMA cartridge was flushed with 10 ml of potassium carbonate followed by 40 ml of water.Reagents and solvents were purchased from Aldrich  without further purification. The precursor, 3-methoxymethyl-16β,17β-epiestriol-v:v:v) as the mobile phase. Analytical HPLC  was used for the quality control of the final product, equipped with a UV/Vis detector preset to 280 nm, an analytical C18 column , and a radioactive detector . The column flow rate was 1 ml/min and was kept at approximately room temperature. The samples were eluted with a mobile phase of 30% acetonitrile:70% water (v:v).For automatic synthesis, CFN-MPS-200 module  was used and modified. Synthesis software was programmed in the Cupid system . Radioactivity was counted in a dose calibrator . The semi-preparative HPLC  equipped with a semi-preparative C18 column , UV/Vis detector , and a radiation detector was used to purify the crude product at a flow rate of 4 ml/min, using 30% acetonitrile:30% ethanol:40% water 18F nuclear reaction, 1.8 ml of target water containing [18O]H2O was irradiated with protons in a Sumitomo HM-10 cyclotron system  to produce [18F]fluoride. The energy of incident protons was 10 MeV, and the beam current was about 60 mA. After the irradiation, [18F]fluoride was transferred to the collection tube and then passed through a Sep-Pak light QMA cartridge. The trapped [18F]fluoride was eluted with 0.9 ml of a mixed aqueous K222/K2CO3 (22 mg of K222 in 0.7 ml of acetonitrile and 5.8 mg of potassium carbonate in 0.2 ml of ultrapure water) and then the mixed solution was dried up. To make sure that the fluorination reaction was in an anhydrous condition, the residue was further dried by azeotropic distillation with another 0.5 ml of anhydrous acetonitrile. Two milligrams of MMSE in 1 ml of anhydrous acetonitrile was added to the completely dried reactor, and the mixture was heated at 115°C for 15 min. Then, the intermediate product was subsequently hydrolyzed by 1.5 ml of 2 M HCl at 120°C for 3.5 min. After cooling to room temperature, 1.5 ml of 5.6% NaHCO3 solution was added to neutralize the reaction mixture. For purification, the crude product was injected into semi-preparative HPLC, and the purification took about 9 min. The purified product was collected in the rotary evaporator to remove solvents and then passed through a sterile 0.22-μm filter with 10 ml of saline containing 1 ml of ethanol.Based on 18F]FES at a flow rate of 1 ml/min using 30% acetonitrile in water by comparing the HPLC spectrum of the 16α-[19F]FES reference standard in the same HPLC conditions. For stability test, 16α-[18F]FES in the final formula was tested 8 h after the synthesis using HPLC.The analytical HPLC system was adapted to measure the radiochemical and chemical purity of 16α-[18F]fluorination was carried out for 5, 10, and 15 min at 105, 115, 125, and 135°C. The overall radiochemical yield was determined by purifying the product by semi-HPLC.To discover how fluorination temperatures and times influence the yield, [18F]FES synthesis processes catalyzed by tetrabutylammonium bicarbonate (TBA·HCO3) were the same compared with those catalyzed by K222 except that [18F]fluoride trapped in the QMA cartridge was eluted by 500 μl of 0.075 M TBA·HCO3 dissolved in ethanol, and fluorination was achieved at 135°C for 10 min.To compare chemical purity and radiochemical yield, 16α-FES before surgery. Patients underwent the surgical operation within 1 month after the PET/CT scan, and definitive diagnosis was determined using postoperative pathohistology analysis. The study protocol was approved by the Sichuan Cancer Hospital Ethics Committee, and informed consent was obtained from all patients before the PET/CT scan.Nine patients  with a clinical diagnosis of breast cancer were enrolled in the study between May 2017 and July 2017. All patients underwent whole-body PET/CT scan with 16α-[18F]FES was infused intravenously over 2 min. The whole-body scanning (2 min per table position) was performed 1 h after administration using a Siemens Biograph mCT-64 PET/CT scanner.All patients fasted 4 h before FES-PET. Approximately a dose of 148–222 MBq (4–6 mCi) 16α-[t test. P-values <0.05 were considered significant. Data analyses were carried out using GraphPad Prism Software Version 5.0 .Means were compared using the two-tailed paired Student's 18F]FES were optimized and successfully performed (18F]FES using the CFN-MPS-200 synthesis system are shown in 2CO3 and TBA·HCO3) were used, respectively, while the purification methods were the same (by semi-preparative HPLC purification). As purification results show in 2CO3 catalyzed) or 3 catalyzed), only one radioactive peak was detected and collected, with a retention time of 7.5 min (using K222/K2CO3) or 7.6 min (using TBA·HCO3). After removing solvents by a rotary evaporator, the decay-corrected radiochemical yield of the final product 16α-[18F]FES was 48.7 ± 0.95%  or 46.7 ± 0.77% .The procedures for the one-pot synthesis of 16α-[erformed . A totalerformed . Hardwar18F]FES was synthesized rapidly in one pot with a high radiochemical yield and purity in this study. Overall, total K222-catalyzed synthesis time was 48 ± 2 min (n = 4) and total TBA-catalyzed synthesis time was 48 ± 3 min (n = 4); both included two azeotropic distillations of [18F]fluoride and acetonitrile, radiofluorination, hydrolysis, neutralization, HPLC purification, and desolventizing by rotary evaporator. In typical synthesis conditions, the six steps took the same time in both K222-catalyzed and TBA-catalyzed methods. Desolventizing by the rotary evaporator can reduce the solvent in the final formula to satisfy clinical use and takes about 10 min. If the synthesis of 16α-[18F]FES was aimed at research and development, omitting the desolventizing step could save 10 min and reduce product loss, which was stained with a rotary evaporator to obtain a high yield. Automated synthesis in commercially available synthesizer systems takes about 80 min, with a 10–20% uncorrected radiochemical yield . Solvent-free 16α-[18F]FES with high radiochemical purity was obtained with HPLC purification and desolventizing by a rotary evaporator and shows significant correlation with ER expression in patients. Moreover, two phase transfer catalysts, Kryptofix 222 and tetrabutylammonium bicarbonate, did not show significant difference in catalytic activity, radiochemical yield, radiochemical purity, and product stability in our study. In conclusion, we present a quick automatic method for synthesizing solvent-free 16α-[18F]FES that could be used in a commercial synthesis module with either Kryptofix 222 or tetrabutylammonium bicarbonate.In this work, 13 times fully automatic synthesis of solvent-free 16α-[The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.The studies involving human participants were reviewed and approved by Sichuan Cancer Hospital Ethic Committee. The patients/participants provided their written informed consent to participate in this study.XJ, YL, XW, TS, ZL, and ZC conceived and designed the study and helped to draft the manuscript. XL, YY, and GZ performed the data collection. YK and JS performed the statistical analysis. All authors read and critically revised the manuscript for intellectual content and approved the final 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.


In this study, we evaluated the proteomic changes in serum of nonhuman primates (NHP) after administering BIO 300 by different routes (po and im). We utilized nanoflow-ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (NanoUPLC-MS/MS) methods for comprehensive global profiling and quantification of serum proteins. The results corroborate previous findings that suggest a very similar metabolic profile following both routes of drug administration. Furthermore, we observed minor alterations in protein levels, 2 hours after drug administration, which relates to the Cmax of BIO 300 for both routes of administration. Taken together, this assessment may provide an insight into the mechanism of radioprotection of BIO 300 and a reasonable illustration of the pharmacodynamics of this radiation countermeasure.Hematopoietic acute radiation syndrome (H-ARS) and delayed effects of acute radiation exposure (DEARE) are detrimental health effects that occur after exposure to high doses of ionizing radiation. BIO 300, a synthetic genistein nanosuspension, was previously proven safe and effective against H-ARS when administered (via the oral ( Furthermore, the constant dependence on nuclear power as an alternative source of energy has led to the spread of nuclear plants worldwide2. Due to their catastrophic potential, nuclear threats and accidents remain a priority for major federal entities and public health agencies3. Accidental exposure to high doses of radiation can lead to a variety of potentially lethal syndromes4. Hematopoietic acute radiation syndrome (H-ARS) is the primary cause of death in humans and occurs within 30–60 days following > 2 Gy total-body irradiation due to bone marrow failure5. Developing new medical countermeasures to combat radiation injury, especially H-ARS, continues to be a primary focus for numerous research institutions6. There are multiple classes of medical countermeasures for radiation exposure. They are defined based on the time of countermeasure administration in relation to radiation exposure. Radioprotectants are agents which are administered prior to radiation exposure to protect individuals. Radiomitigators are administered shortly after radiation exposure, but before the appearance of symptoms of radiation injury. Finally, radiotherapeutics are administered once clinical symptoms of radiation exposure are present. Radioprotectants and radiomitigators can also be used in clinical oncology to minimize the side effects of radiotherapy. Moreover, personnel at risk of unexpected radiation exposure  can benefit greatly from the prophylactic administration of these agents.Nuclear threats and accidents have continued to be a major concern for the United States government. Unsupervised possession of nuclear weapons can lead to a massive loss of life and property8. Amifostine and palifermin are two other FDA approved radioprotectants that act as cytoprotective agents and are also associated with serious side effects10. Researchers have demonstrated the potential toxicity and lethality of using high doses of amifostine13. Clearly there is a need for new and effective medical countermeasures against radiation exposure.The US Food and Drug Administration (US FDA) has approved a small number of radiation countermeasures for H-ARS. Leukocyte growth factors  are radiomitigators for the recovery of radiation-induced bone marrow failure. However, these agents may cause significant adverse effects and thus require close patient monitoring upon administration15. Unfortunately, genistein has high hydrophobicity and low water solubility which leads to poor bioavailability and hinders its medical utility16. This problem was recently resolved by the development of a pharmaceutically acceptable nanosuspension of genistein (BIO 300) which was manufactured by Humanetics Corporation. This nanoparticle formulation utilizes a wet-nanomilling technique, which enhances water solubility through reducing the average particle size by two orders of magnitude, and hence, enhances its bioavailability19.One such molecule is genistein chromen-4-one), a naturally occurring isoflavone found at low levels in soybeans, which has shown efficacy as a radioprotectant against H-ARS in murine models20. These studies revealed a broad scope of pharmacological actions of genistein. For example, some studies highlighted its antioxidant and free radical scavenging ability23, its anti-inflammatory properties25, and its ability to arrest hematopoietic stem cells at the G2/M phase of the cell cycle26 which induces a senescent state and thus reduces the detrimental effects of radiation exposure. In addition, genistein possesses a benzopyran structural scaffold that gives it estrogen-like biological activity. There are two estrogen-binding ligand-dependent intracellular transcription factors: estrogen receptor-α (ERα) and estrogen receptor-β (ERβ)28. Upon ligand activation, these receptors dimerize and translocate to the nucleus and bind to specific DNA transcripts to facilitate site-specific gene transcription. The two transcriptional ERs (α and β) appear to have opposite functions in normal cells. The activation of ERα primarily promotes cellular growth, whereas ERβ negatively regulates ERα and quenches cell proliferation20. Genistein (and BIO 300) demonstrated a selective agonistic activity towards ERβ with a half maximal effective concentration (EC50) of 0.9 nM, which was a 2000-fold greater potency than ERα29. In addition to ERα and ERβ, a third receptor has been identified and termed the G-protein coupled Estrogen Receptor 1 (GPER1). ERα and ERβ are nuclear receptors that are structurally similar, while GPER1 is a less characterized G-protein coupled receptor capable of mediating rapid-translational signal transduction cascades in response to an estrogenic signal31.Although extensively studied, the mechanism of radioprotection by genistein remains only partly understoodpo or im administration as part of the drug’s safety and efficacy evaluation in an NHP model. Transient alterations in phenylalanine, tyrosine, glycerophosphocholine, and glycerophosphoserine reached maximum levels 3–7 days after drug administration and reverted to near-baseline levels 14 days after drug administration. Furthermore, a significant similarity was reported in the metabolic profiles between the serum samples obtained after po and im administration of BIO 30032.In previously published work, a global metabolomic approach with an ultra-performance liquid chromatography (UPLC) quadrupole time-of-flight mass spectrometry (QTOF-MS) platform was used to assess the metabolomic and lipidomic changes induced by BIO 300 following po and im routes). The serum samples were immunodepleted, rebuffered, and analyzed for protein concentration. The protein samples were reduced, alkylated, and digested. Tryptic peptides were desalted and analyzed using NanoUPLC-MS/MS methods for comprehensive profiling and quantification of serum proteins. The methods used in this study involve the depletion of most abundant plasma proteins including albumins and globulins, utilizing Data Dependent Acquisition (DDA) in order to develop a library of identified proteins, and subsequently utilizing a label-free Data Independent Acquisition (DIA) SWATH technique for protein quantification.In the present study, we analyzed serum samples obtained from NHPs at 14 different time points , an unsupervised feature reduction algorithm, was used to measure the degree of difference between sample groups; results indicated mostly overlapping proteomic profiles between the two routes of administration throughout all time points , Beta actin variant (Q53GK6), and I3L3D6 were significantly upregulated following po administration  was downregulated, Protein S100 (B2R4M6) was marginally downregulated, but Putative 26S proteasome regulatory subunit 6b (B9PHK0) was upregulated. The po route demonstrated a similar pattern. Trypsin-1 (E7EQ64) and Apolipoprotein E isoform 1 (A0A0S2Z3D5) were slightly down regulated, while Q53GK6 and Epididymis secretory sperm binding protein Li 78p (V9HVY1) were slightly upregulated , Profilin (I3L3D5) and Beta actin variant (Q53GK6) showed transient changes in serum abundance at 30 min and 1 h in both im and po groups  for H-ARS which are effective after individuals are already exposed to radiation. Nevertheless, there is no FDA approved agent that can be used prophylactically to prevent either ARS or DEARE. Genistein, as well as other radioprotective agents, have been the subject of multiple reviews29. Either individually or in combination with other agents, genistein has been demonstrated to protect various organs41. Despite the poor water solubility, genistein was developed as a nanosuspension formulation (BIO 300) utilizing a wet-nanomilling manufacturing process. This genistein nanosuspension (BIO 300) is currently being evaluated for prevention of H-ARS and has an open Investigational New Drug application. In addition, BIO 300 is currently being assessed for safety and efficacy in a phase 1b/2a clinical trial in non-small cell lung cancer (NSCLC) patients (NCT02567799). This new nanosuspension technology has enabled the administration of genistein through different routes such as po, subcutaneous (sc) or im29. Furthermore, BIO 300 was shown to be effective in mitigating DEARE to the lung (DEARE-lung) in mice when administered daily via oral gavage for up to 6 weeks, starting 24 h after whole-thorax lung irradiation19.Genistein is a radioprotectant that is believed to have anti-inflammatory activity, cell cycle effects, and estrogen-like pharmacological action with a 2,000-fold binding preference for ERβ over ERαim or po administration of BIO 30032. Results of this study revealed that the administration of the drug resulted in modest changes in the metabolic/lipidomic profiles over time. More importantly, the observed changes were most apparent between days 3–7 post BIO 300 administration and this temporal effect was quenched with time. Also, there were no significant differences between the metabolic profiles upon comparing the po to the im routes as indicated by the AUC calculations of the PK studies performed in the same report.In an earlier study, NHPs were used to compare longitudinal changes in molecular metabolomic and lipidomic profiles following a single dose either In this current study, global proteomic profiling of the NHP serum samples was done utilizing a NanoUPLC MS/MS technique. We used a shotgun proteomics approach for global interrogation of alterations in plasma proteomic profiles upon drug administration.im drug administration may reach the bloodstream faster than the drug administered po route. Genistein aglycone levels are ~ 1000 × higher with im administration compared to po administration 30 min after drug delivery32. To further understand the changes happening 0.5 h and 1 h post drug administration, we compared the levels of proteins detected at these time points to the level of the same proteins measured 1 day prior to drug administration.Comparing the two routes of administration at each time point independently, there were few differences. However, the greatest difference was observed at 0.5–1 h post drug administration. This difference might be related to the fact that While a few proteins were found downregulated 0.5 h to 1 h post drug administration, several proteins were upregulated at 0.5 h including Tubulin α chain (B3KT06), cDNA FLJ57036 (B4E3P1), CP protein (A5PL27), Glutathione peroxidase (V9HWN8), Carbonic anhydrase I (V9HWE3), Glutathione S-transferase pi 1 (V9HWE9) and Apolipoprotein E isoform 1 fragment (A0A0S2Z3D5). The levels of these elevated proteins ultimately returned to near normal except for B4E3P1, A5PL27 and V9HWN8. B3KT06, α Tubulin, is a major constituent of microtubules. It binds two molecules of guanosine triphosphate (GTP), one at an exchangeable site on the β chain and one at a non-exchangeable site on the α chain. It has GTPase activity and participates in mitochondrial based processes. B4E3P1 is highly homologous to tropomyosin which is a member of a large family of integral components of actin filaments that play a critical role in regulating the function of actin filaments. A5PL27 is a ceruloplasmin (CP) protein that participates in copper ion binding and iron transport. V9HWN8 is Glutathione peroxidase which is highly secreted in response to oxidative stress. V9HWE9 is Epididymis secretory protein Li 22 that plays an important role in glutathione binding and transfer. Additionally, at 1 h, there were a few upregulated proteins including I3L3D5, Q53GK6, and marginal B4E3P1 and δ-globin (A0N071). Q59EZ3 is an insulin-like growth factor-2 receptor variant that participates in signaling receptor activity and lysosomal transport.43. Nitration of the tyrosine residue is one of the post translational modifications that is usually enhanced upon exposure to ionizing radiation due to the production of reactive nitrogen species like nitrogen dioxide. Nitrotyrosine is an indicator or marker of cell damage, inflammation as well as NO (nitric oxide) production. Inhibiting tyrosine nitration of actin has been a proposed mechanism of action of several radioprotectors like amifostine44. Our results endorse the hypothesis that BIO 300 exerts its radioprotecting effect through the inhibition of Nitrotyrosine of actin. Furthermore, there was evidence of an elevation in the levels of glutathione peroxidase which is a crucial defensive mechanism against oxidative stress produced by irradiation.These changes around the 0.5 to 1 h time points demonstrated an increase in proteins that are involved in the biosynthesis and binding of actin proteins which may play an important role in supporting the infrastructure of cellular membranes against radiation exposure. Several studies reported that radiation promotes changes in the distribution of actinmax of BIO 300 in both routes, minor changes were observed. For the im route, Complement C1r subcomponent (B4DPQ0), which is a major component in the innate immunity system, was downregulated indicating a potential anti-inflammatory activity of the drug. While protein S100 (B2R4M6), which is a low molecular weight damage associated protein, was marginally downregulated, Putative 26S proteasome regulatory subunit 6b (B9PHK0) was upregulated. Also, the po route demonstrated a similar pattern. Trypsin-1 (E7EQ64) and Apolipoprotein E isoform 1 (A0A0S2Z3D5) were slightly downregulated, while Q53GK6 and V9HVY1 were slightly elevated. D0PNI1 is Epididymis luminal protein 4 that has monooxygenase activity and participates in protein domain specific binding. The temporary increase of this protein was associated with a decrease in the levels of phenylalanine and tyrosine at the Cmax of the drug32. These changes returned to near normal levels within the following few hours. This transient elevation in D0PNI1 might be related to the pharmacokinetic effect produced in response to drug absorption into the blood stream.At the Cpo and im routes of administration. Interestingly, we noticed similar patterns for both routes. Most of the protein perturbation occurred earlier in time between 30 min to about 1-h post drug administration but these changes gradually disappeared within a few minutes after Cmax. This pattern was clearly observed in a few proteins e.g., I3L3D5, Q53GK6, A0N071, Pyruvate kinase (B4DRT3), Angiotensinogen variant (Q59EP2) and A0A024R9Q1.Furthermore, we used heatmap-based visualization to examine changes in selected proteins along all the time points for the 45. Furthermore, blocking thrombospondin1 signaling is radioprotective of bone marrow in mice46. Thus, BIO 300′s radioprotective mechanism could be functioning by inhibiting Thrombospondin1 expression. Meanwhile, Alpha-1 antitrypsin (G3V2W1) did not seem to be significantly changed in both routes of administration throughout all time points. This suggests that the anti-inflammatory capability of BIO 300 is not related to the inhibition of protease enzyme activity.Other proteins that showed transient changes in serum abundance included I3L3D5, which is profilin that modulates actin binding and actin cytoskeletal organization, Q53GK6 is β actin variant that is involved in the cytoskeleton structure and A0N071 is a δ-globin that facilitates heme, oxygen and metal binding. B4DRT3 is pyruvate kinase which is involved in kinase activities, magnesium ion binding, and potassium ion binding. Q59EP2 an angiotensinogen variant that regulates systemic arterial blood pressure and X6RJP6 (transgelin-2) which has several biological functions including protein binding were also found to show transient changes in expression post drug administration. Thrombospondin1 (A0A024R9Q1), a cell adhesion molecule was found to decrease in expression 1–2 h post drug administration. Thrombospondin1 plays a role in inducing expression of pro-inflammatory cytokines/genes such as Il-1, IL-6, TNFα and NF-kbpo as well as im administration of BIO 300. The search results against the human and rhesus macaque databases both revealed that protein level was slightly elevated in serum, especially in the im route 30 min to 1 h, meanwhile, this trend was demolished after 1–2 h post-drug administration. Interestingly, gender did not impact the expression pattern of proteins. Proteins D0PNI1, I3L3D5, X6RJP6, B4E3P1, and Q53GK6 showed the same pattern emphasizing the same finding from previous statistical analysis. Q53H26 is transferrin variant that plays an important role in ferric binding and ferric ion transmembrane transport. On the other hand, performing similar analysis on the search results of rhesus macaque database yielded a similar pattern for Transgelin (F6RKI4), F6TIK4 (uncharacterized), Profilin (F7H2A5), Biliverdin reductase (G7NLH4), and C-type lectin domain-containing protein (F7H263). F6RKI4 is a transgelin that facilitates epithelial cell differentiation. F6TIK4 is an uncharacterized protein that is thought to play a role in ATP binding in the synaptic vesicles. F7H2A5 is profilin which regulates actin binding and formation. G7NLH4 is biliverdin reductase B has biliverdin and riboflavin reductase activity and participate in heme catabolism. F7H263 is a C-type lectin domain-containing protein in mannose binding and complement activation.In addition, we examined the longitudinal alterations along all 14 points for proteins that showed clear perturbation at early time points. We used trend line analyses to examine temporal expression of select proteins after Moreover, we noticed a temporal late elevation of a few proteins as a result of drug administration between 8 h and 7 days. The search results against the human database yielded proteins B4DRT3 and Q53H26. Meanwhile, the search results against the rhesus macaque database resulted in two proteins: Apolipoprotein A4 (F7FQN6) and Fructose-bisphosphate aldolase (F6SYK0). F7FQN6 has antioxidant activity and participates in cholesterol biosynthesis, transfer and binding. F6SYK0 is Fructose-bisphosphate aldolase in glycolytic process and fructose-bisphosphate aldolase activity.im and po routes. Our results demonstrate minor changes in proteomic profiles in sera at/around the Cmax of the drug. However, these changes subsided to near normal levels over a short period of time. Furthermore, our longitudinal studies suggested that most of the pharmacokinetic alteration in proteins occurred 30 min to 1 h before Cmax. These alterations suggested that BIO 300 may exert its radioprotective activity through an anti-inflammatory function either by hindering the innate complement system or Thrombospondin 1. Furthermore, these temporal changes suggest that BIO 300 contributes to the elevation of actin by inhibiting its nitration post translation modification. The results of the present study support the advanced development of BIO 300 as an effective medical radiation countermeasure.Taken together, the findings from this study suggest that there were no significant differences in overall serum proteomic profiles resulting from administration of BIO 300 through Macaca mulatta, Chinese sub-strain, two males and two females) 3–7 years of age, weighing 5 to 7 kg, were obtained from National Institutes of Health Animal Center  and maintained in a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC)-International. Animals were quarantined for six weeks prior to initiation of the experiment. Animal housing, health monitoring, care, and enrichment during the experimental period have been described earlier47. Animals were fed a primate diet  twice daily with at least 6 h between feedings  and received drinking water ad libitum. All procedures involving animals were approved (Protocol # P2017-02-005 approved on 23 February 2017) by the Institutional Animal Care and Use Committee  and the Department of Defense Animal Care and Use Review Office (ACURO). This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.A total of four naïve rhesus macaques , 0.2% polysorbate 80 (w/w), 0.18% methylparaben (w/w), and 0.02% propylparaben (w/w)) was used for po dosing32. These two formulations contain the same active pharmaceutical ingredient (synthetic genistein nanoparticles) but differ in their excipients, which have been optimized for the route of administration. A single dose of BIO 300 injectable formulation (50 mg/kg) using a 23 G needle length of 5/8″ attached to a 1 mL syringe was administered im to the four NHPs. The site for injection (thigh quadriceps) was prepared as a surgical site before the injection: hair was clipped using #40 surgical blade and the site was scrubbed at least three times using 4% chlorhexidine and 70% alcohol. The same NHPs used for the im study were used for the po study after a 31-day wash-out period. BIO 300 oral formulation (100 mg/kg) was administered po as a single dose via a nasogastric (NG) tube. NG tube placement was confirmed via digital X-ray prior to drug administration.In this study, two formulations of BIO 300 were used, an oral formulation and an injectable formulation. BIO 300 injectable formulation , 0.2% polysorbate 80 (w/w) in 50 mM phosphate buffered saline (61 mM sodium chloride)) was used for 32. On days when animals were only bled once, the blood draw was conducted between 08:00 AM and 10:00 AM, 1–3 h after animals were fed. A 3 mL disposable luer-lock syringe with a 25-gauge needle was used to collect the desired volume of blood. For serum collection, blood samples were transferred to Capiject serum separator tubes , allowed to clot for 30 min, and then centrifuged . Serum samples were stored at − 70 °C until shipped on dry ice to the Georgetown University Medical Center  for the global proteomic analysis.Blood samples were collected by venipuncture from the saphenous vein of the lower leg after the site was cleaned using a 70% isopropyl alcohol wipe and dried with sterile gauze. All animals were restrained using the pole-and-collar method and placed in a chair for blood collection. On the day of drug administration, animals were bled repeatedly at 0.25, 0.5, 1, 2, 4, 8, and 16 h post-drug administrationSerum samples were diluted tenfold using the load/wash buffer supplied by the Agilent Corporation and the remaining particulates in the diluted serum were removed by centrifugation through a 0.22-μm spin filter (Agilent Cat.#: 5185-5990) for 2 min at 16,000 × g. Multiple Affinity Removal System  columns (4.6 × 50 mm) designed to deplete 14 abundant proteins in serum  were used according to the manufacturer protocol on a Waters Acquity HPLC system. Protein peaks were collected and transferred to Spin Concentrators, 5 K MWCO, 4 mL capacity (Agilent Cat. #: 5185-5991) and centrifuged at 17,172 × g for 45 min at 4 °C. A volume of 2 × 1 mL of water was added to the concentrated sample and centrifuged at 17,172 × g for 45 min at 4 °C. A volume of 1 mL of 50 mM ammonium bicarbonate buffer pH of approximately 8, was used to rebuffer each sample of concentrated proteins. The final protein concentration was determined using a BCA protein assay kit (Thermo Fisher Scientific Cat. #: 23227).18 columns  and eluted using 2 × 500 µL of 50% ACN/H2O acidified by 0.1% formic acid. The collected fractions were evaporated under vacuum and reconstituted for LC–MS for SWATH analysis.Briefly, protein concentration was adjusted to 10 μg/μL in 50 mM ammonium bicarbonate buffer, reduced with 5 mM Dithiothreitol (DTT) for 60 min at 56 °C and alkylated with 15 mM iodoacetamide for 30 min at 37 °C in the dark. The reaction was quenched by addition of another aliquot of 5 mM DTT followed by digestion with (2.5 ng/μL) Trypsin Gold  at 37 °C overnight, to complete the reaction. Trypsin was deactivated by heating at 90 °C for 10 min and samples were allowed to cool down to room temperature then acidified to a pH = 3 using 0.1% trifluoroacetic acid in water. The digested peptides were purified on microspin C18 column (Waters) on an Acquity UPLC instrument with a TUV detector in high pH reversed-phase chromatography mode. Solvent A  and a nonlinear increasing concentration of solvent B  were used to separate peptides. The 60-min separation LC gradient followed this profile: (min: %B) 0:2; 5:2; 10:10; 40:40; 42:90; 45:90; 50:2, and 60: 2. The flow rate was 0.2 mL/min. A total of twelve fractions were obtained in a step-wise concatenation strategy, followed by acidification to a final concentration of 0.1% formic acid and dried down with a SpeedVac. Peptides in each fraction were then analyzed with a nanoAcquity UPLC system (Waters) coupled with a TripleTOF 6600 mass spectrometer (Sciex), with similar settings as shown previously48. Specifically, dried peptides were dissolved into 2% ACN in water plus 0.1% formic acid and loaded onto a C18 Trap column  at 15 µL/min for 4 min. Peptides were then separated with an analytical column  which was temperature controlled at 40 °C. The flow rate was set as 400 nL/min. A 60-min gradient of buffer A  and buffer B (0.1% formic acid in ACN) was used for separation: 1% buffer B at 0 min, 5% buffer B at 1 min, 35% buffer B at 35 min, 99% buffer B at 37 min, 99% buffer B at 40 min, 1% buffer B at 40.1 min, with the column equilibrated with 1% buffer B for 20 min. Data were acquired with the TripleTOF 6600 mass spectrometer using an ion spray voltage of 2.3 kV, GS15 psi, GS2 0, CUR 30 psi and an interface heater temperature of 150 °C. For spectra library generation, the pooled sample  was analyzed, and mass spectra were recorded with Analyst TF 1.7 software in DDA mode. Each cycle consisted of a full scan (m/z 400–1800) and fifty (DIAs) (m/z 100–2000) in the high sensitivity mode for precursors with a 2 + to 5 + charge state. Rolling collision energy was used.First, we established a protein library of all serum proteins detectable with tandem mass-spectrometry (MS/MS) by the analysis of a sample pool from all the subjects . To get a comprehensive ion library, the pooled peptide sample was fractionated with a 2.1 mm × 100 mm column XTerra MS CEach sample from the cohort was acquired individually via label-free SWATH DIA to quantify proteins in the samples. For SWATH acquisition, each of the samples were injected into the same NanoUPLC-MS/MS system but acquired by repeatedly cycling through 32 consecutive 25-Da precursor isolation windows, generating time-resolved fragment ion spectra for all the analytes detectable within the 400–1250 m/z precursor ion scope.49 and the integrated false discovery rate (FDR) analysis function50. The MS/MS data was searched against the NCBI Uniprot database of Homo sapiens downloaded on Oct. 10th, 2019, and NCBI Uniprot database of rhesus macaque downloaded on Oct. 11, 2019. Trypsin was selected as the digesting enzyme. Carbamidomethylation was set as a fixed modification on cysteine. Variable peptide modifications included methionine (M) oxidation. Other search parameters including instrument (TripleTOF 6600), ID Focus , search effort (Thorough), FDR analysis (Yes), and user modified parameter files (No) were optimized accordingly. Proteins were inferred based on the ProGroupTM algorithm associated with the ProteinPilot software. The detected protein threshold in the software was set to the value which corresponded to 1% FDR. Peptides were defined as redundant if they had identical cleavage site(s), amino acid sequence, and modification.For spectral library generation, data files for DDA of the pooled sample fractions were submitted for simultaneous searches using ProteinPilot version 5.0 software (Sciex) utilizing the Paragon, ProGroup algorithmsFor the label-free SWATH quantification, data from each sample was analyzed by PeakView 2.2 (Sciex), with the following settings: (1) Peptide filter: # of peptides per protein: 6; # of transitions per peptide: 6; peptide confidence threshold: 99%; FDR threshold: 1%; (2) XIC Options: XIC extraction window (min): 5; XIC width (ppm): 75. The peak area of each protein was used for protein level quantification. The retention time was calibrated before processing based on a selected set of reference peptides. Quantifiable peptides were carefully chosen, and the peak intensities were normalized based on total ion current (TIC) for further statistical analysis.t-test, whereas, p values of less than 0.05 exclusively were considered significant. Furthermore, p values were corrected for multiple testing using Benjamini–Hochberg procedure which limits the FDRs. Principal-Component Analysis (PCA), Hierarchical Clustering and all statistical analyses were performed using R (version 3.6.1).Spectra were searched against Uniprot human database. Peak intensities of identified proteins were then extracted, filtered, TIC normalized and summarized for protein quantification. The p values were calculated either by Mann–Whitney U test or two-tailed unpaired Student Supplementary information.


Saccharomyces cerevisiae cells with the psf1-100 mutation. This significantly affects DNA replication activity measured in vitro, while in vivo, the psf1-100 mutation reduces replication fidelity by increasing slippage of Pol ε, which manifests as an elevated number of frameshifts. It also increases the occurrence of single-stranded DNA (ssDNA) gaps and the demand for homologous recombination. The psf1-100 mutant shows elevated recombination rates and synthetic lethality with rad52Δ. Additionally, we observe increased participation of DNA polymerase zeta (Pol ζ) in DNA synthesis. We conclude that the impaired interaction between GINS and Pol ε requires enhanced involvement of error-prone Pol ζ, and increased participation of recombination as a rescue mechanism for recovery of impaired replication forks.The CMG complex ) is crucial for both DNA replication initiation and fork progression. The CMG helicase interaction with the leading strand DNA polymerase epsilon (Pol ε) is essential for the preferential loading of Pol ε onto the leading strand, the stimulation of the polymerase, and the modulation of helicase activity. Here, we analyze the consequences of impaired interaction between Pol ε and GINS in  Faithful replication of the genome and precise segregation of sister chromatids are essential for the maintenance of chromosome stability. External or endogenous impediments that stall or slow replication forks are sources of replication stress. Replication stress influences the maintenance of genome integrity and is an important source of various physiological pathologies, including cancer ,2.Precise DNA replication requires coordinated action of the catalytic and noncatalytic proteins forming the replisome complex . In receSaccharomyces cerevisiae [Replication machineries assembled at the replication origin move by the action of 11-subunit CMG helicase, which consists of Cdc45 ,8,9, therevisiae ,30,31,32revisiae ,36,37,38revisiae ,39. Addirevisiae . Among trevisiae ,42, wherrevisiae ,44,45. Rrevisiae . Additiorevisiae ,48,49,50REV3 gene. DRIM occurs as a result of mutations in replicative polymerases or accessory subunits that impair the proper functioning of the replisome [Under circumstances disturbing the replication process, DNA synthesis may be continued by specialized polymerases involved in trans-lesion synthesis (TLS). In yeast cells, Pol ζ (zeta), a TLS polymerase, is composed of the Rev3 subunit with a DNA polymerase domain and the accessory subunit Rev7 . The twoeplisome ,64,65,66Alongside the contribution of error-prone polymerases and various DNA repair mechanisms, homologous recombination (HR) is central to the maintenance of genome stability in all eukaryotic organisms. Through identification of a homologous sequence this mechanism enables the restoration of genetic information and facilitates the repair of DNA breaks or the recovery of stalled replication forks. The highly conserved proteins Rad52 and Rad51 are the major recombinases. Rad52 is the mediator facilitating the recruitment of Rad51 to replication protein A (RPA)-coated single-stranded DNA (ssDNA). This enables the formation of the Rad51/ssDNA nucleofilament involved in searching for and invading the homologous DNA sequence, ssDNA annealing and DNA strand exchange, reviewed in ,68. HR iS. cerevisiae psf1-100 allele constructed in our laboratory [psf1-100 strain exhibits a cold-sensitive phenotype (inability to grow at 18°C) and dumbbell cell morphology with the nucleus in the isthmus between mother and daughter cell. Previously, using two-hybrid analysis and pull-down assays, we showed that the psf1-100 mutation strongly impairs the interaction of Psf1 with the N-terminal part of the Dpb2 subunit of Pol ε [The study of noncatalytic proteins of the CMGE complex is attracting greater attention, because mutations in genes encoding its subunits or changes in their expression have a significant impact on the development of cancer or genetic diseases ,77,78,79boratory . The Psfof Pol ε . TherefoIn eukaryotes, the CMG helicase complex and the leading strand polymerase Pol ε form a 15-subunit assembly called CMGE ,36. To bPsf1-100 and Pol ε.To verify the impact of Psf1-100 on DNA replication processes first, we used purified GINS comprising Psf2, Psf3, Sld5, and Psf1 or Psf1-100 subunits, as well as Pol ε comprising Pol2, Dpb2, Dpb3, and Dpb4 subunits, to analyze the interactions between these two complexes A. After psf1-100 mutation on DNA synthesis, we performed an in vitro replication assay [Psf1 or GINSPsf1-100. When wild-type GINS was used, the maximal (100%) replication activity was reached at a 10 nM GINS concentration. At the same GINS concentration, the replication activity of the replisome with Psf1-100 subunit reached only 50% of the maximal activity observed for the native Psf1. The maximal activity of the mutated replisome was achieved only at 60 nM GINS  regions. As ssDNA is coated with RPA [psf1-100 strain compared to the wild-type strain. Moreover, more mutant cells contained multiple Rfa1 foci  at one CAN1 locus, and the SUP4-o gene (an ochre suppressor) at the second CAN1 locus on the other chromosome V copy. Recombination events are identified through selection of CanR colonies resulting from the formation of can1-100 homozygous cells (lacking SUP4-o) and SUP4-o homozygous cells . The selection of CanR clones enables the calculation of rates of recombination events. We constructed appropriate PSF1 and psf1-100 diploid strains and observed that the rate of wild-type CanR clones formation was 1.51 × 10−5, while for psf1-100 ones, the rate was 10.46 × 10−5 , reviewed in [MMS2 gene to test whether the TS mechanism contributes to the restoration of DNA synthesis in psf1-100 cells. The rate of CanR mutagenesis in the double psf1-100 mms2Δ showed an additive effect (388 × 10−8) compared to the single mutants: 182 × 10−8 for psf1-100 and 282 × 10−8 for mms2Δ  and pif1Δ (328 × 10−8). Next, we tried to combine psf1-100 with deletion of POL32 encoding a subunit of Pol δ required for BIR [psf1-100 pol32Δ mutants by tetrad dissection were unsuccessful  catalytic subunit of Pol ε lacks the proofreading function; thus, Pol ε proofreading does not contribute to error removal in strains carrying this allele [REV3 prevents the introduction of errors by Pol ζ which demonstrated increased participation in DNA synthesis in psf1-100 cells [psf1-100 pol2-4 rev3Δ were compared with spectra of rev3Δ, psf1-100 rev3Δ, and pol2-4 rev3Δ . However, the most pronounced increase in mutation rates in psf1-100 pol2-4 rev3Δ compared to psf1-100 rev3Δ and pol2-4 rev3Δ was +1 insertion with mutagenesis rates 86, 2.5, and 13 × 10−8, respectively, with p-values ≤ 0.05 18, (GT)25, (AACGCAATGCG)4, and (CAACGCAATGCGTTGGATCT)3 [URA3 gene [URA3. Yeast with such modifications are selected on media containing 5-fluoroorotic acid (5-FOA), which is toxic to cells producing the Ura3 protein [psf1-100 than in wild-type cells (psf1-100 was observed for the (G)18, (GT)25, (AACGCAATGCG)4, and (CAACGCAATGCGTTGGATCT)3 sequences with 3.0-, 6.3-, 2.1-, and 4.3-fold changes, respectively 3 ,108. AddRA3 gene . Expansi protein . For thepe cells . A signiectively . The obtSmooth replication fork progression depends on coordinated DNA unwinding and synthesis by the replisome components. In addition to in-depth research into the role of DNA polymerases in controlling DNA replication fidelity, several studies indicate the significant role of other noncatalytic components of the replisome in genome stability ,66,111. psf1-100 allele isolated in our laboratory [Psf1-100 and Pol ε complexes . Previously, we showed, that the observed Pol ζ-dependent errors in psf1-100 cells were not corrected by the MMR mechanism [psf1-100 cells. Moreover, our results show that in addition to enhancing participation of Pol ζ, the psf1-100 mutation also affects the functioning of Pol ε. By combining psf1-100 with the proofreading-deficient pol2-4 allele in the Pol ζ-deficient background, we confirmed slippage of Pol ε during DNA replication, which manifests by an increased number of + 1 frameshift mutations in the double mutant cells  has an psf1-100 strain, inactivation of the latter increases the survival of the psf1-100 strain by approximately 40%, as observed for CFU counts in rev3Δ strain   was used for the selection of URA3 mutants [The is study  are deri mutants . Yeast s mutants . Isolati169, hsdR17 , λ-  were grown at 37 °C in LB medium, supplemented when needed with ampicillin 100 μg/mL . E. coli cells were transformed as described in [Escherichia coli DH5α (endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR nupG Φ80d lacZΔ M15Δ (lacZYA-argF) Uribed in . BacteriPSF1, LEU2) and  cassettes described in [PSF1 locus of the SC765 strain. The presence of the  and  alleles was confirmed by PCR using primers Inprom and dwPSF1  cassette described in [RFA1 locus of the SC766 and SC778 strains. The presence of the  allele was confirmed by PCR, using the primers RFA6231R, RFA7367F and YFP9451R  integration cassette described in [G2-REV3, natNT2) cassette was PCR-amplified with the primers S1_REV3 and S4_REV3  was incubated with rotation with 10 µL of HaloLink Resin in 1× buffer  at 4 °C for 30 min. Next, casein (0.8 mg/mL) and BSA (20 mg/mL) were added, followed by further incubation with rotation at 4 °C for 30 min. Then, the beads were washed two times with 400 µL of 1× buffer, mixed with 2 pmol of Pol ε and 2 mg/mL of BSA in 400 µL of 1× buffer and incubated with rotation at 30 °C for 30 min. Afterwards, the beads were washed two times with 400 µL of 1× buffer and mixed with 50 µL of 1× sample buffer  and incubated at 65 °C for 10 min. The supernatant was mixed with DTT and loading dye and subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Separated proteins were transferred to membranes and incubated with anti-Psf2  and antipARS1 (245 bp ARS1 fragment cloned into the SmaI site of pNEB193) DNA was mixed with the loading complex composed of 11 nM ORC, 23 nM Cdc6, and 50 nM MCM–Cdt1 in a buffer containing 25 mM HEPES-KOH at pH = 7.6, 100 mM Kglutamate, 10 mM magnesium acetate, 0.01% NP-40, 100 μg/mL of BSA, 1 mM DTT, and 5 mM ATP. After 20 min of incubation at 30 °C, DDK was added directly to the reaction to a final concentration of 26 nM, and incubation was continued at 30 °C for 20 min. Next, the replication complex proteins and solutions: were added directly to the reaction: 25 nM Sld3–Sld7, 30 nM Sld2, 30 nM Dpb11, 30 nM GINS, 40 nM Cdc45, 20 nM Polε, 5 nM Mcm10, 100 nM RPA, 3.4 nM CDK, 5 nM Polα, 20 nM RFC, 20 nM PCNA, 10 nM Top2, 10 nM Polδ, 20 mM each NTP, 8 mM dATP, dCTP, and dGTP, 6 mM dTTP, and 1 mM Biotin-16-dUTP (Sigma-Aldrich). After incubation at 30 °C for 20 min, the reaction was terminated by the addition of 1/5 volume of Alkaline stop dye (loading buffer) containing 0.3 N NaOH, 6 mM EDTA, 36% glycerol, and 0.1% Orange G dye. The products were separated on 1% alkaline agarose gels in 0.05 N NaOH and 1 mM EDTA for 75 min at 75 V. DNA was transferred from the gels to Hybond N+ membranes (GE) in 0.5 TBE Buffet for 30 min at 80 V. The membrane was treated with 20× SCC and crosslinked in UV linker. After treatment with 2% ECL Advance Blocking Reagent in TBST for 10 min and two 5 min washes by TBST, the membranes was incubated for 30 min with 0.5 μg/mL of IR-Dye 680 RD streptavidin (LICOR) in TBST with 10% SDS. After washing in TBST with 0.1% SDS, the membrane was scanned on an Odyssey infrared imaging system (LI-COR).CAN1 locus were calculated by dividing the cell count from selective media by the cell count from nonselective media. Each experiment was repeated at least three times. The spontaneous mutation rates were determined as described below.To determine spontaneous mutation frequencies, 10–20 cultures of 2 or 3 independent isolates of each strain were inoculated in 2–20 mL of liquid SD medium, supplemented with the required amino acids and nucleotides. Cultures were grown at 30 °C to the stationary phase. Aliquots of concentrated cultures and appropriate dilutions were plated on selective (containing L-canavanine) and nonselective media, respectively. After 3–5 days of growth at 30 °C, colonies were counted. The frequency of forward mutations at the μ = ƒ/ln(Nμ), where μ is the mutation rate per round of DNA replication, ƒ is the mutant frequency, and N is the total population size [p-values of the differences between the mutation rates of the respective strains, a nonparametric Mann–Whitney U test was used.The mutation rates were calculated using the equation ion size . To calcR colonies selected randomly from each plate was isolated and used for PCR amplification of the CAN1 locus with primers MGCANFF and MGCANRR and DNA sequencing with primers Can_1666, Can_1963, Can_2241, and Can_2465 . After 3–4 days of growth at 30 °C in the darkness, the colonies were counted. The mutant frequency was calculated by dividing the CanR mutant count by the viable cell number. The results represent the median values. The experiment was repeated three times.To determine the UV sensitivity of yeast strains, 10–12 cultures of 2 or 3 independent isolates of each strain were inoculated in 20 mL of liquid SD medium (supplemented with required amino acids and nucleotides) and grown at 30 °C to the logarithmic phase. Aliquots of concentrated cultures and appropriate dilutions were plated (in duplicate) on selective (containing L-canavanine) and nonselective media, respectively, and immediately exposed to specified UV doses of 0, 5 and 15 J/m600 reached 0.4. Next, cells were harvested and resuspended in fresh medium supplemented with α-factor (4 µg/mL). Additionally, α-factor (4 µg/mL) was added after 60–90 min of incubation. After 2–3 h of growth at 30 °C, to release them from α-factor, cells were harvested and washed three times with sterile water, and then resuspended in fresh SD medium and incubated at 30 °C; 1 mL samples were immediately collected and fixed in 70% ethanol. Other samples were taken at the indicated time points and fixed in 70% ethanol.Strains Y1023 and Y1024 were grown until the ODSamples were prepared for flow cytometry, as described previously , with moThe Y1023 and Y1024 strain cells were synchronized with the α-factor (see Synchronization in G1 phase with α-factor) and released from G1-arrest into fresh SD medium and incubated at 30 °C. Samples were taken at the indicated time points and prepared, as described previously . ProteinRAD52–YFP fusion  [RAD51-GFP fusion . The RFA1–YFP fusion was introduced into the native RFA1 locus by gene replacement. Cells were grown at 30 °C to the logarithmic phase in SD liquid medium supplemented with the required amino acids and nitrogenous bases lacking leucine or tryptophan. The Rad52, Rad51, and Rfa1 foci were examined using the Axio Imager M2 fluorescence microscope with the AxioCam MRc5 Digital Camera . Images were analyzed with Axio Vision 4.8 software. The number of cells and Rad52, Rad51, or Rfa1 foci in the cells were counted. To analyze the possible differences between wild-type and psf1-100 strains in foci formation, we used contingency tables, and further applied the χ2 test. For each strain, three biological replicates were analyzed.Strains SC766 and SC778 were transformed with the pWJ1344 plasmid carrying a R, oriC)  or with To determine the viability of the strains Y1012, Y1013, Y1014, Y1017, Y1018, Y1019, Y1020, and Y1006, 3 mL cultures of independent isolates of each strain in liquid SD medium supplemented with the required amino acids and nucleotides were grown at 30 °C to the stationary phase. Yeast viability assessment was performed using two methods. First, to calculate the colony-forming units (CFU), cells were counted in a Neubauer counting chamber, and next, the same number of cells (250 cells) was plated in triplicate on nonselective media. After 4 days of growth at 30 °C, the colonies were counted. The CFU was calculated by dividing the number of colonies formed after plating by the total cell number. Second, to calculate viable cells, 1 mL of each culture was harvested by centrifugation, resuspended in 1 mL PBS containing PI (0.32 μg/mL)  and incubated for 15 min at room temperature in the dark. Next, the samples were again centrifuged, and the pellets were resuspended in 30 μL PBS. Dead PI-stained cells were examined using the Axio Imager M2 fluorescence microscope . Images were analyzed with Axio Vision 4.8. At least 800 cells were examined to count the number of dead and viable cells of each strain, and the average percentage of viable cells was calculated.URA3 mutants) and nonselective media. Colonies were counted after 3–5 days of incubation at 30 °C. The spontaneous mutation rates were determined as described above.The stability of repetitive sequences was determined, as described previously , with mo


Due to low conductivity and large volume expansion/contraction during charge/discharge cycling of Bi2MoO6, effective modification is indispensable to address these issues. In this study, a plate-to-layer Bi2MoO6/Ti3C2Tx (MXene) heterostructure is proposed by electrostatic assembling positive-charged Bi2MoO6 nanoplates on negative-charged MXene nanosheets. MXene nanosheets in the heterostructure act as a highly conductive substrate to load and anchor the Bi2MoO6 nanoplates, so as to improve electronic conductivity and structural stability. When the mass ratio of MXene is optimized to 30%, the Bi2MoO6/MXene heterostructure exhibits high specific capacities of 692 mAh g−1 at 100 mA g−1 after 200 cycles and 545.1 mAh g−1 with 99.6% coulombic efficiency at 1 A g−1 after 1000 cycles. The results provide not only a high-performance lithium storage material, but also an effective strategy that could address the intrinsic issues of various transition metal oxides by anchoring them on MXene nanosheets to form heterostructures and use as anode materials for LIBs. BiThe online version of this article (10.1007/s40820-019-0312-y) contains supplementary material, which is available to authorized users. However, the intrinsic low conductivity and the large volume change during lithiation/delithiation of Bi2MoO6 still require the modification with conductive materials [2MoO6 nanoplates with the Ti3C2Tx MXene nanosheets was achieved by adding the Bi2MoO6 dispersion into the MXene solution and mixing thoroughly under continuous stirring. In order to confirm the successful electrostatic self-assembly of the Bi2MoO6/MXene heterostructure, the zeta potentials of the as-prepared heterostructures coupled with the two components, i.e., the pristine Ti3C2Tx nanosheets and the Bi2MoO6 nanoplates, were measured. As shown in Fig. S5, the as-prepared MXene nanosheets are negatively charged with a zeta potential of − 40.5 mV, which is ascribed to the presence of surface functional groups  [2MoO6 (10.1 mV) mixed with the negative-charged MXene, Bi2MoO6 nanoplates were anchored on the MXene nanosheets based on an electrostatic interaction. Therefore, the zeta potentials of the Bi2MoO6/MXene heterostructures shift down with the increase in the MXene content, demonstrating the effective electrostatic process. In the assembled heterostructure, the highly conductive MXene nanosheets are expected to significantly enhance the conductivity and alleviate the volume expansion/contraction of Bi2MoO6 during lithiation/delithiation, thus improving the rate performance and cycling stability.Figure  reports , 33. Sinig. S3c) . Its straterials , 30. Theand –OH) , 33. Whe2MoO6/MXene heterostructures are shown in Fig. 2MoO6/MXene: 50:50  peak at 10.9° and (131) peak at 28.2° as well as several weak peaks corresponding to the (200), (060), (151), (202), and (062) planes of pristine Bi2MoO6 [2MoO6 and MXene. Moreover, Fig. 2MoO6/MXene-10%, Bi2MoO6/MXene-30%, Bi2MoO6/MXene-50%, and pristine MXene, which is marked by a purple frame in Fig. 2MoO6 nanoplates. The structures of the Bi2MoO6/MXene heterostructures were also characterized by Raman spectroscopy, and the results are shown in Fig. 2MoO6 displays distinct phonon modes in the range of 200–1000 cm−1, signifying the vibrational modes of orthorhombic Bi2MoO6. Specifically, the bands located at 202, 285, 324, 355, and 404 cm−1 can be ascribed to the bending, wagging, and twisting modes of Mo–O bonds, while the bands at 713, 796, and 848 cm−1 correspond to the stretching modes of Mo–O bond [−1 (ω4) corresponding to Eg in-plane vibration of surface functional group atoms, the bands of the pristine MXene show similar locations to the Bi2MoO6, including the bands located at 193 cm−1 (ω2) and 711 cm−1 (ω3) for A 1 g symmetry out-plane vibrations of Ti and C atoms and those at 284 cm−1 (ω5) and 356 cm−1 (ω5) for Eg in-plane vibration of Ti and C atoms [2MoO6/MXene heterostructures display all the characteristic peaks belonging to Bi2MoO6  and MXene (621 cm−1), and the increase in MXene content in the heterostructures leads to the stronger peaks for MXene together with the weaker peaks for Bi2MoO6.XRD analysis was conducted to identify the composition of the samples, as shown in Fig.  Bi2MoO6 –51. Afte.9° and 1 peak at  C atoms , 53. The2MoO6 nanoplates, pristine MXene, and Bi2MoO6/MXene-30%. The XPS survey of the Bi2MoO6/MXene-30% comprises the characteristic peaks of Bi2MoO6 and MXene, such as Ti 2p peak for Ti3C2Tx as well as Bi 4f and Mo 3d peaks for Bi2MoO6 , confirming the strong integration between the Bi2MoO6 nanoplates and the MXene nanosheets. High-resolution XPS spectra of Bi 4f and Mo 3d core levels of Bi2MoO6/MXene-30% and Bi2MoO6 are shown in Fig. f core level of Bi2MoO6/MXene-30% displays two peaks at 159.4 and 164.7 eV, related to Bi 4f7/2 and Bi 4f5/2 of the Bi3+, respectively. Meanwhile, the Mo 3d core level of Bi2MoO6/MXene-30% could be divided into two peaks situated at 232.8 and 235.9 eV, which can be assigned to Mo 3d5/2 and Mo 3d3/2 of Mo6+, respectively. It is noteworthy that the peaks of Bi 4f and Mo 3d core levels of Bi2MoO6/MXene-30% have higher binding energies than those of the Bi2MoO6, implying that the Bi2MoO6 nanoplates become more electrochemically active with the assistance of MXene. As shown in Fig. s core level of Bi2MoO6 is fitted with two components centered at 529.5 and 530.3 eV, which could be ascribed to Bi–O and Mo–O bonds, respectively [2MoO6, the Bi2MoO6/MXene-30% shows an additional peak at 531.6 eV corresponding to Ti–OH bond due to the presence of the MXene. Ti 2p XPS spectra are also showed in Fig. p core level was fitted with seven components, including three doublets (Ti 2p3/2–Ti 2p1/2) and a single peak located at 460.8 eV. The three doublets centered at 454.7/462.2, 455.9/464.4, and 458.7/466.1 eV could be assigned to Ti–C, Ti(II), and Ti–O, respectively, while the single peak might result from TiO2, implying the partial oxidation of the MXene during the construction of the Bi2MoO6/MXene heterostructure [XPS was carried out to evaluate the surface chemical properties of the Biectively , 48. Diftructure , 50.2MoO6/MXene composite electrodes and the Bi2MoO6 electrode were first explored by cyclic voltammetry (CV) for the first three cycles at a scan rate of 0.1 mV s−1 within a voltage range of 0.01–3 V, as shown in Fig. + into layer structure of the Bi2MoO6 crystal, the irreversible transformation from Bi2MoO6 to Bi and Mo metal  of 72.9%. The capacity loss in the first cycle might be attributed to the formation of SEI layer coupled with the irreversible reactions as mentioned above [2MoO6/MXene-30% (72.9%) is lower than those of Bi2MoO6/MXene-10% (76.6%) and Bi2MoO6 (82.6%), but higher than that of Bi2MoO6/MXene-50% (71.2%). The possible reason for this phenomenon is that with higher MXene content, the increased surface area of MXene results in more irreversible reactions between Li+ and the functional groups on the MXene surface, such as –O, –F, and –OH [The galvanostatic charge/discharge curves of the electrodes were conducted at a current density of 100 mA g V Figs. b and S8,ed above , 54, 55. and –OH , 56.2MoO6/MXene composite electrodes and the Bi2MoO6 electrode is given in Fig. −1. The Bi2MoO6/MXene-30% delivers a stable capacity of 692 mAh g−1 after 200 cycles, much higher than those of Bi2MoO6/MXene-50% (617.5 mAh g−1), Bi2MoO6/MXene-10% (497.6 mAh g−1), and the pristine Bi2MoO6 (416.1 mAh g−1). It indicates that the optimized ratio of the composite is beneficial for maximizing the lithium storage capacity of the Bi2MoO6/MXene. Furthermore, a capacity fading occurs in the initial dozens of cycles followed by a capacity reactivation process and an eventual capacity stabilization. The capacity fading results from the mechanical degradation of the electrode structure and the formation of an unstable SEI layer, while the subsequent capacity reactivation and stabilization could be ascribed to a restructuring process as well as stable SEI formation with cycling [The comparison of the cycling performance of the Bi cycling .2MoO6/MXene-30% and the pristine Bi2MoO6 at various current densities from 0.05 to 2 A g−1. As seen, the pristine Bi2MoO6 has a slightly higher capacity of 629.4 mAh g−1 at the small current density while it could only deliver capacities of 375.6, 323.7, and 279.8 mAh g−1 at 0.5, 1, and 2 A g−1, respectively. By contrast, the specific capacity of Bi2MoO6/MXene-30% is measured to be 626.5, 580.6, 522.1, 477, 414.8, and 328.2 mAh g−1 at 0.05, 0.1, 0.2, 0.5, 1, and 2 A g−1, respectively. It demonstrates that the conductive MXene effectively enhances the rate performance of the composite electrode. As displayed in Fig. 2MoO6/MXene-30% exhibits the best rate capability from 0.05 to 2 A g−1 compared with the Bi2MoO6/MXene-50%, Bi2MoO6/MXene-10%, and Bi2MoO6 electrodes. When the current density is turned back from 2 to 0.1 A g−1, the specific capacity of Bi2MoO6/MXene-30% are recovered up to 566.3 mAh g−1, signifying the best reversibility and structural stability compared with other electrodes. Furthermore, to estimate the long-term cycling stability, Bi2MoO6/MXene-30% was charged/discharged for 1000 cycles at a high current density of 1 A g−1 . The impedance behaviors of the Bi2MoO6/MXene-30% and Bi2MoO6 electrodes were explored by the complex model of capacity to confirm the promotion of MXene for rapid diffusion and transportation of lithium ions . After the decoration of MXene, the Bi2MoO6/MXene-30% displays much lower minimal characteristic relaxation time constant τ0 (919 ms) and downtrend of normalized C′(ω) than the pristine Bi2MoO6 electrodes, implying faster transport and diffusion of electrolyte ions in the Bi2MoO6/MXene-30% electrode [To study the origins of the better rate capability of Bikinetics , 59. Thelectrode .Fig. 6El2MoO6/MXene-30%. Figure 2MoO6/MXene-30% at various scan rates from 0.2 to 3 mV s−1. The charge storage mechanism could be analyzed by the following formula, which shows the relationship between the measured current (i) and the scan rate (v):b could be calculated from the slope of the fitted log(i) − log(v) curves [b = 0.5) or a non-diffusion-controlled behavior (b = 1). As shown in Fig. b value of Bi2MoO6/MXene-30% is 0.8987 at 0.58 V in the cathodic scan and 0.8662 at 0.97 V in the anodic scan, indicating a fast charge storage kinetic dominated by pseudocapacitive behavior. This behavior can also be confirmed by quantifying the pseudocapacitive contribution based on Eq. i(v), k1v, k2v1/2, and v are the measured current at a certain potential, the capacitive-dominated current, the diffusion-controlled current, and the corresponding scan rate, respectively. The capacitive-dominated current at a certain scan rate could be obtained via calculating the value of k1 [2MoO6/MXene-30% at various scan rates, in which the shaded portion stands for the capacitive-dominated region, while the non-shaped portion means the diffusion-controlled region. It can be seen that up to 72.8% of the charge is contributed by the pseudocapacitive behavior at 1 mV s−1. Moreover, the pseudocapacitive contribution of the Bi2MoO6/MXene-30% electrode enhances with the scan rate increasing  curves , 61, disue of k1 , 61. Fig2MoO6 could be effectively enhanced by introducing highly conductive MXene as a substrate to fabricate a plate-to-layer Bi2MoO6/MXene heterostructure. In the heterostructure, the MXene nanosheets can promote the charge transport and alleviate the volume change of the Bi2MoO6, leading to high specific capacity, superior rate capability, and excellent long-term cycling stability. The Bi2MoO6/MXene-30% exhibits competitive performance compared with other TMOs-based electrodes, indicating its promising potential as an anode material of LIBs.The above results suggest that the lithium storage property of Bi2MoO6 as an electrode material for LIBs, i.e., low electronic conductivity and huge volume change, we have fabricated Bi2MoO6 nanoplates on highly conductive Ti3C2Tx MXene nanosheets to form a plate-to-layer heterostructure via a simple electrostatic self-assembled method. In the Bi2MoO6/MXene heterostructure, the MXene nanosheets cannot only promote the electron transfer and facilitate the Li+ transport, but also accommodate the volume expansion/contraction of Bi2MoO6 during lithiation/delithiation, endowing the composite electrodes with high conductivity, good structural stability, and excellent electrochemical durability. As a result, the Bi2MoO6/MXene-30% exhibits remarkably enhanced lithium storage properties, presenting a specific capacity of 692 mAh g−1 at 100 mA g−1 after 200 cycles, a superior rate capability of 328.2 mAh g−1 at 2 A g−1 as well as an outstanding cycling durability with a capacity of 545.1 mAh g−1 and 99.6% coulombic efficiency at 1 A g−1 after 1000 cycles. The Bi2MoO6/MXene heterostructure with competitive performance is conceivable to be a promising high-performance anode material for LIBs. Furthermore, various TMOs/Ti3C2Tx composites were suggested to be explored using Ti3C2Tx MXene nanosheets as a conductive substrate to achieve good electrochemical performance and application in energy storage.To solve the problems of BiSupplementary material 1 (PDF 897 kb)Below is the link to the electronic supplementary material.