Multiple tumors are synergistically promoted by c-Met and TRK, and blocking their cross-signalling pathway may give better effects. In this study, we developed a tyrosine kinase inhibitor 1D228, which exhibited excellent anti-tumor activity by targeting c-Met and TRK. Models in vitro, 1D228 showed a significant better inhibition on cancer cell proliferation and migration than the positive drug Tepotinib. Models in vivo, 1D228 showed robust anti-tumor effect on gastric and liver tumor growth with 94.8% and 93.4% of the TGI, respectively, comparing 67.61% and 63.9% of Tepotinib. Importantly, compared with the combination of Larotrectinib and Tepotinib, 1D228 monotherapy in MKN45 xenograft tumor models showed stronger antitumor activity and lower toxicity. Mechanistic studies showed that 1D228 can largely inhibit the phosphorylation of TRKB and c-Met. Interestingly, both kinases, TRKs and c-Met, have been found to be co-expressed at high levels in patients with gastric cancer through IHC. Furthermore, bioinformatics analysis has revealed that both genes are abnormally co-expressed in multiple types of cancer. Cell cycle analysis found that 1D228 induced G0/G1 arrest by inhibiting cyclin D1. Additionally, vascular endothelial cells also showed a pronounced response to 1D228 due to its expression of TRKB and c-Met. 1D228 suppressed the migration and tube formation of endothelial cells, which are the key functions of tumor angiogenesis. Taken together, compound 1D228 may be a promising candidate for the next generation of c-Met and TRK inhibitors for cancer treatment, and offers a novel potential treatment strategy for cancer patients with abnormal expressions of c-Met or NTRK, or simultaneous of them. The c-Mesenchymal-epithelial transition factor (c-Met) is a distinct subfamily of receptor tyrosine kinases (RTKs) that is activated solely by hepatocyte growth factor (HGF) , 2, and In cancer therapy, the tropomyosin receptor kinase (TRK) family of receptor tyrosine kinases is emerging as an important target . A totalVarious protein tyrosine kinases exhibit distinct interferences that significantly contribute to the pathogenesis of human cancer. For instance, in the context of bladder cancer, the reciprocal activation between c-Met and PDGFR, as well as Axl, actively facilitates the progression of this malignancy. Similarly, in large-cell anaplastic rhabdomyosarcoma, the HGF/c-Met pathway and c-Myc collaboratively promote the development of large cell dedifferentiated rhabdomyosarcoma. Numerous studies have demonstrated the synergistic promotion of tumor progression by c-Met and TRK kinases. For instance, in hepatoma HepG2 cells, a synergistic association exists between the HGF/c-Met and TRK signaling pathways, and the simultaneous inhibition of c-Met and TRKB exhibits a synergistic inhibitory effect on hepatoma . In neurModification of approved anti-cancer drugs is one of the popular strategies to improve the drug efficacies. During the whole process in drug design, conformation restriction has become one of the important transformation strategies in the structural optimization of lead compounds. Proper conformation restriction can be used to control the ligand conformation and make the molecule biased to the desired conformation binding with the target proteins as too many free rotation bonds of drug molecules are not conducive for binding , 30. Chi3 was obtained by the coupling reaction of 3-acetylphenylboronic acid with 2-chloro-5-fluoropyrimidine in the present of palladium catalyst. The fluorine atom of intermediate 3 is replaced by N-methylpiperidine-4-methanol in the presence of sodium hydride, and then the hydrogen transfer reaction in the presence of chiral catalyst and DCOONa to afford chiral alcohol 4. Finally, Mitsunobu reaction of chiral alcohol and pyridazinone produced the target product 1D228.The synthesis of 1D228 was performed according to methods in the patent with some modifications . Multiple oncogenes were reduced in expression, including NTRK2, MYC, CEACAM18, AKR1B10, PTPRB, METTL7, etc Fig. . MoreoveNTRK2 is a member of the NTRK protein family. Numerous preclinical and clinical investigations have substantiated the role of NTRK gene fusions in driving cancer  and emer50 values for TRKA, TRKB and TRKC . Furthermore, a comparative analysis was conducted to evaluate the therapeutic effects of 1D228 alone and in combination with Tepotinib and Larotrectinib. The results in Fig. The RNA-sep results showed that 1D228 downregulated a variety of oncogenes, so to further verify whether 1D228 also acts on other targets, we analyzed the inhibition rate of compound 1D228 against 77 TK kinases at a concentration of 500 nM using the HTRF kinase assay Fig. . The resRKC Fig. . The TRKRKC Fig. . AfterwaRKC Fig. . SubsequRKC Fig. . We thenRKC Fig. . MoreoveRKC Fig.  and 1D22RKC Fig. . Additio μM Fig.  and 0.89 μM Fig. , respectFurther, the co-expression of NTRK and c-Met was investigated in gastric cancer tissue microarray samples. We conducted immunohistochemistry (IHC), staining on 80 pairs of gastric cancer and adjacent normal tissues from patients, specifically targeting c-Met and NTRK using specific antibodies. Fig. KEGG enrichment analysis showed that 1D228 was involved in the cell cycle. Thus, we tested the cell cycle changes in vitro. MHCC97H and MKN45 cells were analyzed by flow cytometry. The results showed that 1D228 significantly increased G0/G1 phase in both cancer cells in a dose dependent manner Fig. . Cyclin It is shown that 1D228 had anti-tumor effect on tumor cells, but if this compound also targets other components in tumor tissue is unknown. To investigate this possibility, we focused on one of the typical tumor environment components, blood vessel. Angiogenesis is one of the hall marks of cancer. It plays an essential role in tumor progression in various cancers, which is regulated by many key signaling pathway, such as c-Met, AXL, or TRK, etc. –43. SincWe further compared the therapeutic efficacy of 1D228 alone with Tepotinib and Larotrectinib as a double agent in a gastric cancer nude mouse transplant tumor assay. In a gastric tumor model, 1D228 had a striking inhibitory effect on tumor growth. Under 1D228 treatment, the tumor volume shrank by nearly 90%, and the tumor began to regress after only two treatments, indicating a trend toward tumor elimination. More importantly, the combination of Tepotinib and Larotrectinib showed better tumor inhibition (56% reduction) than treatment alone (20% reduction), but 1D228 showed more significant inhibition than the combination Fig. . The tumCombing multiple drugs to treat cancer patient is a widely used strategy to achieve maximum inhibition of tumor growth and metastasis. However, this approach presents challenges such as complex pharmacokinetic properties, difficulty in dose setting, drug-drug interactions, more adverse effects, and poor patient compliance. The development of single-inhibitor with multi-target drugs is expected to improve efficacy through synergistic effects while reducing side effects. C-Met and TRK are well studied kinases in different cancers, and blocking these kinases has been demonstrated to be an effective strategy for the treatment of hepatocellular carcinoma and sarcomas with NTRK fusion. However, few studies have been investigated the dual inhibitory effect of these two targets in a same tumor model. It would be interesting to study if some tumors have both kinases under hyper activation could be super sensitive to the dual inhibition of these two targets in one shot. In this study, we found that c-Met and TRK were both activated in gastric cancer cells MKN45, indicating that blocking c-Met and TRK could be an effective strategy for treating gastric cancer. The objective of this study was to design and synthesize novel kinase inhibitors targeting c-Met and TRK as dual targets, and to investigate the effects and mechanisms of the dual-target inhibitors in c-Met and TRK high expression tumors.Based on the structure of Tepotinib, we introduced chiral restriction conformation and hydrogen isotope deuterium to improve its metabolic stability, and synthesized a new structure of small molecule inhibitor 1D228. HTRF kinase profile analysis with a pool of 76 kinases showed that the compound 1D228 had high inhibitory bound to c-Met, TRK, AXL, ALK, and MER. Further molecular docking data indicated stable bound to c-Met and TRK. Compared with Tepotinib, 1D228 exhibited more potency on tumor cell inhibition, which may be due to the dual targeting on c-Met and TRK. The liver microsomal stability of 1D228 was significantly improved compared with Tepotinib, especially in monkey and human liver microsomes, suggesting that the compound has good metabolic stability. More surprisingly, the compounds also showed potent anti-tumor activity in vivo. In both gastric and hepatocellular carcinoma models, 1D228 regressed tumors significantly, and the tumors was largely suppressed by 1D228 comparing with the combination treatment of tepotinib and larotrectinib.Upon 1D228 treatment, a serious of oncogenes’ expression were downregulated, and pathway analysis also indicated that the changed genes related to cell cycle, NF-κB, and DNA replication was clustered, showing that 1D228 targeted these pathways. Following experiments verified that 1D228 inhibited cell cycle in G0-G1 phase by inhibiting CyclinD1 expression, caused apoptosis by activating cleaved caspase-3. In addition to cell cycle arrest, 1D228 reversed the EMT by upregulation of E-cadherin expression and downregulation of N-cadherin, which significantly blocked the tumor cell migration. More interestingly, we found that 1D228 also targets blood vessels in both in vitro and in vivo models. Detailed investigation demonstrated that endothelial cells expressed both c-Met and TRK as well, making endothelial cells a target for 1D228. Thus, 1D228 could target both endothelial cells and tumor cells by inhibition of c-Met and TRK signaling, which produced a synergistic anti-tumor effect. This unexpected property of 1D228 makes it a promising candidate for drug development for treating cancers with both c-Met and TRK activation. In a pilot study, we have found double positive of these two kinases with high expression in clinical samples; Furthermore, bioinformatics analysis revealed co-expression of both genes in multiple types of cancer, which implies a close clinical application of 1D228 in the future. Not limited to gastric cancer, other cancers, as HCC or sarcoma patients may also benefit from this compound if double positive of c-Met and TRK are confirmed by pathological analysis.In summary, we identified 1D228 as a dual-targeting small molecule inhibitor of c-Met and NTRK, which exhibited excellent anti-tumor activity by targeting tumor cells and vascular endothelial cells simultaneously. In the nude mouse transplantation tumor model of liver cancer (MHCC97H cell lines) and gastric cancer (MKN45 cell lines), 1D228 provided splendid tumor growth inhibition rates. Considering that liver and gastric cancers are both major diseases that pose a serious threat to people’s health, and there are a large numbers of cancer patients with abnormal expressions of c-Met or NTRK, or simultaneous of them, we believe that as a multi-target kinase inhibitor, 1D228 will play an significant role in the treatment of the aforementioned cancer patients after it is approved.2O mixture at a flow rate of 0.5 mL/min, and was detected at a wavelength of 254 nm. The purity of all biologically evaluated compounds is greater than 95%. The 1H NMR and 13C NMR spectra were recorded using TMS as the internal standard on a Bruker BioSpin GmbH spectrometer at 400 and 100 MHz, respectively, and the coupling constants are reported in hertz. The high-resolution mass spectra were obtained using a Shimadzu LCMS-IT-TOF mass spectrometer.Reagents used in the synthesis were obtained commercially and used without further purification unless otherwise specified. Flash column chromatography was performed using silica gel (200−300 mesh) purchased from Qingdao Haiyang Chemical Co. Ltd. The purity of the samples was determined by high-performance liquid chromatography (HPLC), conducted on a Shimadzu LC-20AT series system with a TC-C18 column . The samples were eluted with a 60:40 acetonitrile/H3: an aqueous solution of sodium carbonate  was added into a solution of 5-fluoro-2-chloropyrimidine  in toluene (13 mL), into which were then added bis(triphenyl phosphine)palladium chloride , acetyl phenyl boronic acid , and ethanol (6.5 mL). Under nitrogen protection, the reaction was stirred at 83 °C for 11 h, and cooled to room temperature. 16 mL ethyl acetate and water (4 mL) were added into the reaction system to separate out the organic phase. The water phase was extracted with 5 mL ethyl acetate once. The organic phase was combined, washed with saturated brine, dried over anhydrous sodium sulfate. The solvent was removed. The crude product was added into ethyl acetate: petroleum ether (1:8), pulped, filtered and dried to get a light yellow solid 0.131 g. Yield 81%. 1HNMR , δ8.98 , 8.70 , 8.59 , 8.09 ,7.60 , 2.71.(1) Preparation of the intermediate (S)-3:(2) Preparation of the intermediate S, S)-CsDPEN  were placed in a 25 mLreaction flask, into which was added water (0.6 mL) and stirred at 40°C under nitrogen protection for 4 h to prepare the catalyst, ready for use.a) Preparation of a chiral catalyst: Dichloro (4-methyl isopropyl benzene) ruthenium (II) dimer , and a chiral ligand  was added batchwise into a solution of 1-methyl piperidine-4-methanol  in N, N-dimethyl formamide (10 mL), and stirred for 15 min. Then a solution of the intermediate 2  in DMF (13 mL) was added dropwise into the above mixture while keeping the reaction temperature (0 °C) basically the same. After dropwise addition, the reaction mixture was slowly warmed to room temperature. Upon the completion of reaction monitored by TLC (about 1.5 h), the reaction solution was slowly poured into 30 mL water (with stirring), into which was added an appropriate amount of dichloromethane and stirred to separate the lower layer. The upper layer was extracted with dichloromethane. After the extraction was completed upon TLC detection, the total used amount of dichloromethane was about 40 mL. The organic phase was combined, washed with water (3*5 mL) to remove DMF as far as possible, and washed with saturated brine to obtain about 40–45 mL solution of the intermediate in dichloromethane, which was kept under nitrogen protection for use.b) Reaction of the intermediate S)-4, enantiomeric excess (ee) determined by HPLC, 98% ee. Analytical conditions: AD-H column, mobile phase, isopropanol 25%, n-hexane 75% (added with 0.1% of diethylamine). The chemical purity of the intermediate (S)-4 was greater than 95%.c) Under nitrogen protection, the above solution of the intermediate in dichloromethane was added into the prepared chiral catalyst solution, into which was then added an aqueous solution of deuterated sodium formate  under nitrogen protection. The reaction was stirred at room temperature for 10 h, until the completion of the reaction detected by TLC. 20 mL water was added into the reaction system and stirred to separate the lower layer. The water layer was extracted twice with an appropriate amount (10 mL) of dichloromethane. The organic phase was combined, washed with saturated brine, dried over anhydrous sodium sulfate, filter over a silica gel column  and eluted to get the intermediate ((R)-1D228: To a reaction flask were added 3--benzonitrile , the intermediate (S)-4 , triphenylphosphine , and tetrahydrofuran , and stirred at 0°C under nitrogen protection for 10 min. Diisopropylazodicarboxylate  was slowly added dropwise, warmed naturally to room temperature, and reacted overnight. 20 mL ethyl acetate and 10 mL water were added into the reaction system, stirred and filtered to separate out the organic layer. The water layer was extracted with 10 mL ethyl acetate. The organic phase was filtered over silica gel, and washed with ethyl acetate until no chromogenic substance was detected by TLC under ultraviolet light. Then it was eluted with ethyl acetate/petroleum ether/triethylamine (200/50/2). The eluent was collected, detected by TLC, and then concentrated to get the target product. Analytical conditions for optical purity: Daicel ADH column (0.46 × 25 cm), n-hexane (added with one thousandth of diethylamine)/ethanol = 80/20, 0.5 mL/min, λ = 254 nm, tR = 40.66 (R), tS = 64.22 (S). Analytical conditions for chemical purity:ace column, CH3CN:H2O:triethylamine = 70:30:0.1.1H NMR  δ 8.67 , 8.50 , 8.28 , 8.20 , 7.98 , 7.69 , 7.56 , 7.43 , 7.02 , 3.96 , 2.92 , 2.30 , 1.97 , 1.93 – 1.82 , 1.52 – 1.41 . 13C NMR  δ 159.22, 157.30, 151.70, 143.94, 141.78, 140.58, 137.89, 136.38, 132.42, 130.29, 129.82, 129.73, 129.64, 129.35, 128.75, 128.64, 127.23, 126.94, 118.57, 113.41, 73.36, 55.32, 46.44, 35.27, 28.94, 19.91. HRMS (ESI) m/z C30H29D1N6O2 [M + H]+, calculated:508.2566; found 508.2553. HPLC purity: 99.0%, retention time: 15.03 min. 97.1% ee. Daicel AD column (0.46 × 25 cm), n-hexane /ethanol = 80/20, 0.5 mL/min, λ = 254 nm, tR = 40.66 (R), tS = 64.22 (S).(3) Synthesis of target The human cancer cell lines  used in this study were purchased from the Guangzhou ginny ou biotechnology co. LTD . The human cancer cell lines  used in this study were purchased from the Wuhan cell bank. Cells were identified by STR profiling, and mycoplasma contamination test were done routinely. A549, ASPC1 and MKN45 Cell lines were cultivated in 1640 containing 10% (v/v) heat-inactivated fetal bovine serum (FBS), 100 units/mL penicillin, and 100 μg/mL streptomycin. HepG2, Hela, MCF7, MDAMB-231, HS746T and MHCC97H Cell lines were cultivated in DMEM containing 10% (v/v) heat-inactivated fetal bovine serum (FBS), 100 units/mL penicillin, and 100 μg/mL streptomycin.3 cells/well) for 24 h, then exposed to different concentrations  of the test compounds for 72 h. After attached cells were incubated with CCK8  for another 1–4 h. Then the absorbance at 450 nm was measured using a multifunction microplate reader , and each experiment was performed at least in triplicate. The cytotoxic effects of each compound were expressed as IC50 values, which represent the drug concentrations required to cause 50% tumour cell growth inhibition, and calculated with GraphPad Prism Software version 5.02 .For cytotoxicity assay, the cells grown in the logarithmic phase were seeded into 96-well plates . Curve fitting and data presentations were performed using Graph Pad Prism version 5.0. Every experiment was repeated at least 3 times.Selectivity of 1D228 for 76 kinases based on HTRF and ADP-Glo methods: The compound was serial diluted from 10 mM stock in DMSO, dilute the test compound to 0.1 mM (200 times the specified test concentration). 2 × ATP & Substrate solution and 2 × kinase & Metal solution were prepared using assay buffer; Transfer 25 nL compound to 384 assay plate by Echo 655; After centrifugation, add 2.5 μL 2 × kinase/metal ion solution 384 assay plate, and incubate at 25 °C for 10 min. 5 μL of Kinase Detection Reagent was added to the well, and incubated for 60 min at 25 °C. 2 × XL665 & Antibody solution were prepared with detection buffer; 5 μL of Kinase Detection Reagent was added to the well, and incubated for 60 min at 25 °C; The fluorescence signals of 620 nm (Cryptate) and 665 nm (XL665) were read by microtiter plate reader; The readout value of reaction control (0.5% DMSO) was set as a 0% inhibition, and the readout value of background (10 µM Positive Control) was set as a 100% inhibition, % Inhibition = 100%-(compound-positive control)/(negative control-positive control)*100%.4 cells/well) and incubated in the presence or absence of compound 1D228, for the cell apoptosis assay, cells were harvested and incubated with 5 μL of Annexin-V/FITC  in binding buffer  at room temperature for 15 min. PI solution was then added to the medium for another 10 min-incubation. Almost 10,000 events were collected for each sample and analysed by flow cytometry . For the cell cycle assay, cells were fixed in ice-cold 70% ethanol overnight. After the ethanol was removed the next day, the cells were re-suspended in ice-cold PBS, treated with RNAse A  at 37 °C for 30 min, and then incubated with the DNA staining solution propidium iodide  at 4 °C for 30 min. Approximately 10,000 events were detected by flow cytometry  at 488 nm. The data regarding the number of cells in different phases of the cell cycle were analysed using EXPO32 ADC analysis software.MHCC97H or MKN45 cells were seeded in 6-well plates  was incubated in a water bath at 37 °C for 200 μl. Each sample was incubated three times in parallel, and an equal volume of pre-chilled acetonitrile was added at preset reaction time points, such as 0, 5, 10, 15, 30 and 60 min. The reaction was terminated by the addition of acetonitrile. The remaining content of the prototype drug in the warm incubation solution was determined by HPLC, HPLC-MC and HPLC-MC/MC.mRNA expression was measured by reverse transcription quantita-tive polymerase chain reaction (RT-qPCR) on RNA isolated from cells. TRIzol reagent  was used to extract RNA from cell lines according to the manufacturer’s protocol. RNA was quantified with a NanoDrop spectrophotometer . RNA (1000 ng) was retro-transcribed to complementary DNA (cDNA) using a High-Capacity cDNA Reverse Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression was used as an internal reference to normalize input cDNA. The threshold cycle value (CT) was determined for each measurement, and mRNA expression was calculated relative to the control using the comparative critical threshold (2 − CT) method, where CT = CTmRNA – CThousekeeping control. Triplicates were performed for each sample.4 cells were seeded in serum-free medium in the top chamber and allowed to migrate for 48 h toward the bottom chamber containing 10% fetal bovine serum medium. Cells were fixed and permeabilized with cold 70% ethanol and methanol and stained with 0.4% crystal violet. The average number of migrating cells was evaluated from at least four independent microscope fields.Migration assay was performed using an 8 μm-pore Boyden chamber . Treated with 1, 5, and 10 nM 1D228 for 48 h. A total of 5 × 104 cells/well, and 4 nM concentration of 1D228 was added after 12 h. The compound-treated MKN45 cells and control strain cells were collected for 24 h at 37 °C in a 5% CO2 incubator, and three samples of each group were collected. RNA-seq and RNA-seq analysis were performed by Bioprofile Technology Company Ltd . Briefly, after RNA extraction, purification and library construction, these libraries were sequenced using Next-Generation Sequencing technology based on Illumina HiSeq sequencing platform with double-end , and then the raw downstream data (Raw Data) were filtered. The filtered high-quality sequences (Clean Data) were compared to the human reference genome. The expression of each gene is calculated based on the results of the alignment. Based on this, the samples are further analyzed for expression differences, enrichment analysis and clustering analysis. We have uploaded RNA-seq data to the Gene Expression Omnibus (GEO) at the NCBI .MKN45 cells were inoculated into large dishes at 7 × 104 cells/dish and grown for 24 h, and the non-migrated cells were scraped off the upper surface of the membrane with a 10 ul pipette. The medium was then replaced with 10% serum 1640 medium and treated with compound 1D228 at the indicated concentrations for another 24 h. After being washed with phosphate buffer solution (PBS), the cell images were immediately detected by a Zeiss LSM 570 laser scanning confocal microscope .MKN45 or ASPC1 cells were plated in a 6-well culture dish at 5 × 10HUVECs/C166 were seeded on 6-well plates and treated with 10 or 100 nM 1D228. After 48 h, cells were detached and transferred to growth factor reduced matrigel (Cultrex-3432-005-01) coated 96-well plates (10000 cells/well). Cells were imaged with microscope.The fixed tumor sections were digested with 20 μg/ml protease K for 5 min. Methanol permeates at room temperature for 30 min; Wash with PBS at room temperature for 30 min; 4 °C wash overnight with 3% milk; Primary antibody was incubated overnight at 4 °C. Wash with 0.3%Triton-100 at 4 °C for 1.5 h; The secondary antibody was incubated at room temperature for 2 h; Incubate with 1.5% milk at room temperature for 1 h and wash overnight with 0.3% Triton-100 at 4 °C. Seal the film and shoot.Mice were dislocated and executed by cervical dislocation and then disinfected by immersion in 75% ethanol aqueous solution, then the tumor tissues were peeled, weighed, and cut by ophthalmic scissors. Collagenase solution was added and shaking was performed on a shaker for 3 min at 1700 rpm, followed by incubation in a 37 °C water bath for 5 min, alternating 10 times. The tumor tissue was digested using collagenase, and then the digested tumor tissue was centrifuged, and the supernatant after centrifugation was discarded to obtain the tissue precipitate. The precipitate was then washed in once using PBS, centrifuged after blowing the precipitate well with a pipette gun, discarded the supernatant, and repeated once. Trypsin solution was added to the collagenase-digested tumor tissue, shaken on a shaker for 10 min, and fetal bovine serum was added to terminate the digestion. The tumor cell suspension after trypsin digestion was filtered through a 30 μm filter, and the filtered tumor cell suspension was centrifuged and the supernatant was discarded; the precipitate was also resuspended and mixed with PBS, and then centrifuged and the supernatant was discarded, and the procedure was repeated once. Trypsin-digested tumor cells were resuspended by adding erythrocyte lysate, incubated at room temperature for 5 min, and the reaction was terminated by adding 5 times the volume of PBS, and the supernatant was discarded by centrifugation. The precipitate was resuspended with 1 mL of 0.5% BSA and transferred to a 1.5 mL EP tube. 10 μL of cells were resuspended with CD45 antibody and 9 times the volume of 0.5% BSA Buffer according to 10 million cells and shaken for 15 min. The cells were washed twice with 500 μL Buffer and the filtered cells were collected and centrifuged to remove the supernatant. After resuspended with CD31 and 9 times the volume of Buffer, shaken for 15 min, passed through the MACS column, Buffer washed twice, the column was taken off and 500 μLBuffer was quickly added to blow the cells into a clean 1.5 mL EP tube, centrifuged and supernatant removed, this cell is the vascular endothelial cell.3) and MHCC97H cells (2 × 103) were seeded into the 6-well plates. After the plates were treated with 1D228  or DMSO (0.32% or 0.64%) for 48 h at 37 °C, then cultuerd in 10% FBS medium, the colonies were fixed with 4% paraformaldehyde and stained with 1% crystal violet solution for 20 min. The colonies were photographed and counted.MKN45 cells (2 × 10Proteins (25–50 ug/lane) were separated on a 4–12% Bis-Tris NuPAGE gel and blotted onto a PVDF membrane . Membranes were probed overnight at 4 °C with primary antibodies against p-c-Met , c-Met , p-AKT , AKT , p-ERK , ERK , GAPDH , p-TRKB , CyclinD1 , cleaved caspase-3 , caspase-3 , N-cadherin , E-cadherin , β-actin , AXL , p-MER , P-AXL , NF-KB , p-ALK , Ki67 . Horseradish peroxidase-conjugated secondary antibodies were from Cell Signaling. Proteins were visualized by enhanced chemiluminescence (Millipore) on ChemiDoc camera (Bio-Rad) and protein expression levels were quantified using the lmageJ software. Full anduncropped western blots are presented in Supplemental File.5 cells were grown in a 10 mm3 confocal culture dish for 24 h and then incubated in the presence or absence of compound 1D228 at theindicated concentrations for another 12 h. After being washed with phosphate buffered solution (PBS) and fixed in 4% prewarmed (37 °C) paraformaldehyde for 15 min, the cells were permeabilized with 0.5% Triton X-100 for 15 min and blocked for 30 min in 10% goatserum. Then, the cells were incubated with Rabit anti-P-MET antibody  at 4 °C overnight. On the next day, cells were washed with PBS three times and incubated with goat anti-Rabit IgG/Alexa-Fluor 488 antibody  for 1 h. After the nuclei were stained with DIPA  in the dark at room temperature for 30 min, the samples were immediately visualized on a Zeiss LSM 570 laser scanning confocal microscope .For the immunofluorescence microscopy, 3 × 102O2. The blocked sections were incubated with anti-Ki67 antibody , anti-c-Met antibody  at 4 °C overnight and then sequentially incubated with a biotin-labeled secondary antibody. The sections were then stained with 3,3′-diaminobenzidine. Finally, the sections were counterstained using hematoxylin and fixed. For each section, three fields of view were randomly selected and photographed under ×200 magnification.Immunohistochemistry (IHC) detected the expression of Ki67, P-c-Met in tumor tissue. The tumor tissue paraffin sections were deparaffinized, and endogenous peroxidase activity was blocked by incubation with 3% Hhttps://www.cancer.gov/ccg/research/genome-sequencing/tcga) and The Genotype-Tissue Expression (GTEx) dataset . We then used VENNY2.1 [https://bioinfogp.cnb.csic.es/tools/venny/), an online analysis site, to visualize the intersection of cancers with significantly (p < 0.05) abnormal expression of MET and NTRK family genes.To determine which cancers specifically express the MET and NTRK families , we downloaded RNA sequencing expression profiles for 32 cancers from The Cancer Genome Atlas (TCGA)  were purchased from Shanghai Xinchao Biological Co., Ltd. and serially sectioned tissue microarrays were selected and incubated and DAB stained with c-Met  and TRK  antibodies, respectively, after dewaxing, antigen repair and sealing steps.7 cells/mL cancer cells grown in logarithmic phase were harvested and resuspended in FBS-free 1640 medium. Then, 100 μL of the cell suspension was subcutaneously injected into the right flank of each mouse. When the tumour volume was reached about 600–800 mm3, it was removed, minced into small pieces of equal volume (2 × 2 × 2 mm3), and transplanted subcutaneously into the nude mice. When the tumour volume was reached about 200–400 mm3, the xenograft tumour-bearing nude mice were randomly allocated to five groups  per group. Each group was dosed by intragastric administration (ig) for 7–14 days. Tumour volume and body weights were recorded every day after drug treatment. At the end of the observation period, the animals were euthanized by cervical dislocation and the tumour bulks were peeled off conformed to the Guide for the Care and Use of Laboratory Animals as published by the US National Institutes of Health  and approved by the Institutional Ethics Review Board of Binzhou Medical University. There was no blinding was done during the animal experiment. No animals were excluded from the analysis.Male BALB/c nude mice  were purchased from Beijing Vital River Laboratory Animal Technology  and allowed to acclimate for 1 week, maintained at constant room temperature and fed a standard rodent chow and water. 1 × 10Female and male Kunming mice  were purchased and housed at the SPF (Beijing) Biotechnology Co. in pathogen-free condition, maintained at constant room temperature and fed a standard rodent chow and water. The mice were randomly divided into experimental group and control group. The animals were fasted overnight before drug administration, and the drug was administered by tail vein injection at a constant volume of 0.4 ml/20 g  in two doses within 24 h, with an interval of 6 h. Acute toxicity of the animals was observed for 7 days after the administration of the drug. The heart, liver, lung and kidney of mice in the 750 mg/kg dose group were taken and fixed for HE staining.p > 0.05 was not significant (ns). *p < 0.05; **p < 0.01; ***p < 0.001.All experiments were repeated three times independently unless specifically indicated. The data was analyzed by GraphPad Prism 7 software. The data was shown as mean ± SD and one-way ANOVA Tukey ‘s multiple comparison test was used to determine the levels of significance between comparison samples. SupplementaI InformationOriginal Data Fileaj-checklist


The strain was also designed to efficiently cleave N-terminal methionine, the obligatory start amino acid for E. coli expressed proteins. 8MTT was purified as a soluble protein from the cytoplasm in a two-column protocol to > 99 % purity, yielding 0.5 g of purified 8MTT/liter of fermentation broth with low endotoxin contamination, and antigenic purity of 3500 Lf/mg protein nitrogen. Mouse immunizations showed 8MTT to be an immunogenic vaccine and effective as a carrier protein for peptide and polysaccharide conjugates. These studies validate 8MTT as commercially viable and, unlike the heterogenous tetanus toxoid, a uniform carrier protein for conjugate vaccines. The development of a recombinant, genetically detoxified toxin produced in E. coli aligns the tetanus vaccine with modern manufacturing, regulatory, standardization, and safety requirements.Tetanus toxoid (TTxd), developed over 100 years ago, is a clinically effective, legacy vaccine against tetanus. Due to the extreme potency of native tetanus toxin, manufacturing and regulatory efforts often focus on TTxd production, standardization, and safety, rather than product modernization. Recently, a genetically detoxified, full-length tetanus toxin protein (8MTT) was reported as a tetanus vaccine alternative to TTxd . Here we describe the production of 8MTT in Gor/Met AccorC. tetani protein contaminants [197), there has been no equivalent for tetanus, which is a larger and more complicated toxin.Although an inexpensive vaccine, TTxd manufacturing has several limitations, including the need for a dedicated biosafety level manufacturing facility, old production protocols, low purity, complex standardization, and local immune reactivity -13. Commaminants . While a197 [197 has fewer and weaker T cell epitopes than TTxd [Carrier proteins are used to enhance the immunogenicity of peptides, glycans and other poorly immunogenic haptens. Only a few carrier proteins are approved for human use, which include TTxd and diphtheria toxoid (DTxd) and CRM197 ,15. Crud197 , which mhan TTxd .E. coli grown in a shake flask. His-8MTT (single chain or trypsin nicked) was not toxic for outbred mice at a dose of 0.6 mg, making His-8MTT > 50 millionfold less toxic than native tetanus toxin [E. coli strain, development of an efficient 2-column purification scheme, and evaluation as a tetanus vaccine and conjugate vaccine carrier. This work facilitates the transition of tetanus vaccine from a chemical toxoid to a modern recombinant vaccine.Earlier studies showed the generation of full-length tetanus toxin with two-point mutations that reduced catalysis and toxicity by ~ 125,000-fold relative to native tetanus toxin ,18. Moreus toxin . His-8MTus toxin . The cur2.E. coli BL21(DE3), Ni-affinity/size exclusion chromatography yielded ~ 5 mg of purified 8MTT-His / L of culture [™E. coli strain [™E. coli expresses soluble, properly folded, disulfide-bonded proteins in the cytoplasm and cleaves the N-terminal methionine from newly synthesized proteins, Gor/Met™ E. coli was engineered by deleting the glutathione reductase gene (gor) to create an oxidative cytoplasm which allows for the expression of soluble, disulfide bonded proteins [tac promoter [™E. coli onto a plasmid under the control of a tac promoter (p8MTT). Thus, IPTG induces 8MTT expression along with the methionine aminopeptidase.Tetanus toxin is a 1315 amino acid single- or di- chain protein with 10 cysteines, 2 sets of disulfide bonds and 6 unpaired thiols (Swiss-Prot: P04958.2) produced as a soluble monomeric protein when expressed in batch culture of  culture . To enhaproteins  and subspromoter . The gen™E. coli (p8MTT) grew to a density of about 10 OD600 in Terrific Broth media, but with yields of < 20 mg of 8MTT /L of culture. We hypothesized that enhanced 8MTT expression in Gor/Met™E. coli required more oxygen than could be provided in a shake flask. Therefore, further optimization of 8MTT expression was performed in a 5 L fermenter, using chemically defined media supplemented with yeast extract + glucose. High expression of soluble 8MTT was observed with IPTG induction between 22 °C and 28 °C, with Gor/Met™E. coli (p8MTT) achieving OD600 densities of 10–100, corresponding to 13–100 g wet cell paste/L fermentation culture, depending on the inducing temperature. Cell productivity (mg 8MTT / g cell paste) was maintained regardless of cell density, i.e., 8MTT yield was proportional to cell density. By SDS-PAGE, the highest cell density yielded ~ 1 g of 8MTT / L fermentation culture.8MTT expression was initially evaluated in shake flasks with IPTG induction between 20 °C and 37 °C. Gor/MetFollowing harvest of the cell mass, subsequent homogenization, and clarification, 8MTT was captured from the lysate onto an anion exchange resin with dextran extender tentacles. 8MTT from the eluant of the anion exchange resin was brought to 2 M NaCl and loaded onto a butyl hydrophobic interaction chromatography (HIC) column. 8MTT was eluted from the HIC column by decreasing the NaCl concentration. 8MTT in the eluant of the HIC column was concentrated, and buffer exchanged into 20 mM HEPES, 200 mM NaCl, pH 7 using tangential flow filtration (50 kDa cutoff membrane) to 10 mg 8MTT/ml. The addition of 200 mM NaCl to the diafiltration buffer increased 8MTT solubility and minimized aggregation. As a final polish, the retentate in the formulation buffer was directly applied to an anion exchange membrane in a flow through mode, followed by sterile filtration of the product. This reduced the endotoxin levels from about 1000 E.U./mg to < 5 E.U./mg of 8MTT protein. Low endotoxin levels are a requirement for injectable products and the purified 8MTT was well under the allowable injection limit .SEC HPLC of purified 8MTT appeared as a single peak with > 99 % purity and SDS PAGE exhibited a single band under both reducing and nonreducing conditions  and 100 kDa Heavy Chain (HC) and are held together by an interchain disulfide bond (C439-C467). By SDS-PAGE, purified 8MTT migrated as a ~ 150 kDa single band in either reducing or nonreducing conditions . Thus, purified 8MTT retained the overall protein structure of native tetanus toxin and, like native tetanus toxin, was resistant to secondary cleavage by trypsin [E. coli does not possess a protease that can cleave 8MTT within the interchain region, rather than the cleavage site not being protease accessible, since trypsin preferentially cleaved 8MTT within the interchain region . Purified 8MTT stored for 1 year at 4 °C or −20 °C (−) and then incubated at room temp or 37 °C for one week showed little degradation .To examine product stability, 8MTT was stored in buffer + 10 % glycerol for one year at −70 °C or −20 °C or 4 °C in buffer without glycerol. In addition, the ability to freeze 8MTT may be beneficial. We have not explored other cryoprotectants or if they are even necessary. N-terminal proteolytic fragment composition. Intact-MS analysis . Thus, the 8MTT mass spectrum is consistent with a protein that lacks an N-terminal methionine and has two disulfide bonds. Thus, the 8MTT mass spectrum is consistent with a protein that lacks an N-terminal methionine and has two disulfide bonds. The higher-mass peak  that is apparent in the deconvoluted spectrum likely represents adducts formed by association of the protein ins and polar components of the solvent in the gas phase, a phenomenon which is common in native MS [m/z region of the mass spectrum, and that the observed charge state distribution was narrow (ranging from + 26 to + 30), indicating that 8MTT in solution maintained a compact conformation with no signs of unfolding and/or misfolding [Purified 8MTT was analyzed by mass spectrometry (MS) to determine intact-mass and analysis , perform™E. coli expression system to cleave the N-terminal methionine from 8MTT. 8MTT was initially analyzed by Orbitrap Fusion  MS with an integrated front-end reversed-phase (C18) NanoLC system, EASY-nLC™ 1000  to obtain terminal peptide fragmentation. This identified a ladder of N-terminal peptides with the first residue of the sequence a Proline  and then boosted twice (day 14 and day 110) with 0.5 μg or 5 μg of 8MTT or TTxd, each absorbed to Alhydrogelas 0.008 . Since et-test), showing 8MTT to be at least as potent a conjugate carrier protein as TTxd for the P0 antigen  since PRP-TTxd is a licensed conjugate vaccine for humans [8MTT and TTxd were next evaluated as conjugate vaccine carriers for polysaccharides, since polysaccharides, as  protein . We chosr humans . Using t3.™E. coli, a novel strain engineered to have an oxidative cytoplasm and to efficiently cleave N-terminal methionine. 8MTT was expressed as a single chain protein without a purification tag and purified in two column steps to > 99 % purity with low endotoxin contamination, yielding 0.5 g of purified 8MTT / liter of fermentation culture, a notable accomplishment given that 8MTT is 150 kDa with ten cysteines, six of which are unpaired. Mass spectrometry confirmed the predicted molecular mass of 8MTT as a monomer and showed that the N terminus did not contain an N-terminal methionine and an immunological determination detected that 8MTT had antigenic purity of 3500 Lf/mg of 8MTT nitrogen by flocculation assay. We found 8MTT equally immunogenic to TTxd in mice as a tetanus vaccine and as a conjugate carrier protein.Tetanus toxin (TT) is one of the most potent toxins known, and tetanus toxoid (TTxd) is a widely used clinical toxoid vaccine. We have developed a genetically detoxified, full-length tetanus toxin (8MTT) that is>50 millionfold less toxic than native tetanus toxin with retention of immunogenic potency relative to TTxd . Here weA fair question is why there is a need for a new tetanus vaccine, given that the current tetanus toxoid is clinically effective, relatively inexpensive, and widely used. We propose that compared with the legacy tetanus toxoid vaccine, genetically detoxified tetanus toxin, 8MTT; (1) is easier, faster, and safer to manufacture, allowing 8MTT to be produced in standard bioprocessing facilities, (2) will be a vaccine of higher purity, which can be characterized/-standardized using modern techniques while minimizing animal use, and (3) is more suitable for use as a carrier protein for conjugate vaccines.C. tetani for toxin production takes 7–10 days [C. tetani tetanus toxin production [E. coli, a BSL1 organism, and produced in ~ 20 h using a standard bioprocessing fermentation facility. Our current efforts yielded about 0.5 g/L of purified 8MTT, using a chemically defined media supplemented with yeast extract. 8MTT yield should increase with further optimization but is already higher than toxoid production in C. tetani, which has seen many decades of process improvement.TTxd manufacturing (WHO/IVB/11.11) requires unique production considerations for laboratory safety and staff and environmental protection . Ferment–10 days . Althougoduction , establioduction ,42. Yieloduction . In contC. tetani. While purity has improved and current manufacturing protocols include tangential flow filtration and ammonium sulfate precipitation, the toxoided vaccine remains contaminated with C. tetani proteins and media components [First described by Ramon ,44, formmponents , and detmponents . With eiin vitro assays for tetanus vaccine antigen content and to reduce animal use, testing tetanus vaccine potency [The WHO requires the demonstration of the absence of toxicity of each TTxd batch, which often is performed in animals. However, the WHO has licensed a chemically inactivated TTxd based on an International Reference Reagent , where a potency . These i potency ,48 and p potency . To dateE. coli strain using chemically defined media and can be characterized using a battery of biophysical, immunological, and biochemical techniques to define the product. We have begun to define the biophysical properties of 8MTT which would be necessary for a batch release. These include identification assays , manufacture safety , aggregation (SEC HPLC), higher-order structure , thermal stability (ITC), along with toxicity assays.The batch release for modern vaccines is based on understanding and controlling the manufacturing process, along with in-process and product testing using state-of-the-art analytical tools, allowing for the manufacture of a consistent product that can be compared to the material used for licensing . Once thTetanus vaccine efficacy for 8MTT is, of course, a critical attribute. The WHO minimum antigenic purity of tetanus toxoid is 1000 Lf units per milligram of protein nitrogen ,52. AnalThe receptor-binding domain of tetanus toxin (THc) has been evaluated as a recombinant tetanus vaccine  and as aMany antigens such as haptens, peptides, and carbohydrates are poorly immunogenic unless chemically linked to a carrier protein. Tetanus toxoid is used as a carrier protein in licensed conjugate vaccines for polysaccharides ,61,62,63™E. coli strain as an additional vaccine strategy and as an alternative to chemical conjugation, since 8MTT protein fusions mediate translocation of biologically active β-lactamase (27-kDa) across an endosome membrane [The TTxd monomer fraction, although “purified,” is nevertheless a heterogeneous and variable product, increasing the challenge of manufacturing and characterizing conjugate vaccines . In contmembrane ,72.4.There is a strong manufacturing and regulatory argument for bringing the tetanus vaccine into the 21st century with a genetically detoxified toxin. Our animal data suggests that 8MTT will be an efficacious stand-alone vaccine for tetanus and as a carrier protein. Still, we recognize the challenge to make the economic case for replacing the toxoid vaccine, as TTxd is a component of many licensed vaccines. Employment as a conjugate vaccine carrier will provide the opportunity to obtain the human data needed to expand the use of 8MTT as a vaccine for the toxin as well.5.™E. coli [™ competent cells. The gene encoding 8MTT on p8MTT was sequenced in multiple sequencing rounds to confirm the nucleotide sequence of 8mtt. Alignment of 8MTT to the published 8MTT sequence [™ strain  containing the p8MTT expression vector was prepared and frozen as the research cell bank and utilized in the production of the master & working cell banks.An expression system was constructed for the production of 8MTT in the cytoplasm of Gor/MetE. coli . Plasmidsequence  confirme™) was expanded and transformed with the p8MTT expression vector with one round of electroporation followed by plating for isolation on LB media with Kanamycin (50 μg/ mL). The next day, three individual colonies were picked, grown in 3 mL of non-inducing MDG Kanamycin medium [2HPO4, 25 mM KH2PO4, 50 mM NH4Cl, 5 mM Na2SO4, 2 mM MgSO4, 0.5 % Glucose, 0.25 % Aspartate, 50 μg/ mL Kanamycin Sulfate, 0.2 X Trace Metals , and 100 μM (NH4)2Fe(SO4)2). After 24 h of culture time at 37 °C resulting in an OD600 of 3, a 60 μl aliquot of the culture was removed and used to inoculate 3 mL of LB media with Kanamycin (50 μg/ mL) for expression analysis while the remaining culture was added 50/50 with 50 % glycerol and stored at −70 °C. Once 8MTT expression was confirmed by SDS-PAGE, as evidenced by banding at the correct molecular weight, the glycerol stocks of positive clones were selected for expansion into 100 mL MDG Kanamycin medium followed by incubation at 37 °C with 250 RPM shaking for 20 h. The culture was cryopreserved with 25 % (v/v) glycerol and aliquoted into 1 mL aliquots, thus creating the Research Cell Bank.One vial of parental cell line (BL21 Gor/Metn medium  (25 mM N4)2SO4, 80 mM K2HPO4, 25 mM Na2HPO4, 5 % glycerol, 40 μM CaCl2, 20 μM MnCl2, 4 μM CoCl2, 4 μM CuCl2, 4 μM NiCl2, 4 μM Na2MoO4, 4 μM H3BO3, 20 μM ZnCl2, 0.4 μM Na2SeO3, 2 mM MgSO4, 0.2 % Trace minerals, 0.1 mM (NH4)2Fe(SO4)2, 50 μg/ml kanamycin, and 0.01 % antifoam in a 5L vessel (New Brunswick Scientific Co. Inc). Fermentation was controlled by a Lab Owl Bioreactor Control System with the following parameters: 37 °C constant temperature, O2 saturation level (DO) set at 25 %, stirring speeds cascading from 300 to 800 rpm controlled by DO, pH 7.2 regulated by adding concentrated NH4OH, and constant air to oxygen (depending on DO) sparging flow rates at 2.5 L/ min. During first four h of growth, the DO slowly decreased and settled at the 25 % set point. The feed media 2SO4, 6 μM Na2SeO3, 6 μM H3BO3i 30 μM ZnCl2, 0.1 mM (NH4)2Fe(SO4)2, and 50 μg/ ml kanamycin) was then added to the fermenter vessel at 1 mL/min. When the bacterial concentration reached an OD600 of 10, the temperature was lowered to 25 °C, followed by the addition of IPTG to a final concentration of 0.5 mM to induce 8MTT production. The feeding rate was then changed to a linear rate from 0.7 mL to 1.8 mL per min in 18 h. The fermentation was stopped when the feed media was completely consumed and the OD600 could reach 100. Cells were harvested by centrifugation at 10,000 × g for 10 min. Approximately 200 g wet pellets per L fermentation culture was obtained. The cells were frozen in flattened plastic bags and stored at −70 °C (frozen cell paste).We used fed-batch fermentation to grow 8MTT transformed bacteria. The seed culture was prepared by inoculating one glycerol vial of 8MTT transformed bacteria stock into 50 mL MDG media  and growg for 30 min followed by filtration with a 0.45 μm PES filter. The clarified soluble lysate was applied to an anion exchange column , washed with 75 mM NaCl in 20 mM Tris, pH 7.0 and 8MTT eluted with 150 mM NaCl in 20 mM Tris, pH 7.0. NaCl was then added to the Q eluant to a final concentration of 2 M and the eluent was applied to a Toyopearl® Butyl-600 M column (Tosoh Biosciences), equilibrated with 2 M NaCl in 20 mM Tris pH 7.0. 8MTT was eluted with 1 M NaCl in 20 mM Tris, pH 7.0. The resulting eluant was concentrated and buffer exchanged into 20 mM HEPES, 200 mM NaCl, pH 7, using tangential flow filtration. The retentate was then passed through a Mustang Q membrane  and sterile filtered. For long term storage, purified 8MTT was made 10 % glycerol, aliquoted and frozen at −20 °C, or −70 °C.Frozen cell paste was suspended in 20 mM Tris pH 7.0 (20 mL/ g paste) and allowed to thaw while stirring. Cells were broken in a Panda GEA homogenizer with a single pass at 16,000 psi. The whole cell lysate was clarified by centrifugation at 22,000 × sEC-HPLC was performed using a Waters Alliance system and a Sepax sEC-150 column , equilibrated with PBS + 0.02 % sodium azide and run at 1 mL/min. Detection was at 280 nm. Primary amine concentrations were determined using trinitrobenzene sulfonic acid (TNBS) [SDS-PAGE was performed with 4–20 % bis-Tris gels (Genscript) followed by protein staining/destaining. d (TNBS) . Lf assa™ 1000 . The protein was reduced with dithiothreitol, alkylated with iodoacetamide and digested with a proteomics-grade trypsin  for 24 h at room temperature using a 1:100 trypsin/substrate ratio. The initial identification of tryptic fragments was carried out by searching the entire complement of peptide ions for the monoisotopic masses falling within 15 ppm of the values that were calculated based on the sequence. The peptide ion identities were then confirmed based on the fragmentation patterns generated by collision-induced dissociation (MS/MS measurements). The details are provided in MS characterization of the purified protein was carried out both at the intact-mass level and the peptide map level. The former was carried out under near-native conditions, following the protein transfer to 150 mM ammonium acetate, pH 7.1 via extensive buffer exchange using an Amicon Ultra-4 30 kDa cutoff centrifugal filter . A SolariX 7  Fourier transform ion cyclotron resonance MS equipped with a conventional ESI source and a 7 T superconducting magnet was used for the intact-mass measurements. The peptide mapping was carried out using an Orbitrap Fusion  MS with an integrated front-end reversed-phase (C18) NanoLC system, EASY-nLCPeptide P0 (AAGGGAAAAKPEESKKEEAK) is derived from an immunogenic region of ribosomal protein P0 of Rhipicephalus spp. that is being evaluated as a broad-spectrum α-tick vaccine [Amblyomma mixtum. P0 was synthesized with an N-terminal cysteine and prepared to > 98 % purity by Peptide 2.0 . 8MTT and TTxd were reacted with a 50-fold molar excess of the maleimide reagent GMBS in 0.1 M HEPES, pH 7.2. At 1 hr, the solution was desalted with an Amicon Ultra 30 kDa cutoff centrifugal device using PBS, pH 6.8. Cysteine-pep tide:maleimide-protein were combined at a 40:1 M ratio at pH 7.2. Following an overnight incubation at 4 °C, unconjugated peptide was removed by extensive dialysis against PBS using a membrane with a 7 kDa cutoff. The product was concentrated using an Amicon device and sterile filtered. PRP polysaccharide  was aminated using CDAP cyanylation reagent, essentially as described [Tetanus toxoid  was fractionated using Super-dex200 size exclusion chromatography to prepare a monomeric fraction.  vaccine . A chemiescribed . In brieescribed  and a reescribed , with a escribed  with rib® such that each dose would contain 1 mg aluminum adjuvant in 100 μl of PBS. Two mouse strains are used; CD-1 mice for the vaccine and polysaccharide immunizations and Balb/c mice which were used for PO immunizations, based upon prior use of this mouse strain with the PO antigen. PO-8MTT and P0-TTxd Immunizations P0 peptide-8MTT and P0 peptide-TTxd conjugates were evaluated in Balb/c mice. For the P0 conjugate study, groups of 8 Balb/c female mice were immunized subcutaneously with 2.5 μg of conjugate (based on protein) on days 0 and 14. Sera from retro-orbital bleeds were taken on days −1 and 28. ELISA plates were coated with P0-BSA, and sera analyzed for anti-P0 IgG (described below). 8MTT, TTxd, PRP-8MTT and PRP-TTxd Immunizations 8MTT, TTxd, PRP-8MTT, PRP-TTxd, or PRP alone were evaluated in ICR mice. For the 8MTT and TTxd and PRP-8MTT and PRP-TTxd studies, groups of 8 ICR female mice were immunized subcutaneously with 0.5 μg or 5 μg of 8MTT, TTxd, PRP-8MTT or PRP-TTxd on days 0, 14 and 110. Sera from retro-orbital bleeds taken on days −1, 28, 44, and 120. Note, 0.5 μg or 5 μg of PRP-8MTT or PRP-TTxd immunizations contained 0.25 μg and 2.5 μg of PRP, respectively. ELISA plates were coated with either 8MTT or TTxd and sera analyzed for anti-tetanus IgG, while ELISA plates were plated with streptavidin/biotinylated PRP, and sera assayed for anti-PRP IgG (described below).Antigens were prepared by combining with AlhydrogelImmunizations was carried out by Noble Life Sciences, German-town, MD in compliance with the current version of the following 1) Animal Welfare Act Regulations (9 CFR); 2) U.S. Public Health Service Office of Laboratory Animal Welfare (OLAW) Policy on Humane Care and Use of Laboratory Animals; 3) Guide for the Care and Use of Laboratory Animals ; and 4) AALAC accreditation.Detection of antibodies reactive with 8MTT or TTxd: Greiner High Bind immunoassay plates were coated with 100 μl per well of 8MTT or TTxd at 1 mg/ ml in PBS. After overnight incubation at room temperature, plates were washed once with 300 μl of PBS and 180 μl/well of Casein Blocking Solution (Surmodics) was added to each well and the plates were incubated for 60 min at room temperature. Plates were washed 3X with PBS containing 0.05 % Tween 20 (PBS-T) and serum from each mouse (diluted in PBS-T from 1:100 to 1:1562500) was added to duplicate wells. The plates were incubated for 60 min at room temperature and washed as noted above. One hundred μl of goat anti-mouse IgG Fc – HRP (Southern Biotechnologies), diluted 1:20000 in PBS-T, was added to each well. The plates were again incubated for 60 min at room temperature and washed with PBS-T. One hundred μL of TMB substrate (Moss Substrates) was added per well and plates incubated in the dark for 15 min. Reactions were stopped by addition of 100 μl of 0.5 N HCl per well and the absorbance at 450 nm were determined using a Molecular Devices Vmax plate reader. The results of 8MTT or TTxd immunizations are shown in Detection of antibodies reactive with P0: anti-peptide P0 antibodies were detected using the above protocol for the 8MTT/TTxd, but with the ELISA plates coated with P0-BSA. Following an overnight incubation, the assay was continued as described above for 8MTT/TTxd assay. The results of P0-8MTT or P0-TTxd immunizations are shown in Detection of antibodies reactive with PRP: Greiner High Bind immunoassay plates were coated with 100 μl per well of streptavidin solution (1 mg/ml). After overnight incubation at room temperature the plates were washed 3X with PBS-T and 100 μl of biotinylated PRP  was added to all wells. Following a 60 min incubation, the assay was continued as described above for 8MTT/TTxd assay. The results of PRP-8MTT or PRP-TTxd immunizations are shown in For each assay, titers were determined using the formula: EXP(((LN(B)-LN(A))/(D-C))*(E-C) + LN(A)), where; E = Titer Point  assuming normal distribution vaccine experiments, using GraphPad Prism 7.Data were analyzed for statistical significance, using a Supplementary Material


In this work, two PdRhNi/C, Pd/C, Rh/C and Ni/C electrocatalysts are synthesized via a one-step impregnation process at ambient pressure and temperature. The catalysts are then applied for ethanol electrooxidation reaction (EOR). Electrochemical evaluation is performed using cyclic voltammetry (CV) and chronoamperometry (CA). Physiochemical characterization is pursued using X-ray diffraction (XRD), transmission electron microscope (TEM), energy-dispersive X-ray spectroscopy (EDX) and X-ray photoelectron spectroscopy (XPS). Unlike Pd/C, the prepared Rh/C and Ni/C do not show any activity for (EOR). The followed protocol produces alloyed dispersed PdRhNi nanoparticles of 3 nm in size. However, the PdRhNi/C samples underperform the monometallic Pd/C, even though the Ni or Rh individual addition to it enhances its activity, as reported in the literature herein. The exact reasons for the low PdRhNi performance are not fully understood. However, a reasonable reference can be given about the lower Pd surface coverage on both PdRhNi samples according to the XPS and EDX results. Furthermore, adding both Rh and Ni to Pd exercises compressive strain on the Pd lattice, noted by the PdRhNi XRD peak shift to higher angles.Direct ethanol fuel cells (DEFCs) need newly designed novel affordable catalysts for commercialization. Additionally, unlike bimetallic systems, trimetallic catalytic systems are not extensively investigated in terms of their catalytic potential toward redox reactions in fuel cells. Furthermore, the Rh potential to break the ethanol rigid C-C bond at low applied potentials, and therefore enhance the DEFC efficiency and CO Furth results ,29.Heightening the impact of a second cocatalyst metal by adding a third metal is at the beginning of laboratory investigation ,33,34,352 (99 wt.%), AgNO3 (97 wt.%) and RhCl3 (99% wt.%) were purchased from Sigma-Aldrich . The solid metal precursors were converted into liquid solutions and stored for frequent use. NABH4 (98 wt.%), KBr (95% wt.%), ethanol (100% absolute), KOH (85 wt.%) and 2-propanol (99%) were also purchased from Sigma-Aldrich . These chemicals were used as received.Vulcan carbon XC72R was procured from Cabot Corp . Metal salts of PdCl4Rh2Ni1/C following their associated Pd:Rh:Ni nominal atomic ratios of 1:1:1 and 4:2:1. Monometallic samples of Pd/C, Rh/C, and Ni/C were prepared for comparison purposes. The catalyst metal loading was fixed at 12 wt.% for all samples. v/v). Prior to that, KBr was added to work as a capping agent and the KBr/Me atomic ratio was 1.5. Then, Vulcan carbon (132 mg each sample) was added into the mixture and sonicated for 30 min. Thereafter, the mixture was transferred to a magnetic stirrer plate. Following this, the NaBH4 solution  was added to the mixture in one portion. The whole mixture was kept under stirring for 30 min, and after that it was left for 24 h for particle precipitation. The mixture was then washed with deionized water and acetone under vacuum filtration. Then, the wet catalyst powder was dried at 120 °C in a vacuum oven overnight.The electrocatalysts were prepared using a quick borohydride reduction following the works of ,37,38 wi2H5OH) solutions. The working electrode was a mirror-shining glassy carbon  one painted with the respective catalyst slurry. The counter and reference electrodes were a Pt wire and Ag/AgCl (sat ’KCl), respectively. To prepare the catalyst slurry, 5 mg of the respective catalyst powder was sonicated in a mixture of 2 mL of ethanol and 25 µL of Nafion ®117 (5 wt.%) for 1 h. A total of 5 µL of the slurry was micro-pipetted on the GCE electrode and left a few minutes to dry; this step was repeated 5 times. The total metal loading on the working electrode for all samples was 85 µg/cm2. The CV scan rate was 50 mV/s, and the 20th cycle was selected for analysis. CA was performed at −0.3 V and +0.1 V vs. NHE.The catalysts were applied for alkaline ethanol oxidation reaction (EOR). The catalytic performance was evaluated by means of cyclic voltammetry (CV) and chronoamperometry (CA). These tests were undertaken in a magnetically stirred 3-electrode cell containing either 1 M KOH or 1 M  with an aluminum X-ray source (72 W). The energy step and step size (to record high-resolution peaks) were 50 eV, 0.1 eV. Argon ions were used to neutralize the charge and CasaXPS (Shirley background and Scofield cross sections) was used to analyze the data with −0.6 energy dependence.X-ray diffraction (XRD) was applied to investigate the catalyst structure using a Bruker D2 Phaser  equipped with Cu Kα radiation at 30 kV and 10 mA. The scan rate applied was 12°/min. The chemical composition was investigated using an energy-dispersive X-ray spectroscopy (EDX) detector attached to a Jeol JSM 6010LA SEM microscope  operating at 20 kV. The catalyst morphology was analyzed using a Philips C100 TEM microscope  with a LaB6 filament operating at 100 kV. X-ray photoelectron spectroscopy (XPS) was performed to analyze surface chemistry. The equipment used was a Thermo-Scientific K-AlphaThe raw research data of this work are available at DOI: 10.17632/hp22gbrn9d.1.4Rh2Ni1/C ink deposited on GCE. The obtained current was divided by the total metal load in the respective catalyst. The Rh voltammogram showed a low potential hydrogen adsorption/absorption peak around −600 mV vs. NHE. A similar peak was also noted with both PdRhNi samples. A little oxidation of Rh occurred towards the forward scan end that was reversed in the backward scan around −100 mV. Similar Rh voltammograms were also reported [2. A minor Ni reduction peak was noted at a much lower potential (−100 mV), which is suggestive of multiple Ni oxidation states’ presence on the catalyst surface. A comparable current rise was noted with the PdRhNi sample in the forward and backward scans. Unlike Rh, the Pd voltammogram did not show a Hads/abs peak around −600 mV, and instead, a OH− adsorption peak was seen around −300 mV. Above 0 mV, the PdO formation occurred incrementally with the applied potential. The PdO was reduced back in the reverse scan around −150 mV. The trimetallic voltammograms shared the features of the three metals, which were Rh Hads/abs, Pd oxidation/reduction and Ni oxidation/reduction following their atomic concentrations.reported . The Ni − species, the adsorbed ethoxy species was oxidized and removed from the Pd active site, increasing the drawn current. This trend continued until around +50 mV, after which the current declined due to the gradual reduction of Pd active sites. In the backward scan, the PdO was reduced back, as shown in 4Rh2Ni1/C. The PdRhNi/C voltammogram shows an intense Ni oxidation/reduction around 600 mV. The effect of Ni added to Pd/C has been repeatedly been reported as beneficial for ethanol oxidation reaction (EOR) [After adding ethanol B, signifon (EOR) ,40,41. Hon (EOR)  found thon (EOR)  prepared4Rh2Ni1/C. The consistent 25° broad peak is attributed to the graphitic Vulcan carbon. The Pd/C pattern shows four peaks of Pd (111), (200), (220) and (311) at approximately 40°, 46°, 68° and 82.5°, respectively . The Rh/C pattern shows only the Rh (111) facet at 41.5°, while other peaks are absent (Rh PDF#05-685). The Ni one shows peaks of Ni(OH)2 at 35° and 60° and a peak of NiO at 44.5°, which were also reported in [4Rh2Ni1/C. The consistent 25° broad peak is attributed to the graphitic Vulcan carbon. The Pd/C pattern shows four peaks of Pd (111), (200), (220) and (311) at approximately 40°, 46°, 68° and 82.5°, respectively . The Rh/C pattern shows only the Rh (111) facet at 41.5°, while other peaks are absent (Rh PDF#05-685). The Ni one shows peaks of Ni(OH)2 at 35° and 60° and a peak of NiO at 44.5°. It is noteworthy that the C peak is more intense than the Rh and Ni ones on Rh/C and Ni/C, respectively, which signifies a weak crystallinity of both Rh and Ni. In this context, it is noteworthy that in previous works, replacing Rh with Au or Ag in PdAuNi/C and PdAgNi/C enhanced the Pd EOR activity [orted in . It is norted in ,43. Unliactivity ,43. Thisτ, nm) is estimated using Scherrer’s Equation (1):K is a constant and equals 0.94, λ is the wavelength (0.154 nm for Cu kα), β is the full width at half maximum of the peak in radians and θ is the half diffraction angle. Utilizing the (111) peak details, the crystallite sizes of Pd, Pd4Rh2Ni1 and PdRhNi are found to be 4 nm, 3.77 nm and 2.46 nm, respectively. It is noteworthy that adding Rh and Ni in Pd4Rh2Ni1 decreased the particle size and that further increasing the Rh and Ni content in PdRhNi/C further decreases the crystallite size. 4Rh2Ni1/C , (200), (220) and (311) facets that are located between the monometallic Pd and the Rh one. The peak shift to higher diffraction angles is suggestive of alloy formation between Pd and Rh. Furthermore, the trimetallic patterns do not show single Ni or Ni oxide peaks, which implies the incorporation of Ni species into the Pd lattice. Moreover, the trimetallic peaks are generally broader than the monometallic Pd/C ones. That is probably due to the smaller crystal size of trimetallic samples. The crystal size  was measured by XPS and EDX (10 kV) for Pd/C, Pd4Rh2Ni1/C and PdRhNi/C.4Rh2Ni1/C and PdRhNi/C. The Pd 3d spectrum of Pd/C , the Rh and Pd concentrations increased 0.23 at.% and 0.08%, respectively, but the Ni one decreased 0.12 at.%. This could be explained by the catalyst surface being overall rich in Rh, while Ni tends to segregate into the higher surface layers. More interestingly, Two samples of trimetallic PdRhNi/C nanoparticles, along with their monometallic Pd/C, Rh/C and Ni/C counterparts, were successfully prepared via a scalable borohydride reduction route. Neither Rh or Ni present sensible activity toward EOR, unlike Pd. Unfortunately, the trimetallic samples underperformed their monometallic Pd/C counterpart on EOR. Although adding Ni or Rh to Pd/C is extensively reported to enhance its EOR activity, their simultaneous addition using the current method gave the opposite outcome. The exact solid reasons for such lower performance are unknown, but some other findings could be drawn up. Neither Rh/C nor Ni/C could activate EOR at the current potential window. Their addition seems to have reduced the Pd surface coverage, reducing the EOR active surface area on the trimetallic samples. Moreover, the Ni and Rh addition shifted the Pd 3d binding energy to higher values, increasing their adsorption strength of EOR intermediates and reducing their CO-like species tolerance.


Chemotherapy with synthetic drugs is the principal approach for toxoplasmosis treatment; however, recent studies reported the limitations and adverse side effects of these chemical drugs.in vitro and in vivo effects of Curcuma longa essential oil (CLE) against the Toxoplasma gondii RH strain.This study aimed to examine the in vitro effect of different concentrations of CLE on T. gondii tachyzoites was assessed by cell viability assay. Flow cytometry and apoptosis analysis were performed, and nitric oxide production by CLE was also evaluated in tachyzoites. BALB/c mice were orally treated with various doses  of CLE for 2 weeks. After the induction of acute toxoplasmosis in the mice, their survival rate and the mean number of peritoneal parasites were checked. The hepatic level of antioxidant enzymes and oxidative stress markers was evaluated by commercial kits. The mRNA expression level of proinflammatory cytokines such as interleukin 1-beta (IL-1β) and interferon-gamma (IFN-γ) was evaluated by quantitative real-time PCR.The T. gondii tachyzoites. It increased the survival rate (ninth day) and reduced the mean number of peritoneal tachyzoites in the infected mice. CLE dependently increased (p < 0.01) the number of necrotic and apoptotic cells as well as NO production. CLE significantly (p < 0.05) reduced the hepatic level of oxidative stress markers but increased (p < 0.001) the antioxidant enzymes and proinflammatory cytokines in the infected mice, with no important toxicity for vital organs.CLE, especially at 50 µg/ml, showed potent inhibitory effects on in vitro inhibitory effects of CLE on T. gondii tachyzoites. The results also exhibited promising in vivo effects of CLE. CLE improved the survival rate of infected mice and reduced the parasite number in them. Although the mechanisms of action of CLE are not clear, our study demonstrated its beneficial effects on acute toxoplasmosis by strengthening the immune system and reducing inflammation and oxidative stress. Still, more studies are required to confirm these results.The findings of this survey revealed the significant  Toxoplasma gondii parasite  are strongly recommended . ChemothToxoplasma effects of various medicinal herbs, e.g., Zataria multiflora, Eurycoma longifolia, Zingiber officinale, Myrtus communis, Berberis vulgaris, Sophora flavescens, Nigella sativa, Piper betle, and Allium cepa, have been investigated  is a well-known spice usually used in foods and traditional medicine  against the T. gondii RH strain.Recent studies showed that features . Turmerifeatures . In addifeatures . Based oThe rhizome of turmeric was purchased in June 2022 from a store in Kerman province, Iran. After identification by a botanist, a voucher example was kept at the Herbarium section of Pharmaceutics Research Center, Kerman, Iran (no. 56658).For the experiment, 250 g of dried and powdered rhizome was used for hydro-distillation technique by the Clevenger-type apparatus. After dehydration of the attained essential oil with anhydrous sodium sulfate, it was stored at 4°C until use . To studT. gondii RH tachyzoites prepared by the Kerman University of Medical Science, Kerman, Iran, were used. Parasites were intraperitoneally (IP) passaged in BALB/c mice. Then, the collected peritoneal tachyzoites were washed with PBS (pH 7.4) and adjusted into 1 × 104 tachyzoites/ml experiments.In this study, 2.J774-A1 cells were prepared from Pasture Institute, Iran, and cultured in RPMI-1640 medium supplemented with 10% inactivated fetal bovine serum (FBS) and pen/strep (100 units/ml) and maintained at 37°C with 5% COin vitro inhibitory effects of CLE on T. gondii tachyzoites, 300 µl of tachyzoites (1 × 106 cells/ml) was exposed to CLE and atovaquone (12.5–50 µg/ml) for 0.5–3 h at 37°C, whereas the selection of these concentrations was based on the primary experiments. Then, 50 µl of MTT -2,5-diphenyltetrazolium bromide, 5 mg/ml) was put in the tested tubes and kept warm at 37°C for 4 h with 5% CO2. Dimethyl sulfoxide (DMSO) was added to the tubes to stop the reaction. Finally, the optical density (OD) of the samples was recorded at 575 nm by an ELISA reader   . The neg6 cells/ml) were exposed to CLE (12.5–50 µg/ml) for 24 h, and the treated parasites were washed with culture medium and phosphate-buffered saline. Then Annexin buffer and FITC-V Annexin conjugate were added. After the addition of propidium iodine solution and incubation for 10 min in a dark place, the level of apoptotic and necrotic cells was determined by a Facscalibur™ Flow Cytometer BD.In order to quantitatively determine the amount of apoptosis of cells, an apoptosis detection kit  was used according to the manufacturer’s instructions; the cells and parasites were stained by the two factors FITC-V Annexin and propidium iodide (PI) and were analyzed using the Sysmex flow cytometry device. Briefly, tachyzoites (1 × 105) were planted in a 96-well cell plate and kept warm at 37°C for 24 h. Then, cells in each well were exposed to 5 × 105 tachyzoites for 24 h. After the supernatant was discarded, artifacts were removed by washing with sterile PBS. Next, the cells were infected by T. gondii and exposed to CLE and atovaquone (12.5–50 µg/ml) for 48 h. The treated cells were smeared and stained via Giemsa and then checked by a light microscope to evaluate the infectivity rate and mean number of parasites in 100 examined cells. The 50% inhibitory concentration (IC50) values were also calculated by Probit test in SPSS software ver. 25.0  were exposed to CLE (12.5–50 µg/ml) for 48 h. Then, supernatants were collected and transferred into a 96-well microplate. Subsequently, Griess reagents A and B (60 μl) were added to wells, with the absorbance of the plates at 540 nm in an ELISA reader (BioTek-ELx800). Non-treated cells and cells treated with lipopolysaccharide (LPS) + interferon-gamma (IFN-γ) were considered negative and positive controls, respectively.The effect of CLE on nitric oxide (NO) creation in macrophage cells was studied using the NO colorimetric kit  according to the manufacturer’s instructions. Cells  were included in this survey. The mice were housed in optimum conditions and heat, with The Sirjan Faculty of Medical Sciences approved this study (No. IR.SIRUMS.REC.1400.007).−1·day−1, and CLE 1.25, 2.5, and 5 mg·kg−1·day−1 for 14 days; the selection of these concentrations was based on the primary experiments. Fifteen days after starting the treatment, the mice in all groups were infected intraperitoneally with parasite solution (100 µl of tachyzoites (1 × 104 tachyzoites/ml) mixed with 100 µl of sterile normal saline).Sixty mice were allocated to five groups (12 mice each), namely orally administrated normal saline, atovaquone 100 mg·kgThe treated animals were daily observed, and the level of survival was noted in the animals in each group. On the third day, after the peritoneal fluids of the tested mice were collected, the number of peritoneal parasites of the mice was microscopically checked .On the third day post-toxoplasmosis induction, six mice from each group were euthanized  to study oxidative stress factors, antioxidant enzymes, and proinflammatory cytokines. The hepatic levels of lipid peroxidation (LPO) and NO in liver homogenates were determined by a biodiagnostic commercial kit  based on the kits’ instructions.The levels of some enzymes involved in antioxidant mechanisms such as glutathione peroxidase (GPx) and superoxide dismutase (SOD) enzyme activity were evaluated using commercial kits  based on the kits’ instructions.via a Mouse IFN-gamma and IL-1 beta ELISA kit  according to the manufacturer’s protocols.The serum level of proinflammatory cytokines, e.g., IFN-γ and interleukin 1-beta (IL-1β), was measured via an RNeasy tissue kit  as explained by the manufacturer’s instructions. The complementary DNA (cDNA) was synthesized by cDNA Synthesis Kits  according to the manufacturer’s guidelines. Sequence primers applied in real-time PCR are shown in −ΔΔCT method via the iQTM5 optical system software  was used to measure the expression level. The housekeeping gene and normalization control were β-actin .A total of 24 healthy mice in four groups were treated orally with normal saline and CLE at doses of 1.25-5 mg·kgntil use . Then, tpost-hoc test was applied to compare the differences among the groups.The data were represented as means ± standard deviation and were analyzed in SPSS 22.0 . One-way analysis of variance (ANOVA) with Tukey’s The yield of yellow essential oil was 2.76% (v/w). As shown in in vitro inhibitory effects on the growth rate of parasites after 0.5–3 h of incubation when compared with the control group. CLE at 25 and 50 µg/ml caused 100% mortality in tachyzoites after 3 and 2 h of incubation, respectively.As depicted in The flow cytometry and apoptosis analysis exhibited that this essential oil dose dependently increased (p < 0.01) the number of necrotic (from 0.25% to 10.1%) and apoptotic cells (from 1.6% to 31.3%), respectively Figure 2T. gondii-infected Vero cells was observed at 50 µg/ml, where it significantly declined (p < 0.001) by 27.3%. Subsequently, at 25 and 50 µg/ml, it reduced (p < 0.05) infectivity to 76.3% and 50.3%, respectively. The intracellular replication of parasites in infected cells considerably decreased (p < 0.001) after treatment with various concentrations of CLE  by 72.4%, 48.5%, and 31.1% after receiving CLE at 1.25, 2.5, and 5 mg·kg−1·day−1, respectively. However, the rate of peritoneal parasites in mice receiving atovaquone 100 mg/kg declined to 32.3%  and NO in the animals, in the infected mice pre-treated with CLE for 2 weeks, oxidative stress levels were significantly (p < 0.05) controlled by reducing the levels of MDA and NO. Toxoplasmosis also decreased the levels of GPx and SOD, whereas in the infected mice pre-treated with CLE for 2 weeks, the levels of GPx and SOD significantly (p < 0.05) increased compared with the control group  increased levels of IFN-γ and IL-1β cytokines on the third day post-infection Figure 7−ΔΔCT method in real-time PCR, the T. gondii-infected mice pre-treated with CLE for 2 weeks showed a significant (p < 0.001) upregulation of IL-1β and IFN-γ mRNA genes  following treatment with various concentrations of CLE. Based on the in vivo assay, the infected mice pre-treated with CLE at 1.25, 2.5, and 5 mg·kg−1·day−1 for 2 weeks showed an increased survival rate and fewer peritoneal parasites.In this study, CLE demonstrated significant (p < 0.001) Echinococcus granulosus protoscoleces; at 50 and 100 μl/ml, it showed the best efficiency  reported that CLE has potent proto-scolicidal effects on ficiency . Magalhãoduction . Recentlvamisole .Based on our findings, the main components were β-turmerone (21.8%), Ar-turmerone (14.7%), and α-turmerone (12.4). In previous studies, the principal constituents of CLE were Ar-turmerone and turmerone . Howeverin vitro anti-Toxoplasma activity of CLE is due to the existence of oxygenated sesquiterpenes such as turmerone and Ar-turmerone.As for the antimicrobial mechanisms of action of oxygenated sesquiterpenes such as turmerone and Ar-turmerone, previous studies have reported that these ingredients display their antimicrobial mechanisms through cell wall destruction and by disrupting the vital intracellular. Consequently, it can be suggested that the considerable S-transferase (GSH), catalase (CAT), SOD, and GPx .FE: Study design and experiments. HM: critical review and validation. YR: Writing the draft and validation. All authors contributed to the article and approved the submitted version.